Research ArticleTNF-120572 Induced the Enhanced Apoptosis of Mesenchymal StemCells in Ankylosing Spondylitis by Overexpressing TRAIL-R2

Zhenhua Liu1 Liangbin Gao1 Peng Wang1 Zhongyu Xie1 Shuizhong Cen1 Yuxi Li1

Xiaohua Wu2 Le Wang3 Hongjun Su2 Wen Deng2 Shan Wang2 Deng Li1 Jinteng Li1

Yi Ouyang1 Yanfeng Wu2 and Huiyong Shen1

1Department of Orthopedics Sun Yat-sen Memorial Hospital Sun Yat-sen University 107 Yan Jiang Road WestGuangzhou 510120 China2Center for Biotherapy Sun Yat-sen Memorial Hospital Sun Yat-sen University 107 Yan Jiang Road West Guangzhou 510120 China3Institute of Orthopaedics and TraumatologyTheThird Affiliated Hospital GuangzhouMedical University Guangzhou 510150 China

Correspondence should be addressed to Yanfeng Wu wuyanfengcn126com and Huiyong Shen shenhuiyongaliyuncom

Received 22 July 2016 Accepted 4 December 2016 Published 15 January 2017

Academic Editor Silvia Brunelli

Copyright copy 2017 Zhenhua Liu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Ankylosing spondylitis (AS) is an autoimmune disease with unknown etiology Dysregulated mesenchymal stem cells (MSCs)apoptosis may contribute to the pathogenesis of autoimmune diseases However apoptosis of MSCs from patients with AS(ASMSCs) has not been investigated yet The present study aims to assess the apoptosis of bone marrow-derived ASMSCs andto investigate the underlying mechanisms of altered ASMSCs apoptosis We successfully induced the apoptosis of ASMSCs andMSCs from healthy donors (HDMSCs) using the combination of tumor necrosis factor alpha (TNF-120572) and cycloheximide (CHX)We found that ASMSCs treated with TNF-120572 and CHX showed higher apoptosis levels compared to HDMSCs During apoptosisASMSCs expressed significantly more TRAIL-R2 which activated both the death receptor pathway and mitochondria pathwayby increasing the expression of FADD cleaved caspase-8 cytosolic cytochrome C and cleaved caspase-3 Inhibiting TRAIL-R2expression using shRNA eliminated the apoptosis differences between HDMSCs and ASMSCs by partially reducing ASMSCsapoptosis butminimally affecting that of HDMSCs Furthermore the expression of FADD cleaved caspase-8 cytosolic cytochromeC and cleaved caspase-3 were comparable between HDMSCs and ASMSCs after TRAIL-R2 inhibitionThese results indicated thatincreased TRAIL-R2 expression results in enhanced ASMSCs apoptosis and may contribute to AS pathogenesis

1 Introduction

Ankylosing spondylitis (AS) is an agnogenic autoimmunedisease characterized by two central pathological changeschronic inflammation and pathological osteogenesis [1]Recent studies have focused on the pathogenesis of AS andseveral risk factors have been reported to be significantlycorrelated with AS including human leukocyte antigen B27(HLA-B27) genetic susceptibility [2] gene polymorphisms[3] environmental triggers [4ndash6] and mechanical stress [7]However none of these risk factors are solely responsible forthe pathogenesis of AS

Mesenchymal stem cells (MSCs) are a heterogeneouspopulation of cells that can be isolated from many tissues

including bone marrow MSCs play a vital role in healthand disease for their immunomodulation potentials self-renewal and multipotent differentiation capacities [8 9]Recent studies have demonstrated that MSCs dysfunctioncontributes to many diseases [10 11] Consistent with thesereports our previous study observed an increased osteogenicdifferentiation capacity of MSCs from AS patients (ASMSCs)compared with MSCs from healthy donors (HDMSCs) [12]whichmay partly account for the pathological osteogenesis inAS However it remains largely unknown whether ASMSCsare involved in the chronic inflammation of AS

Tumor necrosis factor alpha (TNF-120572) is a pleiotropiccytokine involved in a variety of physiological and patho-logical processes ranging from inflammatory cytokine

HindawiStem Cells InternationalVolume 2017 Article ID 4521324 14 pageshttpsdoiorg10115520174521324

2 Stem Cells International

production to cell survival and apoptosis [13] Due to itsability to stimulate inflammation overproduction of TNF-120572has been suggested to contribute to the pathogenesis of AS[14] This hypothesis has been confirmed by the therapeuticeffect observed with TNF-120572 blockers in AS treatment [15 16]In addition to triggering a proinflammatory response TNF-120572also can induce either cell survival or apoptosis Proteininhibitors (such as cycloheximide CHX) can direct TNF-120572to induce the apoptosis of mammalian cells [17] Apoptosisis a normal physiological cell suicide process that plays acrucial role in homeostasis A large quantity of evidencesupports the idea that the defective apoptosis of immunecells can lead to autoimmune diseases [18] Furthermore arecent study indicated that enhanced MSCs apoptosis mayalso contribute to the pathogenesis of autoimmune diseases[19] Thus studying the apoptosis of MSCs may improveour understanding of the pathogenesis of autoimmunediseases including AS However TNF-120572-induced apoptosisof ASMSCs has not been investigated yet

TNF-related apoptosis-inducing ligand receptor 2(TRAIL-R2) is a specific cell surface receptor that belongs tothe TNF receptor superfamily [20] TRAIL-R2 is expressedin many cell types including MSCs [21 22] Upon bindingwith its specific ligand TRAIL TRAIL-R2 initiates therecruitment of Fas-associated protein with death domain(FADD) and signaling molecules (such as caspase-8) toform the death-inducing signaling complex (DISC) It thenactivates downstream caspases (such as caspase-3) and leadsto apoptosis [23]

In this study we investigated the apoptosis of ASMSCsand a possible underlying mechanism Our results showedthat ASMSCsweremore susceptible to TNF-120572CHX-inducedapoptosis than HDMSCs due to increased TRAIL-R2 expres-sion We speculate that the increased expression of TRAIL-R2 results in the enhanced apoptosis of ASMSCs which maycontribute to AS pathogenesis

2 Materials and Methods

21 Recruitment of AS Patients and Healthy Donors Thisstudy was approved by the ethics committee of Sun Yat-sen Memorial Hospital Sun Yat-sen University GuangzhouChina There were 28 healthy donors and 22 patients withAS (diagnosed according to the New York modified crite-ria [24]) who agreed to participate in this study (detailedcharacteristics of the participants are presented in supple-mental Table 1 in Supplementary Material available online athttpsdoiorg10115520174521324) Written informed con-sent was obtained from each participant To minimize theimpact of therapy all patients discontinued any treatment for2 weeks before bone marrow puncture

22 MSCs Isolation Expansion and Identification MSCswere isolated from the iliac crest bonemarrow of all the studysubjects and purified using density gradient centrifugationas previously described [25] After being resuspended inDulbeccorsquos Modified Eaglersquos Medium (DMEM Gibco USA)containing 10 heat-inactivated fetal bovine serum (FBSSijiqing China) MSCs were seeded in 25 cm2 flasks and

cultured in an incubator with 5 CO2at 37∘C To remove

the suspended cells the medium was replaced 48 hourslater and every 3 days thereafter At 80ndash90 confluence theadherent cells were recovered using 025Trypsin containing053mMEDTA (Gibco USA) and replated in 75 cm2 flasks aspassage 1 MSCs were expanded and used at passages 4 for allexperiments Surface markers of MSCs were detected usingflow cytometry (FCM) which was performed on BD Influxcell sorter (BD USA) CD14-APC CD29-PE CD44-FITCCD45-APC CD105-FITC and HLA DR-PerCP antibodieswere used (all from BD USA)

23 Cell Proliferation Assay MSCs at the same passage wereseeded in 96-well plates in DMEM with 10 FBS Mediumwithout cells served as negative control The proliferationability of MSCs was detected using Cell Counting Kit-8(Dojindo Japan) following the manufacturerrsquos instructions

24 Apoptosis Induction MSCs were separately seeded in 6-well plates at a density of 1 times 105 cellswell in DMEMwith 10FBS After 24 hours the medium was removed and MSCswere washed three times with phosphate-buffered saline(PBS) The same sample of MSCs was separately culturedfor 6 hours with four different types of media fresh DMEMwith 10 FBS (control group) fresh DMEM with 10 FBSand 20 ngmL TNF-120572 (TNF-120572 group) fresh DMEMwith 10FBS and 10 120583gmL CHX (CHX group) and fresh DMEMwith 10 FBS 20 ngmL TNF-120572 and 10 120583gmL CHX (TNF-120572CHX group) After 6 hours MSCs were harvested for theexperiments

25 PE-Annexin V and FCM for the Detection of MSCApoptosis Apoptotic cells were stained using PE-AnnexinV Apoptosis Detection Kit I (BD USA) according to themanufacturerrsquos protocol First the cells were washed withPBS and resuspended in binding buffer Second 100 120583L ofthe solution was transferred to a 5mL culture tube Third5 120583L of PE-Annexin V and 5120583L of 7-AAD were added to thecells The mixture was gently vortexed and incubated for 15minutes at room temperature in the dark Then 400120583L ofbinding buffer was added to each tube and FCM analysis ofapoptosis was performed immediately Data were analyzedusing FlowJo software

26 Proteome Profiler Antibody Array Analysis The expres-sion of apoptosis-related proteins in MSCs was detectedusing the Proteome Profiler Human Apoptosis ArrayKit (ARY099 RampD USA) according to the manufacturerrsquosinstructions Briefly equal amounts of protein fromHDMSCsand ASMSCs were incubated with the membranes overnightat 4∘C Then the membranes were washed and incubatedwith reconstituted Detection Antibody Cocktail for 1 hourAfter that the membranes were washed again and incubatedwith Streptavidin-HRP for 30 minutes Finally the proteinexpressions were detected using Chemi Reagent Mix Theintensity of the arrays was analyzed using VisionWorks LSImage Acquisition and Analysis Software (Analytik Jena AGGerman) The intensity of the repeat spots was averaged andthen standardized using the reference spots

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27 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Total RNA of MSCs was extracted using TRIzolreagent (Invitrogen USA) and transcribed into cDNA usingPrimeScript RTMaster Mix (Takara Japan) qRT-PCR wascarried out in triplicate for each sample on a LightCycler 480PCR System (Roche Switzerland) using SYBR Premix ExTaq (Takara Japan) as previously described [26] GAPDHwas used as the housekeeping gene Primers of relatedgenes are shown in supplemental Table 2 The data werestandardized based on GAPDH expression and the 2minusΔΔCtmethod was used to analyze the relative expression of eachgene

28 Western Blot MSCs were lysed and proteins were quan-tified as described [13] Mitochondrial and cytoplasmic pro-teins from MSCs were isolated using the Cell MitochondriaIsolation Kit (Beyotime China) as described [27] Afterboiling with sample loading buffer (Beyotime China) equalamounts of protein extracts were separated and transferredto polyvinylidene fluoride (PVDF) membranes (MilliporeGermany) The membranes were blocked and incubatedovernight at 4∘C with primary antibodies against 120573-actinpro-caspase-3cleaved caspase-3 FADD TRAIL-R2 cleavedcaspase-8 TNFR1 Fas (all from CST USA) and cytochromeC (RampD USA) After washing three times the membraneswere incubated for 1 hour with horseradish peroxidase-(HRP-) conjugated secondary antibody (Santa Cruz USA)ImmobilonWestern Chemiluminescent HRP Substrate (Mil-lipore Germany) was used to detect the antibody-antigencomplexes

29 Lentivirus Construction and Infection Lentivirusesencoding short hairpin RNA (shRNA) for TRAIL-R2 wereconstructed with a target sequence of 51015840-GCAAATATG-GACAGGACTATA-31015840 (Lv-TRAIL-R2) The sequencefor the negative control shRNA was 51015840-TTCTCC-GAACGTGTCACGTTTC-31015840 (Lv-NC) Lentiviruseswere generated by cotransfecting pGLVH1GFPPuro(Gene Pharma China) and packaging plasmids (pGagPolpRev and pVSV-G) into 293T cells Culture supernatantscontaining lentiviruses were filtered and concentrated 72hours after transfection Lentiviruses (1 times 109 TUmL)and Polybrene (5 120583gmL) were added to the medium andincubated with the MSCs for 24 hours at a multiplicityof infection (MOI) of 50 The related experiments wereconducted after 6 hours of apoptosis induction

210 Statistical Analyses Experiments and analyses wereseparately completed by the different authors listed in thisstudy Statistical analysis was performed using SPSS 220software (Chicago USA) The data are expressed as themean plusmn standard deviation (SD) The differences betweentwo groups were determined by Studentrsquos t-test and thedifferences among three or more groups were determinedby ANOVA P values lt 005 were considered signifi-cant

3 Results

31 HDMSCs and ASMSCsHad the SameMorphology Pheno-type and Proliferation Rate As typicalMSCs bothHDMSCsand ASMSCs were plastic-adherent spindle-shaped cells(Figure 1(a)) that constitutively expressed CD29 CD44 andCD105 but not CD14 CD45 or HLA-DR (Figure 1(b))HDMSCs and ASMSCs proliferated similarly in DMEMwith10 FBS for 1ndash7 days (Figure 1(c)) No differences were foundbetween HDMSCs and ASMSCs in morphology phenotypeor proliferation rate

32 ASMSCs Showed Higher Levels of Apoptosis Compared toHDMSCs after Treatment with TNF-120572CHX Apoptotic cellswere stained with phycoerythrin- (PE-) Annexin V7-amino-actinomycin (7-AAD) and identified by flow cytometry(FCM) Negative Annexin V and 7-AAD staining indicatedcells that were not undergoing apoptosis Annexin V-positiveand 7-AAD-negative cells were considered in the early stageapoptosis Annexin V- and 7-AAD-positive cells were inlate-stage apoptosis or already dead Figure 2 shows thattreatment with TNF-120572CHX caused significant apoptosisof HDMSCs and ASMSCs and that the apoptosis rate ofASMSCs was nearly twice as high as that of HDMSCsTNF-120572 or CHX treatment alone did not induce significantapoptosis within 6 hours similar to the control group andno difference between HDMSCs and ASMSCs was found inthese three groupsThese results demonstrated that ASMSCswere more susceptible to apoptosis compared to HDMSCsafter treatment with TNF-120572CHX Considering that TNF-120572or CHX alone cannot induce significant apoptosis of MSCsin 6 hours only the results from the TNF-120572CHX group andthe control group are reported for the following experiments

33 ASMSCs Showed Increased Protein Expression of CleavedCaspase-3 TRAIL-R2 and FADD and Increased Gene Expres-sion of TRAIL-R2 and FADD during Apoptosis To explorethe mechanism involved in the differences in apoptosisbetween HDMSCs and ASMSCs 35 apoptosis-related pro-teins were detected using the Proteome Profiler HumanApoptosis Array Kit The results showed that ASMSCs hadhigher cleaved caspase-3 expression but lower pro-caspase-3expression than HDMSCs after treatment with TNF-120572CHXwhich further confirmed the increased apoptosis of ASMSCsAdditionally significantly higher expression levels of TRAIL-R2 andFADDwere also found inASMSCs thanHDMSCsNosignificant differences in the expression levels of the remain-ing 31 proteins were found between HDMSCs and ASMSCs(Figure 3(a)) qRT-PCR and western blotting assays wereperformed to confirm the results of the Proteome ProfilerHuman Apoptosis Array analysis The mRNA expressionlevels of TRAIL-R2 and FADD were consistent with theresults of the Proteome Profiler Human Apoptosis Arrayanalysis but the mRNA expression levels of total caspase-3were not No significant difference in total caspase-3 mRNAexpression was found between HDMSCs and ASMSCs eitherwith or without TNF-120572CHX treatment (Figure 3(b)) Theprotein expression levels of cleaved caspase-3 pro-caspase-3 TRAIL-R2 and FADD were consistent with the results

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Figure 1 HDMSCs and ASMSCs shared the same morphology phenotype and proliferation capacity (a) Both HDMSCs and ASMSCs wereplastic-adherent and spindle-shaped (b) Both HDMSCs (119899 = 28) and ASMSCs (119899 = 22) were positive for CD29 CD44 and CD105 andnegative for CD14 CD45 and HLA-DR suggesting a typical MSC phenotype (c) The proliferation capacities of HDMSCs (119899 = 28) andASMSCs (119899 = 22) were similar when cultured in DMEM with 10 FBS from 1 to 7 days Optical density (OD) values are presented as themean plusmn SD

of the Proteome Profiler Human Apoptosis Array analysis(Figure 3(c)) Because caspase-3 initiates apoptosis throughits cleaved form the comparable total caspase-3 mRNA levelsbetween HDMSCs and ASMSCs may suggest that ASMSCswere more susceptible to apoptosis compared to HDMSCsvia an increase in the cleavage of caspase-3 rather than anincrease in the mRNA expression of caspase-3

34 The Death Receptor and Mitochondrial Pathways WereBoth Involved in the Apoptosis of MSCs Apoptosis proceedsmainly by the death receptor (DR) pathway and the mito-chondrial pathway The cleavage of caspase-8 and the releaseof cytochrome C from the mitochondria to the cytoplasmrespectively represent the activation of the DR pathway andmitochondrial pathway To determine whether these two

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Figure 2 ASMSCs weremore susceptible to TNF-120572CHX-induced apoptosis compared toHDMSCs (a) Apoptosis of HDMSCs (119899 = 28) andASMSCs (119899 = 22) was determined using FCM Cells in the upper-right and lower-right quadrants represent apoptotic cells the apoptotic ratewas calculated in (b) After a 6-hour culture only treatment with TNF-120572CHX induced significant MSC apoptosis Treatment with controlmedium or with TNF-120572 or CHX alone did not induce significant apoptosis ASMSCs had a higher rate of apoptosis than HDMSCs in theTNF-120572CHX treatment group Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs and ASMSCs indicates119875 lt 005 for the control group compared with the other HDMSC groups $ indicates 119875 lt 005 for the control group compared with the otherASMSC groups

pathways were involved in the altered apoptosis of ASMSCswe measured the mRNA expression levels of total caspase-8 and total cytochrome C as well as the active forms ofthe corresponding proteins cleaved caspase-8 and cytosolic

cytochromeCOur results showed that themRNAexpressionof both total caspase-8 and cytochrome C increased aftertreatment with TNF-120572CHX ASMSCs expressed signifi-cantly higher mRNA level of total caspase-8 than HDMSCs

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Figure 3 ASMSCs had increased protein expression of cleaved caspase-3 TRAIL-R2 and FADD and increased gene expression of TRAIL-R2 and FADD during apoptosis (a) The Proteome Profiler Human Apoptosis Array Kit was used to determine the expression levels of 35apoptosis-related proteins in HDMSCs (119899 = 6) and ASMSCs (119899 = 6) after treatment with TNF-120572CHX The intensity of each pair of spotsrepresents repeated measures of one protein ASMSCs had lower pro-caspase-3 (a1) expression but higher cleaved caspase-3 (a2) TRAIL-R2(a3) and FADD (a4) expression levels than HDMSCs No significant differences in the expression of the remaining 31 proteins were foundbetween HDMSCs and ASMSCs (b) qRT-PCR was performed to confirm the results of the Proteome ProfilerHuman Apoptosis ArrayTheresults of TRAIL-R2 and FADD in HDMSCs (119899 = 28) and ASMSCs (119899 = 22) were consistent with those of the Proteome Profiler HumanApoptosis Array However total caspase-3 mRNA expression was not different between HDMSCs and ASMSCs (c) Western blot analysiswas performed on proteins isolated from HDMSCs (119899 = 28) and ASMSCs (119899 = 22) to further confirm the results of the Proteome ProfilerHuman Apoptosis Array and the results of both methods were in agreement Values are presented as the mean plusmn SD lowast indicates 119875 lt 005between HDMSCs and ASMSCs

while no difference was found between HDMSCs and ASM-SCs in total cytochrome C (Figure 4(a)) The protein expres-sion of both cytosolic cytochrome C and cleaved caspase-8increased after treatment with TNF-120572CHX and comparedwith HDMSCs ASMSCs exhibited much higher expressionof these two proteins (Figure 4(b)) Because cytochrome Cneeds to be released into the cytoplasm for apoptosis tooccur the mRNA expression level of total cytochrome C wasnot as meaningful as the cytosolic protein expression Wealso detected the gene and protein expression of TNFR1 andFas two important receptors that in addition to TRAIL-R2 interact with FADD However although both the gene

and protein expression levels increased after treatment withTNF-120572CHX no difference in gene or protein expressionwas found between the two groups either with or withoutTNF-120572CHX treatment (Figures 4(a) and 4(b))These resultstogether suggest that both the DR pathway and the mito-chondrial pathway are involved in the altered apoptosis ofASMSCs neither TNFR1 nor Fas accounted for the increasedexpression of FADD in the DR pathway

35 Lentiviruses Encoding shRNA-TRAIL-R2 Partly InhibitedtheApoptosis of ASMSCs butHad a Limited Effect on theApop-tosis of HDMSCs Because ASMSCs express more TRAIL-R2

8 Stem Cells International

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Figure 4 The DR and mitochondrial pathways were both involved in MSC apoptosis (a) qRT-PCR was performed to compare the geneexpressions of total caspase-8 total cytochrome C Fas and TNFR1 between HDMSCs (119899 = 28) and ASMSCs (119899 = 22) ASMSCs expressedmuchhigher gene expression levels of total caspase-8 thanHDMSCs after treatmentwithTNF-120572CHXwhile no differencewas found betweenHDMSCs andASMSCs in total cytochromeC Fas or TNFR1 gene expression levels (b) Awestern blot was performed to compare the proteinexpressions of cleaved caspase-8 cytosolic cytochrome C TNFR1 and Fas betweenHDMSCs (119899 = 28) and ASMSCs (119899 = 22) After treatmentwith TNF-120572CHX ASMSCs had higher cleaved caspase-8 and cytosolic cytochrome C protein expression levels than HDMSCs and similarprotein levels of TNFR1 and Fas as HDMSCs Fold changes are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs andASMSCs

than HDMSCs during apoptosis we constructed Lv-TRAIL-R2 to explore the role that TRAIL-R2 plays in the apopto-sis of ASMSCs GFP-positive cells were observed under aconfocal microscope and the transduction efficiencies werecomparable between HDMSCs and ASMSCs (Figure 5(a))Lv-TRAIL-R2 inhibited both gene and protein expression ofTRAIL-R2 inHDMSCs andASMSCs (Figure 5(b))The FCMresults demonstrated that Lv-TRAIL-R2 could reduce theapoptosis of ASMSCs but had a limited effect on the apoptosisof HDMSCs (Figure 6(a)) qRT-PCR showed that Lv-TRAIL-R2 inhibited the expression of FADD and total caspase-8 inASMSCs but had a limited effect on total caspase-3 and totalcytochrome C Meanwhile Lv-TRAIL-R2 did not affect the

expression of the four related aforementioned genes inHDM-SCs After inhibiting TRAIL-R2 expression no apparent dif-ference in gene expression was found between HDMSCs andASMSCs (Figure 6(b)) Additionally Lv-TRAIL-R2 did notaffect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC inHDMSCs but reducedthem in ASMSCs Furthermore no apparent difference inthe protein expression of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochrome C was found betweenHDMSCs andASMSCs after inhibitingTRAIL-R2 expression(Figure 6(c)) Therefore inhibiting TRAIL-R2 expressionpartly decreased the apoptosis of ASMSCs but had a limitedeffect on HDMSCs

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LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

TNF-120572CHX + TNF-120572CHX + TNF-120572CHX+ TNF-120572CHX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

(b)

Figure 5 The transduction efficiencies of lentiviruses encoding shRNA-TRAIL-R2 were comparable in HDMSCs and ASMSCs (a) GFP-positive cells were observed under a confocal microscope (40x) and the transduction efficiencies were comparable between HDMSCs andASMSCs (b)The lentiviruses encoding shRNA-TRAIL-R2 inhibited both the gene and protein expression of TRAIL-R2 inHDMSCs (119899 = 28)and ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs and ASMSCs amp indicates 119875 lt 005 forthe TNF-120572CHXgroup comparedwith otherHDMSCs groups indicates119875 lt 005 for the TNF-120572CHXgroup comparedwith other ASMSCsgroups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative control sequence Lv-TRAIL-R2 refers to MSCs transduced bylentiviruses encoding TRAIL-R2

4 Discussion

Abnormal MSCs are proposed to be involved in the patho-genesis of AS and a better understanding of ASMSCsdysfunctions could improve the understanding of AS Inthis study ASMSCs had morphologies phenotypes and

proliferation rates that were similar to HDMSCs HoweverASMSCs were more susceptible to TNF-120572CHX-inducedapoptosis compared to HDMSCs ASMSCs expressed higherlevels of TRAIL-R2 which activated both the DR and themitochondrial pathways by increasing the expression levelsof FADD cleaved caspase-8 and cytosolic cytochrome C

10 Stem Cells International

Annexin V-PE

103 535

18627500

032 536

17447688

046 456

15317967

050 046

1519753

018 040

1179825

056 950

30895905

079 868

28526201

586027

14987889

HD

MSC

sA

SMSC

s7-

AA

D

104

104

103

103

102

102

101

101

100

104

103

102

101

100

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

104

103

102

101

100

104

103

102

101

100

10010

410

310

210

110

0

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX +

Lv-NC Lv-TRAIL-R2

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX+

LV-NC LV-TRAIL-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX

+

HDMSCsASMSCs

0

20

40

60

Apop

tosis

rate

()

LV-N

C

LV-N

C

LV-N

C

LV-N

C

0

2

4

6

8

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

Total caspase-3 Total caspase-8

0

2

4

6

Fold

chan

ge

Fold

chan

ge

0

2

4

6

8

0

1

2

3

4

Fold

chan

ge

Total cytochrome CFADD

amp

amp amp amp

amp

lowast

lowastlowast

lowast

lowast lowast

(a)

(b)

Figure 6 Continued

Stem Cells International 11

Con

trol

TNF-120572

CH

X

TNF-120572CHX

HDMSCsASMSCs

Con

trol

TNF-120572

CH

X

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Cleaved caspase-3 Cleaved caspase-8

Cytosoliccytochrome CFADD

0

5

10

15

20

Fold

chan

ge

0

5

10

15

Fold

chan

ge

0

10

20

30

Fold

chan

ge

0

20

40

60

80

Fold

chan

ge

amp amp

amp

lowastlowast

lowast lowast

lowastlowast

lowastlowast

35 kD

19 kD17 kD

43 kD41 kD

28 kD

45 kD

17 kD

45 kD

HDMSCs ASMSCs

+++

+

+

minus

minusminus

minusminus

minus

minus

+++

+

+

minus

minusminus

minusminus

minus

minus

Cleaved caspase-3Cleaved caspase-8

FADD

Actin 1

Actin 2

Cytosolic cytochrome C

(c)

Pro-caspase-3

LV-NCLV-TRAIL-R2

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

CTN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+TN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

Figure 6 Lentiviruses encoding shRNA-TRAIL-R2 partly inhibited the apoptosis of ASMSCs while having a limited effect on the apoptosisof HDMSCs (a) The rate of ASMSC apoptosis (119899 = 22) was decreased after Lv-TRAIL-R2 transduction while no apparent changes werefound for HDMSCs (119899 = 28) (b) In ASMSCs (119899 = 22) Lv-TRAIL-R2 reduced FADD and total caspase-8 expression while having a limitedeffect on total caspase-3 and total cytochrome C gene expression In HDMSCs (119899 = 28) Lv-TRAIL-R2 had a limited effect on the expressionof all four genes (c) Lv-TRAIL-R2 did not affect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC in HDMSCs (119899 = 28) but reduced them in ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 betweenHDMSCs and ASMSCs amp indicates 119875 lt 005 for the TNF-120572CHX group compared with other HDMSC groups indicates 119875 lt 005 forthe TNF-120572CHX group compared with other ASMSC groups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative controlsequence Lv-TRAIL-R2 refers to MSCs transduced by lentiviruses encoding TRAIL-R2

Inhibiting TRAIL-R2 expression using shRNA eliminated thedifference in apoptosis between HDMSCs and ASMSCs bydecreasing the apoptosis of ASMSCs

MSCs aremultipotent stromal cells withmultilineage dif-ferentiation capacities and immunomodulatory propertiesOur previous studies suggested that MSCs are implicated inthe pathogenesis and therapy of AS [12 28] We found thatASMSCs possessed an abnormal osteogenic capacity whichmay contribute to the pathogenesis of AS In addition we alsofound that allogenic intravenous infusion of bone marrow-derived MSCs is feasible and safe in the treatment of ASHowever how the in vivo microenvironment in AS interactswith MSCs remains unknown

AS is a complicated autoimmune disease accompaniedby elevated serum levels of various inflammatory cytokines[14 29] Among these cytokines TNF-120572 is one of the most

important players in the pathogenesis of AS PhysiologicallyTNF-120572 is important for a normal response to inflammationbut the overproduction of TNF-120572 can be detrimental Serumlevels and the sacroiliac joint expression of TNF-120572 wereelevated in AS patients including those in the early stages ofAS [30 31] In addition overproduction of TNF-120572 has beensuggested to be related to disease activity and to account forthe pathogenesis of AS [32] The importance of TNF-120572 hasbeen further confirmed by the efficacy of TNF-120572 inhibitorsin the treatment of AS [16] Due to the importance of TNF-120572in AS a study on the interaction between TNF-120572 and MSCsmay help mimic the in vivo microenvironment experiencedby MSCs in AS Previous studies suggest that TNF-120572 affectsMSCs in various ways TNF-120572 can modulate the immuno-suppressive effects of MSCs and suppress their osteogeniccapacity [33] Additionally TNF-120572 can alter homeostasis

12 Stem Cells International

in MSCs by regulating the balance between apoptosis andsurvival [13] ConverselyMSCs affect the production of TNF-120572 by causing macrophages to adopt an anti-inflammatoryphenotype [34] Both the dysfunction of MSCs and theoverproduction of TNF-120572 may lead to immune disordersA recent study suggested that TNF-120572-induced apoptosis ofMSCs may contribute to the pathogenesis of SLE [19] In ourstudy we found that ASMSCs were more susceptible to TNF-120572-induced apoptosis compared to HDMSCs as evidencedby an increased apoptosis rate and the higher expression ofcleaved caspase-3 protein However how the dysregulationof apoptosis contributes to the pathogenesis of AS remainsuncertain

Apoptosis also defined as programmed cell death iscritical to the pathogenesis of various diseases includingautoimmune diseases [18] The maintenance of immunetolerance and immune homeostasis is largely dependent onapoptosis Defective apoptosis of immune cells may result inthe defective clearance of autoreactive cells or the delayedelimination of autoantigens while excessive apoptosis ofimmunosuppressive cells (such as MSCs) may also lead toimmune hyperactivation Therefore both conditions maycontribute to autoimmune disease [19 35] In patients withAS increased apoptosis of ASMSCs may result in decreasesin cell numbers and immunosuppressive potential eventuallycontributing to autoimmune disorders Moreover becauseMSCs are able to reduce TNF-120572 production increasedMSCs apoptosis may also lead to TNF-120572 overproductionFurthermore TNF-120572 overproduction induces further MSCapoptosis forming a vicious cycle that leads to chronicinflammation in AS Thus increased ASMSC apoptosis maybe critical for AS pathogenesis and further study of themechanism of dysregulated apoptosis in ASMSCs may helpto elucidate the pathogenesis of AS

TRAIL-R2 is a specific cell surface receptor engagedin apoptosis TRAIL-R2-mediated apoptosis has arousedattention due to its role in preferentially killing tumor cellswhile sparing normal cells In our study we observed asignificant increased expression of TRAIL-R2 in ASMSCsafter treatment with TNF-120572CHX Inhibiting TRAIL-R2expression partly decreased the apoptosis of ASMSCs buthad a limited effect on the apoptosis of HDMSCs whichsuggests that TRAIL-R2 overexpression is the main cause ofthe increased apoptosis in ASMSCs However this result isconfusing for two reasons First TRAIL-R2 is reported to bemainly activated by its specific ligand TRAIL [20] Howeverthe results in our study indicated that TRAIL-R2 in MSCscould be activated by TNF-120572CHX as well In fact a similarresult was previously reported but the mechanism remainsunclear [36] Considering the overexpression of TRAIL-R2 can lead to apoptosis even without its ligand [22] wespeculate that TNF-120572maymediate its proapoptotic effects notonly through its own receptor (TNFR1) but also by increasingthe expression of TRAIL-R2 Second bone marrow-derivedadult MSCs are reportedly resistant to TRAIL-R2-mediatedapoptosis [21] However in our study HDMSCs were resis-tant to TRAIL-R2-mediated apoptosis while ASMSCs werenot Multiple mechanisms of TRAIL-R2-mediated apoptosisresistance such as dysregulation of antagonistic receptors

abnormal O-glycosylation and overexpression of the inhi-bition adaptors have been identified [37] Further studiesare needed to illustrate the associated specific mechanismunderlying the difference in resistance of HDMSCs andASMSCs to TRAIL-R2 Additionally based on the findingthat the TRAIL-R2 pathway did not lead to the apoptosis ofnormal cells ASMSCs could be considered abnormal cellsThus it is reasonable to presume that allogenic rather thanautologous MSC transplantation might be preferable for AStreatment

In this study we demonstrated that TRAIL-R2 upregu-lation resulted in enhanced apoptosis of ASMSCs Increasedapoptosis of ASMSCs may decrease the immunosuppressivepotential of these cells and potentially contributing to ASpathogenesis However some limitations remain in thisstudy The questions of how TNF-120572 activates the expressionof TRAIL-R2 why HDMSCs are resistant to TRAIL-R2-mediated apoptosis while ASMSCs are not and whether ourresults can be repeated in vivo remain unanswered Futurestudies should focus on these questions and further elucidatethe mechanism of AS

Competing Interests

The authors have no potential conflict of interests to declare

Authorsrsquo Contributions

Zhenhua Liu LiangbinGao andPengWanghave contributedequally to this work

Acknowledgments

The authors thank Dr Liang Zhang for insightful discussionThis study was supported by the National Natural ScienceFoundation of China (81401850) the Science and Tech-nology Project of Guangdong Province (2015B0202280012015B090903059) and the Engineering Technology ResearchCenter for Comprehensive Diagnosis and Treatment ofAnkylosing Spondylitis of Guangdong Higher EducationInstitutes (GCZX-A1301)

References

[1] M Dougados and D Baeten ldquoSpondyloarthritisrdquo The Lancetvol 377 no 9783 pp 2127ndash2137 2011

[2] P Bowness ldquoHLA-B27rdquo Annual Review of Immunology vol 33pp 29ndash48 2015

[3] J D Reveille A-M Sims P Danoy et al ldquoGenome-wideassociation study of ankylosing spondylitis identifies non-MHCsusceptibility locirdquo Nature Genetics vol 42 no 2 pp 123ndash1272010

[4] J D Taurog J A Richardson J T Croft et al ldquoThe germfreestate prevents development of gut and joint inflammatorydisease in HLA-B27 transgenic ratsrdquo Journal of ExperimentalMedicine vol 180 no 6 pp 2359ndash2364 1994

[5] M-E Costello F Ciccia D Willner et al ldquoBrief Reportintestinal dysbiosis in ankylosing spondylitisrdquo Arthritis andRheumatology vol 67 no 3 pp 686ndash691 2015

Stem Cells International 13

[6] T Rashid and A Ebringer ldquoAnkylosing spondylitis is linked toKlebsiellamdashthe evidencerdquo Clinical Rheumatology vol 26 no 6pp 858ndash864 2007

[7] P Jacques and D McGonagle ldquoThe role of mechanical stress inthe pathogenesis of spondyloarthritis and how to combat itrdquoBest Practice and Research Clinical Rheumatology vol 28 no5 pp 703ndash710 2014

[8] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008

[9] M C Stewart and A A Stewart ldquoMesenchymal stem cellscharacteristics sources and mechanisms of actionrdquo VeterinaryClinics of North AmericamdashEquine Practice vol 27 no 2 pp243ndash261 2011

[10] L Y Sun H Y Zhang X B Feng Y Y Hou LW Lu and L MFan ldquoAbnormality of bone marrow-derived mesenchymal stemcells in patients with systemic lupus erythematosusrdquo Lupus vol16 no 2 pp 121ndash128 2007

[11] J A Perez-Simon S Tabera M E Sarasquete et al ldquoMes-enchymal stem cells are functionally abnormal in patients withimmune thrombocytopenic purpurardquo Cytotherapy vol 11 no6 pp 698ndash705 2009

[12] Z Xie P Wang Y Li et al ldquoImbalance between bone morpho-genetic protein 2 and noggin induces abnormal osteogenic dif-ferentiation of mesenchymal stem cells in ankylosing spondyli-tisrdquo Arthritis and Rheumatology vol 68 no 2 pp 430ndash4402016

[13] D Brenner H Blaser and T W Mak ldquoRegulation of tumournecrosis factor signalling live or let dierdquo Nature ReviewsImmunology vol 15 no 6 pp 362ndash374 2015

[14] J Gratacos A Collado X Filella et al ldquoSerum cytokines(IL-6 TNF-120572 IL-1 120573 and IFN-120574) in ankylosing spondylitis aclose correlation between serum IL-6 and disease activity andseverityrdquo British Journal of Rheumatology vol 33 no 10 pp927ndash931 1994

[15] N Barkham L C Coates H Keen et al ldquoDouble-blindplacebo-controlled trial of etanercept in the prevention of workdisability in ankylosing spondylitisrdquo Annals of the RheumaticDiseases vol 69 no 11 pp 1926ndash1928 2010

[16] J Callhoff J Sieper AWeiss A Zink and J Listing ldquoEfficacy ofTNF120572 blockers in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis ameta-analysisrdquoAnnals ofthe Rheumatic Diseases vol 74 no 6 pp 1241ndash1248 2015

[17] L Wang F Du and X Wang ldquoTNF-120572 induces two distinctcaspase-8 activation pathwaysrdquoCell vol 133 no 4 pp 693ndash7032008

[18] B Favaloro N Allocati V Graziano C Di Ilio and V DeLaurenzi ldquoRole of apoptosis in diseaserdquo Aging vol 4 no 5 pp330ndash349 2012

[19] X Li L Liu D Meng et al ldquoEnhanced apoptosis and senes-cence of bone-marrow-derived mesenchymal stem cells inpatients with systemic lupus erythematosusrdquo Stem Cells andDevelopment vol 21 no 13 pp 2387ndash2394 2012

[20] G R Screaton J Mongkolsapaya X-N Xu A E Cowper A JMcMichael and J I Bell ldquoTRICK2 a new alternatively splicedreceptor that transduces the cytotoxic signal from TRAILrdquoCurrent Biology vol 7 no 9 pp 693ndash696 1997

[21] E Szegezdi A OrsquoReilly Y Davy et al ldquoStem cells are resistantto TRAIL receptor-mediated apoptosisrdquo Journal of Cellular andMolecular Medicine vol 13 no 11-12 pp 4409ndash4414 2009

[22] H Walczak M A Degli-Esposti R S Johnson et al ldquoTRAIL-R2 a novel apoptosis-mediating receptor for TRAILrdquo EMBOJournal vol 16 no 17 pp 5386ndash5397 1997

[23] J-L Bodmer N Holler S Reynard et al ldquoTRAIL receptor-2signals apoptosis through FADD and caspase-8rdquo Nature CellBiology vol 2 no 4 pp 241ndash243 2000

[24] S Van Der Linden H A Valkenburg and A Cats ldquoEvaluationof diagnostic criteria for ankylosing spondylitis A proposal formodification of the New York criteriardquo Arthritis and Rheuma-tism vol 27 no 4 pp 361ndash368 1984

[25] D C Colter R Class C M DiGirolamo and D J ProckopldquoRapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrowrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3213ndash3218 2000

[26] D Li P Wang Y Li et al ldquoAll-Trans Retinoic Acid Improvesthe Effects of Bone Marrow-Derived Mesenchymal Stem Cellson the Treatment of Ankylosing Spondylitis An InVitro StudyrdquoStem Cells International vol 2015 Article ID 484528 10 pages2015

[27] K Fu H Ren Y Wang E Fei H Wang and G Wang ldquoDJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8recruitment to FADDrdquo Oncogene vol 31 no 10 pp 1311ndash13222012

[28] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cell intravenous infusion in active ankylos-ing spondylitis patients who failed NSAIDs a 20-week clinicaltrialrdquo Cell Transplantation vol 23 no 10 pp 1293ndash1303 2014

[29] Y Mei F Pan J Gao et al ldquoIncreased serum IL-17 and IL-23 inthe patient with ankylosing spondylitisrdquoClinical Rheumatologyvol 30 no 2 pp 269ndash273 2011

[30] R J Francois L Neure J Sieper and J Braun ldquoImmunohis-tological examination of open sacroiliac biopsies of patientswith ankylosing spondylitis detection of tumour necrosis factor120572 in two patients with early disease and transforming growthfactor 120573 in three more advanced casesrdquoAnnals of the RheumaticDiseases vol 65 no 6 pp 713ndash720 2006

[31] J Braun M Bollow L Neure et al ldquoUse of immunohistologicand in situ hybridization techniques in the examination ofsacroiliac joint biopsy specimens from patients with ankylosingspondylitisrdquo Arthritis and Rheumatism vol 38 no 4 pp 499ndash505 1995

[32] U Lange J Teichmann and H Stracke ldquoCorrelation betweenplasma TNF-alpha IGF-1 biochemical markers of bonemetabolismmarkers of inflammationdisease activity and clin-ical manifestations in ankylosing spondylitisrdquo European Journalof Medical Research vol 5 no 12 pp 507ndash511 2000

[33] L Zhao J Huang H Zhang et al ldquoTumor necrosis factorinhibits mesenchymal stem cell differentiation into osteoblastsvia the ubiquitin E3 ligase Wwp1rdquo Stem Cells vol 29 no 10 pp1601ndash1610 2011

[34] L Chiossone R Conte G M Spaggiari et al ldquoMesenchymalstromal cells induce peculiar alternatively activated macro-phages capable of dampening both innate and adaptive immuneresponsesrdquo STEM CELLS vol 34 no 7 pp 1909ndash1921 2016

[35] S Nagata ldquoApoptosis and autoimmune diseasesrdquo Annals of theNew York Academy of Sciences vol 1209 no 1 pp 10ndash16 2010

[36] M S Sheikh T F Burns Y Huang et al ldquop53-dependent and-independent regulation of the death receptor KILLERDR5gene expression in response to genotoxic stress and tumor

14 Stem Cells International

necrosis factorrdquo Cancer Research vol 58 no 8 pp 1593ndash15981998

[37] S Shirley A Morizot and O Micheau ldquoRegulating TRAILreceptor-induced cell death at the membrane a deadly discus-sionrdquo Recent Patents on Anti-Cancer Drug Discovery vol 6 no3 pp 311ndash323 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 Stem Cells International

ASMSCs

ASMSCs

(a)

(c) (b)

HDMSCs

HDMSCs

CD29

Cou

ntCD

44CD

105

CD14

CD45

HLA

-DR

HDMSCsASMSCs

OD

val

ue (4

50

nm)

Comp-PE-A CD29 PE-A Comp-PE-A CD29 PE-A

Comp-FITC-A CD44 FITC-A Comp-FITC-A CD44 FITC-A

Comp-FITC-A CD105 FITC-A Comp-FITC-A CD105 FITC-A

Comp-APC-A CD14 APC-A Comp-APC-A CD14 APC-A

Comp-APC-A CD45 APC-A Comp-APC-A CD45 APC-A

Comp-PerCP-AHLA-DR PerCP-A Comp-PerCP-AHLA-DR PerCP-A

0

0

102

103

104

105

Cou

nt

0

0

102

103

104

105

Cou

nt

0

0

102

103

104

105

Cou

nt

0

0

102

103

104

105

Cou

nt

0

0

102

103

104

105

Cou

nt

0

0

102

103

104

105

Cou

nt

0

0

102

103

104

105

Cou

nt

0

0

102

103

104

105

Cou

nt

0

0

102

103

104

105

Cou

nt

0

0

102

103

104

105

Cou

nt

0

0

102

103

104

105

Cou

nt

0

0

102

103

104

105

0

1

2

3

4

5

3 5 71Days of culture (d)

Figure 1 HDMSCs and ASMSCs shared the same morphology phenotype and proliferation capacity (a) Both HDMSCs and ASMSCs wereplastic-adherent and spindle-shaped (b) Both HDMSCs (119899 = 28) and ASMSCs (119899 = 22) were positive for CD29 CD44 and CD105 andnegative for CD14 CD45 and HLA-DR suggesting a typical MSC phenotype (c) The proliferation capacities of HDMSCs (119899 = 28) andASMSCs (119899 = 22) were similar when cultured in DMEM with 10 FBS from 1 to 7 days Optical density (OD) values are presented as themean plusmn SD

of the Proteome Profiler Human Apoptosis Array analysis(Figure 3(c)) Because caspase-3 initiates apoptosis throughits cleaved form the comparable total caspase-3 mRNA levelsbetween HDMSCs and ASMSCs may suggest that ASMSCswere more susceptible to apoptosis compared to HDMSCsvia an increase in the cleavage of caspase-3 rather than anincrease in the mRNA expression of caspase-3

34 The Death Receptor and Mitochondrial Pathways WereBoth Involved in the Apoptosis of MSCs Apoptosis proceedsmainly by the death receptor (DR) pathway and the mito-chondrial pathway The cleavage of caspase-8 and the releaseof cytochrome C from the mitochondria to the cytoplasmrespectively represent the activation of the DR pathway andmitochondrial pathway To determine whether these two

Stem Cells International 5

024 028

1559793

001 038

2699692

030 015

1529803

027 296

2167517

055

198

006

9741

036

359

011

9594

023

191

007

9780

027 607

33316035

HD

MSC

sA

SMSC

s

Annexin V-PE

7-A

AD

Control CHX TNF-120572CHX

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

TNF-120572

(a)

HDMSCsASMSCs

Control TNF-120572 CHX TNF-120572CHX

lowast

$

0

20

40

60

Apop

tosis

rate

()

(b)

Figure 2 ASMSCs weremore susceptible to TNF-120572CHX-induced apoptosis compared toHDMSCs (a) Apoptosis of HDMSCs (119899 = 28) andASMSCs (119899 = 22) was determined using FCM Cells in the upper-right and lower-right quadrants represent apoptotic cells the apoptotic ratewas calculated in (b) After a 6-hour culture only treatment with TNF-120572CHX induced significant MSC apoptosis Treatment with controlmedium or with TNF-120572 or CHX alone did not induce significant apoptosis ASMSCs had a higher rate of apoptosis than HDMSCs in theTNF-120572CHX treatment group Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs and ASMSCs indicates119875 lt 005 for the control group compared with the other HDMSC groups $ indicates 119875 lt 005 for the control group compared with the otherASMSC groups

pathways were involved in the altered apoptosis of ASMSCswe measured the mRNA expression levels of total caspase-8 and total cytochrome C as well as the active forms ofthe corresponding proteins cleaved caspase-8 and cytosolic

cytochromeCOur results showed that themRNAexpressionof both total caspase-8 and cytochrome C increased aftertreatment with TNF-120572CHX ASMSCs expressed signifi-cantly higher mRNA level of total caspase-8 than HDMSCs

6 Stem Cells International

HO

-1H

MO

X1H

SP32

HO

-2H

MO

X2

HSP

60H

SP70

PON

2

XIA

P

FADD

Total caspase-3 TRAIL-R2

BAD

BAX

BCL-

2BC

L-X

Pro-

casp

ase-

3Cl

eave

d ca

spas

e-3

Cata

lase

CIA

P-1

CIA

P-2

Clas

pin

Clus

terin

Cyto

chro

me C

TRA

IL R

1D

R4TR

AIL

R2

DR5

FAD

DFA

STN

FSF6

CD

95H

IF-1

a

HSP

27

HTR

A2

Om

iLi

vin

P21

CIP1

CD

KN1A

P27

Kip1

Phos

pho-

p53(

S15)

Phos

pho-

p53(

S46)

Phos

pho-

p53(

S392

)Ph

osph

o-Ra

d17(

S635

)SM

ACD

iabl

oSu

rviv

inTN

FR1

TNFR

SF1A

00

05

10

15

1

1

2

2

3

3

4

4

1 2

3 4

Mea

n re

lativ

e int

ensit

y

HDMSCsASMSCs

(a)

HDMSCs

HDMSCs

ASMSCs

ASMSCs

HDMSCsASMSCs

HDMSCsASMSCs

lowast

lowast

Control TNF-120572CHX Control TNF-120572CHX

Control TNF-120572CHX

0

2

4

6

8

Relat

ive m

RNA

expr

essio

nFo

ld ch

ange

0

2

4

6

Fold

chan

ge

(b)

0

2

4

6

8

Fold

chan

ge

Figure 3 Continued

Stem Cells International 7

Cleaved caspase-3

Pro-caspase-3

Actin

35 kD

19 kD17 kD

48 kD

40 kD

28 kD

45 kD

TRAIL-R2

FADD

HDMSCs ASMSCs

+

+

minus

minus

+

+

minus

minus

TNF-120572

CHX

Cleaved caspase-3

0

5

10

15

20

Fold

chan

ge

0

1

2

3

4

Fold

chan

ge

0

5

10

15

Fold

chan

ge

FADDTRAIL-R2

Control TNF-120572CHX Control TNF-120572CHX Control TNF-120572CHX

HDMSCsASMSCs

HDMSCsASMSCs

HDMSCsASMSCs

(c)

lowastlowast

lowast

Figure 3 ASMSCs had increased protein expression of cleaved caspase-3 TRAIL-R2 and FADD and increased gene expression of TRAIL-R2 and FADD during apoptosis (a) The Proteome Profiler Human Apoptosis Array Kit was used to determine the expression levels of 35apoptosis-related proteins in HDMSCs (119899 = 6) and ASMSCs (119899 = 6) after treatment with TNF-120572CHX The intensity of each pair of spotsrepresents repeated measures of one protein ASMSCs had lower pro-caspase-3 (a1) expression but higher cleaved caspase-3 (a2) TRAIL-R2(a3) and FADD (a4) expression levels than HDMSCs No significant differences in the expression of the remaining 31 proteins were foundbetween HDMSCs and ASMSCs (b) qRT-PCR was performed to confirm the results of the Proteome ProfilerHuman Apoptosis ArrayTheresults of TRAIL-R2 and FADD in HDMSCs (119899 = 28) and ASMSCs (119899 = 22) were consistent with those of the Proteome Profiler HumanApoptosis Array However total caspase-3 mRNA expression was not different between HDMSCs and ASMSCs (c) Western blot analysiswas performed on proteins isolated from HDMSCs (119899 = 28) and ASMSCs (119899 = 22) to further confirm the results of the Proteome ProfilerHuman Apoptosis Array and the results of both methods were in agreement Values are presented as the mean plusmn SD lowast indicates 119875 lt 005between HDMSCs and ASMSCs

while no difference was found between HDMSCs and ASM-SCs in total cytochrome C (Figure 4(a)) The protein expres-sion of both cytosolic cytochrome C and cleaved caspase-8increased after treatment with TNF-120572CHX and comparedwith HDMSCs ASMSCs exhibited much higher expressionof these two proteins (Figure 4(b)) Because cytochrome Cneeds to be released into the cytoplasm for apoptosis tooccur the mRNA expression level of total cytochrome C wasnot as meaningful as the cytosolic protein expression Wealso detected the gene and protein expression of TNFR1 andFas two important receptors that in addition to TRAIL-R2 interact with FADD However although both the gene

and protein expression levels increased after treatment withTNF-120572CHX no difference in gene or protein expressionwas found between the two groups either with or withoutTNF-120572CHX treatment (Figures 4(a) and 4(b))These resultstogether suggest that both the DR pathway and the mito-chondrial pathway are involved in the altered apoptosis ofASMSCs neither TNFR1 nor Fas accounted for the increasedexpression of FADD in the DR pathway

35 Lentiviruses Encoding shRNA-TRAIL-R2 Partly InhibitedtheApoptosis of ASMSCs butHad a Limited Effect on theApop-tosis of HDMSCs Because ASMSCs express more TRAIL-R2

8 Stem Cells International

Fas TNFR1Total cytochrome CTotal caspase-8

0

2

4

6

Relat

ive m

RNA

expr

essio

nFo

ld ch

ange

0

1

2

3

4

Fold

chan

ge

0

1

2

3

Fold

chan

ge

0

1

2

3

Fold

chan

ge

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

lowast

(a)

0

20

40

60

Fold

chan

ge

000510152025

Fold

chan

ge

000510152025

Fold

chan

ge

0

10

20

30

Fold

chan

ge

Cleaved caspase-8

Control TNF-120572CHX Control TNF-120572CHX

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

HDMSCsASMSCs

HDMSCsASMSCs

Cytosolic cytochrome C

FasTNFR1

43 kD41 kD

55 kD

45 kD

45 kD

17 kD

HDMSCs ASMSCs

+

+

minus

minus

+

+

minus

minus

TNF-120572CHX

40sim50 kD

Cleaved caspase-8

TNFR1

Fas

Actin 1

Actin 2

Cytosolic cytochrome C

lowast lowast

(b)

Figure 4 The DR and mitochondrial pathways were both involved in MSC apoptosis (a) qRT-PCR was performed to compare the geneexpressions of total caspase-8 total cytochrome C Fas and TNFR1 between HDMSCs (119899 = 28) and ASMSCs (119899 = 22) ASMSCs expressedmuchhigher gene expression levels of total caspase-8 thanHDMSCs after treatmentwithTNF-120572CHXwhile no differencewas found betweenHDMSCs andASMSCs in total cytochromeC Fas or TNFR1 gene expression levels (b) Awestern blot was performed to compare the proteinexpressions of cleaved caspase-8 cytosolic cytochrome C TNFR1 and Fas betweenHDMSCs (119899 = 28) and ASMSCs (119899 = 22) After treatmentwith TNF-120572CHX ASMSCs had higher cleaved caspase-8 and cytosolic cytochrome C protein expression levels than HDMSCs and similarprotein levels of TNFR1 and Fas as HDMSCs Fold changes are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs andASMSCs

than HDMSCs during apoptosis we constructed Lv-TRAIL-R2 to explore the role that TRAIL-R2 plays in the apopto-sis of ASMSCs GFP-positive cells were observed under aconfocal microscope and the transduction efficiencies werecomparable between HDMSCs and ASMSCs (Figure 5(a))Lv-TRAIL-R2 inhibited both gene and protein expression ofTRAIL-R2 inHDMSCs andASMSCs (Figure 5(b))The FCMresults demonstrated that Lv-TRAIL-R2 could reduce theapoptosis of ASMSCs but had a limited effect on the apoptosisof HDMSCs (Figure 6(a)) qRT-PCR showed that Lv-TRAIL-R2 inhibited the expression of FADD and total caspase-8 inASMSCs but had a limited effect on total caspase-3 and totalcytochrome C Meanwhile Lv-TRAIL-R2 did not affect the

expression of the four related aforementioned genes inHDM-SCs After inhibiting TRAIL-R2 expression no apparent dif-ference in gene expression was found between HDMSCs andASMSCs (Figure 6(b)) Additionally Lv-TRAIL-R2 did notaffect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC inHDMSCs but reducedthem in ASMSCs Furthermore no apparent difference inthe protein expression of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochrome C was found betweenHDMSCs andASMSCs after inhibitingTRAIL-R2 expression(Figure 6(c)) Therefore inhibiting TRAIL-R2 expressionpartly decreased the apoptosis of ASMSCs but had a limitedeffect on HDMSCs

Stem Cells International 9

HDMSCs ASMSCs

(a)

HDMSCsASMSCs

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

Con

trol

TNF-

120572C

HX

Control TNF-120572CHX Lv-NC Lv-TRAIL-R2 Control TNF-120572CHX Lv-NC Lv-TRAIL-R2

TRAIL R2

TRAIL-R2 TRAIL-R2

Actin

48 kD

40 kD

45 kD

0

1

2

3

4

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

lowast

amp

amp

amp

amp

lowast

lowastlowast

0

1

2

3

4

5

Relat

ive p

rote

in ex

pres

sion

Fold

chan

ge

HDMSCs ASMSCs

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

TNF-120572CHX + TNF-120572CHX + TNF-120572CHX+ TNF-120572CHX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

(b)

Figure 5 The transduction efficiencies of lentiviruses encoding shRNA-TRAIL-R2 were comparable in HDMSCs and ASMSCs (a) GFP-positive cells were observed under a confocal microscope (40x) and the transduction efficiencies were comparable between HDMSCs andASMSCs (b)The lentiviruses encoding shRNA-TRAIL-R2 inhibited both the gene and protein expression of TRAIL-R2 inHDMSCs (119899 = 28)and ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs and ASMSCs amp indicates 119875 lt 005 forthe TNF-120572CHXgroup comparedwith otherHDMSCs groups indicates119875 lt 005 for the TNF-120572CHXgroup comparedwith other ASMSCsgroups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative control sequence Lv-TRAIL-R2 refers to MSCs transduced bylentiviruses encoding TRAIL-R2

4 Discussion

Abnormal MSCs are proposed to be involved in the patho-genesis of AS and a better understanding of ASMSCsdysfunctions could improve the understanding of AS Inthis study ASMSCs had morphologies phenotypes and

proliferation rates that were similar to HDMSCs HoweverASMSCs were more susceptible to TNF-120572CHX-inducedapoptosis compared to HDMSCs ASMSCs expressed higherlevels of TRAIL-R2 which activated both the DR and themitochondrial pathways by increasing the expression levelsof FADD cleaved caspase-8 and cytosolic cytochrome C

10 Stem Cells International

Annexin V-PE

103 535

18627500

032 536

17447688

046 456

15317967

050 046

1519753

018 040

1179825

056 950

30895905

079 868

28526201

586027

14987889

HD

MSC

sA

SMSC

s7-

AA

D

104

104

103

103

102

102

101

101

100

104

103

102

101

100

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

104

103

102

101

100

104

103

102

101

100

10010

410

310

210

110

0

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX +

Lv-NC Lv-TRAIL-R2

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX+

LV-NC LV-TRAIL-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX

+

HDMSCsASMSCs

0

20

40

60

Apop

tosis

rate

()

LV-N

C

LV-N

C

LV-N

C

LV-N

C

0

2

4

6

8

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

Total caspase-3 Total caspase-8

0

2

4

6

Fold

chan

ge

Fold

chan

ge

0

2

4

6

8

0

1

2

3

4

Fold

chan

ge

Total cytochrome CFADD

amp

amp amp amp

amp

lowast

lowastlowast

lowast

lowast lowast

(a)

(b)

Figure 6 Continued

Stem Cells International 11

Con

trol

TNF-120572

CH

X

TNF-120572CHX

HDMSCsASMSCs

Con

trol

TNF-120572

CH

X

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Cleaved caspase-3 Cleaved caspase-8

Cytosoliccytochrome CFADD

0

5

10

15

20

Fold

chan

ge

0

5

10

15

Fold

chan

ge

0

10

20

30

Fold

chan

ge

0

20

40

60

80

Fold

chan

ge

amp amp

amp

lowastlowast

lowast lowast

lowastlowast

lowastlowast

35 kD

19 kD17 kD

43 kD41 kD

28 kD

45 kD

17 kD

45 kD

HDMSCs ASMSCs

+++

+

+

minus

minusminus

minusminus

minus

minus

+++

+

+

minus

minusminus

minusminus

minus

minus

Cleaved caspase-3Cleaved caspase-8

FADD

Actin 1

Actin 2

Cytosolic cytochrome C

(c)

Pro-caspase-3

LV-NCLV-TRAIL-R2

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

CTN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+TN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

Figure 6 Lentiviruses encoding shRNA-TRAIL-R2 partly inhibited the apoptosis of ASMSCs while having a limited effect on the apoptosisof HDMSCs (a) The rate of ASMSC apoptosis (119899 = 22) was decreased after Lv-TRAIL-R2 transduction while no apparent changes werefound for HDMSCs (119899 = 28) (b) In ASMSCs (119899 = 22) Lv-TRAIL-R2 reduced FADD and total caspase-8 expression while having a limitedeffect on total caspase-3 and total cytochrome C gene expression In HDMSCs (119899 = 28) Lv-TRAIL-R2 had a limited effect on the expressionof all four genes (c) Lv-TRAIL-R2 did not affect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC in HDMSCs (119899 = 28) but reduced them in ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 betweenHDMSCs and ASMSCs amp indicates 119875 lt 005 for the TNF-120572CHX group compared with other HDMSC groups indicates 119875 lt 005 forthe TNF-120572CHX group compared with other ASMSC groups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative controlsequence Lv-TRAIL-R2 refers to MSCs transduced by lentiviruses encoding TRAIL-R2

Inhibiting TRAIL-R2 expression using shRNA eliminated thedifference in apoptosis between HDMSCs and ASMSCs bydecreasing the apoptosis of ASMSCs

MSCs aremultipotent stromal cells withmultilineage dif-ferentiation capacities and immunomodulatory propertiesOur previous studies suggested that MSCs are implicated inthe pathogenesis and therapy of AS [12 28] We found thatASMSCs possessed an abnormal osteogenic capacity whichmay contribute to the pathogenesis of AS In addition we alsofound that allogenic intravenous infusion of bone marrow-derived MSCs is feasible and safe in the treatment of ASHowever how the in vivo microenvironment in AS interactswith MSCs remains unknown

AS is a complicated autoimmune disease accompaniedby elevated serum levels of various inflammatory cytokines[14 29] Among these cytokines TNF-120572 is one of the most

important players in the pathogenesis of AS PhysiologicallyTNF-120572 is important for a normal response to inflammationbut the overproduction of TNF-120572 can be detrimental Serumlevels and the sacroiliac joint expression of TNF-120572 wereelevated in AS patients including those in the early stages ofAS [30 31] In addition overproduction of TNF-120572 has beensuggested to be related to disease activity and to account forthe pathogenesis of AS [32] The importance of TNF-120572 hasbeen further confirmed by the efficacy of TNF-120572 inhibitorsin the treatment of AS [16] Due to the importance of TNF-120572in AS a study on the interaction between TNF-120572 and MSCsmay help mimic the in vivo microenvironment experiencedby MSCs in AS Previous studies suggest that TNF-120572 affectsMSCs in various ways TNF-120572 can modulate the immuno-suppressive effects of MSCs and suppress their osteogeniccapacity [33] Additionally TNF-120572 can alter homeostasis

12 Stem Cells International

in MSCs by regulating the balance between apoptosis andsurvival [13] ConverselyMSCs affect the production of TNF-120572 by causing macrophages to adopt an anti-inflammatoryphenotype [34] Both the dysfunction of MSCs and theoverproduction of TNF-120572 may lead to immune disordersA recent study suggested that TNF-120572-induced apoptosis ofMSCs may contribute to the pathogenesis of SLE [19] In ourstudy we found that ASMSCs were more susceptible to TNF-120572-induced apoptosis compared to HDMSCs as evidencedby an increased apoptosis rate and the higher expression ofcleaved caspase-3 protein However how the dysregulationof apoptosis contributes to the pathogenesis of AS remainsuncertain

Apoptosis also defined as programmed cell death iscritical to the pathogenesis of various diseases includingautoimmune diseases [18] The maintenance of immunetolerance and immune homeostasis is largely dependent onapoptosis Defective apoptosis of immune cells may result inthe defective clearance of autoreactive cells or the delayedelimination of autoantigens while excessive apoptosis ofimmunosuppressive cells (such as MSCs) may also lead toimmune hyperactivation Therefore both conditions maycontribute to autoimmune disease [19 35] In patients withAS increased apoptosis of ASMSCs may result in decreasesin cell numbers and immunosuppressive potential eventuallycontributing to autoimmune disorders Moreover becauseMSCs are able to reduce TNF-120572 production increasedMSCs apoptosis may also lead to TNF-120572 overproductionFurthermore TNF-120572 overproduction induces further MSCapoptosis forming a vicious cycle that leads to chronicinflammation in AS Thus increased ASMSC apoptosis maybe critical for AS pathogenesis and further study of themechanism of dysregulated apoptosis in ASMSCs may helpto elucidate the pathogenesis of AS

TRAIL-R2 is a specific cell surface receptor engagedin apoptosis TRAIL-R2-mediated apoptosis has arousedattention due to its role in preferentially killing tumor cellswhile sparing normal cells In our study we observed asignificant increased expression of TRAIL-R2 in ASMSCsafter treatment with TNF-120572CHX Inhibiting TRAIL-R2expression partly decreased the apoptosis of ASMSCs buthad a limited effect on the apoptosis of HDMSCs whichsuggests that TRAIL-R2 overexpression is the main cause ofthe increased apoptosis in ASMSCs However this result isconfusing for two reasons First TRAIL-R2 is reported to bemainly activated by its specific ligand TRAIL [20] Howeverthe results in our study indicated that TRAIL-R2 in MSCscould be activated by TNF-120572CHX as well In fact a similarresult was previously reported but the mechanism remainsunclear [36] Considering the overexpression of TRAIL-R2 can lead to apoptosis even without its ligand [22] wespeculate that TNF-120572maymediate its proapoptotic effects notonly through its own receptor (TNFR1) but also by increasingthe expression of TRAIL-R2 Second bone marrow-derivedadult MSCs are reportedly resistant to TRAIL-R2-mediatedapoptosis [21] However in our study HDMSCs were resis-tant to TRAIL-R2-mediated apoptosis while ASMSCs werenot Multiple mechanisms of TRAIL-R2-mediated apoptosisresistance such as dysregulation of antagonistic receptors

abnormal O-glycosylation and overexpression of the inhi-bition adaptors have been identified [37] Further studiesare needed to illustrate the associated specific mechanismunderlying the difference in resistance of HDMSCs andASMSCs to TRAIL-R2 Additionally based on the findingthat the TRAIL-R2 pathway did not lead to the apoptosis ofnormal cells ASMSCs could be considered abnormal cellsThus it is reasonable to presume that allogenic rather thanautologous MSC transplantation might be preferable for AStreatment

In this study we demonstrated that TRAIL-R2 upregu-lation resulted in enhanced apoptosis of ASMSCs Increasedapoptosis of ASMSCs may decrease the immunosuppressivepotential of these cells and potentially contributing to ASpathogenesis However some limitations remain in thisstudy The questions of how TNF-120572 activates the expressionof TRAIL-R2 why HDMSCs are resistant to TRAIL-R2-mediated apoptosis while ASMSCs are not and whether ourresults can be repeated in vivo remain unanswered Futurestudies should focus on these questions and further elucidatethe mechanism of AS

Competing Interests

The authors have no potential conflict of interests to declare

Authorsrsquo Contributions

Zhenhua Liu LiangbinGao andPengWanghave contributedequally to this work

Acknowledgments

The authors thank Dr Liang Zhang for insightful discussionThis study was supported by the National Natural ScienceFoundation of China (81401850) the Science and Tech-nology Project of Guangdong Province (2015B0202280012015B090903059) and the Engineering Technology ResearchCenter for Comprehensive Diagnosis and Treatment ofAnkylosing Spondylitis of Guangdong Higher EducationInstitutes (GCZX-A1301)

References

[1] M Dougados and D Baeten ldquoSpondyloarthritisrdquo The Lancetvol 377 no 9783 pp 2127ndash2137 2011

[2] P Bowness ldquoHLA-B27rdquo Annual Review of Immunology vol 33pp 29ndash48 2015

[3] J D Reveille A-M Sims P Danoy et al ldquoGenome-wideassociation study of ankylosing spondylitis identifies non-MHCsusceptibility locirdquo Nature Genetics vol 42 no 2 pp 123ndash1272010

[4] J D Taurog J A Richardson J T Croft et al ldquoThe germfreestate prevents development of gut and joint inflammatorydisease in HLA-B27 transgenic ratsrdquo Journal of ExperimentalMedicine vol 180 no 6 pp 2359ndash2364 1994

[5] M-E Costello F Ciccia D Willner et al ldquoBrief Reportintestinal dysbiosis in ankylosing spondylitisrdquo Arthritis andRheumatology vol 67 no 3 pp 686ndash691 2015

Stem Cells International 13

[6] T Rashid and A Ebringer ldquoAnkylosing spondylitis is linked toKlebsiellamdashthe evidencerdquo Clinical Rheumatology vol 26 no 6pp 858ndash864 2007

[7] P Jacques and D McGonagle ldquoThe role of mechanical stress inthe pathogenesis of spondyloarthritis and how to combat itrdquoBest Practice and Research Clinical Rheumatology vol 28 no5 pp 703ndash710 2014

[8] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008

[9] M C Stewart and A A Stewart ldquoMesenchymal stem cellscharacteristics sources and mechanisms of actionrdquo VeterinaryClinics of North AmericamdashEquine Practice vol 27 no 2 pp243ndash261 2011

[10] L Y Sun H Y Zhang X B Feng Y Y Hou LW Lu and L MFan ldquoAbnormality of bone marrow-derived mesenchymal stemcells in patients with systemic lupus erythematosusrdquo Lupus vol16 no 2 pp 121ndash128 2007

[11] J A Perez-Simon S Tabera M E Sarasquete et al ldquoMes-enchymal stem cells are functionally abnormal in patients withimmune thrombocytopenic purpurardquo Cytotherapy vol 11 no6 pp 698ndash705 2009

[12] Z Xie P Wang Y Li et al ldquoImbalance between bone morpho-genetic protein 2 and noggin induces abnormal osteogenic dif-ferentiation of mesenchymal stem cells in ankylosing spondyli-tisrdquo Arthritis and Rheumatology vol 68 no 2 pp 430ndash4402016

[13] D Brenner H Blaser and T W Mak ldquoRegulation of tumournecrosis factor signalling live or let dierdquo Nature ReviewsImmunology vol 15 no 6 pp 362ndash374 2015

[14] J Gratacos A Collado X Filella et al ldquoSerum cytokines(IL-6 TNF-120572 IL-1 120573 and IFN-120574) in ankylosing spondylitis aclose correlation between serum IL-6 and disease activity andseverityrdquo British Journal of Rheumatology vol 33 no 10 pp927ndash931 1994

[15] N Barkham L C Coates H Keen et al ldquoDouble-blindplacebo-controlled trial of etanercept in the prevention of workdisability in ankylosing spondylitisrdquo Annals of the RheumaticDiseases vol 69 no 11 pp 1926ndash1928 2010

[16] J Callhoff J Sieper AWeiss A Zink and J Listing ldquoEfficacy ofTNF120572 blockers in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis ameta-analysisrdquoAnnals ofthe Rheumatic Diseases vol 74 no 6 pp 1241ndash1248 2015

[17] L Wang F Du and X Wang ldquoTNF-120572 induces two distinctcaspase-8 activation pathwaysrdquoCell vol 133 no 4 pp 693ndash7032008

[18] B Favaloro N Allocati V Graziano C Di Ilio and V DeLaurenzi ldquoRole of apoptosis in diseaserdquo Aging vol 4 no 5 pp330ndash349 2012

[19] X Li L Liu D Meng et al ldquoEnhanced apoptosis and senes-cence of bone-marrow-derived mesenchymal stem cells inpatients with systemic lupus erythematosusrdquo Stem Cells andDevelopment vol 21 no 13 pp 2387ndash2394 2012

[20] G R Screaton J Mongkolsapaya X-N Xu A E Cowper A JMcMichael and J I Bell ldquoTRICK2 a new alternatively splicedreceptor that transduces the cytotoxic signal from TRAILrdquoCurrent Biology vol 7 no 9 pp 693ndash696 1997

[21] E Szegezdi A OrsquoReilly Y Davy et al ldquoStem cells are resistantto TRAIL receptor-mediated apoptosisrdquo Journal of Cellular andMolecular Medicine vol 13 no 11-12 pp 4409ndash4414 2009

[22] H Walczak M A Degli-Esposti R S Johnson et al ldquoTRAIL-R2 a novel apoptosis-mediating receptor for TRAILrdquo EMBOJournal vol 16 no 17 pp 5386ndash5397 1997

[23] J-L Bodmer N Holler S Reynard et al ldquoTRAIL receptor-2signals apoptosis through FADD and caspase-8rdquo Nature CellBiology vol 2 no 4 pp 241ndash243 2000

[24] S Van Der Linden H A Valkenburg and A Cats ldquoEvaluationof diagnostic criteria for ankylosing spondylitis A proposal formodification of the New York criteriardquo Arthritis and Rheuma-tism vol 27 no 4 pp 361ndash368 1984

[25] D C Colter R Class C M DiGirolamo and D J ProckopldquoRapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrowrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3213ndash3218 2000

[26] D Li P Wang Y Li et al ldquoAll-Trans Retinoic Acid Improvesthe Effects of Bone Marrow-Derived Mesenchymal Stem Cellson the Treatment of Ankylosing Spondylitis An InVitro StudyrdquoStem Cells International vol 2015 Article ID 484528 10 pages2015

[27] K Fu H Ren Y Wang E Fei H Wang and G Wang ldquoDJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8recruitment to FADDrdquo Oncogene vol 31 no 10 pp 1311ndash13222012

[28] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cell intravenous infusion in active ankylos-ing spondylitis patients who failed NSAIDs a 20-week clinicaltrialrdquo Cell Transplantation vol 23 no 10 pp 1293ndash1303 2014

[29] Y Mei F Pan J Gao et al ldquoIncreased serum IL-17 and IL-23 inthe patient with ankylosing spondylitisrdquoClinical Rheumatologyvol 30 no 2 pp 269ndash273 2011

[30] R J Francois L Neure J Sieper and J Braun ldquoImmunohis-tological examination of open sacroiliac biopsies of patientswith ankylosing spondylitis detection of tumour necrosis factor120572 in two patients with early disease and transforming growthfactor 120573 in three more advanced casesrdquoAnnals of the RheumaticDiseases vol 65 no 6 pp 713ndash720 2006

[31] J Braun M Bollow L Neure et al ldquoUse of immunohistologicand in situ hybridization techniques in the examination ofsacroiliac joint biopsy specimens from patients with ankylosingspondylitisrdquo Arthritis and Rheumatism vol 38 no 4 pp 499ndash505 1995

[32] U Lange J Teichmann and H Stracke ldquoCorrelation betweenplasma TNF-alpha IGF-1 biochemical markers of bonemetabolismmarkers of inflammationdisease activity and clin-ical manifestations in ankylosing spondylitisrdquo European Journalof Medical Research vol 5 no 12 pp 507ndash511 2000

[33] L Zhao J Huang H Zhang et al ldquoTumor necrosis factorinhibits mesenchymal stem cell differentiation into osteoblastsvia the ubiquitin E3 ligase Wwp1rdquo Stem Cells vol 29 no 10 pp1601ndash1610 2011

[34] L Chiossone R Conte G M Spaggiari et al ldquoMesenchymalstromal cells induce peculiar alternatively activated macro-phages capable of dampening both innate and adaptive immuneresponsesrdquo STEM CELLS vol 34 no 7 pp 1909ndash1921 2016

[35] S Nagata ldquoApoptosis and autoimmune diseasesrdquo Annals of theNew York Academy of Sciences vol 1209 no 1 pp 10ndash16 2010

[36] M S Sheikh T F Burns Y Huang et al ldquop53-dependent and-independent regulation of the death receptor KILLERDR5gene expression in response to genotoxic stress and tumor

14 Stem Cells International

necrosis factorrdquo Cancer Research vol 58 no 8 pp 1593ndash15981998

[37] S Shirley A Morizot and O Micheau ldquoRegulating TRAILreceptor-induced cell death at the membrane a deadly discus-sionrdquo Recent Patents on Anti-Cancer Drug Discovery vol 6 no3 pp 311ndash323 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 5

024 028

1559793

001 038

2699692

030 015

1529803

027 296

2167517

055

198

006

9741

036

359

011

9594

023

191

007

9780

027 607

33316035

HD

MSC

sA

SMSC

s

Annexin V-PE

7-A

AD

Control CHX TNF-120572CHX

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

104

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103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

100

TNF-120572

(a)

HDMSCsASMSCs

Control TNF-120572 CHX TNF-120572CHX

lowast

$

0

20

40

60

Apop

tosis

rate

()

(b)

Figure 2 ASMSCs weremore susceptible to TNF-120572CHX-induced apoptosis compared toHDMSCs (a) Apoptosis of HDMSCs (119899 = 28) andASMSCs (119899 = 22) was determined using FCM Cells in the upper-right and lower-right quadrants represent apoptotic cells the apoptotic ratewas calculated in (b) After a 6-hour culture only treatment with TNF-120572CHX induced significant MSC apoptosis Treatment with controlmedium or with TNF-120572 or CHX alone did not induce significant apoptosis ASMSCs had a higher rate of apoptosis than HDMSCs in theTNF-120572CHX treatment group Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs and ASMSCs indicates119875 lt 005 for the control group compared with the other HDMSC groups $ indicates 119875 lt 005 for the control group compared with the otherASMSC groups

pathways were involved in the altered apoptosis of ASMSCswe measured the mRNA expression levels of total caspase-8 and total cytochrome C as well as the active forms ofthe corresponding proteins cleaved caspase-8 and cytosolic

cytochromeCOur results showed that themRNAexpressionof both total caspase-8 and cytochrome C increased aftertreatment with TNF-120572CHX ASMSCs expressed signifi-cantly higher mRNA level of total caspase-8 than HDMSCs

6 Stem Cells International

HO

-1H

MO

X1H

SP32

HO

-2H

MO

X2

HSP

60H

SP70

PON

2

XIA

P

FADD

Total caspase-3 TRAIL-R2

BAD

BAX

BCL-

2BC

L-X

Pro-

casp

ase-

3Cl

eave

d ca

spas

e-3

Cata

lase

CIA

P-1

CIA

P-2

Clas

pin

Clus

terin

Cyto

chro

me C

TRA

IL R

1D

R4TR

AIL

R2

DR5

FAD

DFA

STN

FSF6

CD

95H

IF-1

a

HSP

27

HTR

A2

Om

iLi

vin

P21

CIP1

CD

KN1A

P27

Kip1

Phos

pho-

p53(

S15)

Phos

pho-

p53(

S46)

Phos

pho-

p53(

S392

)Ph

osph

o-Ra

d17(

S635

)SM

ACD

iabl

oSu

rviv

inTN

FR1

TNFR

SF1A

00

05

10

15

1

1

2

2

3

3

4

4

1 2

3 4

Mea

n re

lativ

e int

ensit

y

HDMSCsASMSCs

(a)

HDMSCs

HDMSCs

ASMSCs

ASMSCs

HDMSCsASMSCs

HDMSCsASMSCs

lowast

lowast

Control TNF-120572CHX Control TNF-120572CHX

Control TNF-120572CHX

0

2

4

6

8

Relat

ive m

RNA

expr

essio

nFo

ld ch

ange

0

2

4

6

Fold

chan

ge

(b)

0

2

4

6

8

Fold

chan

ge

Figure 3 Continued

Stem Cells International 7

Cleaved caspase-3

Pro-caspase-3

Actin

35 kD

19 kD17 kD

48 kD

40 kD

28 kD

45 kD

TRAIL-R2

FADD

HDMSCs ASMSCs

+

+

minus

minus

+

+

minus

minus

TNF-120572

CHX

Cleaved caspase-3

0

5

10

15

20

Fold

chan

ge

0

1

2

3

4

Fold

chan

ge

0

5

10

15

Fold

chan

ge

FADDTRAIL-R2

Control TNF-120572CHX Control TNF-120572CHX Control TNF-120572CHX

HDMSCsASMSCs

HDMSCsASMSCs

HDMSCsASMSCs

(c)

lowastlowast

lowast

Figure 3 ASMSCs had increased protein expression of cleaved caspase-3 TRAIL-R2 and FADD and increased gene expression of TRAIL-R2 and FADD during apoptosis (a) The Proteome Profiler Human Apoptosis Array Kit was used to determine the expression levels of 35apoptosis-related proteins in HDMSCs (119899 = 6) and ASMSCs (119899 = 6) after treatment with TNF-120572CHX The intensity of each pair of spotsrepresents repeated measures of one protein ASMSCs had lower pro-caspase-3 (a1) expression but higher cleaved caspase-3 (a2) TRAIL-R2(a3) and FADD (a4) expression levels than HDMSCs No significant differences in the expression of the remaining 31 proteins were foundbetween HDMSCs and ASMSCs (b) qRT-PCR was performed to confirm the results of the Proteome ProfilerHuman Apoptosis ArrayTheresults of TRAIL-R2 and FADD in HDMSCs (119899 = 28) and ASMSCs (119899 = 22) were consistent with those of the Proteome Profiler HumanApoptosis Array However total caspase-3 mRNA expression was not different between HDMSCs and ASMSCs (c) Western blot analysiswas performed on proteins isolated from HDMSCs (119899 = 28) and ASMSCs (119899 = 22) to further confirm the results of the Proteome ProfilerHuman Apoptosis Array and the results of both methods were in agreement Values are presented as the mean plusmn SD lowast indicates 119875 lt 005between HDMSCs and ASMSCs

while no difference was found between HDMSCs and ASM-SCs in total cytochrome C (Figure 4(a)) The protein expres-sion of both cytosolic cytochrome C and cleaved caspase-8increased after treatment with TNF-120572CHX and comparedwith HDMSCs ASMSCs exhibited much higher expressionof these two proteins (Figure 4(b)) Because cytochrome Cneeds to be released into the cytoplasm for apoptosis tooccur the mRNA expression level of total cytochrome C wasnot as meaningful as the cytosolic protein expression Wealso detected the gene and protein expression of TNFR1 andFas two important receptors that in addition to TRAIL-R2 interact with FADD However although both the gene

and protein expression levels increased after treatment withTNF-120572CHX no difference in gene or protein expressionwas found between the two groups either with or withoutTNF-120572CHX treatment (Figures 4(a) and 4(b))These resultstogether suggest that both the DR pathway and the mito-chondrial pathway are involved in the altered apoptosis ofASMSCs neither TNFR1 nor Fas accounted for the increasedexpression of FADD in the DR pathway

35 Lentiviruses Encoding shRNA-TRAIL-R2 Partly InhibitedtheApoptosis of ASMSCs butHad a Limited Effect on theApop-tosis of HDMSCs Because ASMSCs express more TRAIL-R2

8 Stem Cells International

Fas TNFR1Total cytochrome CTotal caspase-8

0

2

4

6

Relat

ive m

RNA

expr

essio

nFo

ld ch

ange

0

1

2

3

4

Fold

chan

ge

0

1

2

3

Fold

chan

ge

0

1

2

3

Fold

chan

ge

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

lowast

(a)

0

20

40

60

Fold

chan

ge

000510152025

Fold

chan

ge

000510152025

Fold

chan

ge

0

10

20

30

Fold

chan

ge

Cleaved caspase-8

Control TNF-120572CHX Control TNF-120572CHX

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

HDMSCsASMSCs

HDMSCsASMSCs

Cytosolic cytochrome C

FasTNFR1

43 kD41 kD

55 kD

45 kD

45 kD

17 kD

HDMSCs ASMSCs

+

+

minus

minus

+

+

minus

minus

TNF-120572CHX

40sim50 kD

Cleaved caspase-8

TNFR1

Fas

Actin 1

Actin 2

Cytosolic cytochrome C

lowast lowast

(b)

Figure 4 The DR and mitochondrial pathways were both involved in MSC apoptosis (a) qRT-PCR was performed to compare the geneexpressions of total caspase-8 total cytochrome C Fas and TNFR1 between HDMSCs (119899 = 28) and ASMSCs (119899 = 22) ASMSCs expressedmuchhigher gene expression levels of total caspase-8 thanHDMSCs after treatmentwithTNF-120572CHXwhile no differencewas found betweenHDMSCs andASMSCs in total cytochromeC Fas or TNFR1 gene expression levels (b) Awestern blot was performed to compare the proteinexpressions of cleaved caspase-8 cytosolic cytochrome C TNFR1 and Fas betweenHDMSCs (119899 = 28) and ASMSCs (119899 = 22) After treatmentwith TNF-120572CHX ASMSCs had higher cleaved caspase-8 and cytosolic cytochrome C protein expression levels than HDMSCs and similarprotein levels of TNFR1 and Fas as HDMSCs Fold changes are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs andASMSCs

than HDMSCs during apoptosis we constructed Lv-TRAIL-R2 to explore the role that TRAIL-R2 plays in the apopto-sis of ASMSCs GFP-positive cells were observed under aconfocal microscope and the transduction efficiencies werecomparable between HDMSCs and ASMSCs (Figure 5(a))Lv-TRAIL-R2 inhibited both gene and protein expression ofTRAIL-R2 inHDMSCs andASMSCs (Figure 5(b))The FCMresults demonstrated that Lv-TRAIL-R2 could reduce theapoptosis of ASMSCs but had a limited effect on the apoptosisof HDMSCs (Figure 6(a)) qRT-PCR showed that Lv-TRAIL-R2 inhibited the expression of FADD and total caspase-8 inASMSCs but had a limited effect on total caspase-3 and totalcytochrome C Meanwhile Lv-TRAIL-R2 did not affect the

expression of the four related aforementioned genes inHDM-SCs After inhibiting TRAIL-R2 expression no apparent dif-ference in gene expression was found between HDMSCs andASMSCs (Figure 6(b)) Additionally Lv-TRAIL-R2 did notaffect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC inHDMSCs but reducedthem in ASMSCs Furthermore no apparent difference inthe protein expression of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochrome C was found betweenHDMSCs andASMSCs after inhibitingTRAIL-R2 expression(Figure 6(c)) Therefore inhibiting TRAIL-R2 expressionpartly decreased the apoptosis of ASMSCs but had a limitedeffect on HDMSCs

Stem Cells International 9

HDMSCs ASMSCs

(a)

HDMSCsASMSCs

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

Con

trol

TNF-

120572C

HX

Control TNF-120572CHX Lv-NC Lv-TRAIL-R2 Control TNF-120572CHX Lv-NC Lv-TRAIL-R2

TRAIL R2

TRAIL-R2 TRAIL-R2

Actin

48 kD

40 kD

45 kD

0

1

2

3

4

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

lowast

amp

amp

amp

amp

lowast

lowastlowast

0

1

2

3

4

5

Relat

ive p

rote

in ex

pres

sion

Fold

chan

ge

HDMSCs ASMSCs

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

TNF-120572CHX + TNF-120572CHX + TNF-120572CHX+ TNF-120572CHX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

(b)

Figure 5 The transduction efficiencies of lentiviruses encoding shRNA-TRAIL-R2 were comparable in HDMSCs and ASMSCs (a) GFP-positive cells were observed under a confocal microscope (40x) and the transduction efficiencies were comparable between HDMSCs andASMSCs (b)The lentiviruses encoding shRNA-TRAIL-R2 inhibited both the gene and protein expression of TRAIL-R2 inHDMSCs (119899 = 28)and ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs and ASMSCs amp indicates 119875 lt 005 forthe TNF-120572CHXgroup comparedwith otherHDMSCs groups indicates119875 lt 005 for the TNF-120572CHXgroup comparedwith other ASMSCsgroups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative control sequence Lv-TRAIL-R2 refers to MSCs transduced bylentiviruses encoding TRAIL-R2

4 Discussion

Abnormal MSCs are proposed to be involved in the patho-genesis of AS and a better understanding of ASMSCsdysfunctions could improve the understanding of AS Inthis study ASMSCs had morphologies phenotypes and

proliferation rates that were similar to HDMSCs HoweverASMSCs were more susceptible to TNF-120572CHX-inducedapoptosis compared to HDMSCs ASMSCs expressed higherlevels of TRAIL-R2 which activated both the DR and themitochondrial pathways by increasing the expression levelsof FADD cleaved caspase-8 and cytosolic cytochrome C

10 Stem Cells International

Annexin V-PE

103 535

18627500

032 536

17447688

046 456

15317967

050 046

1519753

018 040

1179825

056 950

30895905

079 868

28526201

586027

14987889

HD

MSC

sA

SMSC

s7-

AA

D

104

104

103

103

102

102

101

101

100

104

103

102

101

100

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

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103

103

102

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101

101

100

100

104

103

102

101

100

104

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103

103

102

102

101

101

100

100

104

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103

103

102

102

101

101

100

104

103

102

101

100

104

103

102

101

100

10010

410

310

210

110

0

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX +

Lv-NC Lv-TRAIL-R2

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX+

LV-NC LV-TRAIL-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX

+

HDMSCsASMSCs

0

20

40

60

Apop

tosis

rate

()

LV-N

C

LV-N

C

LV-N

C

LV-N

C

0

2

4

6

8

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

Total caspase-3 Total caspase-8

0

2

4

6

Fold

chan

ge

Fold

chan

ge

0

2

4

6

8

0

1

2

3

4

Fold

chan

ge

Total cytochrome CFADD

amp

amp amp amp

amp

lowast

lowastlowast

lowast

lowast lowast

(a)

(b)

Figure 6 Continued

Stem Cells International 11

Con

trol

TNF-120572

CH

X

TNF-120572CHX

HDMSCsASMSCs

Con

trol

TNF-120572

CH

X

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Cleaved caspase-3 Cleaved caspase-8

Cytosoliccytochrome CFADD

0

5

10

15

20

Fold

chan

ge

0

5

10

15

Fold

chan

ge

0

10

20

30

Fold

chan

ge

0

20

40

60

80

Fold

chan

ge

amp amp

amp

lowastlowast

lowast lowast

lowastlowast

lowastlowast

35 kD

19 kD17 kD

43 kD41 kD

28 kD

45 kD

17 kD

45 kD

HDMSCs ASMSCs

+++

+

+

minus

minusminus

minusminus

minus

minus

+++

+

+

minus

minusminus

minusminus

minus

minus

Cleaved caspase-3Cleaved caspase-8

FADD

Actin 1

Actin 2

Cytosolic cytochrome C

(c)

Pro-caspase-3

LV-NCLV-TRAIL-R2

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

CTN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+TN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

Figure 6 Lentiviruses encoding shRNA-TRAIL-R2 partly inhibited the apoptosis of ASMSCs while having a limited effect on the apoptosisof HDMSCs (a) The rate of ASMSC apoptosis (119899 = 22) was decreased after Lv-TRAIL-R2 transduction while no apparent changes werefound for HDMSCs (119899 = 28) (b) In ASMSCs (119899 = 22) Lv-TRAIL-R2 reduced FADD and total caspase-8 expression while having a limitedeffect on total caspase-3 and total cytochrome C gene expression In HDMSCs (119899 = 28) Lv-TRAIL-R2 had a limited effect on the expressionof all four genes (c) Lv-TRAIL-R2 did not affect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC in HDMSCs (119899 = 28) but reduced them in ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 betweenHDMSCs and ASMSCs amp indicates 119875 lt 005 for the TNF-120572CHX group compared with other HDMSC groups indicates 119875 lt 005 forthe TNF-120572CHX group compared with other ASMSC groups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative controlsequence Lv-TRAIL-R2 refers to MSCs transduced by lentiviruses encoding TRAIL-R2

Inhibiting TRAIL-R2 expression using shRNA eliminated thedifference in apoptosis between HDMSCs and ASMSCs bydecreasing the apoptosis of ASMSCs

MSCs aremultipotent stromal cells withmultilineage dif-ferentiation capacities and immunomodulatory propertiesOur previous studies suggested that MSCs are implicated inthe pathogenesis and therapy of AS [12 28] We found thatASMSCs possessed an abnormal osteogenic capacity whichmay contribute to the pathogenesis of AS In addition we alsofound that allogenic intravenous infusion of bone marrow-derived MSCs is feasible and safe in the treatment of ASHowever how the in vivo microenvironment in AS interactswith MSCs remains unknown

AS is a complicated autoimmune disease accompaniedby elevated serum levels of various inflammatory cytokines[14 29] Among these cytokines TNF-120572 is one of the most

important players in the pathogenesis of AS PhysiologicallyTNF-120572 is important for a normal response to inflammationbut the overproduction of TNF-120572 can be detrimental Serumlevels and the sacroiliac joint expression of TNF-120572 wereelevated in AS patients including those in the early stages ofAS [30 31] In addition overproduction of TNF-120572 has beensuggested to be related to disease activity and to account forthe pathogenesis of AS [32] The importance of TNF-120572 hasbeen further confirmed by the efficacy of TNF-120572 inhibitorsin the treatment of AS [16] Due to the importance of TNF-120572in AS a study on the interaction between TNF-120572 and MSCsmay help mimic the in vivo microenvironment experiencedby MSCs in AS Previous studies suggest that TNF-120572 affectsMSCs in various ways TNF-120572 can modulate the immuno-suppressive effects of MSCs and suppress their osteogeniccapacity [33] Additionally TNF-120572 can alter homeostasis

12 Stem Cells International

in MSCs by regulating the balance between apoptosis andsurvival [13] ConverselyMSCs affect the production of TNF-120572 by causing macrophages to adopt an anti-inflammatoryphenotype [34] Both the dysfunction of MSCs and theoverproduction of TNF-120572 may lead to immune disordersA recent study suggested that TNF-120572-induced apoptosis ofMSCs may contribute to the pathogenesis of SLE [19] In ourstudy we found that ASMSCs were more susceptible to TNF-120572-induced apoptosis compared to HDMSCs as evidencedby an increased apoptosis rate and the higher expression ofcleaved caspase-3 protein However how the dysregulationof apoptosis contributes to the pathogenesis of AS remainsuncertain

Apoptosis also defined as programmed cell death iscritical to the pathogenesis of various diseases includingautoimmune diseases [18] The maintenance of immunetolerance and immune homeostasis is largely dependent onapoptosis Defective apoptosis of immune cells may result inthe defective clearance of autoreactive cells or the delayedelimination of autoantigens while excessive apoptosis ofimmunosuppressive cells (such as MSCs) may also lead toimmune hyperactivation Therefore both conditions maycontribute to autoimmune disease [19 35] In patients withAS increased apoptosis of ASMSCs may result in decreasesin cell numbers and immunosuppressive potential eventuallycontributing to autoimmune disorders Moreover becauseMSCs are able to reduce TNF-120572 production increasedMSCs apoptosis may also lead to TNF-120572 overproductionFurthermore TNF-120572 overproduction induces further MSCapoptosis forming a vicious cycle that leads to chronicinflammation in AS Thus increased ASMSC apoptosis maybe critical for AS pathogenesis and further study of themechanism of dysregulated apoptosis in ASMSCs may helpto elucidate the pathogenesis of AS

TRAIL-R2 is a specific cell surface receptor engagedin apoptosis TRAIL-R2-mediated apoptosis has arousedattention due to its role in preferentially killing tumor cellswhile sparing normal cells In our study we observed asignificant increased expression of TRAIL-R2 in ASMSCsafter treatment with TNF-120572CHX Inhibiting TRAIL-R2expression partly decreased the apoptosis of ASMSCs buthad a limited effect on the apoptosis of HDMSCs whichsuggests that TRAIL-R2 overexpression is the main cause ofthe increased apoptosis in ASMSCs However this result isconfusing for two reasons First TRAIL-R2 is reported to bemainly activated by its specific ligand TRAIL [20] Howeverthe results in our study indicated that TRAIL-R2 in MSCscould be activated by TNF-120572CHX as well In fact a similarresult was previously reported but the mechanism remainsunclear [36] Considering the overexpression of TRAIL-R2 can lead to apoptosis even without its ligand [22] wespeculate that TNF-120572maymediate its proapoptotic effects notonly through its own receptor (TNFR1) but also by increasingthe expression of TRAIL-R2 Second bone marrow-derivedadult MSCs are reportedly resistant to TRAIL-R2-mediatedapoptosis [21] However in our study HDMSCs were resis-tant to TRAIL-R2-mediated apoptosis while ASMSCs werenot Multiple mechanisms of TRAIL-R2-mediated apoptosisresistance such as dysregulation of antagonistic receptors

abnormal O-glycosylation and overexpression of the inhi-bition adaptors have been identified [37] Further studiesare needed to illustrate the associated specific mechanismunderlying the difference in resistance of HDMSCs andASMSCs to TRAIL-R2 Additionally based on the findingthat the TRAIL-R2 pathway did not lead to the apoptosis ofnormal cells ASMSCs could be considered abnormal cellsThus it is reasonable to presume that allogenic rather thanautologous MSC transplantation might be preferable for AStreatment

In this study we demonstrated that TRAIL-R2 upregu-lation resulted in enhanced apoptosis of ASMSCs Increasedapoptosis of ASMSCs may decrease the immunosuppressivepotential of these cells and potentially contributing to ASpathogenesis However some limitations remain in thisstudy The questions of how TNF-120572 activates the expressionof TRAIL-R2 why HDMSCs are resistant to TRAIL-R2-mediated apoptosis while ASMSCs are not and whether ourresults can be repeated in vivo remain unanswered Futurestudies should focus on these questions and further elucidatethe mechanism of AS

Competing Interests

The authors have no potential conflict of interests to declare

Authorsrsquo Contributions

Zhenhua Liu LiangbinGao andPengWanghave contributedequally to this work

Acknowledgments

The authors thank Dr Liang Zhang for insightful discussionThis study was supported by the National Natural ScienceFoundation of China (81401850) the Science and Tech-nology Project of Guangdong Province (2015B0202280012015B090903059) and the Engineering Technology ResearchCenter for Comprehensive Diagnosis and Treatment ofAnkylosing Spondylitis of Guangdong Higher EducationInstitutes (GCZX-A1301)

References

[1] M Dougados and D Baeten ldquoSpondyloarthritisrdquo The Lancetvol 377 no 9783 pp 2127ndash2137 2011

[2] P Bowness ldquoHLA-B27rdquo Annual Review of Immunology vol 33pp 29ndash48 2015

[3] J D Reveille A-M Sims P Danoy et al ldquoGenome-wideassociation study of ankylosing spondylitis identifies non-MHCsusceptibility locirdquo Nature Genetics vol 42 no 2 pp 123ndash1272010

[4] J D Taurog J A Richardson J T Croft et al ldquoThe germfreestate prevents development of gut and joint inflammatorydisease in HLA-B27 transgenic ratsrdquo Journal of ExperimentalMedicine vol 180 no 6 pp 2359ndash2364 1994

[5] M-E Costello F Ciccia D Willner et al ldquoBrief Reportintestinal dysbiosis in ankylosing spondylitisrdquo Arthritis andRheumatology vol 67 no 3 pp 686ndash691 2015

Stem Cells International 13

[6] T Rashid and A Ebringer ldquoAnkylosing spondylitis is linked toKlebsiellamdashthe evidencerdquo Clinical Rheumatology vol 26 no 6pp 858ndash864 2007

[7] P Jacques and D McGonagle ldquoThe role of mechanical stress inthe pathogenesis of spondyloarthritis and how to combat itrdquoBest Practice and Research Clinical Rheumatology vol 28 no5 pp 703ndash710 2014

[8] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008

[9] M C Stewart and A A Stewart ldquoMesenchymal stem cellscharacteristics sources and mechanisms of actionrdquo VeterinaryClinics of North AmericamdashEquine Practice vol 27 no 2 pp243ndash261 2011

[10] L Y Sun H Y Zhang X B Feng Y Y Hou LW Lu and L MFan ldquoAbnormality of bone marrow-derived mesenchymal stemcells in patients with systemic lupus erythematosusrdquo Lupus vol16 no 2 pp 121ndash128 2007

[11] J A Perez-Simon S Tabera M E Sarasquete et al ldquoMes-enchymal stem cells are functionally abnormal in patients withimmune thrombocytopenic purpurardquo Cytotherapy vol 11 no6 pp 698ndash705 2009

[12] Z Xie P Wang Y Li et al ldquoImbalance between bone morpho-genetic protein 2 and noggin induces abnormal osteogenic dif-ferentiation of mesenchymal stem cells in ankylosing spondyli-tisrdquo Arthritis and Rheumatology vol 68 no 2 pp 430ndash4402016

[13] D Brenner H Blaser and T W Mak ldquoRegulation of tumournecrosis factor signalling live or let dierdquo Nature ReviewsImmunology vol 15 no 6 pp 362ndash374 2015

[14] J Gratacos A Collado X Filella et al ldquoSerum cytokines(IL-6 TNF-120572 IL-1 120573 and IFN-120574) in ankylosing spondylitis aclose correlation between serum IL-6 and disease activity andseverityrdquo British Journal of Rheumatology vol 33 no 10 pp927ndash931 1994

[15] N Barkham L C Coates H Keen et al ldquoDouble-blindplacebo-controlled trial of etanercept in the prevention of workdisability in ankylosing spondylitisrdquo Annals of the RheumaticDiseases vol 69 no 11 pp 1926ndash1928 2010

[16] J Callhoff J Sieper AWeiss A Zink and J Listing ldquoEfficacy ofTNF120572 blockers in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis ameta-analysisrdquoAnnals ofthe Rheumatic Diseases vol 74 no 6 pp 1241ndash1248 2015

[17] L Wang F Du and X Wang ldquoTNF-120572 induces two distinctcaspase-8 activation pathwaysrdquoCell vol 133 no 4 pp 693ndash7032008

[18] B Favaloro N Allocati V Graziano C Di Ilio and V DeLaurenzi ldquoRole of apoptosis in diseaserdquo Aging vol 4 no 5 pp330ndash349 2012

[19] X Li L Liu D Meng et al ldquoEnhanced apoptosis and senes-cence of bone-marrow-derived mesenchymal stem cells inpatients with systemic lupus erythematosusrdquo Stem Cells andDevelopment vol 21 no 13 pp 2387ndash2394 2012

[20] G R Screaton J Mongkolsapaya X-N Xu A E Cowper A JMcMichael and J I Bell ldquoTRICK2 a new alternatively splicedreceptor that transduces the cytotoxic signal from TRAILrdquoCurrent Biology vol 7 no 9 pp 693ndash696 1997

[21] E Szegezdi A OrsquoReilly Y Davy et al ldquoStem cells are resistantto TRAIL receptor-mediated apoptosisrdquo Journal of Cellular andMolecular Medicine vol 13 no 11-12 pp 4409ndash4414 2009

[22] H Walczak M A Degli-Esposti R S Johnson et al ldquoTRAIL-R2 a novel apoptosis-mediating receptor for TRAILrdquo EMBOJournal vol 16 no 17 pp 5386ndash5397 1997

[23] J-L Bodmer N Holler S Reynard et al ldquoTRAIL receptor-2signals apoptosis through FADD and caspase-8rdquo Nature CellBiology vol 2 no 4 pp 241ndash243 2000

[24] S Van Der Linden H A Valkenburg and A Cats ldquoEvaluationof diagnostic criteria for ankylosing spondylitis A proposal formodification of the New York criteriardquo Arthritis and Rheuma-tism vol 27 no 4 pp 361ndash368 1984

[25] D C Colter R Class C M DiGirolamo and D J ProckopldquoRapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrowrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3213ndash3218 2000

[26] D Li P Wang Y Li et al ldquoAll-Trans Retinoic Acid Improvesthe Effects of Bone Marrow-Derived Mesenchymal Stem Cellson the Treatment of Ankylosing Spondylitis An InVitro StudyrdquoStem Cells International vol 2015 Article ID 484528 10 pages2015

[27] K Fu H Ren Y Wang E Fei H Wang and G Wang ldquoDJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8recruitment to FADDrdquo Oncogene vol 31 no 10 pp 1311ndash13222012

[28] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cell intravenous infusion in active ankylos-ing spondylitis patients who failed NSAIDs a 20-week clinicaltrialrdquo Cell Transplantation vol 23 no 10 pp 1293ndash1303 2014

[29] Y Mei F Pan J Gao et al ldquoIncreased serum IL-17 and IL-23 inthe patient with ankylosing spondylitisrdquoClinical Rheumatologyvol 30 no 2 pp 269ndash273 2011

[30] R J Francois L Neure J Sieper and J Braun ldquoImmunohis-tological examination of open sacroiliac biopsies of patientswith ankylosing spondylitis detection of tumour necrosis factor120572 in two patients with early disease and transforming growthfactor 120573 in three more advanced casesrdquoAnnals of the RheumaticDiseases vol 65 no 6 pp 713ndash720 2006

[31] J Braun M Bollow L Neure et al ldquoUse of immunohistologicand in situ hybridization techniques in the examination ofsacroiliac joint biopsy specimens from patients with ankylosingspondylitisrdquo Arthritis and Rheumatism vol 38 no 4 pp 499ndash505 1995

[32] U Lange J Teichmann and H Stracke ldquoCorrelation betweenplasma TNF-alpha IGF-1 biochemical markers of bonemetabolismmarkers of inflammationdisease activity and clin-ical manifestations in ankylosing spondylitisrdquo European Journalof Medical Research vol 5 no 12 pp 507ndash511 2000

[33] L Zhao J Huang H Zhang et al ldquoTumor necrosis factorinhibits mesenchymal stem cell differentiation into osteoblastsvia the ubiquitin E3 ligase Wwp1rdquo Stem Cells vol 29 no 10 pp1601ndash1610 2011

[34] L Chiossone R Conte G M Spaggiari et al ldquoMesenchymalstromal cells induce peculiar alternatively activated macro-phages capable of dampening both innate and adaptive immuneresponsesrdquo STEM CELLS vol 34 no 7 pp 1909ndash1921 2016

[35] S Nagata ldquoApoptosis and autoimmune diseasesrdquo Annals of theNew York Academy of Sciences vol 1209 no 1 pp 10ndash16 2010

[36] M S Sheikh T F Burns Y Huang et al ldquop53-dependent and-independent regulation of the death receptor KILLERDR5gene expression in response to genotoxic stress and tumor

14 Stem Cells International

necrosis factorrdquo Cancer Research vol 58 no 8 pp 1593ndash15981998

[37] S Shirley A Morizot and O Micheau ldquoRegulating TRAILreceptor-induced cell death at the membrane a deadly discus-sionrdquo Recent Patents on Anti-Cancer Drug Discovery vol 6 no3 pp 311ndash323 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 Stem Cells International

HO

-1H

MO

X1H

SP32

HO

-2H

MO

X2

HSP

60H

SP70

PON

2

XIA

P

FADD

Total caspase-3 TRAIL-R2

BAD

BAX

BCL-

2BC

L-X

Pro-

casp

ase-

3Cl

eave

d ca

spas

e-3

Cata

lase

CIA

P-1

CIA

P-2

Clas

pin

Clus

terin

Cyto

chro

me C

TRA

IL R

1D

R4TR

AIL

R2

DR5

FAD

DFA

STN

FSF6

CD

95H

IF-1

a

HSP

27

HTR

A2

Om

iLi

vin

P21

CIP1

CD

KN1A

P27

Kip1

Phos

pho-

p53(

S15)

Phos

pho-

p53(

S46)

Phos

pho-

p53(

S392

)Ph

osph

o-Ra

d17(

S635

)SM

ACD

iabl

oSu

rviv

inTN

FR1

TNFR

SF1A

00

05

10

15

1

1

2

2

3

3

4

4

1 2

3 4

Mea

n re

lativ

e int

ensit

y

HDMSCsASMSCs

(a)

HDMSCs

HDMSCs

ASMSCs

ASMSCs

HDMSCsASMSCs

HDMSCsASMSCs

lowast

lowast

Control TNF-120572CHX Control TNF-120572CHX

Control TNF-120572CHX

0

2

4

6

8

Relat

ive m

RNA

expr

essio

nFo

ld ch

ange

0

2

4

6

Fold

chan

ge

(b)

0

2

4

6

8

Fold

chan

ge

Figure 3 Continued

Stem Cells International 7

Cleaved caspase-3

Pro-caspase-3

Actin

35 kD

19 kD17 kD

48 kD

40 kD

28 kD

45 kD

TRAIL-R2

FADD

HDMSCs ASMSCs

+

+

minus

minus

+

+

minus

minus

TNF-120572

CHX

Cleaved caspase-3

0

5

10

15

20

Fold

chan

ge

0

1

2

3

4

Fold

chan

ge

0

5

10

15

Fold

chan

ge

FADDTRAIL-R2

Control TNF-120572CHX Control TNF-120572CHX Control TNF-120572CHX

HDMSCsASMSCs

HDMSCsASMSCs

HDMSCsASMSCs

(c)

lowastlowast

lowast

Figure 3 ASMSCs had increased protein expression of cleaved caspase-3 TRAIL-R2 and FADD and increased gene expression of TRAIL-R2 and FADD during apoptosis (a) The Proteome Profiler Human Apoptosis Array Kit was used to determine the expression levels of 35apoptosis-related proteins in HDMSCs (119899 = 6) and ASMSCs (119899 = 6) after treatment with TNF-120572CHX The intensity of each pair of spotsrepresents repeated measures of one protein ASMSCs had lower pro-caspase-3 (a1) expression but higher cleaved caspase-3 (a2) TRAIL-R2(a3) and FADD (a4) expression levels than HDMSCs No significant differences in the expression of the remaining 31 proteins were foundbetween HDMSCs and ASMSCs (b) qRT-PCR was performed to confirm the results of the Proteome ProfilerHuman Apoptosis ArrayTheresults of TRAIL-R2 and FADD in HDMSCs (119899 = 28) and ASMSCs (119899 = 22) were consistent with those of the Proteome Profiler HumanApoptosis Array However total caspase-3 mRNA expression was not different between HDMSCs and ASMSCs (c) Western blot analysiswas performed on proteins isolated from HDMSCs (119899 = 28) and ASMSCs (119899 = 22) to further confirm the results of the Proteome ProfilerHuman Apoptosis Array and the results of both methods were in agreement Values are presented as the mean plusmn SD lowast indicates 119875 lt 005between HDMSCs and ASMSCs

while no difference was found between HDMSCs and ASM-SCs in total cytochrome C (Figure 4(a)) The protein expres-sion of both cytosolic cytochrome C and cleaved caspase-8increased after treatment with TNF-120572CHX and comparedwith HDMSCs ASMSCs exhibited much higher expressionof these two proteins (Figure 4(b)) Because cytochrome Cneeds to be released into the cytoplasm for apoptosis tooccur the mRNA expression level of total cytochrome C wasnot as meaningful as the cytosolic protein expression Wealso detected the gene and protein expression of TNFR1 andFas two important receptors that in addition to TRAIL-R2 interact with FADD However although both the gene

and protein expression levels increased after treatment withTNF-120572CHX no difference in gene or protein expressionwas found between the two groups either with or withoutTNF-120572CHX treatment (Figures 4(a) and 4(b))These resultstogether suggest that both the DR pathway and the mito-chondrial pathway are involved in the altered apoptosis ofASMSCs neither TNFR1 nor Fas accounted for the increasedexpression of FADD in the DR pathway

35 Lentiviruses Encoding shRNA-TRAIL-R2 Partly InhibitedtheApoptosis of ASMSCs butHad a Limited Effect on theApop-tosis of HDMSCs Because ASMSCs express more TRAIL-R2

8 Stem Cells International

Fas TNFR1Total cytochrome CTotal caspase-8

0

2

4

6

Relat

ive m

RNA

expr

essio

nFo

ld ch

ange

0

1

2

3

4

Fold

chan

ge

0

1

2

3

Fold

chan

ge

0

1

2

3

Fold

chan

ge

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

lowast

(a)

0

20

40

60

Fold

chan

ge

000510152025

Fold

chan

ge

000510152025

Fold

chan

ge

0

10

20

30

Fold

chan

ge

Cleaved caspase-8

Control TNF-120572CHX Control TNF-120572CHX

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

HDMSCsASMSCs

HDMSCsASMSCs

Cytosolic cytochrome C

FasTNFR1

43 kD41 kD

55 kD

45 kD

45 kD

17 kD

HDMSCs ASMSCs

+

+

minus

minus

+

+

minus

minus

TNF-120572CHX

40sim50 kD

Cleaved caspase-8

TNFR1

Fas

Actin 1

Actin 2

Cytosolic cytochrome C

lowast lowast

(b)

Figure 4 The DR and mitochondrial pathways were both involved in MSC apoptosis (a) qRT-PCR was performed to compare the geneexpressions of total caspase-8 total cytochrome C Fas and TNFR1 between HDMSCs (119899 = 28) and ASMSCs (119899 = 22) ASMSCs expressedmuchhigher gene expression levels of total caspase-8 thanHDMSCs after treatmentwithTNF-120572CHXwhile no differencewas found betweenHDMSCs andASMSCs in total cytochromeC Fas or TNFR1 gene expression levels (b) Awestern blot was performed to compare the proteinexpressions of cleaved caspase-8 cytosolic cytochrome C TNFR1 and Fas betweenHDMSCs (119899 = 28) and ASMSCs (119899 = 22) After treatmentwith TNF-120572CHX ASMSCs had higher cleaved caspase-8 and cytosolic cytochrome C protein expression levels than HDMSCs and similarprotein levels of TNFR1 and Fas as HDMSCs Fold changes are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs andASMSCs

than HDMSCs during apoptosis we constructed Lv-TRAIL-R2 to explore the role that TRAIL-R2 plays in the apopto-sis of ASMSCs GFP-positive cells were observed under aconfocal microscope and the transduction efficiencies werecomparable between HDMSCs and ASMSCs (Figure 5(a))Lv-TRAIL-R2 inhibited both gene and protein expression ofTRAIL-R2 inHDMSCs andASMSCs (Figure 5(b))The FCMresults demonstrated that Lv-TRAIL-R2 could reduce theapoptosis of ASMSCs but had a limited effect on the apoptosisof HDMSCs (Figure 6(a)) qRT-PCR showed that Lv-TRAIL-R2 inhibited the expression of FADD and total caspase-8 inASMSCs but had a limited effect on total caspase-3 and totalcytochrome C Meanwhile Lv-TRAIL-R2 did not affect the

expression of the four related aforementioned genes inHDM-SCs After inhibiting TRAIL-R2 expression no apparent dif-ference in gene expression was found between HDMSCs andASMSCs (Figure 6(b)) Additionally Lv-TRAIL-R2 did notaffect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC inHDMSCs but reducedthem in ASMSCs Furthermore no apparent difference inthe protein expression of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochrome C was found betweenHDMSCs andASMSCs after inhibitingTRAIL-R2 expression(Figure 6(c)) Therefore inhibiting TRAIL-R2 expressionpartly decreased the apoptosis of ASMSCs but had a limitedeffect on HDMSCs

Stem Cells International 9

HDMSCs ASMSCs

(a)

HDMSCsASMSCs

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

Con

trol

TNF-

120572C

HX

Control TNF-120572CHX Lv-NC Lv-TRAIL-R2 Control TNF-120572CHX Lv-NC Lv-TRAIL-R2

TRAIL R2

TRAIL-R2 TRAIL-R2

Actin

48 kD

40 kD

45 kD

0

1

2

3

4

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

lowast

amp

amp

amp

amp

lowast

lowastlowast

0

1

2

3

4

5

Relat

ive p

rote

in ex

pres

sion

Fold

chan

ge

HDMSCs ASMSCs

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

TNF-120572CHX + TNF-120572CHX + TNF-120572CHX+ TNF-120572CHX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

(b)

Figure 5 The transduction efficiencies of lentiviruses encoding shRNA-TRAIL-R2 were comparable in HDMSCs and ASMSCs (a) GFP-positive cells were observed under a confocal microscope (40x) and the transduction efficiencies were comparable between HDMSCs andASMSCs (b)The lentiviruses encoding shRNA-TRAIL-R2 inhibited both the gene and protein expression of TRAIL-R2 inHDMSCs (119899 = 28)and ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs and ASMSCs amp indicates 119875 lt 005 forthe TNF-120572CHXgroup comparedwith otherHDMSCs groups indicates119875 lt 005 for the TNF-120572CHXgroup comparedwith other ASMSCsgroups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative control sequence Lv-TRAIL-R2 refers to MSCs transduced bylentiviruses encoding TRAIL-R2

4 Discussion

Abnormal MSCs are proposed to be involved in the patho-genesis of AS and a better understanding of ASMSCsdysfunctions could improve the understanding of AS Inthis study ASMSCs had morphologies phenotypes and

proliferation rates that were similar to HDMSCs HoweverASMSCs were more susceptible to TNF-120572CHX-inducedapoptosis compared to HDMSCs ASMSCs expressed higherlevels of TRAIL-R2 which activated both the DR and themitochondrial pathways by increasing the expression levelsof FADD cleaved caspase-8 and cytosolic cytochrome C

10 Stem Cells International

Annexin V-PE

103 535

18627500

032 536

17447688

046 456

15317967

050 046

1519753

018 040

1179825

056 950

30895905

079 868

28526201

586027

14987889

HD

MSC

sA

SMSC

s7-

AA

D

104

104

103

103

102

102

101

101

100

104

103

102

101

100

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

104

103

102

101

100

104

103

102

101

100

10010

410

310

210

110

0

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX +

Lv-NC Lv-TRAIL-R2

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX+

LV-NC LV-TRAIL-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX

+

HDMSCsASMSCs

0

20

40

60

Apop

tosis

rate

()

LV-N

C

LV-N

C

LV-N

C

LV-N

C

0

2

4

6

8

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

Total caspase-3 Total caspase-8

0

2

4

6

Fold

chan

ge

Fold

chan

ge

0

2

4

6

8

0

1

2

3

4

Fold

chan

ge

Total cytochrome CFADD

amp

amp amp amp

amp

lowast

lowastlowast

lowast

lowast lowast

(a)

(b)

Figure 6 Continued

Stem Cells International 11

Con

trol

TNF-120572

CH

X

TNF-120572CHX

HDMSCsASMSCs

Con

trol

TNF-120572

CH

X

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Cleaved caspase-3 Cleaved caspase-8

Cytosoliccytochrome CFADD

0

5

10

15

20

Fold

chan

ge

0

5

10

15

Fold

chan

ge

0

10

20

30

Fold

chan

ge

0

20

40

60

80

Fold

chan

ge

amp amp

amp

lowastlowast

lowast lowast

lowastlowast

lowastlowast

35 kD

19 kD17 kD

43 kD41 kD

28 kD

45 kD

17 kD

45 kD

HDMSCs ASMSCs

+++

+

+

minus

minusminus

minusminus

minus

minus

+++

+

+

minus

minusminus

minusminus

minus

minus

Cleaved caspase-3Cleaved caspase-8

FADD

Actin 1

Actin 2

Cytosolic cytochrome C

(c)

Pro-caspase-3

LV-NCLV-TRAIL-R2

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

CTN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+TN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

Figure 6 Lentiviruses encoding shRNA-TRAIL-R2 partly inhibited the apoptosis of ASMSCs while having a limited effect on the apoptosisof HDMSCs (a) The rate of ASMSC apoptosis (119899 = 22) was decreased after Lv-TRAIL-R2 transduction while no apparent changes werefound for HDMSCs (119899 = 28) (b) In ASMSCs (119899 = 22) Lv-TRAIL-R2 reduced FADD and total caspase-8 expression while having a limitedeffect on total caspase-3 and total cytochrome C gene expression In HDMSCs (119899 = 28) Lv-TRAIL-R2 had a limited effect on the expressionof all four genes (c) Lv-TRAIL-R2 did not affect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC in HDMSCs (119899 = 28) but reduced them in ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 betweenHDMSCs and ASMSCs amp indicates 119875 lt 005 for the TNF-120572CHX group compared with other HDMSC groups indicates 119875 lt 005 forthe TNF-120572CHX group compared with other ASMSC groups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative controlsequence Lv-TRAIL-R2 refers to MSCs transduced by lentiviruses encoding TRAIL-R2

Inhibiting TRAIL-R2 expression using shRNA eliminated thedifference in apoptosis between HDMSCs and ASMSCs bydecreasing the apoptosis of ASMSCs

MSCs aremultipotent stromal cells withmultilineage dif-ferentiation capacities and immunomodulatory propertiesOur previous studies suggested that MSCs are implicated inthe pathogenesis and therapy of AS [12 28] We found thatASMSCs possessed an abnormal osteogenic capacity whichmay contribute to the pathogenesis of AS In addition we alsofound that allogenic intravenous infusion of bone marrow-derived MSCs is feasible and safe in the treatment of ASHowever how the in vivo microenvironment in AS interactswith MSCs remains unknown

AS is a complicated autoimmune disease accompaniedby elevated serum levels of various inflammatory cytokines[14 29] Among these cytokines TNF-120572 is one of the most

important players in the pathogenesis of AS PhysiologicallyTNF-120572 is important for a normal response to inflammationbut the overproduction of TNF-120572 can be detrimental Serumlevels and the sacroiliac joint expression of TNF-120572 wereelevated in AS patients including those in the early stages ofAS [30 31] In addition overproduction of TNF-120572 has beensuggested to be related to disease activity and to account forthe pathogenesis of AS [32] The importance of TNF-120572 hasbeen further confirmed by the efficacy of TNF-120572 inhibitorsin the treatment of AS [16] Due to the importance of TNF-120572in AS a study on the interaction between TNF-120572 and MSCsmay help mimic the in vivo microenvironment experiencedby MSCs in AS Previous studies suggest that TNF-120572 affectsMSCs in various ways TNF-120572 can modulate the immuno-suppressive effects of MSCs and suppress their osteogeniccapacity [33] Additionally TNF-120572 can alter homeostasis

12 Stem Cells International

in MSCs by regulating the balance between apoptosis andsurvival [13] ConverselyMSCs affect the production of TNF-120572 by causing macrophages to adopt an anti-inflammatoryphenotype [34] Both the dysfunction of MSCs and theoverproduction of TNF-120572 may lead to immune disordersA recent study suggested that TNF-120572-induced apoptosis ofMSCs may contribute to the pathogenesis of SLE [19] In ourstudy we found that ASMSCs were more susceptible to TNF-120572-induced apoptosis compared to HDMSCs as evidencedby an increased apoptosis rate and the higher expression ofcleaved caspase-3 protein However how the dysregulationof apoptosis contributes to the pathogenesis of AS remainsuncertain

Apoptosis also defined as programmed cell death iscritical to the pathogenesis of various diseases includingautoimmune diseases [18] The maintenance of immunetolerance and immune homeostasis is largely dependent onapoptosis Defective apoptosis of immune cells may result inthe defective clearance of autoreactive cells or the delayedelimination of autoantigens while excessive apoptosis ofimmunosuppressive cells (such as MSCs) may also lead toimmune hyperactivation Therefore both conditions maycontribute to autoimmune disease [19 35] In patients withAS increased apoptosis of ASMSCs may result in decreasesin cell numbers and immunosuppressive potential eventuallycontributing to autoimmune disorders Moreover becauseMSCs are able to reduce TNF-120572 production increasedMSCs apoptosis may also lead to TNF-120572 overproductionFurthermore TNF-120572 overproduction induces further MSCapoptosis forming a vicious cycle that leads to chronicinflammation in AS Thus increased ASMSC apoptosis maybe critical for AS pathogenesis and further study of themechanism of dysregulated apoptosis in ASMSCs may helpto elucidate the pathogenesis of AS

TRAIL-R2 is a specific cell surface receptor engagedin apoptosis TRAIL-R2-mediated apoptosis has arousedattention due to its role in preferentially killing tumor cellswhile sparing normal cells In our study we observed asignificant increased expression of TRAIL-R2 in ASMSCsafter treatment with TNF-120572CHX Inhibiting TRAIL-R2expression partly decreased the apoptosis of ASMSCs buthad a limited effect on the apoptosis of HDMSCs whichsuggests that TRAIL-R2 overexpression is the main cause ofthe increased apoptosis in ASMSCs However this result isconfusing for two reasons First TRAIL-R2 is reported to bemainly activated by its specific ligand TRAIL [20] Howeverthe results in our study indicated that TRAIL-R2 in MSCscould be activated by TNF-120572CHX as well In fact a similarresult was previously reported but the mechanism remainsunclear [36] Considering the overexpression of TRAIL-R2 can lead to apoptosis even without its ligand [22] wespeculate that TNF-120572maymediate its proapoptotic effects notonly through its own receptor (TNFR1) but also by increasingthe expression of TRAIL-R2 Second bone marrow-derivedadult MSCs are reportedly resistant to TRAIL-R2-mediatedapoptosis [21] However in our study HDMSCs were resis-tant to TRAIL-R2-mediated apoptosis while ASMSCs werenot Multiple mechanisms of TRAIL-R2-mediated apoptosisresistance such as dysregulation of antagonistic receptors

abnormal O-glycosylation and overexpression of the inhi-bition adaptors have been identified [37] Further studiesare needed to illustrate the associated specific mechanismunderlying the difference in resistance of HDMSCs andASMSCs to TRAIL-R2 Additionally based on the findingthat the TRAIL-R2 pathway did not lead to the apoptosis ofnormal cells ASMSCs could be considered abnormal cellsThus it is reasonable to presume that allogenic rather thanautologous MSC transplantation might be preferable for AStreatment

In this study we demonstrated that TRAIL-R2 upregu-lation resulted in enhanced apoptosis of ASMSCs Increasedapoptosis of ASMSCs may decrease the immunosuppressivepotential of these cells and potentially contributing to ASpathogenesis However some limitations remain in thisstudy The questions of how TNF-120572 activates the expressionof TRAIL-R2 why HDMSCs are resistant to TRAIL-R2-mediated apoptosis while ASMSCs are not and whether ourresults can be repeated in vivo remain unanswered Futurestudies should focus on these questions and further elucidatethe mechanism of AS

Competing Interests

The authors have no potential conflict of interests to declare

Authorsrsquo Contributions

Zhenhua Liu LiangbinGao andPengWanghave contributedequally to this work

Acknowledgments

The authors thank Dr Liang Zhang for insightful discussionThis study was supported by the National Natural ScienceFoundation of China (81401850) the Science and Tech-nology Project of Guangdong Province (2015B0202280012015B090903059) and the Engineering Technology ResearchCenter for Comprehensive Diagnosis and Treatment ofAnkylosing Spondylitis of Guangdong Higher EducationInstitutes (GCZX-A1301)

References

[1] M Dougados and D Baeten ldquoSpondyloarthritisrdquo The Lancetvol 377 no 9783 pp 2127ndash2137 2011

[2] P Bowness ldquoHLA-B27rdquo Annual Review of Immunology vol 33pp 29ndash48 2015

[3] J D Reveille A-M Sims P Danoy et al ldquoGenome-wideassociation study of ankylosing spondylitis identifies non-MHCsusceptibility locirdquo Nature Genetics vol 42 no 2 pp 123ndash1272010

[4] J D Taurog J A Richardson J T Croft et al ldquoThe germfreestate prevents development of gut and joint inflammatorydisease in HLA-B27 transgenic ratsrdquo Journal of ExperimentalMedicine vol 180 no 6 pp 2359ndash2364 1994

[5] M-E Costello F Ciccia D Willner et al ldquoBrief Reportintestinal dysbiosis in ankylosing spondylitisrdquo Arthritis andRheumatology vol 67 no 3 pp 686ndash691 2015

Stem Cells International 13

[6] T Rashid and A Ebringer ldquoAnkylosing spondylitis is linked toKlebsiellamdashthe evidencerdquo Clinical Rheumatology vol 26 no 6pp 858ndash864 2007

[7] P Jacques and D McGonagle ldquoThe role of mechanical stress inthe pathogenesis of spondyloarthritis and how to combat itrdquoBest Practice and Research Clinical Rheumatology vol 28 no5 pp 703ndash710 2014

[8] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008

[9] M C Stewart and A A Stewart ldquoMesenchymal stem cellscharacteristics sources and mechanisms of actionrdquo VeterinaryClinics of North AmericamdashEquine Practice vol 27 no 2 pp243ndash261 2011

[10] L Y Sun H Y Zhang X B Feng Y Y Hou LW Lu and L MFan ldquoAbnormality of bone marrow-derived mesenchymal stemcells in patients with systemic lupus erythematosusrdquo Lupus vol16 no 2 pp 121ndash128 2007

[11] J A Perez-Simon S Tabera M E Sarasquete et al ldquoMes-enchymal stem cells are functionally abnormal in patients withimmune thrombocytopenic purpurardquo Cytotherapy vol 11 no6 pp 698ndash705 2009

[12] Z Xie P Wang Y Li et al ldquoImbalance between bone morpho-genetic protein 2 and noggin induces abnormal osteogenic dif-ferentiation of mesenchymal stem cells in ankylosing spondyli-tisrdquo Arthritis and Rheumatology vol 68 no 2 pp 430ndash4402016

[13] D Brenner H Blaser and T W Mak ldquoRegulation of tumournecrosis factor signalling live or let dierdquo Nature ReviewsImmunology vol 15 no 6 pp 362ndash374 2015

[14] J Gratacos A Collado X Filella et al ldquoSerum cytokines(IL-6 TNF-120572 IL-1 120573 and IFN-120574) in ankylosing spondylitis aclose correlation between serum IL-6 and disease activity andseverityrdquo British Journal of Rheumatology vol 33 no 10 pp927ndash931 1994

[15] N Barkham L C Coates H Keen et al ldquoDouble-blindplacebo-controlled trial of etanercept in the prevention of workdisability in ankylosing spondylitisrdquo Annals of the RheumaticDiseases vol 69 no 11 pp 1926ndash1928 2010

[16] J Callhoff J Sieper AWeiss A Zink and J Listing ldquoEfficacy ofTNF120572 blockers in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis ameta-analysisrdquoAnnals ofthe Rheumatic Diseases vol 74 no 6 pp 1241ndash1248 2015

[17] L Wang F Du and X Wang ldquoTNF-120572 induces two distinctcaspase-8 activation pathwaysrdquoCell vol 133 no 4 pp 693ndash7032008

[18] B Favaloro N Allocati V Graziano C Di Ilio and V DeLaurenzi ldquoRole of apoptosis in diseaserdquo Aging vol 4 no 5 pp330ndash349 2012

[19] X Li L Liu D Meng et al ldquoEnhanced apoptosis and senes-cence of bone-marrow-derived mesenchymal stem cells inpatients with systemic lupus erythematosusrdquo Stem Cells andDevelopment vol 21 no 13 pp 2387ndash2394 2012

[20] G R Screaton J Mongkolsapaya X-N Xu A E Cowper A JMcMichael and J I Bell ldquoTRICK2 a new alternatively splicedreceptor that transduces the cytotoxic signal from TRAILrdquoCurrent Biology vol 7 no 9 pp 693ndash696 1997

[21] E Szegezdi A OrsquoReilly Y Davy et al ldquoStem cells are resistantto TRAIL receptor-mediated apoptosisrdquo Journal of Cellular andMolecular Medicine vol 13 no 11-12 pp 4409ndash4414 2009

[22] H Walczak M A Degli-Esposti R S Johnson et al ldquoTRAIL-R2 a novel apoptosis-mediating receptor for TRAILrdquo EMBOJournal vol 16 no 17 pp 5386ndash5397 1997

[23] J-L Bodmer N Holler S Reynard et al ldquoTRAIL receptor-2signals apoptosis through FADD and caspase-8rdquo Nature CellBiology vol 2 no 4 pp 241ndash243 2000

[24] S Van Der Linden H A Valkenburg and A Cats ldquoEvaluationof diagnostic criteria for ankylosing spondylitis A proposal formodification of the New York criteriardquo Arthritis and Rheuma-tism vol 27 no 4 pp 361ndash368 1984

[25] D C Colter R Class C M DiGirolamo and D J ProckopldquoRapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrowrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3213ndash3218 2000

[26] D Li P Wang Y Li et al ldquoAll-Trans Retinoic Acid Improvesthe Effects of Bone Marrow-Derived Mesenchymal Stem Cellson the Treatment of Ankylosing Spondylitis An InVitro StudyrdquoStem Cells International vol 2015 Article ID 484528 10 pages2015

[27] K Fu H Ren Y Wang E Fei H Wang and G Wang ldquoDJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8recruitment to FADDrdquo Oncogene vol 31 no 10 pp 1311ndash13222012

[28] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cell intravenous infusion in active ankylos-ing spondylitis patients who failed NSAIDs a 20-week clinicaltrialrdquo Cell Transplantation vol 23 no 10 pp 1293ndash1303 2014

[29] Y Mei F Pan J Gao et al ldquoIncreased serum IL-17 and IL-23 inthe patient with ankylosing spondylitisrdquoClinical Rheumatologyvol 30 no 2 pp 269ndash273 2011

[30] R J Francois L Neure J Sieper and J Braun ldquoImmunohis-tological examination of open sacroiliac biopsies of patientswith ankylosing spondylitis detection of tumour necrosis factor120572 in two patients with early disease and transforming growthfactor 120573 in three more advanced casesrdquoAnnals of the RheumaticDiseases vol 65 no 6 pp 713ndash720 2006

[31] J Braun M Bollow L Neure et al ldquoUse of immunohistologicand in situ hybridization techniques in the examination ofsacroiliac joint biopsy specimens from patients with ankylosingspondylitisrdquo Arthritis and Rheumatism vol 38 no 4 pp 499ndash505 1995

[32] U Lange J Teichmann and H Stracke ldquoCorrelation betweenplasma TNF-alpha IGF-1 biochemical markers of bonemetabolismmarkers of inflammationdisease activity and clin-ical manifestations in ankylosing spondylitisrdquo European Journalof Medical Research vol 5 no 12 pp 507ndash511 2000

[33] L Zhao J Huang H Zhang et al ldquoTumor necrosis factorinhibits mesenchymal stem cell differentiation into osteoblastsvia the ubiquitin E3 ligase Wwp1rdquo Stem Cells vol 29 no 10 pp1601ndash1610 2011

[34] L Chiossone R Conte G M Spaggiari et al ldquoMesenchymalstromal cells induce peculiar alternatively activated macro-phages capable of dampening both innate and adaptive immuneresponsesrdquo STEM CELLS vol 34 no 7 pp 1909ndash1921 2016

[35] S Nagata ldquoApoptosis and autoimmune diseasesrdquo Annals of theNew York Academy of Sciences vol 1209 no 1 pp 10ndash16 2010

[36] M S Sheikh T F Burns Y Huang et al ldquop53-dependent and-independent regulation of the death receptor KILLERDR5gene expression in response to genotoxic stress and tumor

14 Stem Cells International

necrosis factorrdquo Cancer Research vol 58 no 8 pp 1593ndash15981998

[37] S Shirley A Morizot and O Micheau ldquoRegulating TRAILreceptor-induced cell death at the membrane a deadly discus-sionrdquo Recent Patents on Anti-Cancer Drug Discovery vol 6 no3 pp 311ndash323 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 7

Cleaved caspase-3

Pro-caspase-3

Actin

35 kD

19 kD17 kD

48 kD

40 kD

28 kD

45 kD

TRAIL-R2

FADD

HDMSCs ASMSCs

+

+

minus

minus

+

+

minus

minus

TNF-120572

CHX

Cleaved caspase-3

0

5

10

15

20

Fold

chan

ge

0

1

2

3

4

Fold

chan

ge

0

5

10

15

Fold

chan

ge

FADDTRAIL-R2

Control TNF-120572CHX Control TNF-120572CHX Control TNF-120572CHX

HDMSCsASMSCs

HDMSCsASMSCs

HDMSCsASMSCs

(c)

lowastlowast

lowast

Figure 3 ASMSCs had increased protein expression of cleaved caspase-3 TRAIL-R2 and FADD and increased gene expression of TRAIL-R2 and FADD during apoptosis (a) The Proteome Profiler Human Apoptosis Array Kit was used to determine the expression levels of 35apoptosis-related proteins in HDMSCs (119899 = 6) and ASMSCs (119899 = 6) after treatment with TNF-120572CHX The intensity of each pair of spotsrepresents repeated measures of one protein ASMSCs had lower pro-caspase-3 (a1) expression but higher cleaved caspase-3 (a2) TRAIL-R2(a3) and FADD (a4) expression levels than HDMSCs No significant differences in the expression of the remaining 31 proteins were foundbetween HDMSCs and ASMSCs (b) qRT-PCR was performed to confirm the results of the Proteome ProfilerHuman Apoptosis ArrayTheresults of TRAIL-R2 and FADD in HDMSCs (119899 = 28) and ASMSCs (119899 = 22) were consistent with those of the Proteome Profiler HumanApoptosis Array However total caspase-3 mRNA expression was not different between HDMSCs and ASMSCs (c) Western blot analysiswas performed on proteins isolated from HDMSCs (119899 = 28) and ASMSCs (119899 = 22) to further confirm the results of the Proteome ProfilerHuman Apoptosis Array and the results of both methods were in agreement Values are presented as the mean plusmn SD lowast indicates 119875 lt 005between HDMSCs and ASMSCs

while no difference was found between HDMSCs and ASM-SCs in total cytochrome C (Figure 4(a)) The protein expres-sion of both cytosolic cytochrome C and cleaved caspase-8increased after treatment with TNF-120572CHX and comparedwith HDMSCs ASMSCs exhibited much higher expressionof these two proteins (Figure 4(b)) Because cytochrome Cneeds to be released into the cytoplasm for apoptosis tooccur the mRNA expression level of total cytochrome C wasnot as meaningful as the cytosolic protein expression Wealso detected the gene and protein expression of TNFR1 andFas two important receptors that in addition to TRAIL-R2 interact with FADD However although both the gene

and protein expression levels increased after treatment withTNF-120572CHX no difference in gene or protein expressionwas found between the two groups either with or withoutTNF-120572CHX treatment (Figures 4(a) and 4(b))These resultstogether suggest that both the DR pathway and the mito-chondrial pathway are involved in the altered apoptosis ofASMSCs neither TNFR1 nor Fas accounted for the increasedexpression of FADD in the DR pathway

35 Lentiviruses Encoding shRNA-TRAIL-R2 Partly InhibitedtheApoptosis of ASMSCs butHad a Limited Effect on theApop-tosis of HDMSCs Because ASMSCs express more TRAIL-R2

8 Stem Cells International

Fas TNFR1Total cytochrome CTotal caspase-8

0

2

4

6

Relat

ive m

RNA

expr

essio

nFo

ld ch

ange

0

1

2

3

4

Fold

chan

ge

0

1

2

3

Fold

chan

ge

0

1

2

3

Fold

chan

ge

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

lowast

(a)

0

20

40

60

Fold

chan

ge

000510152025

Fold

chan

ge

000510152025

Fold

chan

ge

0

10

20

30

Fold

chan

ge

Cleaved caspase-8

Control TNF-120572CHX Control TNF-120572CHX

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

HDMSCsASMSCs

HDMSCsASMSCs

Cytosolic cytochrome C

FasTNFR1

43 kD41 kD

55 kD

45 kD

45 kD

17 kD

HDMSCs ASMSCs

+

+

minus

minus

+

+

minus

minus

TNF-120572CHX

40sim50 kD

Cleaved caspase-8

TNFR1

Fas

Actin 1

Actin 2

Cytosolic cytochrome C

lowast lowast

(b)

Figure 4 The DR and mitochondrial pathways were both involved in MSC apoptosis (a) qRT-PCR was performed to compare the geneexpressions of total caspase-8 total cytochrome C Fas and TNFR1 between HDMSCs (119899 = 28) and ASMSCs (119899 = 22) ASMSCs expressedmuchhigher gene expression levels of total caspase-8 thanHDMSCs after treatmentwithTNF-120572CHXwhile no differencewas found betweenHDMSCs andASMSCs in total cytochromeC Fas or TNFR1 gene expression levels (b) Awestern blot was performed to compare the proteinexpressions of cleaved caspase-8 cytosolic cytochrome C TNFR1 and Fas betweenHDMSCs (119899 = 28) and ASMSCs (119899 = 22) After treatmentwith TNF-120572CHX ASMSCs had higher cleaved caspase-8 and cytosolic cytochrome C protein expression levels than HDMSCs and similarprotein levels of TNFR1 and Fas as HDMSCs Fold changes are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs andASMSCs

than HDMSCs during apoptosis we constructed Lv-TRAIL-R2 to explore the role that TRAIL-R2 plays in the apopto-sis of ASMSCs GFP-positive cells were observed under aconfocal microscope and the transduction efficiencies werecomparable between HDMSCs and ASMSCs (Figure 5(a))Lv-TRAIL-R2 inhibited both gene and protein expression ofTRAIL-R2 inHDMSCs andASMSCs (Figure 5(b))The FCMresults demonstrated that Lv-TRAIL-R2 could reduce theapoptosis of ASMSCs but had a limited effect on the apoptosisof HDMSCs (Figure 6(a)) qRT-PCR showed that Lv-TRAIL-R2 inhibited the expression of FADD and total caspase-8 inASMSCs but had a limited effect on total caspase-3 and totalcytochrome C Meanwhile Lv-TRAIL-R2 did not affect the

expression of the four related aforementioned genes inHDM-SCs After inhibiting TRAIL-R2 expression no apparent dif-ference in gene expression was found between HDMSCs andASMSCs (Figure 6(b)) Additionally Lv-TRAIL-R2 did notaffect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC inHDMSCs but reducedthem in ASMSCs Furthermore no apparent difference inthe protein expression of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochrome C was found betweenHDMSCs andASMSCs after inhibitingTRAIL-R2 expression(Figure 6(c)) Therefore inhibiting TRAIL-R2 expressionpartly decreased the apoptosis of ASMSCs but had a limitedeffect on HDMSCs

Stem Cells International 9

HDMSCs ASMSCs

(a)

HDMSCsASMSCs

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

Con

trol

TNF-

120572C

HX

Control TNF-120572CHX Lv-NC Lv-TRAIL-R2 Control TNF-120572CHX Lv-NC Lv-TRAIL-R2

TRAIL R2

TRAIL-R2 TRAIL-R2

Actin

48 kD

40 kD

45 kD

0

1

2

3

4

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

lowast

amp

amp

amp

amp

lowast

lowastlowast

0

1

2

3

4

5

Relat

ive p

rote

in ex

pres

sion

Fold

chan

ge

HDMSCs ASMSCs

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

TNF-120572CHX + TNF-120572CHX + TNF-120572CHX+ TNF-120572CHX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

(b)

Figure 5 The transduction efficiencies of lentiviruses encoding shRNA-TRAIL-R2 were comparable in HDMSCs and ASMSCs (a) GFP-positive cells were observed under a confocal microscope (40x) and the transduction efficiencies were comparable between HDMSCs andASMSCs (b)The lentiviruses encoding shRNA-TRAIL-R2 inhibited both the gene and protein expression of TRAIL-R2 inHDMSCs (119899 = 28)and ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs and ASMSCs amp indicates 119875 lt 005 forthe TNF-120572CHXgroup comparedwith otherHDMSCs groups indicates119875 lt 005 for the TNF-120572CHXgroup comparedwith other ASMSCsgroups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative control sequence Lv-TRAIL-R2 refers to MSCs transduced bylentiviruses encoding TRAIL-R2

4 Discussion

Abnormal MSCs are proposed to be involved in the patho-genesis of AS and a better understanding of ASMSCsdysfunctions could improve the understanding of AS Inthis study ASMSCs had morphologies phenotypes and

proliferation rates that were similar to HDMSCs HoweverASMSCs were more susceptible to TNF-120572CHX-inducedapoptosis compared to HDMSCs ASMSCs expressed higherlevels of TRAIL-R2 which activated both the DR and themitochondrial pathways by increasing the expression levelsof FADD cleaved caspase-8 and cytosolic cytochrome C

10 Stem Cells International

Annexin V-PE

103 535

18627500

032 536

17447688

046 456

15317967

050 046

1519753

018 040

1179825

056 950

30895905

079 868

28526201

586027

14987889

HD

MSC

sA

SMSC

s7-

AA

D

104

104

103

103

102

102

101

101

100

104

103

102

101

100

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

104

103

102

101

100

104

103

102

101

100

10010

410

310

210

110

0

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX +

Lv-NC Lv-TRAIL-R2

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX+

LV-NC LV-TRAIL-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX

+

HDMSCsASMSCs

0

20

40

60

Apop

tosis

rate

()

LV-N

C

LV-N

C

LV-N

C

LV-N

C

0

2

4

6

8

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

Total caspase-3 Total caspase-8

0

2

4

6

Fold

chan

ge

Fold

chan

ge

0

2

4

6

8

0

1

2

3

4

Fold

chan

ge

Total cytochrome CFADD

amp

amp amp amp

amp

lowast

lowastlowast

lowast

lowast lowast

(a)

(b)

Figure 6 Continued

Stem Cells International 11

Con

trol

TNF-120572

CH

X

TNF-120572CHX

HDMSCsASMSCs

Con

trol

TNF-120572

CH

X

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Cleaved caspase-3 Cleaved caspase-8

Cytosoliccytochrome CFADD

0

5

10

15

20

Fold

chan

ge

0

5

10

15

Fold

chan

ge

0

10

20

30

Fold

chan

ge

0

20

40

60

80

Fold

chan

ge

amp amp

amp

lowastlowast

lowast lowast

lowastlowast

lowastlowast

35 kD

19 kD17 kD

43 kD41 kD

28 kD

45 kD

17 kD

45 kD

HDMSCs ASMSCs

+++

+

+

minus

minusminus

minusminus

minus

minus

+++

+

+

minus

minusminus

minusminus

minus

minus

Cleaved caspase-3Cleaved caspase-8

FADD

Actin 1

Actin 2

Cytosolic cytochrome C

(c)

Pro-caspase-3

LV-NCLV-TRAIL-R2

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

CTN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+TN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

Figure 6 Lentiviruses encoding shRNA-TRAIL-R2 partly inhibited the apoptosis of ASMSCs while having a limited effect on the apoptosisof HDMSCs (a) The rate of ASMSC apoptosis (119899 = 22) was decreased after Lv-TRAIL-R2 transduction while no apparent changes werefound for HDMSCs (119899 = 28) (b) In ASMSCs (119899 = 22) Lv-TRAIL-R2 reduced FADD and total caspase-8 expression while having a limitedeffect on total caspase-3 and total cytochrome C gene expression In HDMSCs (119899 = 28) Lv-TRAIL-R2 had a limited effect on the expressionof all four genes (c) Lv-TRAIL-R2 did not affect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC in HDMSCs (119899 = 28) but reduced them in ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 betweenHDMSCs and ASMSCs amp indicates 119875 lt 005 for the TNF-120572CHX group compared with other HDMSC groups indicates 119875 lt 005 forthe TNF-120572CHX group compared with other ASMSC groups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative controlsequence Lv-TRAIL-R2 refers to MSCs transduced by lentiviruses encoding TRAIL-R2

Inhibiting TRAIL-R2 expression using shRNA eliminated thedifference in apoptosis between HDMSCs and ASMSCs bydecreasing the apoptosis of ASMSCs

MSCs aremultipotent stromal cells withmultilineage dif-ferentiation capacities and immunomodulatory propertiesOur previous studies suggested that MSCs are implicated inthe pathogenesis and therapy of AS [12 28] We found thatASMSCs possessed an abnormal osteogenic capacity whichmay contribute to the pathogenesis of AS In addition we alsofound that allogenic intravenous infusion of bone marrow-derived MSCs is feasible and safe in the treatment of ASHowever how the in vivo microenvironment in AS interactswith MSCs remains unknown

AS is a complicated autoimmune disease accompaniedby elevated serum levels of various inflammatory cytokines[14 29] Among these cytokines TNF-120572 is one of the most

important players in the pathogenesis of AS PhysiologicallyTNF-120572 is important for a normal response to inflammationbut the overproduction of TNF-120572 can be detrimental Serumlevels and the sacroiliac joint expression of TNF-120572 wereelevated in AS patients including those in the early stages ofAS [30 31] In addition overproduction of TNF-120572 has beensuggested to be related to disease activity and to account forthe pathogenesis of AS [32] The importance of TNF-120572 hasbeen further confirmed by the efficacy of TNF-120572 inhibitorsin the treatment of AS [16] Due to the importance of TNF-120572in AS a study on the interaction between TNF-120572 and MSCsmay help mimic the in vivo microenvironment experiencedby MSCs in AS Previous studies suggest that TNF-120572 affectsMSCs in various ways TNF-120572 can modulate the immuno-suppressive effects of MSCs and suppress their osteogeniccapacity [33] Additionally TNF-120572 can alter homeostasis

12 Stem Cells International

in MSCs by regulating the balance between apoptosis andsurvival [13] ConverselyMSCs affect the production of TNF-120572 by causing macrophages to adopt an anti-inflammatoryphenotype [34] Both the dysfunction of MSCs and theoverproduction of TNF-120572 may lead to immune disordersA recent study suggested that TNF-120572-induced apoptosis ofMSCs may contribute to the pathogenesis of SLE [19] In ourstudy we found that ASMSCs were more susceptible to TNF-120572-induced apoptosis compared to HDMSCs as evidencedby an increased apoptosis rate and the higher expression ofcleaved caspase-3 protein However how the dysregulationof apoptosis contributes to the pathogenesis of AS remainsuncertain

Apoptosis also defined as programmed cell death iscritical to the pathogenesis of various diseases includingautoimmune diseases [18] The maintenance of immunetolerance and immune homeostasis is largely dependent onapoptosis Defective apoptosis of immune cells may result inthe defective clearance of autoreactive cells or the delayedelimination of autoantigens while excessive apoptosis ofimmunosuppressive cells (such as MSCs) may also lead toimmune hyperactivation Therefore both conditions maycontribute to autoimmune disease [19 35] In patients withAS increased apoptosis of ASMSCs may result in decreasesin cell numbers and immunosuppressive potential eventuallycontributing to autoimmune disorders Moreover becauseMSCs are able to reduce TNF-120572 production increasedMSCs apoptosis may also lead to TNF-120572 overproductionFurthermore TNF-120572 overproduction induces further MSCapoptosis forming a vicious cycle that leads to chronicinflammation in AS Thus increased ASMSC apoptosis maybe critical for AS pathogenesis and further study of themechanism of dysregulated apoptosis in ASMSCs may helpto elucidate the pathogenesis of AS

TRAIL-R2 is a specific cell surface receptor engagedin apoptosis TRAIL-R2-mediated apoptosis has arousedattention due to its role in preferentially killing tumor cellswhile sparing normal cells In our study we observed asignificant increased expression of TRAIL-R2 in ASMSCsafter treatment with TNF-120572CHX Inhibiting TRAIL-R2expression partly decreased the apoptosis of ASMSCs buthad a limited effect on the apoptosis of HDMSCs whichsuggests that TRAIL-R2 overexpression is the main cause ofthe increased apoptosis in ASMSCs However this result isconfusing for two reasons First TRAIL-R2 is reported to bemainly activated by its specific ligand TRAIL [20] Howeverthe results in our study indicated that TRAIL-R2 in MSCscould be activated by TNF-120572CHX as well In fact a similarresult was previously reported but the mechanism remainsunclear [36] Considering the overexpression of TRAIL-R2 can lead to apoptosis even without its ligand [22] wespeculate that TNF-120572maymediate its proapoptotic effects notonly through its own receptor (TNFR1) but also by increasingthe expression of TRAIL-R2 Second bone marrow-derivedadult MSCs are reportedly resistant to TRAIL-R2-mediatedapoptosis [21] However in our study HDMSCs were resis-tant to TRAIL-R2-mediated apoptosis while ASMSCs werenot Multiple mechanisms of TRAIL-R2-mediated apoptosisresistance such as dysregulation of antagonistic receptors

abnormal O-glycosylation and overexpression of the inhi-bition adaptors have been identified [37] Further studiesare needed to illustrate the associated specific mechanismunderlying the difference in resistance of HDMSCs andASMSCs to TRAIL-R2 Additionally based on the findingthat the TRAIL-R2 pathway did not lead to the apoptosis ofnormal cells ASMSCs could be considered abnormal cellsThus it is reasonable to presume that allogenic rather thanautologous MSC transplantation might be preferable for AStreatment

In this study we demonstrated that TRAIL-R2 upregu-lation resulted in enhanced apoptosis of ASMSCs Increasedapoptosis of ASMSCs may decrease the immunosuppressivepotential of these cells and potentially contributing to ASpathogenesis However some limitations remain in thisstudy The questions of how TNF-120572 activates the expressionof TRAIL-R2 why HDMSCs are resistant to TRAIL-R2-mediated apoptosis while ASMSCs are not and whether ourresults can be repeated in vivo remain unanswered Futurestudies should focus on these questions and further elucidatethe mechanism of AS

Competing Interests

The authors have no potential conflict of interests to declare

Authorsrsquo Contributions

Zhenhua Liu LiangbinGao andPengWanghave contributedequally to this work

Acknowledgments

The authors thank Dr Liang Zhang for insightful discussionThis study was supported by the National Natural ScienceFoundation of China (81401850) the Science and Tech-nology Project of Guangdong Province (2015B0202280012015B090903059) and the Engineering Technology ResearchCenter for Comprehensive Diagnosis and Treatment ofAnkylosing Spondylitis of Guangdong Higher EducationInstitutes (GCZX-A1301)

References

[1] M Dougados and D Baeten ldquoSpondyloarthritisrdquo The Lancetvol 377 no 9783 pp 2127ndash2137 2011

[2] P Bowness ldquoHLA-B27rdquo Annual Review of Immunology vol 33pp 29ndash48 2015

[3] J D Reveille A-M Sims P Danoy et al ldquoGenome-wideassociation study of ankylosing spondylitis identifies non-MHCsusceptibility locirdquo Nature Genetics vol 42 no 2 pp 123ndash1272010

[4] J D Taurog J A Richardson J T Croft et al ldquoThe germfreestate prevents development of gut and joint inflammatorydisease in HLA-B27 transgenic ratsrdquo Journal of ExperimentalMedicine vol 180 no 6 pp 2359ndash2364 1994

[5] M-E Costello F Ciccia D Willner et al ldquoBrief Reportintestinal dysbiosis in ankylosing spondylitisrdquo Arthritis andRheumatology vol 67 no 3 pp 686ndash691 2015

Stem Cells International 13

[6] T Rashid and A Ebringer ldquoAnkylosing spondylitis is linked toKlebsiellamdashthe evidencerdquo Clinical Rheumatology vol 26 no 6pp 858ndash864 2007

[7] P Jacques and D McGonagle ldquoThe role of mechanical stress inthe pathogenesis of spondyloarthritis and how to combat itrdquoBest Practice and Research Clinical Rheumatology vol 28 no5 pp 703ndash710 2014

[8] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008

[9] M C Stewart and A A Stewart ldquoMesenchymal stem cellscharacteristics sources and mechanisms of actionrdquo VeterinaryClinics of North AmericamdashEquine Practice vol 27 no 2 pp243ndash261 2011

[10] L Y Sun H Y Zhang X B Feng Y Y Hou LW Lu and L MFan ldquoAbnormality of bone marrow-derived mesenchymal stemcells in patients with systemic lupus erythematosusrdquo Lupus vol16 no 2 pp 121ndash128 2007

[11] J A Perez-Simon S Tabera M E Sarasquete et al ldquoMes-enchymal stem cells are functionally abnormal in patients withimmune thrombocytopenic purpurardquo Cytotherapy vol 11 no6 pp 698ndash705 2009

[12] Z Xie P Wang Y Li et al ldquoImbalance between bone morpho-genetic protein 2 and noggin induces abnormal osteogenic dif-ferentiation of mesenchymal stem cells in ankylosing spondyli-tisrdquo Arthritis and Rheumatology vol 68 no 2 pp 430ndash4402016

[13] D Brenner H Blaser and T W Mak ldquoRegulation of tumournecrosis factor signalling live or let dierdquo Nature ReviewsImmunology vol 15 no 6 pp 362ndash374 2015

[14] J Gratacos A Collado X Filella et al ldquoSerum cytokines(IL-6 TNF-120572 IL-1 120573 and IFN-120574) in ankylosing spondylitis aclose correlation between serum IL-6 and disease activity andseverityrdquo British Journal of Rheumatology vol 33 no 10 pp927ndash931 1994

[15] N Barkham L C Coates H Keen et al ldquoDouble-blindplacebo-controlled trial of etanercept in the prevention of workdisability in ankylosing spondylitisrdquo Annals of the RheumaticDiseases vol 69 no 11 pp 1926ndash1928 2010

[16] J Callhoff J Sieper AWeiss A Zink and J Listing ldquoEfficacy ofTNF120572 blockers in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis ameta-analysisrdquoAnnals ofthe Rheumatic Diseases vol 74 no 6 pp 1241ndash1248 2015

[17] L Wang F Du and X Wang ldquoTNF-120572 induces two distinctcaspase-8 activation pathwaysrdquoCell vol 133 no 4 pp 693ndash7032008

[18] B Favaloro N Allocati V Graziano C Di Ilio and V DeLaurenzi ldquoRole of apoptosis in diseaserdquo Aging vol 4 no 5 pp330ndash349 2012

[19] X Li L Liu D Meng et al ldquoEnhanced apoptosis and senes-cence of bone-marrow-derived mesenchymal stem cells inpatients with systemic lupus erythematosusrdquo Stem Cells andDevelopment vol 21 no 13 pp 2387ndash2394 2012

[20] G R Screaton J Mongkolsapaya X-N Xu A E Cowper A JMcMichael and J I Bell ldquoTRICK2 a new alternatively splicedreceptor that transduces the cytotoxic signal from TRAILrdquoCurrent Biology vol 7 no 9 pp 693ndash696 1997

[21] E Szegezdi A OrsquoReilly Y Davy et al ldquoStem cells are resistantto TRAIL receptor-mediated apoptosisrdquo Journal of Cellular andMolecular Medicine vol 13 no 11-12 pp 4409ndash4414 2009

[22] H Walczak M A Degli-Esposti R S Johnson et al ldquoTRAIL-R2 a novel apoptosis-mediating receptor for TRAILrdquo EMBOJournal vol 16 no 17 pp 5386ndash5397 1997

[23] J-L Bodmer N Holler S Reynard et al ldquoTRAIL receptor-2signals apoptosis through FADD and caspase-8rdquo Nature CellBiology vol 2 no 4 pp 241ndash243 2000

[24] S Van Der Linden H A Valkenburg and A Cats ldquoEvaluationof diagnostic criteria for ankylosing spondylitis A proposal formodification of the New York criteriardquo Arthritis and Rheuma-tism vol 27 no 4 pp 361ndash368 1984

[25] D C Colter R Class C M DiGirolamo and D J ProckopldquoRapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrowrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3213ndash3218 2000

[26] D Li P Wang Y Li et al ldquoAll-Trans Retinoic Acid Improvesthe Effects of Bone Marrow-Derived Mesenchymal Stem Cellson the Treatment of Ankylosing Spondylitis An InVitro StudyrdquoStem Cells International vol 2015 Article ID 484528 10 pages2015

[27] K Fu H Ren Y Wang E Fei H Wang and G Wang ldquoDJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8recruitment to FADDrdquo Oncogene vol 31 no 10 pp 1311ndash13222012

[28] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cell intravenous infusion in active ankylos-ing spondylitis patients who failed NSAIDs a 20-week clinicaltrialrdquo Cell Transplantation vol 23 no 10 pp 1293ndash1303 2014

[29] Y Mei F Pan J Gao et al ldquoIncreased serum IL-17 and IL-23 inthe patient with ankylosing spondylitisrdquoClinical Rheumatologyvol 30 no 2 pp 269ndash273 2011

[30] R J Francois L Neure J Sieper and J Braun ldquoImmunohis-tological examination of open sacroiliac biopsies of patientswith ankylosing spondylitis detection of tumour necrosis factor120572 in two patients with early disease and transforming growthfactor 120573 in three more advanced casesrdquoAnnals of the RheumaticDiseases vol 65 no 6 pp 713ndash720 2006

[31] J Braun M Bollow L Neure et al ldquoUse of immunohistologicand in situ hybridization techniques in the examination ofsacroiliac joint biopsy specimens from patients with ankylosingspondylitisrdquo Arthritis and Rheumatism vol 38 no 4 pp 499ndash505 1995

[32] U Lange J Teichmann and H Stracke ldquoCorrelation betweenplasma TNF-alpha IGF-1 biochemical markers of bonemetabolismmarkers of inflammationdisease activity and clin-ical manifestations in ankylosing spondylitisrdquo European Journalof Medical Research vol 5 no 12 pp 507ndash511 2000

[33] L Zhao J Huang H Zhang et al ldquoTumor necrosis factorinhibits mesenchymal stem cell differentiation into osteoblastsvia the ubiquitin E3 ligase Wwp1rdquo Stem Cells vol 29 no 10 pp1601ndash1610 2011

[34] L Chiossone R Conte G M Spaggiari et al ldquoMesenchymalstromal cells induce peculiar alternatively activated macro-phages capable of dampening both innate and adaptive immuneresponsesrdquo STEM CELLS vol 34 no 7 pp 1909ndash1921 2016

[35] S Nagata ldquoApoptosis and autoimmune diseasesrdquo Annals of theNew York Academy of Sciences vol 1209 no 1 pp 10ndash16 2010

[36] M S Sheikh T F Burns Y Huang et al ldquop53-dependent and-independent regulation of the death receptor KILLERDR5gene expression in response to genotoxic stress and tumor

14 Stem Cells International

necrosis factorrdquo Cancer Research vol 58 no 8 pp 1593ndash15981998

[37] S Shirley A Morizot and O Micheau ldquoRegulating TRAILreceptor-induced cell death at the membrane a deadly discus-sionrdquo Recent Patents on Anti-Cancer Drug Discovery vol 6 no3 pp 311ndash323 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 Stem Cells International

Fas TNFR1Total cytochrome CTotal caspase-8

0

2

4

6

Relat

ive m

RNA

expr

essio

nFo

ld ch

ange

0

1

2

3

4

Fold

chan

ge

0

1

2

3

Fold

chan

ge

0

1

2

3

Fold

chan

ge

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

lowast

(a)

0

20

40

60

Fold

chan

ge

000510152025

Fold

chan

ge

000510152025

Fold

chan

ge

0

10

20

30

Fold

chan

ge

Cleaved caspase-8

Control TNF-120572CHX Control TNF-120572CHX

Control TNF-120572CHX

HDMSCsASMSCs

Control TNF-120572CHX

HDMSCsASMSCs

HDMSCsASMSCs

HDMSCsASMSCs

Cytosolic cytochrome C

FasTNFR1

43 kD41 kD

55 kD

45 kD

45 kD

17 kD

HDMSCs ASMSCs

+

+

minus

minus

+

+

minus

minus

TNF-120572CHX

40sim50 kD

Cleaved caspase-8

TNFR1

Fas

Actin 1

Actin 2

Cytosolic cytochrome C

lowast lowast

(b)

Figure 4 The DR and mitochondrial pathways were both involved in MSC apoptosis (a) qRT-PCR was performed to compare the geneexpressions of total caspase-8 total cytochrome C Fas and TNFR1 between HDMSCs (119899 = 28) and ASMSCs (119899 = 22) ASMSCs expressedmuchhigher gene expression levels of total caspase-8 thanHDMSCs after treatmentwithTNF-120572CHXwhile no differencewas found betweenHDMSCs andASMSCs in total cytochromeC Fas or TNFR1 gene expression levels (b) Awestern blot was performed to compare the proteinexpressions of cleaved caspase-8 cytosolic cytochrome C TNFR1 and Fas betweenHDMSCs (119899 = 28) and ASMSCs (119899 = 22) After treatmentwith TNF-120572CHX ASMSCs had higher cleaved caspase-8 and cytosolic cytochrome C protein expression levels than HDMSCs and similarprotein levels of TNFR1 and Fas as HDMSCs Fold changes are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs andASMSCs

than HDMSCs during apoptosis we constructed Lv-TRAIL-R2 to explore the role that TRAIL-R2 plays in the apopto-sis of ASMSCs GFP-positive cells were observed under aconfocal microscope and the transduction efficiencies werecomparable between HDMSCs and ASMSCs (Figure 5(a))Lv-TRAIL-R2 inhibited both gene and protein expression ofTRAIL-R2 inHDMSCs andASMSCs (Figure 5(b))The FCMresults demonstrated that Lv-TRAIL-R2 could reduce theapoptosis of ASMSCs but had a limited effect on the apoptosisof HDMSCs (Figure 6(a)) qRT-PCR showed that Lv-TRAIL-R2 inhibited the expression of FADD and total caspase-8 inASMSCs but had a limited effect on total caspase-3 and totalcytochrome C Meanwhile Lv-TRAIL-R2 did not affect the

expression of the four related aforementioned genes inHDM-SCs After inhibiting TRAIL-R2 expression no apparent dif-ference in gene expression was found between HDMSCs andASMSCs (Figure 6(b)) Additionally Lv-TRAIL-R2 did notaffect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC inHDMSCs but reducedthem in ASMSCs Furthermore no apparent difference inthe protein expression of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochrome C was found betweenHDMSCs andASMSCs after inhibitingTRAIL-R2 expression(Figure 6(c)) Therefore inhibiting TRAIL-R2 expressionpartly decreased the apoptosis of ASMSCs but had a limitedeffect on HDMSCs

Stem Cells International 9

HDMSCs ASMSCs

(a)

HDMSCsASMSCs

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

Con

trol

TNF-

120572C

HX

Control TNF-120572CHX Lv-NC Lv-TRAIL-R2 Control TNF-120572CHX Lv-NC Lv-TRAIL-R2

TRAIL R2

TRAIL-R2 TRAIL-R2

Actin

48 kD

40 kD

45 kD

0

1

2

3

4

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

lowast

amp

amp

amp

amp

lowast

lowastlowast

0

1

2

3

4

5

Relat

ive p

rote

in ex

pres

sion

Fold

chan

ge

HDMSCs ASMSCs

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

TNF-120572CHX + TNF-120572CHX + TNF-120572CHX+ TNF-120572CHX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

(b)

Figure 5 The transduction efficiencies of lentiviruses encoding shRNA-TRAIL-R2 were comparable in HDMSCs and ASMSCs (a) GFP-positive cells were observed under a confocal microscope (40x) and the transduction efficiencies were comparable between HDMSCs andASMSCs (b)The lentiviruses encoding shRNA-TRAIL-R2 inhibited both the gene and protein expression of TRAIL-R2 inHDMSCs (119899 = 28)and ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs and ASMSCs amp indicates 119875 lt 005 forthe TNF-120572CHXgroup comparedwith otherHDMSCs groups indicates119875 lt 005 for the TNF-120572CHXgroup comparedwith other ASMSCsgroups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative control sequence Lv-TRAIL-R2 refers to MSCs transduced bylentiviruses encoding TRAIL-R2

4 Discussion

Abnormal MSCs are proposed to be involved in the patho-genesis of AS and a better understanding of ASMSCsdysfunctions could improve the understanding of AS Inthis study ASMSCs had morphologies phenotypes and

proliferation rates that were similar to HDMSCs HoweverASMSCs were more susceptible to TNF-120572CHX-inducedapoptosis compared to HDMSCs ASMSCs expressed higherlevels of TRAIL-R2 which activated both the DR and themitochondrial pathways by increasing the expression levelsof FADD cleaved caspase-8 and cytosolic cytochrome C

10 Stem Cells International

Annexin V-PE

103 535

18627500

032 536

17447688

046 456

15317967

050 046

1519753

018 040

1179825

056 950

30895905

079 868

28526201

586027

14987889

HD

MSC

sA

SMSC

s7-

AA

D

104

104

103

103

102

102

101

101

100

104

103

102

101

100

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

104

103

102

101

100

104

103

102

101

100

10010

410

310

210

110

0

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX +

Lv-NC Lv-TRAIL-R2

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX+

LV-NC LV-TRAIL-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX

+

HDMSCsASMSCs

0

20

40

60

Apop

tosis

rate

()

LV-N

C

LV-N

C

LV-N

C

LV-N

C

0

2

4

6

8

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

Total caspase-3 Total caspase-8

0

2

4

6

Fold

chan

ge

Fold

chan

ge

0

2

4

6

8

0

1

2

3

4

Fold

chan

ge

Total cytochrome CFADD

amp

amp amp amp

amp

lowast

lowastlowast

lowast

lowast lowast

(a)

(b)

Figure 6 Continued

Stem Cells International 11

Con

trol

TNF-120572

CH

X

TNF-120572CHX

HDMSCsASMSCs

Con

trol

TNF-120572

CH

X

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Cleaved caspase-3 Cleaved caspase-8

Cytosoliccytochrome CFADD

0

5

10

15

20

Fold

chan

ge

0

5

10

15

Fold

chan

ge

0

10

20

30

Fold

chan

ge

0

20

40

60

80

Fold

chan

ge

amp amp

amp

lowastlowast

lowast lowast

lowastlowast

lowastlowast

35 kD

19 kD17 kD

43 kD41 kD

28 kD

45 kD

17 kD

45 kD

HDMSCs ASMSCs

+++

+

+

minus

minusminus

minusminus

minus

minus

+++

+

+

minus

minusminus

minusminus

minus

minus

Cleaved caspase-3Cleaved caspase-8

FADD

Actin 1

Actin 2

Cytosolic cytochrome C

(c)

Pro-caspase-3

LV-NCLV-TRAIL-R2

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

CTN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+TN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

Figure 6 Lentiviruses encoding shRNA-TRAIL-R2 partly inhibited the apoptosis of ASMSCs while having a limited effect on the apoptosisof HDMSCs (a) The rate of ASMSC apoptosis (119899 = 22) was decreased after Lv-TRAIL-R2 transduction while no apparent changes werefound for HDMSCs (119899 = 28) (b) In ASMSCs (119899 = 22) Lv-TRAIL-R2 reduced FADD and total caspase-8 expression while having a limitedeffect on total caspase-3 and total cytochrome C gene expression In HDMSCs (119899 = 28) Lv-TRAIL-R2 had a limited effect on the expressionof all four genes (c) Lv-TRAIL-R2 did not affect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC in HDMSCs (119899 = 28) but reduced them in ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 betweenHDMSCs and ASMSCs amp indicates 119875 lt 005 for the TNF-120572CHX group compared with other HDMSC groups indicates 119875 lt 005 forthe TNF-120572CHX group compared with other ASMSC groups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative controlsequence Lv-TRAIL-R2 refers to MSCs transduced by lentiviruses encoding TRAIL-R2

Inhibiting TRAIL-R2 expression using shRNA eliminated thedifference in apoptosis between HDMSCs and ASMSCs bydecreasing the apoptosis of ASMSCs

MSCs aremultipotent stromal cells withmultilineage dif-ferentiation capacities and immunomodulatory propertiesOur previous studies suggested that MSCs are implicated inthe pathogenesis and therapy of AS [12 28] We found thatASMSCs possessed an abnormal osteogenic capacity whichmay contribute to the pathogenesis of AS In addition we alsofound that allogenic intravenous infusion of bone marrow-derived MSCs is feasible and safe in the treatment of ASHowever how the in vivo microenvironment in AS interactswith MSCs remains unknown

AS is a complicated autoimmune disease accompaniedby elevated serum levels of various inflammatory cytokines[14 29] Among these cytokines TNF-120572 is one of the most

important players in the pathogenesis of AS PhysiologicallyTNF-120572 is important for a normal response to inflammationbut the overproduction of TNF-120572 can be detrimental Serumlevels and the sacroiliac joint expression of TNF-120572 wereelevated in AS patients including those in the early stages ofAS [30 31] In addition overproduction of TNF-120572 has beensuggested to be related to disease activity and to account forthe pathogenesis of AS [32] The importance of TNF-120572 hasbeen further confirmed by the efficacy of TNF-120572 inhibitorsin the treatment of AS [16] Due to the importance of TNF-120572in AS a study on the interaction between TNF-120572 and MSCsmay help mimic the in vivo microenvironment experiencedby MSCs in AS Previous studies suggest that TNF-120572 affectsMSCs in various ways TNF-120572 can modulate the immuno-suppressive effects of MSCs and suppress their osteogeniccapacity [33] Additionally TNF-120572 can alter homeostasis

12 Stem Cells International

in MSCs by regulating the balance between apoptosis andsurvival [13] ConverselyMSCs affect the production of TNF-120572 by causing macrophages to adopt an anti-inflammatoryphenotype [34] Both the dysfunction of MSCs and theoverproduction of TNF-120572 may lead to immune disordersA recent study suggested that TNF-120572-induced apoptosis ofMSCs may contribute to the pathogenesis of SLE [19] In ourstudy we found that ASMSCs were more susceptible to TNF-120572-induced apoptosis compared to HDMSCs as evidencedby an increased apoptosis rate and the higher expression ofcleaved caspase-3 protein However how the dysregulationof apoptosis contributes to the pathogenesis of AS remainsuncertain

Apoptosis also defined as programmed cell death iscritical to the pathogenesis of various diseases includingautoimmune diseases [18] The maintenance of immunetolerance and immune homeostasis is largely dependent onapoptosis Defective apoptosis of immune cells may result inthe defective clearance of autoreactive cells or the delayedelimination of autoantigens while excessive apoptosis ofimmunosuppressive cells (such as MSCs) may also lead toimmune hyperactivation Therefore both conditions maycontribute to autoimmune disease [19 35] In patients withAS increased apoptosis of ASMSCs may result in decreasesin cell numbers and immunosuppressive potential eventuallycontributing to autoimmune disorders Moreover becauseMSCs are able to reduce TNF-120572 production increasedMSCs apoptosis may also lead to TNF-120572 overproductionFurthermore TNF-120572 overproduction induces further MSCapoptosis forming a vicious cycle that leads to chronicinflammation in AS Thus increased ASMSC apoptosis maybe critical for AS pathogenesis and further study of themechanism of dysregulated apoptosis in ASMSCs may helpto elucidate the pathogenesis of AS

TRAIL-R2 is a specific cell surface receptor engagedin apoptosis TRAIL-R2-mediated apoptosis has arousedattention due to its role in preferentially killing tumor cellswhile sparing normal cells In our study we observed asignificant increased expression of TRAIL-R2 in ASMSCsafter treatment with TNF-120572CHX Inhibiting TRAIL-R2expression partly decreased the apoptosis of ASMSCs buthad a limited effect on the apoptosis of HDMSCs whichsuggests that TRAIL-R2 overexpression is the main cause ofthe increased apoptosis in ASMSCs However this result isconfusing for two reasons First TRAIL-R2 is reported to bemainly activated by its specific ligand TRAIL [20] Howeverthe results in our study indicated that TRAIL-R2 in MSCscould be activated by TNF-120572CHX as well In fact a similarresult was previously reported but the mechanism remainsunclear [36] Considering the overexpression of TRAIL-R2 can lead to apoptosis even without its ligand [22] wespeculate that TNF-120572maymediate its proapoptotic effects notonly through its own receptor (TNFR1) but also by increasingthe expression of TRAIL-R2 Second bone marrow-derivedadult MSCs are reportedly resistant to TRAIL-R2-mediatedapoptosis [21] However in our study HDMSCs were resis-tant to TRAIL-R2-mediated apoptosis while ASMSCs werenot Multiple mechanisms of TRAIL-R2-mediated apoptosisresistance such as dysregulation of antagonistic receptors

abnormal O-glycosylation and overexpression of the inhi-bition adaptors have been identified [37] Further studiesare needed to illustrate the associated specific mechanismunderlying the difference in resistance of HDMSCs andASMSCs to TRAIL-R2 Additionally based on the findingthat the TRAIL-R2 pathway did not lead to the apoptosis ofnormal cells ASMSCs could be considered abnormal cellsThus it is reasonable to presume that allogenic rather thanautologous MSC transplantation might be preferable for AStreatment

In this study we demonstrated that TRAIL-R2 upregu-lation resulted in enhanced apoptosis of ASMSCs Increasedapoptosis of ASMSCs may decrease the immunosuppressivepotential of these cells and potentially contributing to ASpathogenesis However some limitations remain in thisstudy The questions of how TNF-120572 activates the expressionof TRAIL-R2 why HDMSCs are resistant to TRAIL-R2-mediated apoptosis while ASMSCs are not and whether ourresults can be repeated in vivo remain unanswered Futurestudies should focus on these questions and further elucidatethe mechanism of AS

Competing Interests

The authors have no potential conflict of interests to declare

Authorsrsquo Contributions

Zhenhua Liu LiangbinGao andPengWanghave contributedequally to this work

Acknowledgments

The authors thank Dr Liang Zhang for insightful discussionThis study was supported by the National Natural ScienceFoundation of China (81401850) the Science and Tech-nology Project of Guangdong Province (2015B0202280012015B090903059) and the Engineering Technology ResearchCenter for Comprehensive Diagnosis and Treatment ofAnkylosing Spondylitis of Guangdong Higher EducationInstitutes (GCZX-A1301)

References

[1] M Dougados and D Baeten ldquoSpondyloarthritisrdquo The Lancetvol 377 no 9783 pp 2127ndash2137 2011

[2] P Bowness ldquoHLA-B27rdquo Annual Review of Immunology vol 33pp 29ndash48 2015

[3] J D Reveille A-M Sims P Danoy et al ldquoGenome-wideassociation study of ankylosing spondylitis identifies non-MHCsusceptibility locirdquo Nature Genetics vol 42 no 2 pp 123ndash1272010

[4] J D Taurog J A Richardson J T Croft et al ldquoThe germfreestate prevents development of gut and joint inflammatorydisease in HLA-B27 transgenic ratsrdquo Journal of ExperimentalMedicine vol 180 no 6 pp 2359ndash2364 1994

[5] M-E Costello F Ciccia D Willner et al ldquoBrief Reportintestinal dysbiosis in ankylosing spondylitisrdquo Arthritis andRheumatology vol 67 no 3 pp 686ndash691 2015

Stem Cells International 13

[6] T Rashid and A Ebringer ldquoAnkylosing spondylitis is linked toKlebsiellamdashthe evidencerdquo Clinical Rheumatology vol 26 no 6pp 858ndash864 2007

[7] P Jacques and D McGonagle ldquoThe role of mechanical stress inthe pathogenesis of spondyloarthritis and how to combat itrdquoBest Practice and Research Clinical Rheumatology vol 28 no5 pp 703ndash710 2014

[8] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008

[9] M C Stewart and A A Stewart ldquoMesenchymal stem cellscharacteristics sources and mechanisms of actionrdquo VeterinaryClinics of North AmericamdashEquine Practice vol 27 no 2 pp243ndash261 2011

[10] L Y Sun H Y Zhang X B Feng Y Y Hou LW Lu and L MFan ldquoAbnormality of bone marrow-derived mesenchymal stemcells in patients with systemic lupus erythematosusrdquo Lupus vol16 no 2 pp 121ndash128 2007

[11] J A Perez-Simon S Tabera M E Sarasquete et al ldquoMes-enchymal stem cells are functionally abnormal in patients withimmune thrombocytopenic purpurardquo Cytotherapy vol 11 no6 pp 698ndash705 2009

[12] Z Xie P Wang Y Li et al ldquoImbalance between bone morpho-genetic protein 2 and noggin induces abnormal osteogenic dif-ferentiation of mesenchymal stem cells in ankylosing spondyli-tisrdquo Arthritis and Rheumatology vol 68 no 2 pp 430ndash4402016

[13] D Brenner H Blaser and T W Mak ldquoRegulation of tumournecrosis factor signalling live or let dierdquo Nature ReviewsImmunology vol 15 no 6 pp 362ndash374 2015

[14] J Gratacos A Collado X Filella et al ldquoSerum cytokines(IL-6 TNF-120572 IL-1 120573 and IFN-120574) in ankylosing spondylitis aclose correlation between serum IL-6 and disease activity andseverityrdquo British Journal of Rheumatology vol 33 no 10 pp927ndash931 1994

[15] N Barkham L C Coates H Keen et al ldquoDouble-blindplacebo-controlled trial of etanercept in the prevention of workdisability in ankylosing spondylitisrdquo Annals of the RheumaticDiseases vol 69 no 11 pp 1926ndash1928 2010

[16] J Callhoff J Sieper AWeiss A Zink and J Listing ldquoEfficacy ofTNF120572 blockers in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis ameta-analysisrdquoAnnals ofthe Rheumatic Diseases vol 74 no 6 pp 1241ndash1248 2015

[17] L Wang F Du and X Wang ldquoTNF-120572 induces two distinctcaspase-8 activation pathwaysrdquoCell vol 133 no 4 pp 693ndash7032008

[18] B Favaloro N Allocati V Graziano C Di Ilio and V DeLaurenzi ldquoRole of apoptosis in diseaserdquo Aging vol 4 no 5 pp330ndash349 2012

[19] X Li L Liu D Meng et al ldquoEnhanced apoptosis and senes-cence of bone-marrow-derived mesenchymal stem cells inpatients with systemic lupus erythematosusrdquo Stem Cells andDevelopment vol 21 no 13 pp 2387ndash2394 2012

[20] G R Screaton J Mongkolsapaya X-N Xu A E Cowper A JMcMichael and J I Bell ldquoTRICK2 a new alternatively splicedreceptor that transduces the cytotoxic signal from TRAILrdquoCurrent Biology vol 7 no 9 pp 693ndash696 1997

[21] E Szegezdi A OrsquoReilly Y Davy et al ldquoStem cells are resistantto TRAIL receptor-mediated apoptosisrdquo Journal of Cellular andMolecular Medicine vol 13 no 11-12 pp 4409ndash4414 2009

[22] H Walczak M A Degli-Esposti R S Johnson et al ldquoTRAIL-R2 a novel apoptosis-mediating receptor for TRAILrdquo EMBOJournal vol 16 no 17 pp 5386ndash5397 1997

[23] J-L Bodmer N Holler S Reynard et al ldquoTRAIL receptor-2signals apoptosis through FADD and caspase-8rdquo Nature CellBiology vol 2 no 4 pp 241ndash243 2000

[24] S Van Der Linden H A Valkenburg and A Cats ldquoEvaluationof diagnostic criteria for ankylosing spondylitis A proposal formodification of the New York criteriardquo Arthritis and Rheuma-tism vol 27 no 4 pp 361ndash368 1984

[25] D C Colter R Class C M DiGirolamo and D J ProckopldquoRapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrowrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3213ndash3218 2000

[26] D Li P Wang Y Li et al ldquoAll-Trans Retinoic Acid Improvesthe Effects of Bone Marrow-Derived Mesenchymal Stem Cellson the Treatment of Ankylosing Spondylitis An InVitro StudyrdquoStem Cells International vol 2015 Article ID 484528 10 pages2015

[27] K Fu H Ren Y Wang E Fei H Wang and G Wang ldquoDJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8recruitment to FADDrdquo Oncogene vol 31 no 10 pp 1311ndash13222012

[28] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cell intravenous infusion in active ankylos-ing spondylitis patients who failed NSAIDs a 20-week clinicaltrialrdquo Cell Transplantation vol 23 no 10 pp 1293ndash1303 2014

[29] Y Mei F Pan J Gao et al ldquoIncreased serum IL-17 and IL-23 inthe patient with ankylosing spondylitisrdquoClinical Rheumatologyvol 30 no 2 pp 269ndash273 2011

[30] R J Francois L Neure J Sieper and J Braun ldquoImmunohis-tological examination of open sacroiliac biopsies of patientswith ankylosing spondylitis detection of tumour necrosis factor120572 in two patients with early disease and transforming growthfactor 120573 in three more advanced casesrdquoAnnals of the RheumaticDiseases vol 65 no 6 pp 713ndash720 2006

[31] J Braun M Bollow L Neure et al ldquoUse of immunohistologicand in situ hybridization techniques in the examination ofsacroiliac joint biopsy specimens from patients with ankylosingspondylitisrdquo Arthritis and Rheumatism vol 38 no 4 pp 499ndash505 1995

[32] U Lange J Teichmann and H Stracke ldquoCorrelation betweenplasma TNF-alpha IGF-1 biochemical markers of bonemetabolismmarkers of inflammationdisease activity and clin-ical manifestations in ankylosing spondylitisrdquo European Journalof Medical Research vol 5 no 12 pp 507ndash511 2000

[33] L Zhao J Huang H Zhang et al ldquoTumor necrosis factorinhibits mesenchymal stem cell differentiation into osteoblastsvia the ubiquitin E3 ligase Wwp1rdquo Stem Cells vol 29 no 10 pp1601ndash1610 2011

[34] L Chiossone R Conte G M Spaggiari et al ldquoMesenchymalstromal cells induce peculiar alternatively activated macro-phages capable of dampening both innate and adaptive immuneresponsesrdquo STEM CELLS vol 34 no 7 pp 1909ndash1921 2016

[35] S Nagata ldquoApoptosis and autoimmune diseasesrdquo Annals of theNew York Academy of Sciences vol 1209 no 1 pp 10ndash16 2010

[36] M S Sheikh T F Burns Y Huang et al ldquop53-dependent and-independent regulation of the death receptor KILLERDR5gene expression in response to genotoxic stress and tumor

14 Stem Cells International

necrosis factorrdquo Cancer Research vol 58 no 8 pp 1593ndash15981998

[37] S Shirley A Morizot and O Micheau ldquoRegulating TRAILreceptor-induced cell death at the membrane a deadly discus-sionrdquo Recent Patents on Anti-Cancer Drug Discovery vol 6 no3 pp 311ndash323 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 9

HDMSCs ASMSCs

(a)

HDMSCsASMSCs

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

Con

trol

TNF-

120572C

HX

Control TNF-120572CHX Lv-NC Lv-TRAIL-R2 Control TNF-120572CHX Lv-NC Lv-TRAIL-R2

TRAIL R2

TRAIL-R2 TRAIL-R2

Actin

48 kD

40 kD

45 kD

0

1

2

3

4

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

lowast

amp

amp

amp

amp

lowast

lowastlowast

0

1

2

3

4

5

Relat

ive p

rote

in ex

pres

sion

Fold

chan

ge

HDMSCs ASMSCs

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

TNF-120572CHX + TNF-120572CHX + TNF-120572CHX+ TNF-120572CHX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

TNF-

120572C

HX+

(b)

Figure 5 The transduction efficiencies of lentiviruses encoding shRNA-TRAIL-R2 were comparable in HDMSCs and ASMSCs (a) GFP-positive cells were observed under a confocal microscope (40x) and the transduction efficiencies were comparable between HDMSCs andASMSCs (b)The lentiviruses encoding shRNA-TRAIL-R2 inhibited both the gene and protein expression of TRAIL-R2 inHDMSCs (119899 = 28)and ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 between HDMSCs and ASMSCs amp indicates 119875 lt 005 forthe TNF-120572CHXgroup comparedwith otherHDMSCs groups indicates119875 lt 005 for the TNF-120572CHXgroup comparedwith other ASMSCsgroups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative control sequence Lv-TRAIL-R2 refers to MSCs transduced bylentiviruses encoding TRAIL-R2

4 Discussion

Abnormal MSCs are proposed to be involved in the patho-genesis of AS and a better understanding of ASMSCsdysfunctions could improve the understanding of AS Inthis study ASMSCs had morphologies phenotypes and

proliferation rates that were similar to HDMSCs HoweverASMSCs were more susceptible to TNF-120572CHX-inducedapoptosis compared to HDMSCs ASMSCs expressed higherlevels of TRAIL-R2 which activated both the DR and themitochondrial pathways by increasing the expression levelsof FADD cleaved caspase-8 and cytosolic cytochrome C

10 Stem Cells International

Annexin V-PE

103 535

18627500

032 536

17447688

046 456

15317967

050 046

1519753

018 040

1179825

056 950

30895905

079 868

28526201

586027

14987889

HD

MSC

sA

SMSC

s7-

AA

D

104

104

103

103

102

102

101

101

100

104

103

102

101

100

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

104

103

102

101

100

104

103

102

101

100

10010

410

310

210

110

0

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX +

Lv-NC Lv-TRAIL-R2

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX+

LV-NC LV-TRAIL-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX

+

HDMSCsASMSCs

0

20

40

60

Apop

tosis

rate

()

LV-N

C

LV-N

C

LV-N

C

LV-N

C

0

2

4

6

8

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

Total caspase-3 Total caspase-8

0

2

4

6

Fold

chan

ge

Fold

chan

ge

0

2

4

6

8

0

1

2

3

4

Fold

chan

ge

Total cytochrome CFADD

amp

amp amp amp

amp

lowast

lowastlowast

lowast

lowast lowast

(a)

(b)

Figure 6 Continued

Stem Cells International 11

Con

trol

TNF-120572

CH

X

TNF-120572CHX

HDMSCsASMSCs

Con

trol

TNF-120572

CH

X

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Cleaved caspase-3 Cleaved caspase-8

Cytosoliccytochrome CFADD

0

5

10

15

20

Fold

chan

ge

0

5

10

15

Fold

chan

ge

0

10

20

30

Fold

chan

ge

0

20

40

60

80

Fold

chan

ge

amp amp

amp

lowastlowast

lowast lowast

lowastlowast

lowastlowast

35 kD

19 kD17 kD

43 kD41 kD

28 kD

45 kD

17 kD

45 kD

HDMSCs ASMSCs

+++

+

+

minus

minusminus

minusminus

minus

minus

+++

+

+

minus

minusminus

minusminus

minus

minus

Cleaved caspase-3Cleaved caspase-8

FADD

Actin 1

Actin 2

Cytosolic cytochrome C

(c)

Pro-caspase-3

LV-NCLV-TRAIL-R2

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

CTN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+TN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

Figure 6 Lentiviruses encoding shRNA-TRAIL-R2 partly inhibited the apoptosis of ASMSCs while having a limited effect on the apoptosisof HDMSCs (a) The rate of ASMSC apoptosis (119899 = 22) was decreased after Lv-TRAIL-R2 transduction while no apparent changes werefound for HDMSCs (119899 = 28) (b) In ASMSCs (119899 = 22) Lv-TRAIL-R2 reduced FADD and total caspase-8 expression while having a limitedeffect on total caspase-3 and total cytochrome C gene expression In HDMSCs (119899 = 28) Lv-TRAIL-R2 had a limited effect on the expressionof all four genes (c) Lv-TRAIL-R2 did not affect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC in HDMSCs (119899 = 28) but reduced them in ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 betweenHDMSCs and ASMSCs amp indicates 119875 lt 005 for the TNF-120572CHX group compared with other HDMSC groups indicates 119875 lt 005 forthe TNF-120572CHX group compared with other ASMSC groups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative controlsequence Lv-TRAIL-R2 refers to MSCs transduced by lentiviruses encoding TRAIL-R2

Inhibiting TRAIL-R2 expression using shRNA eliminated thedifference in apoptosis between HDMSCs and ASMSCs bydecreasing the apoptosis of ASMSCs

MSCs aremultipotent stromal cells withmultilineage dif-ferentiation capacities and immunomodulatory propertiesOur previous studies suggested that MSCs are implicated inthe pathogenesis and therapy of AS [12 28] We found thatASMSCs possessed an abnormal osteogenic capacity whichmay contribute to the pathogenesis of AS In addition we alsofound that allogenic intravenous infusion of bone marrow-derived MSCs is feasible and safe in the treatment of ASHowever how the in vivo microenvironment in AS interactswith MSCs remains unknown

AS is a complicated autoimmune disease accompaniedby elevated serum levels of various inflammatory cytokines[14 29] Among these cytokines TNF-120572 is one of the most

important players in the pathogenesis of AS PhysiologicallyTNF-120572 is important for a normal response to inflammationbut the overproduction of TNF-120572 can be detrimental Serumlevels and the sacroiliac joint expression of TNF-120572 wereelevated in AS patients including those in the early stages ofAS [30 31] In addition overproduction of TNF-120572 has beensuggested to be related to disease activity and to account forthe pathogenesis of AS [32] The importance of TNF-120572 hasbeen further confirmed by the efficacy of TNF-120572 inhibitorsin the treatment of AS [16] Due to the importance of TNF-120572in AS a study on the interaction between TNF-120572 and MSCsmay help mimic the in vivo microenvironment experiencedby MSCs in AS Previous studies suggest that TNF-120572 affectsMSCs in various ways TNF-120572 can modulate the immuno-suppressive effects of MSCs and suppress their osteogeniccapacity [33] Additionally TNF-120572 can alter homeostasis

12 Stem Cells International

in MSCs by regulating the balance between apoptosis andsurvival [13] ConverselyMSCs affect the production of TNF-120572 by causing macrophages to adopt an anti-inflammatoryphenotype [34] Both the dysfunction of MSCs and theoverproduction of TNF-120572 may lead to immune disordersA recent study suggested that TNF-120572-induced apoptosis ofMSCs may contribute to the pathogenesis of SLE [19] In ourstudy we found that ASMSCs were more susceptible to TNF-120572-induced apoptosis compared to HDMSCs as evidencedby an increased apoptosis rate and the higher expression ofcleaved caspase-3 protein However how the dysregulationof apoptosis contributes to the pathogenesis of AS remainsuncertain

Apoptosis also defined as programmed cell death iscritical to the pathogenesis of various diseases includingautoimmune diseases [18] The maintenance of immunetolerance and immune homeostasis is largely dependent onapoptosis Defective apoptosis of immune cells may result inthe defective clearance of autoreactive cells or the delayedelimination of autoantigens while excessive apoptosis ofimmunosuppressive cells (such as MSCs) may also lead toimmune hyperactivation Therefore both conditions maycontribute to autoimmune disease [19 35] In patients withAS increased apoptosis of ASMSCs may result in decreasesin cell numbers and immunosuppressive potential eventuallycontributing to autoimmune disorders Moreover becauseMSCs are able to reduce TNF-120572 production increasedMSCs apoptosis may also lead to TNF-120572 overproductionFurthermore TNF-120572 overproduction induces further MSCapoptosis forming a vicious cycle that leads to chronicinflammation in AS Thus increased ASMSC apoptosis maybe critical for AS pathogenesis and further study of themechanism of dysregulated apoptosis in ASMSCs may helpto elucidate the pathogenesis of AS

TRAIL-R2 is a specific cell surface receptor engagedin apoptosis TRAIL-R2-mediated apoptosis has arousedattention due to its role in preferentially killing tumor cellswhile sparing normal cells In our study we observed asignificant increased expression of TRAIL-R2 in ASMSCsafter treatment with TNF-120572CHX Inhibiting TRAIL-R2expression partly decreased the apoptosis of ASMSCs buthad a limited effect on the apoptosis of HDMSCs whichsuggests that TRAIL-R2 overexpression is the main cause ofthe increased apoptosis in ASMSCs However this result isconfusing for two reasons First TRAIL-R2 is reported to bemainly activated by its specific ligand TRAIL [20] Howeverthe results in our study indicated that TRAIL-R2 in MSCscould be activated by TNF-120572CHX as well In fact a similarresult was previously reported but the mechanism remainsunclear [36] Considering the overexpression of TRAIL-R2 can lead to apoptosis even without its ligand [22] wespeculate that TNF-120572maymediate its proapoptotic effects notonly through its own receptor (TNFR1) but also by increasingthe expression of TRAIL-R2 Second bone marrow-derivedadult MSCs are reportedly resistant to TRAIL-R2-mediatedapoptosis [21] However in our study HDMSCs were resis-tant to TRAIL-R2-mediated apoptosis while ASMSCs werenot Multiple mechanisms of TRAIL-R2-mediated apoptosisresistance such as dysregulation of antagonistic receptors

abnormal O-glycosylation and overexpression of the inhi-bition adaptors have been identified [37] Further studiesare needed to illustrate the associated specific mechanismunderlying the difference in resistance of HDMSCs andASMSCs to TRAIL-R2 Additionally based on the findingthat the TRAIL-R2 pathway did not lead to the apoptosis ofnormal cells ASMSCs could be considered abnormal cellsThus it is reasonable to presume that allogenic rather thanautologous MSC transplantation might be preferable for AStreatment

In this study we demonstrated that TRAIL-R2 upregu-lation resulted in enhanced apoptosis of ASMSCs Increasedapoptosis of ASMSCs may decrease the immunosuppressivepotential of these cells and potentially contributing to ASpathogenesis However some limitations remain in thisstudy The questions of how TNF-120572 activates the expressionof TRAIL-R2 why HDMSCs are resistant to TRAIL-R2-mediated apoptosis while ASMSCs are not and whether ourresults can be repeated in vivo remain unanswered Futurestudies should focus on these questions and further elucidatethe mechanism of AS

Competing Interests

The authors have no potential conflict of interests to declare

Authorsrsquo Contributions

Zhenhua Liu LiangbinGao andPengWanghave contributedequally to this work

Acknowledgments

The authors thank Dr Liang Zhang for insightful discussionThis study was supported by the National Natural ScienceFoundation of China (81401850) the Science and Tech-nology Project of Guangdong Province (2015B0202280012015B090903059) and the Engineering Technology ResearchCenter for Comprehensive Diagnosis and Treatment ofAnkylosing Spondylitis of Guangdong Higher EducationInstitutes (GCZX-A1301)

References

[1] M Dougados and D Baeten ldquoSpondyloarthritisrdquo The Lancetvol 377 no 9783 pp 2127ndash2137 2011

[2] P Bowness ldquoHLA-B27rdquo Annual Review of Immunology vol 33pp 29ndash48 2015

[3] J D Reveille A-M Sims P Danoy et al ldquoGenome-wideassociation study of ankylosing spondylitis identifies non-MHCsusceptibility locirdquo Nature Genetics vol 42 no 2 pp 123ndash1272010

[4] J D Taurog J A Richardson J T Croft et al ldquoThe germfreestate prevents development of gut and joint inflammatorydisease in HLA-B27 transgenic ratsrdquo Journal of ExperimentalMedicine vol 180 no 6 pp 2359ndash2364 1994

[5] M-E Costello F Ciccia D Willner et al ldquoBrief Reportintestinal dysbiosis in ankylosing spondylitisrdquo Arthritis andRheumatology vol 67 no 3 pp 686ndash691 2015

Stem Cells International 13

[6] T Rashid and A Ebringer ldquoAnkylosing spondylitis is linked toKlebsiellamdashthe evidencerdquo Clinical Rheumatology vol 26 no 6pp 858ndash864 2007

[7] P Jacques and D McGonagle ldquoThe role of mechanical stress inthe pathogenesis of spondyloarthritis and how to combat itrdquoBest Practice and Research Clinical Rheumatology vol 28 no5 pp 703ndash710 2014

[8] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008

[9] M C Stewart and A A Stewart ldquoMesenchymal stem cellscharacteristics sources and mechanisms of actionrdquo VeterinaryClinics of North AmericamdashEquine Practice vol 27 no 2 pp243ndash261 2011

[10] L Y Sun H Y Zhang X B Feng Y Y Hou LW Lu and L MFan ldquoAbnormality of bone marrow-derived mesenchymal stemcells in patients with systemic lupus erythematosusrdquo Lupus vol16 no 2 pp 121ndash128 2007

[11] J A Perez-Simon S Tabera M E Sarasquete et al ldquoMes-enchymal stem cells are functionally abnormal in patients withimmune thrombocytopenic purpurardquo Cytotherapy vol 11 no6 pp 698ndash705 2009

[12] Z Xie P Wang Y Li et al ldquoImbalance between bone morpho-genetic protein 2 and noggin induces abnormal osteogenic dif-ferentiation of mesenchymal stem cells in ankylosing spondyli-tisrdquo Arthritis and Rheumatology vol 68 no 2 pp 430ndash4402016

[13] D Brenner H Blaser and T W Mak ldquoRegulation of tumournecrosis factor signalling live or let dierdquo Nature ReviewsImmunology vol 15 no 6 pp 362ndash374 2015

[14] J Gratacos A Collado X Filella et al ldquoSerum cytokines(IL-6 TNF-120572 IL-1 120573 and IFN-120574) in ankylosing spondylitis aclose correlation between serum IL-6 and disease activity andseverityrdquo British Journal of Rheumatology vol 33 no 10 pp927ndash931 1994

[15] N Barkham L C Coates H Keen et al ldquoDouble-blindplacebo-controlled trial of etanercept in the prevention of workdisability in ankylosing spondylitisrdquo Annals of the RheumaticDiseases vol 69 no 11 pp 1926ndash1928 2010

[16] J Callhoff J Sieper AWeiss A Zink and J Listing ldquoEfficacy ofTNF120572 blockers in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis ameta-analysisrdquoAnnals ofthe Rheumatic Diseases vol 74 no 6 pp 1241ndash1248 2015

[17] L Wang F Du and X Wang ldquoTNF-120572 induces two distinctcaspase-8 activation pathwaysrdquoCell vol 133 no 4 pp 693ndash7032008

[18] B Favaloro N Allocati V Graziano C Di Ilio and V DeLaurenzi ldquoRole of apoptosis in diseaserdquo Aging vol 4 no 5 pp330ndash349 2012

[19] X Li L Liu D Meng et al ldquoEnhanced apoptosis and senes-cence of bone-marrow-derived mesenchymal stem cells inpatients with systemic lupus erythematosusrdquo Stem Cells andDevelopment vol 21 no 13 pp 2387ndash2394 2012

[20] G R Screaton J Mongkolsapaya X-N Xu A E Cowper A JMcMichael and J I Bell ldquoTRICK2 a new alternatively splicedreceptor that transduces the cytotoxic signal from TRAILrdquoCurrent Biology vol 7 no 9 pp 693ndash696 1997

[21] E Szegezdi A OrsquoReilly Y Davy et al ldquoStem cells are resistantto TRAIL receptor-mediated apoptosisrdquo Journal of Cellular andMolecular Medicine vol 13 no 11-12 pp 4409ndash4414 2009

[22] H Walczak M A Degli-Esposti R S Johnson et al ldquoTRAIL-R2 a novel apoptosis-mediating receptor for TRAILrdquo EMBOJournal vol 16 no 17 pp 5386ndash5397 1997

[23] J-L Bodmer N Holler S Reynard et al ldquoTRAIL receptor-2signals apoptosis through FADD and caspase-8rdquo Nature CellBiology vol 2 no 4 pp 241ndash243 2000

[24] S Van Der Linden H A Valkenburg and A Cats ldquoEvaluationof diagnostic criteria for ankylosing spondylitis A proposal formodification of the New York criteriardquo Arthritis and Rheuma-tism vol 27 no 4 pp 361ndash368 1984

[25] D C Colter R Class C M DiGirolamo and D J ProckopldquoRapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrowrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3213ndash3218 2000

[26] D Li P Wang Y Li et al ldquoAll-Trans Retinoic Acid Improvesthe Effects of Bone Marrow-Derived Mesenchymal Stem Cellson the Treatment of Ankylosing Spondylitis An InVitro StudyrdquoStem Cells International vol 2015 Article ID 484528 10 pages2015

[27] K Fu H Ren Y Wang E Fei H Wang and G Wang ldquoDJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8recruitment to FADDrdquo Oncogene vol 31 no 10 pp 1311ndash13222012

[28] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cell intravenous infusion in active ankylos-ing spondylitis patients who failed NSAIDs a 20-week clinicaltrialrdquo Cell Transplantation vol 23 no 10 pp 1293ndash1303 2014

[29] Y Mei F Pan J Gao et al ldquoIncreased serum IL-17 and IL-23 inthe patient with ankylosing spondylitisrdquoClinical Rheumatologyvol 30 no 2 pp 269ndash273 2011

[30] R J Francois L Neure J Sieper and J Braun ldquoImmunohis-tological examination of open sacroiliac biopsies of patientswith ankylosing spondylitis detection of tumour necrosis factor120572 in two patients with early disease and transforming growthfactor 120573 in three more advanced casesrdquoAnnals of the RheumaticDiseases vol 65 no 6 pp 713ndash720 2006

[31] J Braun M Bollow L Neure et al ldquoUse of immunohistologicand in situ hybridization techniques in the examination ofsacroiliac joint biopsy specimens from patients with ankylosingspondylitisrdquo Arthritis and Rheumatism vol 38 no 4 pp 499ndash505 1995

[32] U Lange J Teichmann and H Stracke ldquoCorrelation betweenplasma TNF-alpha IGF-1 biochemical markers of bonemetabolismmarkers of inflammationdisease activity and clin-ical manifestations in ankylosing spondylitisrdquo European Journalof Medical Research vol 5 no 12 pp 507ndash511 2000

[33] L Zhao J Huang H Zhang et al ldquoTumor necrosis factorinhibits mesenchymal stem cell differentiation into osteoblastsvia the ubiquitin E3 ligase Wwp1rdquo Stem Cells vol 29 no 10 pp1601ndash1610 2011

[34] L Chiossone R Conte G M Spaggiari et al ldquoMesenchymalstromal cells induce peculiar alternatively activated macro-phages capable of dampening both innate and adaptive immuneresponsesrdquo STEM CELLS vol 34 no 7 pp 1909ndash1921 2016

[35] S Nagata ldquoApoptosis and autoimmune diseasesrdquo Annals of theNew York Academy of Sciences vol 1209 no 1 pp 10ndash16 2010

[36] M S Sheikh T F Burns Y Huang et al ldquop53-dependent and-independent regulation of the death receptor KILLERDR5gene expression in response to genotoxic stress and tumor

14 Stem Cells International

necrosis factorrdquo Cancer Research vol 58 no 8 pp 1593ndash15981998

[37] S Shirley A Morizot and O Micheau ldquoRegulating TRAILreceptor-induced cell death at the membrane a deadly discus-sionrdquo Recent Patents on Anti-Cancer Drug Discovery vol 6 no3 pp 311ndash323 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 Stem Cells International

Annexin V-PE

103 535

18627500

032 536

17447688

046 456

15317967

050 046

1519753

018 040

1179825

056 950

30895905

079 868

28526201

586027

14987889

HD

MSC

sA

SMSC

s7-

AA

D

104

104

103

103

102

102

101

101

100

104

103

102

101

100

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

104

103

102

101

100

104

103

102

101

100

10010

410

310

210

110

0

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX +

Lv-NC Lv-TRAIL-R2

Control TNF-120572CHXTNF-120572CHX + TNF-120572CHX+

LV-NC LV-TRAIL-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

LV-T

RAIL

-R2

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX+

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

TNF-

120572C

HX +

TNF-

120572C

HX

+

HDMSCsASMSCs

0

20

40

60

Apop

tosis

rate

()

LV-N

C

LV-N

C

LV-N

C

LV-N

C

0

2

4

6

8

Relat

ive m

RNA

expr

essio

n Fo

ld ch

ange

Total caspase-3 Total caspase-8

0

2

4

6

Fold

chan

ge

Fold

chan

ge

0

2

4

6

8

0

1

2

3

4

Fold

chan

ge

Total cytochrome CFADD

amp

amp amp amp

amp

lowast

lowastlowast

lowast

lowast lowast

(a)

(b)

Figure 6 Continued

Stem Cells International 11

Con

trol

TNF-120572

CH

X

TNF-120572CHX

HDMSCsASMSCs

Con

trol

TNF-120572

CH

X

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Cleaved caspase-3 Cleaved caspase-8

Cytosoliccytochrome CFADD

0

5

10

15

20

Fold

chan

ge

0

5

10

15

Fold

chan

ge

0

10

20

30

Fold

chan

ge

0

20

40

60

80

Fold

chan

ge

amp amp

amp

lowastlowast

lowast lowast

lowastlowast

lowastlowast

35 kD

19 kD17 kD

43 kD41 kD

28 kD

45 kD

17 kD

45 kD

HDMSCs ASMSCs

+++

+

+

minus

minusminus

minusminus

minus

minus

+++

+

+

minus

minusminus

minusminus

minus

minus

Cleaved caspase-3Cleaved caspase-8

FADD

Actin 1

Actin 2

Cytosolic cytochrome C

(c)

Pro-caspase-3

LV-NCLV-TRAIL-R2

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

CTN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+TN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

Figure 6 Lentiviruses encoding shRNA-TRAIL-R2 partly inhibited the apoptosis of ASMSCs while having a limited effect on the apoptosisof HDMSCs (a) The rate of ASMSC apoptosis (119899 = 22) was decreased after Lv-TRAIL-R2 transduction while no apparent changes werefound for HDMSCs (119899 = 28) (b) In ASMSCs (119899 = 22) Lv-TRAIL-R2 reduced FADD and total caspase-8 expression while having a limitedeffect on total caspase-3 and total cytochrome C gene expression In HDMSCs (119899 = 28) Lv-TRAIL-R2 had a limited effect on the expressionof all four genes (c) Lv-TRAIL-R2 did not affect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC in HDMSCs (119899 = 28) but reduced them in ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 betweenHDMSCs and ASMSCs amp indicates 119875 lt 005 for the TNF-120572CHX group compared with other HDMSC groups indicates 119875 lt 005 forthe TNF-120572CHX group compared with other ASMSC groups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative controlsequence Lv-TRAIL-R2 refers to MSCs transduced by lentiviruses encoding TRAIL-R2

Inhibiting TRAIL-R2 expression using shRNA eliminated thedifference in apoptosis between HDMSCs and ASMSCs bydecreasing the apoptosis of ASMSCs

MSCs aremultipotent stromal cells withmultilineage dif-ferentiation capacities and immunomodulatory propertiesOur previous studies suggested that MSCs are implicated inthe pathogenesis and therapy of AS [12 28] We found thatASMSCs possessed an abnormal osteogenic capacity whichmay contribute to the pathogenesis of AS In addition we alsofound that allogenic intravenous infusion of bone marrow-derived MSCs is feasible and safe in the treatment of ASHowever how the in vivo microenvironment in AS interactswith MSCs remains unknown

AS is a complicated autoimmune disease accompaniedby elevated serum levels of various inflammatory cytokines[14 29] Among these cytokines TNF-120572 is one of the most

important players in the pathogenesis of AS PhysiologicallyTNF-120572 is important for a normal response to inflammationbut the overproduction of TNF-120572 can be detrimental Serumlevels and the sacroiliac joint expression of TNF-120572 wereelevated in AS patients including those in the early stages ofAS [30 31] In addition overproduction of TNF-120572 has beensuggested to be related to disease activity and to account forthe pathogenesis of AS [32] The importance of TNF-120572 hasbeen further confirmed by the efficacy of TNF-120572 inhibitorsin the treatment of AS [16] Due to the importance of TNF-120572in AS a study on the interaction between TNF-120572 and MSCsmay help mimic the in vivo microenvironment experiencedby MSCs in AS Previous studies suggest that TNF-120572 affectsMSCs in various ways TNF-120572 can modulate the immuno-suppressive effects of MSCs and suppress their osteogeniccapacity [33] Additionally TNF-120572 can alter homeostasis

12 Stem Cells International

in MSCs by regulating the balance between apoptosis andsurvival [13] ConverselyMSCs affect the production of TNF-120572 by causing macrophages to adopt an anti-inflammatoryphenotype [34] Both the dysfunction of MSCs and theoverproduction of TNF-120572 may lead to immune disordersA recent study suggested that TNF-120572-induced apoptosis ofMSCs may contribute to the pathogenesis of SLE [19] In ourstudy we found that ASMSCs were more susceptible to TNF-120572-induced apoptosis compared to HDMSCs as evidencedby an increased apoptosis rate and the higher expression ofcleaved caspase-3 protein However how the dysregulationof apoptosis contributes to the pathogenesis of AS remainsuncertain

Apoptosis also defined as programmed cell death iscritical to the pathogenesis of various diseases includingautoimmune diseases [18] The maintenance of immunetolerance and immune homeostasis is largely dependent onapoptosis Defective apoptosis of immune cells may result inthe defective clearance of autoreactive cells or the delayedelimination of autoantigens while excessive apoptosis ofimmunosuppressive cells (such as MSCs) may also lead toimmune hyperactivation Therefore both conditions maycontribute to autoimmune disease [19 35] In patients withAS increased apoptosis of ASMSCs may result in decreasesin cell numbers and immunosuppressive potential eventuallycontributing to autoimmune disorders Moreover becauseMSCs are able to reduce TNF-120572 production increasedMSCs apoptosis may also lead to TNF-120572 overproductionFurthermore TNF-120572 overproduction induces further MSCapoptosis forming a vicious cycle that leads to chronicinflammation in AS Thus increased ASMSC apoptosis maybe critical for AS pathogenesis and further study of themechanism of dysregulated apoptosis in ASMSCs may helpto elucidate the pathogenesis of AS

TRAIL-R2 is a specific cell surface receptor engagedin apoptosis TRAIL-R2-mediated apoptosis has arousedattention due to its role in preferentially killing tumor cellswhile sparing normal cells In our study we observed asignificant increased expression of TRAIL-R2 in ASMSCsafter treatment with TNF-120572CHX Inhibiting TRAIL-R2expression partly decreased the apoptosis of ASMSCs buthad a limited effect on the apoptosis of HDMSCs whichsuggests that TRAIL-R2 overexpression is the main cause ofthe increased apoptosis in ASMSCs However this result isconfusing for two reasons First TRAIL-R2 is reported to bemainly activated by its specific ligand TRAIL [20] Howeverthe results in our study indicated that TRAIL-R2 in MSCscould be activated by TNF-120572CHX as well In fact a similarresult was previously reported but the mechanism remainsunclear [36] Considering the overexpression of TRAIL-R2 can lead to apoptosis even without its ligand [22] wespeculate that TNF-120572maymediate its proapoptotic effects notonly through its own receptor (TNFR1) but also by increasingthe expression of TRAIL-R2 Second bone marrow-derivedadult MSCs are reportedly resistant to TRAIL-R2-mediatedapoptosis [21] However in our study HDMSCs were resis-tant to TRAIL-R2-mediated apoptosis while ASMSCs werenot Multiple mechanisms of TRAIL-R2-mediated apoptosisresistance such as dysregulation of antagonistic receptors

abnormal O-glycosylation and overexpression of the inhi-bition adaptors have been identified [37] Further studiesare needed to illustrate the associated specific mechanismunderlying the difference in resistance of HDMSCs andASMSCs to TRAIL-R2 Additionally based on the findingthat the TRAIL-R2 pathway did not lead to the apoptosis ofnormal cells ASMSCs could be considered abnormal cellsThus it is reasonable to presume that allogenic rather thanautologous MSC transplantation might be preferable for AStreatment

In this study we demonstrated that TRAIL-R2 upregu-lation resulted in enhanced apoptosis of ASMSCs Increasedapoptosis of ASMSCs may decrease the immunosuppressivepotential of these cells and potentially contributing to ASpathogenesis However some limitations remain in thisstudy The questions of how TNF-120572 activates the expressionof TRAIL-R2 why HDMSCs are resistant to TRAIL-R2-mediated apoptosis while ASMSCs are not and whether ourresults can be repeated in vivo remain unanswered Futurestudies should focus on these questions and further elucidatethe mechanism of AS

Competing Interests

The authors have no potential conflict of interests to declare

Authorsrsquo Contributions

Zhenhua Liu LiangbinGao andPengWanghave contributedequally to this work

Acknowledgments

The authors thank Dr Liang Zhang for insightful discussionThis study was supported by the National Natural ScienceFoundation of China (81401850) the Science and Tech-nology Project of Guangdong Province (2015B0202280012015B090903059) and the Engineering Technology ResearchCenter for Comprehensive Diagnosis and Treatment ofAnkylosing Spondylitis of Guangdong Higher EducationInstitutes (GCZX-A1301)

References

[1] M Dougados and D Baeten ldquoSpondyloarthritisrdquo The Lancetvol 377 no 9783 pp 2127ndash2137 2011

[2] P Bowness ldquoHLA-B27rdquo Annual Review of Immunology vol 33pp 29ndash48 2015

[3] J D Reveille A-M Sims P Danoy et al ldquoGenome-wideassociation study of ankylosing spondylitis identifies non-MHCsusceptibility locirdquo Nature Genetics vol 42 no 2 pp 123ndash1272010

[4] J D Taurog J A Richardson J T Croft et al ldquoThe germfreestate prevents development of gut and joint inflammatorydisease in HLA-B27 transgenic ratsrdquo Journal of ExperimentalMedicine vol 180 no 6 pp 2359ndash2364 1994

[5] M-E Costello F Ciccia D Willner et al ldquoBrief Reportintestinal dysbiosis in ankylosing spondylitisrdquo Arthritis andRheumatology vol 67 no 3 pp 686ndash691 2015

Stem Cells International 13

[6] T Rashid and A Ebringer ldquoAnkylosing spondylitis is linked toKlebsiellamdashthe evidencerdquo Clinical Rheumatology vol 26 no 6pp 858ndash864 2007

[7] P Jacques and D McGonagle ldquoThe role of mechanical stress inthe pathogenesis of spondyloarthritis and how to combat itrdquoBest Practice and Research Clinical Rheumatology vol 28 no5 pp 703ndash710 2014

[8] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008

[9] M C Stewart and A A Stewart ldquoMesenchymal stem cellscharacteristics sources and mechanisms of actionrdquo VeterinaryClinics of North AmericamdashEquine Practice vol 27 no 2 pp243ndash261 2011

[10] L Y Sun H Y Zhang X B Feng Y Y Hou LW Lu and L MFan ldquoAbnormality of bone marrow-derived mesenchymal stemcells in patients with systemic lupus erythematosusrdquo Lupus vol16 no 2 pp 121ndash128 2007

[11] J A Perez-Simon S Tabera M E Sarasquete et al ldquoMes-enchymal stem cells are functionally abnormal in patients withimmune thrombocytopenic purpurardquo Cytotherapy vol 11 no6 pp 698ndash705 2009

[12] Z Xie P Wang Y Li et al ldquoImbalance between bone morpho-genetic protein 2 and noggin induces abnormal osteogenic dif-ferentiation of mesenchymal stem cells in ankylosing spondyli-tisrdquo Arthritis and Rheumatology vol 68 no 2 pp 430ndash4402016

[13] D Brenner H Blaser and T W Mak ldquoRegulation of tumournecrosis factor signalling live or let dierdquo Nature ReviewsImmunology vol 15 no 6 pp 362ndash374 2015

[14] J Gratacos A Collado X Filella et al ldquoSerum cytokines(IL-6 TNF-120572 IL-1 120573 and IFN-120574) in ankylosing spondylitis aclose correlation between serum IL-6 and disease activity andseverityrdquo British Journal of Rheumatology vol 33 no 10 pp927ndash931 1994

[15] N Barkham L C Coates H Keen et al ldquoDouble-blindplacebo-controlled trial of etanercept in the prevention of workdisability in ankylosing spondylitisrdquo Annals of the RheumaticDiseases vol 69 no 11 pp 1926ndash1928 2010

[16] J Callhoff J Sieper AWeiss A Zink and J Listing ldquoEfficacy ofTNF120572 blockers in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis ameta-analysisrdquoAnnals ofthe Rheumatic Diseases vol 74 no 6 pp 1241ndash1248 2015

[17] L Wang F Du and X Wang ldquoTNF-120572 induces two distinctcaspase-8 activation pathwaysrdquoCell vol 133 no 4 pp 693ndash7032008

[18] B Favaloro N Allocati V Graziano C Di Ilio and V DeLaurenzi ldquoRole of apoptosis in diseaserdquo Aging vol 4 no 5 pp330ndash349 2012

[19] X Li L Liu D Meng et al ldquoEnhanced apoptosis and senes-cence of bone-marrow-derived mesenchymal stem cells inpatients with systemic lupus erythematosusrdquo Stem Cells andDevelopment vol 21 no 13 pp 2387ndash2394 2012

[20] G R Screaton J Mongkolsapaya X-N Xu A E Cowper A JMcMichael and J I Bell ldquoTRICK2 a new alternatively splicedreceptor that transduces the cytotoxic signal from TRAILrdquoCurrent Biology vol 7 no 9 pp 693ndash696 1997

[21] E Szegezdi A OrsquoReilly Y Davy et al ldquoStem cells are resistantto TRAIL receptor-mediated apoptosisrdquo Journal of Cellular andMolecular Medicine vol 13 no 11-12 pp 4409ndash4414 2009

[22] H Walczak M A Degli-Esposti R S Johnson et al ldquoTRAIL-R2 a novel apoptosis-mediating receptor for TRAILrdquo EMBOJournal vol 16 no 17 pp 5386ndash5397 1997

[23] J-L Bodmer N Holler S Reynard et al ldquoTRAIL receptor-2signals apoptosis through FADD and caspase-8rdquo Nature CellBiology vol 2 no 4 pp 241ndash243 2000

[24] S Van Der Linden H A Valkenburg and A Cats ldquoEvaluationof diagnostic criteria for ankylosing spondylitis A proposal formodification of the New York criteriardquo Arthritis and Rheuma-tism vol 27 no 4 pp 361ndash368 1984

[25] D C Colter R Class C M DiGirolamo and D J ProckopldquoRapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrowrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3213ndash3218 2000

[26] D Li P Wang Y Li et al ldquoAll-Trans Retinoic Acid Improvesthe Effects of Bone Marrow-Derived Mesenchymal Stem Cellson the Treatment of Ankylosing Spondylitis An InVitro StudyrdquoStem Cells International vol 2015 Article ID 484528 10 pages2015

[27] K Fu H Ren Y Wang E Fei H Wang and G Wang ldquoDJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8recruitment to FADDrdquo Oncogene vol 31 no 10 pp 1311ndash13222012

[28] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cell intravenous infusion in active ankylos-ing spondylitis patients who failed NSAIDs a 20-week clinicaltrialrdquo Cell Transplantation vol 23 no 10 pp 1293ndash1303 2014

[29] Y Mei F Pan J Gao et al ldquoIncreased serum IL-17 and IL-23 inthe patient with ankylosing spondylitisrdquoClinical Rheumatologyvol 30 no 2 pp 269ndash273 2011

[30] R J Francois L Neure J Sieper and J Braun ldquoImmunohis-tological examination of open sacroiliac biopsies of patientswith ankylosing spondylitis detection of tumour necrosis factor120572 in two patients with early disease and transforming growthfactor 120573 in three more advanced casesrdquoAnnals of the RheumaticDiseases vol 65 no 6 pp 713ndash720 2006

[31] J Braun M Bollow L Neure et al ldquoUse of immunohistologicand in situ hybridization techniques in the examination ofsacroiliac joint biopsy specimens from patients with ankylosingspondylitisrdquo Arthritis and Rheumatism vol 38 no 4 pp 499ndash505 1995

[32] U Lange J Teichmann and H Stracke ldquoCorrelation betweenplasma TNF-alpha IGF-1 biochemical markers of bonemetabolismmarkers of inflammationdisease activity and clin-ical manifestations in ankylosing spondylitisrdquo European Journalof Medical Research vol 5 no 12 pp 507ndash511 2000

[33] L Zhao J Huang H Zhang et al ldquoTumor necrosis factorinhibits mesenchymal stem cell differentiation into osteoblastsvia the ubiquitin E3 ligase Wwp1rdquo Stem Cells vol 29 no 10 pp1601ndash1610 2011

[34] L Chiossone R Conte G M Spaggiari et al ldquoMesenchymalstromal cells induce peculiar alternatively activated macro-phages capable of dampening both innate and adaptive immuneresponsesrdquo STEM CELLS vol 34 no 7 pp 1909ndash1921 2016

[35] S Nagata ldquoApoptosis and autoimmune diseasesrdquo Annals of theNew York Academy of Sciences vol 1209 no 1 pp 10ndash16 2010

[36] M S Sheikh T F Burns Y Huang et al ldquop53-dependent and-independent regulation of the death receptor KILLERDR5gene expression in response to genotoxic stress and tumor

14 Stem Cells International

necrosis factorrdquo Cancer Research vol 58 no 8 pp 1593ndash15981998

[37] S Shirley A Morizot and O Micheau ldquoRegulating TRAILreceptor-induced cell death at the membrane a deadly discus-sionrdquo Recent Patents on Anti-Cancer Drug Discovery vol 6 no3 pp 311ndash323 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 11

Con

trol

TNF-120572

CH

X

TNF-120572CHX

HDMSCsASMSCs

Con

trol

TNF-120572

CH

X

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Con

trol

TNF-

120572C

HX

HDMSCsASMSCs

Cleaved caspase-3 Cleaved caspase-8

Cytosoliccytochrome CFADD

0

5

10

15

20

Fold

chan

ge

0

5

10

15

Fold

chan

ge

0

10

20

30

Fold

chan

ge

0

20

40

60

80

Fold

chan

ge

amp amp

amp

lowastlowast

lowast lowast

lowastlowast

lowastlowast

35 kD

19 kD17 kD

43 kD41 kD

28 kD

45 kD

17 kD

45 kD

HDMSCs ASMSCs

+++

+

+

minus

minusminus

minusminus

minus

minus

+++

+

+

minus

minusminus

minusminus

minus

minus

Cleaved caspase-3Cleaved caspase-8

FADD

Actin 1

Actin 2

Cytosolic cytochrome C

(c)

Pro-caspase-3

LV-NCLV-TRAIL-R2

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

C

LV-T

RAIL

-R2

LV-N

CTN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+TN

F-120572

CH

X+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

TNF-

120572C

HX

+

Figure 6 Lentiviruses encoding shRNA-TRAIL-R2 partly inhibited the apoptosis of ASMSCs while having a limited effect on the apoptosisof HDMSCs (a) The rate of ASMSC apoptosis (119899 = 22) was decreased after Lv-TRAIL-R2 transduction while no apparent changes werefound for HDMSCs (119899 = 28) (b) In ASMSCs (119899 = 22) Lv-TRAIL-R2 reduced FADD and total caspase-8 expression while having a limitedeffect on total caspase-3 and total cytochrome C gene expression In HDMSCs (119899 = 28) Lv-TRAIL-R2 had a limited effect on the expressionof all four genes (c) Lv-TRAIL-R2 did not affect the protein levels of cleaved caspase-3 cleaved caspase-8 FADD or cytosolic cytochromeC in HDMSCs (119899 = 28) but reduced them in ASMSCs (119899 = 22) Values are presented as the mean plusmn SD lowast indicates 119875 lt 005 betweenHDMSCs and ASMSCs amp indicates 119875 lt 005 for the TNF-120572CHX group compared with other HDMSC groups indicates 119875 lt 005 forthe TNF-120572CHX group compared with other ASMSC groups Lv-NC refers to MSCs transduced by lentiviruses encoding a negative controlsequence Lv-TRAIL-R2 refers to MSCs transduced by lentiviruses encoding TRAIL-R2

Inhibiting TRAIL-R2 expression using shRNA eliminated thedifference in apoptosis between HDMSCs and ASMSCs bydecreasing the apoptosis of ASMSCs

MSCs aremultipotent stromal cells withmultilineage dif-ferentiation capacities and immunomodulatory propertiesOur previous studies suggested that MSCs are implicated inthe pathogenesis and therapy of AS [12 28] We found thatASMSCs possessed an abnormal osteogenic capacity whichmay contribute to the pathogenesis of AS In addition we alsofound that allogenic intravenous infusion of bone marrow-derived MSCs is feasible and safe in the treatment of ASHowever how the in vivo microenvironment in AS interactswith MSCs remains unknown

AS is a complicated autoimmune disease accompaniedby elevated serum levels of various inflammatory cytokines[14 29] Among these cytokines TNF-120572 is one of the most

important players in the pathogenesis of AS PhysiologicallyTNF-120572 is important for a normal response to inflammationbut the overproduction of TNF-120572 can be detrimental Serumlevels and the sacroiliac joint expression of TNF-120572 wereelevated in AS patients including those in the early stages ofAS [30 31] In addition overproduction of TNF-120572 has beensuggested to be related to disease activity and to account forthe pathogenesis of AS [32] The importance of TNF-120572 hasbeen further confirmed by the efficacy of TNF-120572 inhibitorsin the treatment of AS [16] Due to the importance of TNF-120572in AS a study on the interaction between TNF-120572 and MSCsmay help mimic the in vivo microenvironment experiencedby MSCs in AS Previous studies suggest that TNF-120572 affectsMSCs in various ways TNF-120572 can modulate the immuno-suppressive effects of MSCs and suppress their osteogeniccapacity [33] Additionally TNF-120572 can alter homeostasis

12 Stem Cells International

in MSCs by regulating the balance between apoptosis andsurvival [13] ConverselyMSCs affect the production of TNF-120572 by causing macrophages to adopt an anti-inflammatoryphenotype [34] Both the dysfunction of MSCs and theoverproduction of TNF-120572 may lead to immune disordersA recent study suggested that TNF-120572-induced apoptosis ofMSCs may contribute to the pathogenesis of SLE [19] In ourstudy we found that ASMSCs were more susceptible to TNF-120572-induced apoptosis compared to HDMSCs as evidencedby an increased apoptosis rate and the higher expression ofcleaved caspase-3 protein However how the dysregulationof apoptosis contributes to the pathogenesis of AS remainsuncertain

Apoptosis also defined as programmed cell death iscritical to the pathogenesis of various diseases includingautoimmune diseases [18] The maintenance of immunetolerance and immune homeostasis is largely dependent onapoptosis Defective apoptosis of immune cells may result inthe defective clearance of autoreactive cells or the delayedelimination of autoantigens while excessive apoptosis ofimmunosuppressive cells (such as MSCs) may also lead toimmune hyperactivation Therefore both conditions maycontribute to autoimmune disease [19 35] In patients withAS increased apoptosis of ASMSCs may result in decreasesin cell numbers and immunosuppressive potential eventuallycontributing to autoimmune disorders Moreover becauseMSCs are able to reduce TNF-120572 production increasedMSCs apoptosis may also lead to TNF-120572 overproductionFurthermore TNF-120572 overproduction induces further MSCapoptosis forming a vicious cycle that leads to chronicinflammation in AS Thus increased ASMSC apoptosis maybe critical for AS pathogenesis and further study of themechanism of dysregulated apoptosis in ASMSCs may helpto elucidate the pathogenesis of AS

TRAIL-R2 is a specific cell surface receptor engagedin apoptosis TRAIL-R2-mediated apoptosis has arousedattention due to its role in preferentially killing tumor cellswhile sparing normal cells In our study we observed asignificant increased expression of TRAIL-R2 in ASMSCsafter treatment with TNF-120572CHX Inhibiting TRAIL-R2expression partly decreased the apoptosis of ASMSCs buthad a limited effect on the apoptosis of HDMSCs whichsuggests that TRAIL-R2 overexpression is the main cause ofthe increased apoptosis in ASMSCs However this result isconfusing for two reasons First TRAIL-R2 is reported to bemainly activated by its specific ligand TRAIL [20] Howeverthe results in our study indicated that TRAIL-R2 in MSCscould be activated by TNF-120572CHX as well In fact a similarresult was previously reported but the mechanism remainsunclear [36] Considering the overexpression of TRAIL-R2 can lead to apoptosis even without its ligand [22] wespeculate that TNF-120572maymediate its proapoptotic effects notonly through its own receptor (TNFR1) but also by increasingthe expression of TRAIL-R2 Second bone marrow-derivedadult MSCs are reportedly resistant to TRAIL-R2-mediatedapoptosis [21] However in our study HDMSCs were resis-tant to TRAIL-R2-mediated apoptosis while ASMSCs werenot Multiple mechanisms of TRAIL-R2-mediated apoptosisresistance such as dysregulation of antagonistic receptors

abnormal O-glycosylation and overexpression of the inhi-bition adaptors have been identified [37] Further studiesare needed to illustrate the associated specific mechanismunderlying the difference in resistance of HDMSCs andASMSCs to TRAIL-R2 Additionally based on the findingthat the TRAIL-R2 pathway did not lead to the apoptosis ofnormal cells ASMSCs could be considered abnormal cellsThus it is reasonable to presume that allogenic rather thanautologous MSC transplantation might be preferable for AStreatment

In this study we demonstrated that TRAIL-R2 upregu-lation resulted in enhanced apoptosis of ASMSCs Increasedapoptosis of ASMSCs may decrease the immunosuppressivepotential of these cells and potentially contributing to ASpathogenesis However some limitations remain in thisstudy The questions of how TNF-120572 activates the expressionof TRAIL-R2 why HDMSCs are resistant to TRAIL-R2-mediated apoptosis while ASMSCs are not and whether ourresults can be repeated in vivo remain unanswered Futurestudies should focus on these questions and further elucidatethe mechanism of AS

Competing Interests

The authors have no potential conflict of interests to declare

Authorsrsquo Contributions

Zhenhua Liu LiangbinGao andPengWanghave contributedequally to this work

Acknowledgments

The authors thank Dr Liang Zhang for insightful discussionThis study was supported by the National Natural ScienceFoundation of China (81401850) the Science and Tech-nology Project of Guangdong Province (2015B0202280012015B090903059) and the Engineering Technology ResearchCenter for Comprehensive Diagnosis and Treatment ofAnkylosing Spondylitis of Guangdong Higher EducationInstitutes (GCZX-A1301)

References

[1] M Dougados and D Baeten ldquoSpondyloarthritisrdquo The Lancetvol 377 no 9783 pp 2127ndash2137 2011

[2] P Bowness ldquoHLA-B27rdquo Annual Review of Immunology vol 33pp 29ndash48 2015

[3] J D Reveille A-M Sims P Danoy et al ldquoGenome-wideassociation study of ankylosing spondylitis identifies non-MHCsusceptibility locirdquo Nature Genetics vol 42 no 2 pp 123ndash1272010

[4] J D Taurog J A Richardson J T Croft et al ldquoThe germfreestate prevents development of gut and joint inflammatorydisease in HLA-B27 transgenic ratsrdquo Journal of ExperimentalMedicine vol 180 no 6 pp 2359ndash2364 1994

[5] M-E Costello F Ciccia D Willner et al ldquoBrief Reportintestinal dysbiosis in ankylosing spondylitisrdquo Arthritis andRheumatology vol 67 no 3 pp 686ndash691 2015

Stem Cells International 13

[6] T Rashid and A Ebringer ldquoAnkylosing spondylitis is linked toKlebsiellamdashthe evidencerdquo Clinical Rheumatology vol 26 no 6pp 858ndash864 2007

[7] P Jacques and D McGonagle ldquoThe role of mechanical stress inthe pathogenesis of spondyloarthritis and how to combat itrdquoBest Practice and Research Clinical Rheumatology vol 28 no5 pp 703ndash710 2014

[8] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008

[9] M C Stewart and A A Stewart ldquoMesenchymal stem cellscharacteristics sources and mechanisms of actionrdquo VeterinaryClinics of North AmericamdashEquine Practice vol 27 no 2 pp243ndash261 2011

[10] L Y Sun H Y Zhang X B Feng Y Y Hou LW Lu and L MFan ldquoAbnormality of bone marrow-derived mesenchymal stemcells in patients with systemic lupus erythematosusrdquo Lupus vol16 no 2 pp 121ndash128 2007

[11] J A Perez-Simon S Tabera M E Sarasquete et al ldquoMes-enchymal stem cells are functionally abnormal in patients withimmune thrombocytopenic purpurardquo Cytotherapy vol 11 no6 pp 698ndash705 2009

[12] Z Xie P Wang Y Li et al ldquoImbalance between bone morpho-genetic protein 2 and noggin induces abnormal osteogenic dif-ferentiation of mesenchymal stem cells in ankylosing spondyli-tisrdquo Arthritis and Rheumatology vol 68 no 2 pp 430ndash4402016

[13] D Brenner H Blaser and T W Mak ldquoRegulation of tumournecrosis factor signalling live or let dierdquo Nature ReviewsImmunology vol 15 no 6 pp 362ndash374 2015

[14] J Gratacos A Collado X Filella et al ldquoSerum cytokines(IL-6 TNF-120572 IL-1 120573 and IFN-120574) in ankylosing spondylitis aclose correlation between serum IL-6 and disease activity andseverityrdquo British Journal of Rheumatology vol 33 no 10 pp927ndash931 1994

[15] N Barkham L C Coates H Keen et al ldquoDouble-blindplacebo-controlled trial of etanercept in the prevention of workdisability in ankylosing spondylitisrdquo Annals of the RheumaticDiseases vol 69 no 11 pp 1926ndash1928 2010

[16] J Callhoff J Sieper AWeiss A Zink and J Listing ldquoEfficacy ofTNF120572 blockers in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis ameta-analysisrdquoAnnals ofthe Rheumatic Diseases vol 74 no 6 pp 1241ndash1248 2015

[17] L Wang F Du and X Wang ldquoTNF-120572 induces two distinctcaspase-8 activation pathwaysrdquoCell vol 133 no 4 pp 693ndash7032008

[18] B Favaloro N Allocati V Graziano C Di Ilio and V DeLaurenzi ldquoRole of apoptosis in diseaserdquo Aging vol 4 no 5 pp330ndash349 2012

[19] X Li L Liu D Meng et al ldquoEnhanced apoptosis and senes-cence of bone-marrow-derived mesenchymal stem cells inpatients with systemic lupus erythematosusrdquo Stem Cells andDevelopment vol 21 no 13 pp 2387ndash2394 2012

[20] G R Screaton J Mongkolsapaya X-N Xu A E Cowper A JMcMichael and J I Bell ldquoTRICK2 a new alternatively splicedreceptor that transduces the cytotoxic signal from TRAILrdquoCurrent Biology vol 7 no 9 pp 693ndash696 1997

[21] E Szegezdi A OrsquoReilly Y Davy et al ldquoStem cells are resistantto TRAIL receptor-mediated apoptosisrdquo Journal of Cellular andMolecular Medicine vol 13 no 11-12 pp 4409ndash4414 2009

[22] H Walczak M A Degli-Esposti R S Johnson et al ldquoTRAIL-R2 a novel apoptosis-mediating receptor for TRAILrdquo EMBOJournal vol 16 no 17 pp 5386ndash5397 1997

[23] J-L Bodmer N Holler S Reynard et al ldquoTRAIL receptor-2signals apoptosis through FADD and caspase-8rdquo Nature CellBiology vol 2 no 4 pp 241ndash243 2000

[24] S Van Der Linden H A Valkenburg and A Cats ldquoEvaluationof diagnostic criteria for ankylosing spondylitis A proposal formodification of the New York criteriardquo Arthritis and Rheuma-tism vol 27 no 4 pp 361ndash368 1984

[25] D C Colter R Class C M DiGirolamo and D J ProckopldquoRapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrowrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3213ndash3218 2000

[26] D Li P Wang Y Li et al ldquoAll-Trans Retinoic Acid Improvesthe Effects of Bone Marrow-Derived Mesenchymal Stem Cellson the Treatment of Ankylosing Spondylitis An InVitro StudyrdquoStem Cells International vol 2015 Article ID 484528 10 pages2015

[27] K Fu H Ren Y Wang E Fei H Wang and G Wang ldquoDJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8recruitment to FADDrdquo Oncogene vol 31 no 10 pp 1311ndash13222012

[28] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cell intravenous infusion in active ankylos-ing spondylitis patients who failed NSAIDs a 20-week clinicaltrialrdquo Cell Transplantation vol 23 no 10 pp 1293ndash1303 2014

[29] Y Mei F Pan J Gao et al ldquoIncreased serum IL-17 and IL-23 inthe patient with ankylosing spondylitisrdquoClinical Rheumatologyvol 30 no 2 pp 269ndash273 2011

[30] R J Francois L Neure J Sieper and J Braun ldquoImmunohis-tological examination of open sacroiliac biopsies of patientswith ankylosing spondylitis detection of tumour necrosis factor120572 in two patients with early disease and transforming growthfactor 120573 in three more advanced casesrdquoAnnals of the RheumaticDiseases vol 65 no 6 pp 713ndash720 2006

[31] J Braun M Bollow L Neure et al ldquoUse of immunohistologicand in situ hybridization techniques in the examination ofsacroiliac joint biopsy specimens from patients with ankylosingspondylitisrdquo Arthritis and Rheumatism vol 38 no 4 pp 499ndash505 1995

[32] U Lange J Teichmann and H Stracke ldquoCorrelation betweenplasma TNF-alpha IGF-1 biochemical markers of bonemetabolismmarkers of inflammationdisease activity and clin-ical manifestations in ankylosing spondylitisrdquo European Journalof Medical Research vol 5 no 12 pp 507ndash511 2000

[33] L Zhao J Huang H Zhang et al ldquoTumor necrosis factorinhibits mesenchymal stem cell differentiation into osteoblastsvia the ubiquitin E3 ligase Wwp1rdquo Stem Cells vol 29 no 10 pp1601ndash1610 2011

[34] L Chiossone R Conte G M Spaggiari et al ldquoMesenchymalstromal cells induce peculiar alternatively activated macro-phages capable of dampening both innate and adaptive immuneresponsesrdquo STEM CELLS vol 34 no 7 pp 1909ndash1921 2016

[35] S Nagata ldquoApoptosis and autoimmune diseasesrdquo Annals of theNew York Academy of Sciences vol 1209 no 1 pp 10ndash16 2010

[36] M S Sheikh T F Burns Y Huang et al ldquop53-dependent and-independent regulation of the death receptor KILLERDR5gene expression in response to genotoxic stress and tumor

14 Stem Cells International

necrosis factorrdquo Cancer Research vol 58 no 8 pp 1593ndash15981998

[37] S Shirley A Morizot and O Micheau ldquoRegulating TRAILreceptor-induced cell death at the membrane a deadly discus-sionrdquo Recent Patents on Anti-Cancer Drug Discovery vol 6 no3 pp 311ndash323 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 Stem Cells International

in MSCs by regulating the balance between apoptosis andsurvival [13] ConverselyMSCs affect the production of TNF-120572 by causing macrophages to adopt an anti-inflammatoryphenotype [34] Both the dysfunction of MSCs and theoverproduction of TNF-120572 may lead to immune disordersA recent study suggested that TNF-120572-induced apoptosis ofMSCs may contribute to the pathogenesis of SLE [19] In ourstudy we found that ASMSCs were more susceptible to TNF-120572-induced apoptosis compared to HDMSCs as evidencedby an increased apoptosis rate and the higher expression ofcleaved caspase-3 protein However how the dysregulationof apoptosis contributes to the pathogenesis of AS remainsuncertain

Apoptosis also defined as programmed cell death iscritical to the pathogenesis of various diseases includingautoimmune diseases [18] The maintenance of immunetolerance and immune homeostasis is largely dependent onapoptosis Defective apoptosis of immune cells may result inthe defective clearance of autoreactive cells or the delayedelimination of autoantigens while excessive apoptosis ofimmunosuppressive cells (such as MSCs) may also lead toimmune hyperactivation Therefore both conditions maycontribute to autoimmune disease [19 35] In patients withAS increased apoptosis of ASMSCs may result in decreasesin cell numbers and immunosuppressive potential eventuallycontributing to autoimmune disorders Moreover becauseMSCs are able to reduce TNF-120572 production increasedMSCs apoptosis may also lead to TNF-120572 overproductionFurthermore TNF-120572 overproduction induces further MSCapoptosis forming a vicious cycle that leads to chronicinflammation in AS Thus increased ASMSC apoptosis maybe critical for AS pathogenesis and further study of themechanism of dysregulated apoptosis in ASMSCs may helpto elucidate the pathogenesis of AS

TRAIL-R2 is a specific cell surface receptor engagedin apoptosis TRAIL-R2-mediated apoptosis has arousedattention due to its role in preferentially killing tumor cellswhile sparing normal cells In our study we observed asignificant increased expression of TRAIL-R2 in ASMSCsafter treatment with TNF-120572CHX Inhibiting TRAIL-R2expression partly decreased the apoptosis of ASMSCs buthad a limited effect on the apoptosis of HDMSCs whichsuggests that TRAIL-R2 overexpression is the main cause ofthe increased apoptosis in ASMSCs However this result isconfusing for two reasons First TRAIL-R2 is reported to bemainly activated by its specific ligand TRAIL [20] Howeverthe results in our study indicated that TRAIL-R2 in MSCscould be activated by TNF-120572CHX as well In fact a similarresult was previously reported but the mechanism remainsunclear [36] Considering the overexpression of TRAIL-R2 can lead to apoptosis even without its ligand [22] wespeculate that TNF-120572maymediate its proapoptotic effects notonly through its own receptor (TNFR1) but also by increasingthe expression of TRAIL-R2 Second bone marrow-derivedadult MSCs are reportedly resistant to TRAIL-R2-mediatedapoptosis [21] However in our study HDMSCs were resis-tant to TRAIL-R2-mediated apoptosis while ASMSCs werenot Multiple mechanisms of TRAIL-R2-mediated apoptosisresistance such as dysregulation of antagonistic receptors

abnormal O-glycosylation and overexpression of the inhi-bition adaptors have been identified [37] Further studiesare needed to illustrate the associated specific mechanismunderlying the difference in resistance of HDMSCs andASMSCs to TRAIL-R2 Additionally based on the findingthat the TRAIL-R2 pathway did not lead to the apoptosis ofnormal cells ASMSCs could be considered abnormal cellsThus it is reasonable to presume that allogenic rather thanautologous MSC transplantation might be preferable for AStreatment

In this study we demonstrated that TRAIL-R2 upregu-lation resulted in enhanced apoptosis of ASMSCs Increasedapoptosis of ASMSCs may decrease the immunosuppressivepotential of these cells and potentially contributing to ASpathogenesis However some limitations remain in thisstudy The questions of how TNF-120572 activates the expressionof TRAIL-R2 why HDMSCs are resistant to TRAIL-R2-mediated apoptosis while ASMSCs are not and whether ourresults can be repeated in vivo remain unanswered Futurestudies should focus on these questions and further elucidatethe mechanism of AS

Competing Interests

The authors have no potential conflict of interests to declare

Authorsrsquo Contributions

Zhenhua Liu LiangbinGao andPengWanghave contributedequally to this work

Acknowledgments

The authors thank Dr Liang Zhang for insightful discussionThis study was supported by the National Natural ScienceFoundation of China (81401850) the Science and Tech-nology Project of Guangdong Province (2015B0202280012015B090903059) and the Engineering Technology ResearchCenter for Comprehensive Diagnosis and Treatment ofAnkylosing Spondylitis of Guangdong Higher EducationInstitutes (GCZX-A1301)

References

[1] M Dougados and D Baeten ldquoSpondyloarthritisrdquo The Lancetvol 377 no 9783 pp 2127ndash2137 2011

[2] P Bowness ldquoHLA-B27rdquo Annual Review of Immunology vol 33pp 29ndash48 2015

[3] J D Reveille A-M Sims P Danoy et al ldquoGenome-wideassociation study of ankylosing spondylitis identifies non-MHCsusceptibility locirdquo Nature Genetics vol 42 no 2 pp 123ndash1272010

[4] J D Taurog J A Richardson J T Croft et al ldquoThe germfreestate prevents development of gut and joint inflammatorydisease in HLA-B27 transgenic ratsrdquo Journal of ExperimentalMedicine vol 180 no 6 pp 2359ndash2364 1994

[5] M-E Costello F Ciccia D Willner et al ldquoBrief Reportintestinal dysbiosis in ankylosing spondylitisrdquo Arthritis andRheumatology vol 67 no 3 pp 686ndash691 2015

Stem Cells International 13

[6] T Rashid and A Ebringer ldquoAnkylosing spondylitis is linked toKlebsiellamdashthe evidencerdquo Clinical Rheumatology vol 26 no 6pp 858ndash864 2007

[7] P Jacques and D McGonagle ldquoThe role of mechanical stress inthe pathogenesis of spondyloarthritis and how to combat itrdquoBest Practice and Research Clinical Rheumatology vol 28 no5 pp 703ndash710 2014

[8] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008

[9] M C Stewart and A A Stewart ldquoMesenchymal stem cellscharacteristics sources and mechanisms of actionrdquo VeterinaryClinics of North AmericamdashEquine Practice vol 27 no 2 pp243ndash261 2011

[10] L Y Sun H Y Zhang X B Feng Y Y Hou LW Lu and L MFan ldquoAbnormality of bone marrow-derived mesenchymal stemcells in patients with systemic lupus erythematosusrdquo Lupus vol16 no 2 pp 121ndash128 2007

[11] J A Perez-Simon S Tabera M E Sarasquete et al ldquoMes-enchymal stem cells are functionally abnormal in patients withimmune thrombocytopenic purpurardquo Cytotherapy vol 11 no6 pp 698ndash705 2009

[12] Z Xie P Wang Y Li et al ldquoImbalance between bone morpho-genetic protein 2 and noggin induces abnormal osteogenic dif-ferentiation of mesenchymal stem cells in ankylosing spondyli-tisrdquo Arthritis and Rheumatology vol 68 no 2 pp 430ndash4402016

[13] D Brenner H Blaser and T W Mak ldquoRegulation of tumournecrosis factor signalling live or let dierdquo Nature ReviewsImmunology vol 15 no 6 pp 362ndash374 2015

[14] J Gratacos A Collado X Filella et al ldquoSerum cytokines(IL-6 TNF-120572 IL-1 120573 and IFN-120574) in ankylosing spondylitis aclose correlation between serum IL-6 and disease activity andseverityrdquo British Journal of Rheumatology vol 33 no 10 pp927ndash931 1994

[15] N Barkham L C Coates H Keen et al ldquoDouble-blindplacebo-controlled trial of etanercept in the prevention of workdisability in ankylosing spondylitisrdquo Annals of the RheumaticDiseases vol 69 no 11 pp 1926ndash1928 2010

[16] J Callhoff J Sieper AWeiss A Zink and J Listing ldquoEfficacy ofTNF120572 blockers in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis ameta-analysisrdquoAnnals ofthe Rheumatic Diseases vol 74 no 6 pp 1241ndash1248 2015

[17] L Wang F Du and X Wang ldquoTNF-120572 induces two distinctcaspase-8 activation pathwaysrdquoCell vol 133 no 4 pp 693ndash7032008

[18] B Favaloro N Allocati V Graziano C Di Ilio and V DeLaurenzi ldquoRole of apoptosis in diseaserdquo Aging vol 4 no 5 pp330ndash349 2012

[19] X Li L Liu D Meng et al ldquoEnhanced apoptosis and senes-cence of bone-marrow-derived mesenchymal stem cells inpatients with systemic lupus erythematosusrdquo Stem Cells andDevelopment vol 21 no 13 pp 2387ndash2394 2012

[20] G R Screaton J Mongkolsapaya X-N Xu A E Cowper A JMcMichael and J I Bell ldquoTRICK2 a new alternatively splicedreceptor that transduces the cytotoxic signal from TRAILrdquoCurrent Biology vol 7 no 9 pp 693ndash696 1997

[21] E Szegezdi A OrsquoReilly Y Davy et al ldquoStem cells are resistantto TRAIL receptor-mediated apoptosisrdquo Journal of Cellular andMolecular Medicine vol 13 no 11-12 pp 4409ndash4414 2009

[22] H Walczak M A Degli-Esposti R S Johnson et al ldquoTRAIL-R2 a novel apoptosis-mediating receptor for TRAILrdquo EMBOJournal vol 16 no 17 pp 5386ndash5397 1997

[23] J-L Bodmer N Holler S Reynard et al ldquoTRAIL receptor-2signals apoptosis through FADD and caspase-8rdquo Nature CellBiology vol 2 no 4 pp 241ndash243 2000

[24] S Van Der Linden H A Valkenburg and A Cats ldquoEvaluationof diagnostic criteria for ankylosing spondylitis A proposal formodification of the New York criteriardquo Arthritis and Rheuma-tism vol 27 no 4 pp 361ndash368 1984

[25] D C Colter R Class C M DiGirolamo and D J ProckopldquoRapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrowrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3213ndash3218 2000

[26] D Li P Wang Y Li et al ldquoAll-Trans Retinoic Acid Improvesthe Effects of Bone Marrow-Derived Mesenchymal Stem Cellson the Treatment of Ankylosing Spondylitis An InVitro StudyrdquoStem Cells International vol 2015 Article ID 484528 10 pages2015

[27] K Fu H Ren Y Wang E Fei H Wang and G Wang ldquoDJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8recruitment to FADDrdquo Oncogene vol 31 no 10 pp 1311ndash13222012

[28] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cell intravenous infusion in active ankylos-ing spondylitis patients who failed NSAIDs a 20-week clinicaltrialrdquo Cell Transplantation vol 23 no 10 pp 1293ndash1303 2014

[29] Y Mei F Pan J Gao et al ldquoIncreased serum IL-17 and IL-23 inthe patient with ankylosing spondylitisrdquoClinical Rheumatologyvol 30 no 2 pp 269ndash273 2011

[30] R J Francois L Neure J Sieper and J Braun ldquoImmunohis-tological examination of open sacroiliac biopsies of patientswith ankylosing spondylitis detection of tumour necrosis factor120572 in two patients with early disease and transforming growthfactor 120573 in three more advanced casesrdquoAnnals of the RheumaticDiseases vol 65 no 6 pp 713ndash720 2006

[31] J Braun M Bollow L Neure et al ldquoUse of immunohistologicand in situ hybridization techniques in the examination ofsacroiliac joint biopsy specimens from patients with ankylosingspondylitisrdquo Arthritis and Rheumatism vol 38 no 4 pp 499ndash505 1995

[32] U Lange J Teichmann and H Stracke ldquoCorrelation betweenplasma TNF-alpha IGF-1 biochemical markers of bonemetabolismmarkers of inflammationdisease activity and clin-ical manifestations in ankylosing spondylitisrdquo European Journalof Medical Research vol 5 no 12 pp 507ndash511 2000

[33] L Zhao J Huang H Zhang et al ldquoTumor necrosis factorinhibits mesenchymal stem cell differentiation into osteoblastsvia the ubiquitin E3 ligase Wwp1rdquo Stem Cells vol 29 no 10 pp1601ndash1610 2011

[34] L Chiossone R Conte G M Spaggiari et al ldquoMesenchymalstromal cells induce peculiar alternatively activated macro-phages capable of dampening both innate and adaptive immuneresponsesrdquo STEM CELLS vol 34 no 7 pp 1909ndash1921 2016

[35] S Nagata ldquoApoptosis and autoimmune diseasesrdquo Annals of theNew York Academy of Sciences vol 1209 no 1 pp 10ndash16 2010

[36] M S Sheikh T F Burns Y Huang et al ldquop53-dependent and-independent regulation of the death receptor KILLERDR5gene expression in response to genotoxic stress and tumor

14 Stem Cells International

necrosis factorrdquo Cancer Research vol 58 no 8 pp 1593ndash15981998

[37] S Shirley A Morizot and O Micheau ldquoRegulating TRAILreceptor-induced cell death at the membrane a deadly discus-sionrdquo Recent Patents on Anti-Cancer Drug Discovery vol 6 no3 pp 311ndash323 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 13

[6] T Rashid and A Ebringer ldquoAnkylosing spondylitis is linked toKlebsiellamdashthe evidencerdquo Clinical Rheumatology vol 26 no 6pp 858ndash864 2007

[7] P Jacques and D McGonagle ldquoThe role of mechanical stress inthe pathogenesis of spondyloarthritis and how to combat itrdquoBest Practice and Research Clinical Rheumatology vol 28 no5 pp 703ndash710 2014

[8] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008

[9] M C Stewart and A A Stewart ldquoMesenchymal stem cellscharacteristics sources and mechanisms of actionrdquo VeterinaryClinics of North AmericamdashEquine Practice vol 27 no 2 pp243ndash261 2011

[10] L Y Sun H Y Zhang X B Feng Y Y Hou LW Lu and L MFan ldquoAbnormality of bone marrow-derived mesenchymal stemcells in patients with systemic lupus erythematosusrdquo Lupus vol16 no 2 pp 121ndash128 2007

[11] J A Perez-Simon S Tabera M E Sarasquete et al ldquoMes-enchymal stem cells are functionally abnormal in patients withimmune thrombocytopenic purpurardquo Cytotherapy vol 11 no6 pp 698ndash705 2009

[12] Z Xie P Wang Y Li et al ldquoImbalance between bone morpho-genetic protein 2 and noggin induces abnormal osteogenic dif-ferentiation of mesenchymal stem cells in ankylosing spondyli-tisrdquo Arthritis and Rheumatology vol 68 no 2 pp 430ndash4402016

[13] D Brenner H Blaser and T W Mak ldquoRegulation of tumournecrosis factor signalling live or let dierdquo Nature ReviewsImmunology vol 15 no 6 pp 362ndash374 2015

[14] J Gratacos A Collado X Filella et al ldquoSerum cytokines(IL-6 TNF-120572 IL-1 120573 and IFN-120574) in ankylosing spondylitis aclose correlation between serum IL-6 and disease activity andseverityrdquo British Journal of Rheumatology vol 33 no 10 pp927ndash931 1994

[15] N Barkham L C Coates H Keen et al ldquoDouble-blindplacebo-controlled trial of etanercept in the prevention of workdisability in ankylosing spondylitisrdquo Annals of the RheumaticDiseases vol 69 no 11 pp 1926ndash1928 2010

[16] J Callhoff J Sieper AWeiss A Zink and J Listing ldquoEfficacy ofTNF120572 blockers in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis ameta-analysisrdquoAnnals ofthe Rheumatic Diseases vol 74 no 6 pp 1241ndash1248 2015

[17] L Wang F Du and X Wang ldquoTNF-120572 induces two distinctcaspase-8 activation pathwaysrdquoCell vol 133 no 4 pp 693ndash7032008

[18] B Favaloro N Allocati V Graziano C Di Ilio and V DeLaurenzi ldquoRole of apoptosis in diseaserdquo Aging vol 4 no 5 pp330ndash349 2012

[19] X Li L Liu D Meng et al ldquoEnhanced apoptosis and senes-cence of bone-marrow-derived mesenchymal stem cells inpatients with systemic lupus erythematosusrdquo Stem Cells andDevelopment vol 21 no 13 pp 2387ndash2394 2012

[20] G R Screaton J Mongkolsapaya X-N Xu A E Cowper A JMcMichael and J I Bell ldquoTRICK2 a new alternatively splicedreceptor that transduces the cytotoxic signal from TRAILrdquoCurrent Biology vol 7 no 9 pp 693ndash696 1997

[21] E Szegezdi A OrsquoReilly Y Davy et al ldquoStem cells are resistantto TRAIL receptor-mediated apoptosisrdquo Journal of Cellular andMolecular Medicine vol 13 no 11-12 pp 4409ndash4414 2009

[22] H Walczak M A Degli-Esposti R S Johnson et al ldquoTRAIL-R2 a novel apoptosis-mediating receptor for TRAILrdquo EMBOJournal vol 16 no 17 pp 5386ndash5397 1997

[23] J-L Bodmer N Holler S Reynard et al ldquoTRAIL receptor-2signals apoptosis through FADD and caspase-8rdquo Nature CellBiology vol 2 no 4 pp 241ndash243 2000

[24] S Van Der Linden H A Valkenburg and A Cats ldquoEvaluationof diagnostic criteria for ankylosing spondylitis A proposal formodification of the New York criteriardquo Arthritis and Rheuma-tism vol 27 no 4 pp 361ndash368 1984

[25] D C Colter R Class C M DiGirolamo and D J ProckopldquoRapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrowrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3213ndash3218 2000

[26] D Li P Wang Y Li et al ldquoAll-Trans Retinoic Acid Improvesthe Effects of Bone Marrow-Derived Mesenchymal Stem Cellson the Treatment of Ankylosing Spondylitis An InVitro StudyrdquoStem Cells International vol 2015 Article ID 484528 10 pages2015

[27] K Fu H Ren Y Wang E Fei H Wang and G Wang ldquoDJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8recruitment to FADDrdquo Oncogene vol 31 no 10 pp 1311ndash13222012

[28] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cell intravenous infusion in active ankylos-ing spondylitis patients who failed NSAIDs a 20-week clinicaltrialrdquo Cell Transplantation vol 23 no 10 pp 1293ndash1303 2014

[29] Y Mei F Pan J Gao et al ldquoIncreased serum IL-17 and IL-23 inthe patient with ankylosing spondylitisrdquoClinical Rheumatologyvol 30 no 2 pp 269ndash273 2011

[30] R J Francois L Neure J Sieper and J Braun ldquoImmunohis-tological examination of open sacroiliac biopsies of patientswith ankylosing spondylitis detection of tumour necrosis factor120572 in two patients with early disease and transforming growthfactor 120573 in three more advanced casesrdquoAnnals of the RheumaticDiseases vol 65 no 6 pp 713ndash720 2006

[31] J Braun M Bollow L Neure et al ldquoUse of immunohistologicand in situ hybridization techniques in the examination ofsacroiliac joint biopsy specimens from patients with ankylosingspondylitisrdquo Arthritis and Rheumatism vol 38 no 4 pp 499ndash505 1995

[32] U Lange J Teichmann and H Stracke ldquoCorrelation betweenplasma TNF-alpha IGF-1 biochemical markers of bonemetabolismmarkers of inflammationdisease activity and clin-ical manifestations in ankylosing spondylitisrdquo European Journalof Medical Research vol 5 no 12 pp 507ndash511 2000

[33] L Zhao J Huang H Zhang et al ldquoTumor necrosis factorinhibits mesenchymal stem cell differentiation into osteoblastsvia the ubiquitin E3 ligase Wwp1rdquo Stem Cells vol 29 no 10 pp1601ndash1610 2011

[34] L Chiossone R Conte G M Spaggiari et al ldquoMesenchymalstromal cells induce peculiar alternatively activated macro-phages capable of dampening both innate and adaptive immuneresponsesrdquo STEM CELLS vol 34 no 7 pp 1909ndash1921 2016

[35] S Nagata ldquoApoptosis and autoimmune diseasesrdquo Annals of theNew York Academy of Sciences vol 1209 no 1 pp 10ndash16 2010

[36] M S Sheikh T F Burns Y Huang et al ldquop53-dependent and-independent regulation of the death receptor KILLERDR5gene expression in response to genotoxic stress and tumor

14 Stem Cells International

necrosis factorrdquo Cancer Research vol 58 no 8 pp 1593ndash15981998

[37] S Shirley A Morizot and O Micheau ldquoRegulating TRAILreceptor-induced cell death at the membrane a deadly discus-sionrdquo Recent Patents on Anti-Cancer Drug Discovery vol 6 no3 pp 311ndash323 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

14 Stem Cells International

necrosis factorrdquo Cancer Research vol 58 no 8 pp 1593ndash15981998

[37] S Shirley A Morizot and O Micheau ldquoRegulating TRAILreceptor-induced cell death at the membrane a deadly discus-sionrdquo Recent Patents on Anti-Cancer Drug Discovery vol 6 no3 pp 311ndash323 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology