Lactobacillus Probiotic Protects Intestinal Epithelium

8/13/2019 Lactobacillus Probiotic Protects Intestinal Epithelium

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ORIGINAL ARTICLE

Lactobacillus probiotic protects intestinal epitheliumfrom radiation injury in a TLR-2/cyclo-oxygenase-2-dependent manner

Matthew A Ciorba,1 Terrence E Riehl,1 M Suprada Rao,1 Clara Moon,2 Xueping Ee,1

Gerardo M Nava,2 Monica R Walker,2 Jeffrey M Marinshaw,1

Thaddeus S Stappenbeck,2 William F Stenson1

ABSTRACTBackground The small intestinal epithelium is highlysensitive to radiation and is a major site of injury duringradiation therapy and environmental overexposure.

Objective To examine probiotic bacteria as potentialradioprotective agents in the intestine.

Methods 8-week-old C57BL/6 wild-type or knockoutmice were administered probiotic by gavage for 3 daysbefore 12 Gy whole body radiation. The intestine wasevaluated for cell-positional apoptosis (6 h) and cryptsurvival (84 h).

Results Gavage of 53107 Lactobacillus rhamnosus GG(LGG) improved crypt survival about twofold (p


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radiation injury. Lipopolysaccharide (LPS), a TLR-4 ligand, isradioprotective in the mouse intestine through a mechanism thatinvolves prostaglandin E2 (PGE2) synthesis through cyclo-oxygenase-2 (COX-2).10Administration of TLR-5 ligands,agellinor CBL13502 (a polypeptide drug derived from salmonellaagellin) before irradiation protected both mice and monkeysfrom gastrointestinal (GI) and haematopoietic acute radiationsyndromes.11e13

Probiotic therapies have been clinically evaluated and used astreatment for human inammatory bowel diseases, pouchitis,irritable bowel syndrome and antibiotic-associated diar-rhoea.14e16 For inammatory bowel diseases, most probioticstudies have been largely descriptive, although recent investi-gations in mice describe anti-apoptotic and anti-inammatorymechanisms for the probiotic, Lactobacillus rhamnosus GG(LGG).17 18

There have been preliminary studies of probiotic use in theprevention or treatment of radiation injury in the intestine.19 Inexperimental models of radiation injury, lactobacillus ora mixture of lactobacillus and bidobacterium given to rodentsbefore and after radiation resulted in improved histology20 anddecreased endotoxaemia and sepsis.2 1 2 2 No assessment wasmade of epithelial crypt survival or apoptosis, nor was anymechanism described. In humans, probiotics have been used tocontrol diarrhoea after radiation exposure has begun, showinga trend towards benet.23e25 Treatment with a probioticcombination of Lactobacillus acidophilus plus Bidobacteriumbidum was compared with placebo in patients receiving radia-tion therapy with weekly cisplatin for cervical cancer.26 Thecombination of probiotics resulted in less diarrhoea. Sinceradiotherapy is a planned event and prophylactic therapy isfeasible, we sought to determine if prophylactic probiotictreatment affects the intestinal epithelial response to radiationas assessed by apoptosis and crypt survival.

METHODS AND MATERIALSMice

All mice used were on the C57Bl/6 background. Wild-type (WT),TLR-2/ and TLR-4/ mice were purchased from JacksonLaboratories (Bar Harbour, Maine, USA) and bred in house.MyD88/and COX-2/ mice are maintained in our facility aspreviously described.4 Experiments with WT mice wereconrmed both on those purchased directly from JacksonLaboratories and those bred in house. Comparator groups werematched for age and sex, typically as littermates. Female micewere preferentially used. WT mice (phosphate-buffered saline(PBS) vs LGG treated) were tested concurrently as positivecontrols with all knockout mouse experiments. All mice weremaintained on a 12 h light/dark schedule in a temperature-

controlled specic pathogen free facility and fed standard labo-ratory mouse chow. Animal procedures and protocols wereconducted in accordance with the institutional review board at

Washington University School of Medicine. Whole body irradi-ation of mice was carried out in a Gammacell 40 137Cs irradiator(Atomic Energy of Canada Ltd, Chalk River, Ontario, Canada) ata dose rate of 80.7 cGy/min and a total dose of 12 Gy.

Apoptosis and crypt survival in mouse small intestineAfter 12 Gy whole body irradiation, mice were killed at 6 h(apoptosis studies) or 84 h (crypt survival studies). The entiresmall intestine was harvested and used to collect tissue for RNAor protein determination or histology. The proximal jejunum and

distal ileum were

xed for histology in 10% formalin with PBS. Aminimum of six 5 mm segments were taken from each mouse and

parafn embedded for analysis. Apoptosis was scored on a cell-positional basis by light microscopic analysis (H&E and TUNEL(terminal deoxynucleotidyl transferase dUTP nick end labeling))

of 100 half-crypt sections per mouse with at least four mice ineach group.27 All crypts chosen were at least 20 cells in height,with cell position 1 located at the crypt base. Crypt survival wasmeasured as described previously using a modication of themicrocolony assay.10 28 For these experiments, 120 mg/kgbromodeoxyuridine (BrdU, Sigma) and 12 mg/kg uorodeoxyur-idine (Sigma) was injected 90 min before death to label S-phasecells identied by immunohistochemistry. The viability ofa surviving crypt was conrmed by incorporation of BrdU intove or more epithelial cells within each regenerative crypt.

Probiotics and conditioned mediumLactobacillusspp. were purchased from American Tissue Culture

Collection (ATCC, Manassas, Virginia, USA). Lactobacillusrhamnosus GG (LGG) (ATCC 53103), Lactobacillus acidophilus

2 4 6 8 10 12 14 16 18 20

0

10

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40

PBS

LGG

LGG-CM

p


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(ATCC 393) and Lactobacillus casei (ATCC 4356) were culturedaccording to the manufacturers recommendations in MRSbroth (Fisher Scientic, Pittsburg, Pennsylvania, USA). Bacteriawere cultured at 378C to reach log phase growth as determinedby a 0.5A600reading,

29 then immediately centrifuged at 12003gfor 10 min and washed twice with cold PBS. Bacteria werebrought up in volumes to reach the desired number of bacteriaper 200 ml for murine gavage. Concentrations of live bacteria

were conrmed by serial dilutions and plating for colonyforming unit counts. Commercially marketed forms of theprobiotic supplements LGG (Culturelle; Amerit, Cromwell,Connecticut, USA) and Bidobacterium infantis 35624 (Proctor &Gamble, Cincinnati, Ohio, USA) were tested separately.

Heat-killed bacteria were prepared as previously describedexposing LGG in PBS to boiling temperature for 30 min followedby a PBS wash.30 LGG conditioned culture medium (LGG-CM)was prepared by growing LGG in MRS broth to log phasegrowth. The cultured broth was then centrifuged (15003g for10 min) to pellet the majority of bacteria before allowing theculture to again reach log phase growth. After a second centri-fugation, the CM pH was adjusted to that of freshly preparedcontrol MRS broth. Both preparations were sterile ltered(0.2 mm) before use.

Organ-specific mesenchymal stem cells (MSCs)Small intestinal and colon MSCs were prepared as previouslydescribed.31 In vitro stimulation of MSCs was performed witha commercial TLR-2 ligand (Pam3CSK4, catalogue numberTlr1-pms; Invivogen, San Diego, California, USA) at theconcentrations described.

Protein and nucleic acid analysisWhole tissue RNA expression was analysed by quantitativereverse transcription (qRT)-PCR as previously described.3 2 3 3

qRT-PCR primer sequences are described in online supplemen-

tary table 1. Formalin-xed parafn-embedded jejunal tissueswere used for immunouorescence using high-temperaturecitrate-based antigen retrieval. COX-2-expressing cells weredetected using a mouse monoclonal antibody (BD Biosciences,San Diego, California, USA; catalogue number 610204; diluted1:50) followed by AlexaFluor 594 goat anti-mouse (MolecularProbes, Carlsbad, California, USA; catalogue number 11032;diluted 1:250). The surfaces of epithelial cells were labelled usingrabbit anti-human b-catenin (Santa Cruz Biotechnology, SantaCruz, California, USA; catalogue number sc-7199; diluted 1:100)followed by AlexaFluor 488 donkey anti-rabbit (MolecularProbes; catalogue number 21206; diluted 1:250). Photomicro-graphs were taken at 2003 magnication and used to obtaincounts of COX-2-expressing stromal cells in the crypt and villus

No irradiation

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Figure 2 Crypt survival after radiation is improved by live Lactobacillusprobiotics and Lactobacillus rhamnosus GG (LGG) culture mediumsupernatant (LGG-CM). Mice were gavaged with probiotic speciesdescribed or LGG-CM for 3 days before radiation (IR) with 12 Gy. Eighty-

four hours after radiation exposure, mice were injected with

bromodeoxyuridine (BrdU). Intestinal tissues were harvested 90 minlater and fixed for BrdU staining. (A) A representative jejunal cross-section from an untreated mouse is compared with sections from miceexposed to radiation which received pretreatment with either phosphate-buffered saline (PBS) (control) or LGG. (B) Dose finding comparisonshowed no additional benefit at bacterial loads over 13108, with a doseof 53107 being used for subsequent experiments. *p


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regions. At this magnication, each picture covered a length of340 mm. Data are the mean number of cells per picture (340 mmlength) for 20 locations along the lengths of the jejunal tissuesections from ve mice. This encompassed >800 COX-2-expressing cells in more than 120 crypt/villus structures. ThePGE2 assay was performed as previously described

33 using anELISA (Cayman Chemical, Ann Arbor, Michigan, USA; cata-

logue number 514010) according to the manufacturer s protocol.Flow cytometry was also performed as previously describedwith identical antibodies.31

To evaluate the effect that LGG gavage has on small intestinalbacterial composition, we used a genomic DNA-based approachas previously described.34 A complete methodological descrip-tion is available in supplementary information online. Briey,ileal contents and mucosal scrapings were obtained from groupsof mice 12 h after the last of three gavages with PBS or livewashed LGG in PBS. Bacterial DNA concentrations were esti-mated by quantitative PCR (qPCR) assays using validatedprimers targeting total bacteria (rpoB) and family-specicprimers targeting 16S rRNA.35 For quantication of LGG,

a qPCR assay was standardised using validated strain-speci

cprimers.36 37

Statistical analysisThe two-sided, non-paired Student t test was used formost comparisons between treatment and control groupsincluding crypt survival, positional apoptosis, bacterial concen-trations, and weight loss by day. Survival data were assessedusing the ManteleCox log rank test. Data are expressed asmean6SEM unless otherwise noted. Prism GraphPad Software(LaJolla, California, USA) was used for data analysis andgraph generation.

RESULTSLGG and LGG-CM diminish radiation-induced apoptosis ina position-dependent fashionIn WT mice, 12 Gy of radiation induced small intestinal epithelialcell apoptosis in a position-dependent fashion (gure 1A).

Assessment of apoptosis by H&E or TUNEL assay gave equalresults (gure 1B). Six hours after irradiation, the apoptoticindex was >30% in positions 4e5 and fell off gradually to below10% at position 12. Mice pretreated with LGG or LGG-CMshowed a signicant reduction in position-dependent apoptosis.LGG and LGG-CM reduced apoptosis at position 4 from 33% to17% and 20%, respectively. Position 4 is the position of thepurported quiescent epithelial stem cell.38 The effects of LGG

and LGG-CM in the proximal jejunum were similar. There wasno signicant difference in the apoptotic index between control

groups gavaged with PBS or uncultured MRS medium (data forMRS not shown). The radioprotection of LGG or LGG-CM wasapparent both in mice purchased from a commercial vendor andthose with lifetime exposure to our institutional housingfacility.

Lactobacillusprobiotics and LGG-CM enhance intestinal cryptsurvivalIn the absence of radiation, there were about 120 crypts percross-jejunal section. Eighty-four hours after a single dose of12 Gy radiation in WT mice there averaged 15 surviving cryptsper cross-section. Pretreatment with LGG increased the numberof surviving crypts approximately twofold (gure 2A). Indoseeresponse studies, LGG offered some protection at a dose of13106 bacteria per treatment, with improved efcacy at doses of$13107 bacteria per treatment (gure 2B). Pretreatment withL

casei and L acidophilus also increased crypt survival (gure 2C).Observed crypt survival was highest in the LGG group, althoughthe difference between Lactobacillus spp. was not signicant.

Pretreatment with LGG signicantly increased the number ofsurviving crypts along the length of the small intestine, both in

the proximal jejunum and distal ileum (gure 2D). In contrast,the administration of LGG after irradiation had no effect on thenumber of surviving crypts. Pretreatment with LGG superna-tant increased the number of surviving crypts from 15 to 29. Incontrast with the increase in crypt survival with live LGG orwith LGG-CM, there was no signicant increase in cryptsurvival with heat-killed LGG. To determine if the effects of theLGG supernatant could be active systemically as well as topi-cally, LGG-CM was administered at equal dosing intraperito-neally to mice before radiation. Although mice tolerated thisinjection without adverse outcome, no benet to crypt survivalwas observed (data not shown).

Administration of a commercially available LGG product(Culturelle; 1/500th of a capsule dissolved in PBS) resulted ina level of radioprotection similar to that produced by livecultured LGG and signicantly greater than PBS control (table 1).This benet was not offered with equal dosing of a non-lactobacillus-containing commercially available probiotic (Binfantis 35624; Align), suggesting that Lactobacillus may be thefavoured probiotic genus for limiting radiation-induced smallintestinal injury.

LGG before radiation improves survival, diminishes weight loss,and reduces intestinal crypt lossSmall intestinal epithelial damage and weight loss are compo-nents of the radiation GI syndrome. Irradiated mice consistentlylost weight after irradiation, reaching 70% of their starting

weight by 7 days (gure 3A). Mice pretreated with LGG lostweight for 5 days after irradiation and then began to regain theirlost weight before reaching a plateau and ultimately losingweight again. In mice, death is a standard end point measured inthe study of acute GI radiation syndrome and typically occurswithin the rst 10 days after exposure.1 In our experiments,irradiated control mice began to die at 6 days after irradiationand mortality was 100% by day 11 (gure 3B). In contrast, micepretreated with LGG began to die at 10 days after irradiation andsome survived to day 14. Death after day 10 is described as mostattributable to the combined effects of GI toxicity and bonemarrow failure. Histological examination at day 7, when thegreatest separation in weight curves was observed, revealed

a larger number of villi in LGG-pretreated mice than in irradiatedcontrols (gure 3C,D).

Table 1 Crypt survival after pretreatment withprobiotics

Treatment Crypt survival p Value

PBS control 1.0060.15

LGG (cultured/washed) 1.9860.23


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LGG treatment and small intestinal microbial compositionSince change in commensal bacteria composition is a potentialmechanism by which probiotics exert their effect, we examinedthe impact of LGG gavage on small intestinal microbialcomposition using a genomic-based approach. As the biological

end point of improved epithelial crypt survival was identied inthe ileum, this intestinal segment was analysed using theluminal contents and mucosal scrapings of mice treated withPBS or LGG. No signicant changes were found in total bacteria(rpoB gene copy 2.86 (PBS treated) vs 2.84 (LGG treated)log10 ng/g intestinal sample, p>0.05), nor was a shift in bacte-rial family composition found (gure 4). In the LGG treatmentgroup, a detectable level of LGG was found in three of sevenmouse ileums at 12 h after the third daily gavage.

The increased crypt survival seen with the administration of LGGis dependent on TLR-2, MyD88 and COX-2 signallingHaving found that administration of LGG to WT mice results indiminished radiation-induced apoptosis and increased crypt

survival, we next sought to dene a signalling pathway for theseeffects. Mice lacking MyD88 signalling exhibited similar baselineapoptosis to WT mice, but modestly higher crypt survival.

Administration of LGG or LGG-CM to MyD88/ mice had noeffect on either crypt survival or apoptosis (gure 5A,B). Thissuggests that the radioprotective effects of LGG in WT mice aremediated through a MyD88-dependent mechanism. Lactobacilliare Gram-positive bacteria exhibiting a cell well containinglipoteichoic acid, but lacking agella or LPS. Thus we chose toinvestigate whether TLR-2 was involved in Lactobacillus-medi-ated radioprotection. In TLR-2/ mice, prophylactic LGGtreatment offered no reduction in epithelial cell positionalapoptosis or improvement in crypt survival, suggesting that the

radioprotective effects of LGG in WT mice are mediated through

P B S

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30p0.05) after LGG treatment. N7 per group. LGG wasdetected in 0% of PBS-treated mice and 43% of LGG-treated mice.Lachno-Rumino, LachnospiraceaeeRuminococcaceae; UDL, underdetection limit (1.38 pg DNA/ml).

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TLR-2 (gure 5C,D). As a negative control, we examined thespecicity of the TLR-2/MyD88 pathway by conrmingthe presence of LGG-mediated intestinal radioprotection inTLR-4/ mice (mean crypt survival/jejunal cross-section13.262.1 vs 22.663.1 for PBS vs LGG; p0.02). We suspectedthat TLR-4 signalling would not play a role in the radioprotec-tive effects of LGG because LGG is a Gram-positive organismand does not make LPS.

We have previously demonstrated that the radioprotectiveeffects of LPS are mediated through the induction of COX-2expression and the synthesis of PGE2.

10 We sought to determineif this pathway is involved in the radioprotective effects of LGG.

At baseline, radiation-induced apoptosis in mice with disruptedCOX-2 was modestly diminished compared with WT mice(gure 5E). The administration of LGG to COX-2/ miceresulted in a small but signicant further decrease in apoptosisonly at position 5 (from 28% to 22%), a reduction less strikingthan that seen in the WT mice. Baseline crypt survival afterradiation in COX-2/ mice was similar to that seen in WTmice, and administration of LGG to COX-2/ mice did notimprove crypt survival (gure 5F). These results suggest that theradioprotective effects of LGG on crypt survival are mediated atleast in part through COX-2.

Having found that signalling through COX-2 is important tothe radioprotective effects of LGG, we next sought to determineif administration of LGG resulted in an increase in COX-2expression. Administration of LGG by gavage did not result inincreases in intestinal COX-2 mRNA or intestinal PGE2 levels(gure 6A,B). We previously demonstrated that in DSS-inducedcolitis, COX-2-expressing MSCs in the distal colon migrate frompositions in the lamina propria that are adjacent to the upperparts of the crypts to positions in the lamina propria adjacent tothe more rapidly proliferating epithelial cells in the lowercrypts.4 In light of this observation, we sought to determine ifthe administration of LGG resulted in the migration of COX-2-

expressing cells in the small intestine. Since the effect of LGGwas prophylactic in preventing injury, we examined unirradiatedmice for this shift. The LGG dosing schedule used here was thesame as that used to demonstrate radioprotection. While therewas no difference in the number of COX-2-expressing stromalcells after LGG, there was a shift in cell localisation from the villito the crypt region (gure 6C,D). In PBS-gavaged mice, we found80% of COX-2-expressing cells in the lamina propria of the villi,with 20% residing in the lamina propria surrounding the crypts.

After LGG, however, 62% of the COX-2-expressing cells wereassociated with the villi, and 38% were in the crypt region(gure 6E). Thus we observed a highly signicant (p10 total/group over two ormore experiments). (C) TLR-2/ mice exhibit similar epithelial cryptapoptosis index with LGG treatment to PBS (n4/group). (D) Cryptsurvival is not improved by LGG in TLR-2/ mice (n9/group over twoseparate experiments). (E) COX-2/ mice were evaluated forapopotosis (n7/group) and (F) surviving crypts (n9/group). FSC,

forward scatter. *p#0.05 vs LGG.

(Continued)

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ndings support the feasible link between LGG, TLR-2 signallingand COX-2-dependent radioprotection.

We next sought to further characterise the identied COX-2-positive cells of the lamina propria. By immunouorescence,most of the COX-2-positive cells were CD44 positive and manywere positive for CD29 and CD106 (gure 7A). This pattern ofexpression is consistent with these COX-2-positive cells being

MSCs. We had previously demonstrated that colonic MSCs hadhigh levels of COX-2 expression.31 We now isolated small

intestinal MSCs and found that they also had high levels ofCOX-2 expression (gure 7B) and by ow cytometry werepositive for MSC markers CD29, CD44 and CD106, and nega-tive for cell surface markers CD45 (haematopoietic), F4/80(macrophage) and PECAM (endothelial) (gure 7C). qRT-PCRwas used to identify TLR expression on these MSCs, with TLRs2, 3, 4 and 5 showing the highest expression levels (gure 7D).No PCR product was observed on gel electrophoresis for murine

TLR-13. In vitro exposure of the MSCs to a TLR-2 ligandresulted in increased tumour necrosis factor a and COX-2mRNA, demonstrating functional relevance to the TLR-2expression (gure 7E).

DISCUSSIONAdministration of probioticLactobacillus spp. before irradiationresults in decreased epithelial apoptosis and increased cryptsurvival in the mouse small intestine. In this study, the greatestprotection was offered by administration of LGG, althoughgavage with other Lactobacillus spp. or LGG-CM also showedsignicant benet. Administration of aBidobacteriumsp. was notradioprotective in this model. The radioprotective effects mediatedby LGG required intact signalling of TLR-2, MyD88 and COX-2.LGG was protective when given before, but not after, radiation,suggesting that LGG acts by preventing radiation-induced injuryrather than enhancing repair.

Peptidoglycan and lipoteichoic acid, components of the cellwall of Gram-positive bacteria including LGG, are TLR-2 ligands.The nding that LGG-CM is also radioprotective suggests thatLGG peptidoglycan or lipoteichoic acid is released into themedium and is capable of binding TLR-2. This is the rstdemonstration that a probiotic-based biological effect is medi-ated through TLR-2. The only previous demonstration of thebenecial effects of a probiotic being mediated through TLRsignalling was the report that anti-inammatory effects of

VSL#3, a mixture of different bacteria, are mediated through

TLR-9.39

Heat shock proteins, reactive oxygen species and AKTactivation have all been implicated in mediating the biologicaleffects of LGG.18 30 40 Whether TLR-2 activation is also involvedin mediating these effects has not been addressed. The positionalcurves for radiation-induced apoptosis were different in theTLR-2/ and MyD88/ mice, raising the possibility that otherTLRs may also play a role in the host response to radiation.

Yan et al described two novel proteins secreted by LGG, p40and p75, that induce AKTactivation and inhibit tumour necrosisfactor-induced apoptosis in human and mouse epithelial celllines and in mouse colonic explants.17 30While it is possible thatthese proteins contribute to radioprotection, it is unlikely thatone or both of these proteins is solely responsible for thedecrease in radiation-induced apoptosis and increased crypt

survival induced by LGG. The radioprotective effects seen withLGG are mediated by MyD88 and COX-2. The role of TLRs andCOX-2 in mediating the effects of p40 and p75 has not beenaddressed. However, it was observed that the p40 and p75proteins were expressed and cytokine protection was exhibitedby both LGG and L casei, although not by L acidophilus. Incontrast, L acidophilus was effective at reducing radiation-induced epithelial injury in the present study. The explanationfor possible genus specicity for lactobacilli in radioprotection isnot known. While bidobacteria are also Gram-positive bacteria,the species we tested was not radioprotective. LGG-CM isprotective and the radioprotective effects of LGG are TLR-2dependent. This suggests that LGG may release a TLR-2 ligand

into the medium. The failure of bi

dobacterium, another Gram-positive bacterium, may be related to the failure to release

BA

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a TLR-2 ligand, adherence properties, differential positioning ofthe bacterium with respect to the intestinal epithelium and themucous layer, or differential signalling by its lipoteichoic acid-based outer membrane.41e43 The nding of differential efcacyamong specic probiotic species and strains has been reportedfor other conditions.44 Dosage, potency and/or host handling areother possible explanations for the differences in efcacy.

Radioprotective effects of LGG are mediated through COX-2.

PGE2, which can be produced through either COX-1 or COX-2,is radioprotective in the intestine.10 The radioprotective effectsof LPS, acting through TLR-4, are dependent on COX-2 andPGE2.

10 Parenteral administration of LPS induces increased COX-2 expression in the intestine. Although LGG also acts through aCOX-2-dependent mechanism, gavage with LGG affectedneither the expression of COX-2 nor the level of PGE2 in thesmall intestine. LGG did not affect the total number of COX-2-expressing cells in the intestine; however, in response to LGG,the number of COX-2-expressing cells associated with the cryptsincreased.

In the normal colon, there is a population of cells in thelamina propria near the upper crypts that constitutively expressCOX-2.4 These cells also express CD44, CD29, CD105 andCD106, a pattern consistent with MSCs.31 In DSS-inducedcolitis, there are COX-2-dependent protective effects thatpreserve epithelial proliferation, even though DSS-colitis is notassociated with increased COX-2 expression or increased PGE2production. In the DSS model, COX-2-expressing MSCsmigrated from near the upper crypt epithelium to near the lowercrypt epithelial cells. This process is MyD88 dependent. Ourinterpretation of these ndings was that the migration of COX-2-expressing cells allowed a high concentration of PGE2 in thearea immediately surrounding the proliferating crypt cells andthat PGE2 supported epithelial cell proliferation. In the presentstudy, we observed a similar pattern now in the small intestine.The small intestinal MSCs that constitutively express high

levels of COX-2 migrated in response to LGG in a TLR-2-dependent fashion. The migration of these COX-2-positive cellswas associated with diminished epithelial apoptosis andincreased crypt survival in response to radiation injury.

These migrating cells express COX-2 and surface proteinsassociated with MSCs. The constitutive expression of high levelsof COX-2 is specic to intestinal and colonic MSCs, as bonemarrow-derived MSCs expressed little COX-2 (gure 6B). Thehigh level of COX-2 expression in colonic MSCs is not TLRdependent, but dependent on broblast growth factor 9 (FGF9)produced by epithelial cells.31 In the present study, the migrationof these intestinal MSCs was found to be TLR-2 specic. HowTLR-2 signalling is involved in their migration is not clear. It ispossible that the TLR-2 signalling occurs in the MSCs, as these

cells functionally express the gene; however, it also possible thatthe TLR-2 ligand binds to a cell in or near the crypt, and this cellproduces a chemotactic factor that promotes the migration ofthe MSCs towards the crypt. PGE2 produced by these COX-2-expressing MSCs would act to protect adjacent epithelial stem

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Figure 7 Characterization of small intestinal cyclo-oxygenase 2 (COX-2)-positive cells. (A) Immunofluoresence co-staining of jejunal COX-2-

expressing cells and mesenchymal stem cell (MSC) surface markers. (B)

COX-2 quantitative reverse transcription-PCR of small intestinal (SI) andcolonic (C) MSCs relative to bone marrow-derived (BM) MSCs. (C) Flowcytometry for cell surface markers of isolated COX-2-expressing MSCs.Gating based on isotype control. (D) mRNA expression of murine TLRligands relative to actin (log10) in isolated SI-MSCs. (E) Tumour necrosisfactor (TNF)a and COX-2 mRNA expression in MSCs after stimulationwith a TLR-2 ligand (Pam3CSK4) for 18 h. FSC, forward scatter

*p


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cells from radiation-induced apoptosis. The close physicalproximity of the PGE2-producing MSCs and the epithelial stemcells is important because PGE2 is unstable and therefore onlyacts over short distances. We have now demonstrated TLR-dependent migration of COX-2-positive MSCs as being involvedin DSS-colitis and radioprotective in the small intestine. Thisraises the possibility that TLR-dependent migration of COX-2-expressing MSCs may be a generalised mechanism for epithelial

protection in the intestine and colon.These studies were carried out using 12 Gy of total body

irradiation, with epithelial apoptosis, crypt survival, weight lossand duration of survival used as biological end points. This is thestandard protocol for intestinal radioprotection studies in themouse. As no animal model exactly mirrors the human disease,certain distinctions should be noted. Humans undergoing radi-ation therapy for abdominal malignancy receive lower, frac-tionated doses of radiation to the intestine. The major clinicaleffects in humansddiarrhoea and vomitingdtypically developduring the second week of radiotherapy (cumulative radiationdose of 15e20 Gy).3 Mice do not typically develop diarrhoea orvomiting in response to radiation but exhibit weight loss and, ifthe enterocyte depletion is high, death over the rst 10 days.Nonetheless, this study has relevance for radioprotection inpatients in that radiation-induced diarrhoea is a product ofradiation-induced epithelial apoptosis in the intestine, and otheragents that reduce radiation-induced apoptosis are also effectiveradioprotective agents.1 1 4 5 Furthermore, agents demonstratedto be radioprotective in mice using this radiation protocol arealso radioprotective in primates.11

This study raises the possibility of using probiotics asradioprotective agents in man and provides insight into theadministrative route, timing and mechanism of probiotic-mediated intestinal radioprotection. As radioprotectants,probiotics have the advantage of providing wide luminalcoverage and act locally at the mucosal surface to protect the

normal epithelium without having a systemic distribution.Although probiotics such as LGG46 have an excellent humansafety prole, if viable bacteria were used in human trials, closemonitoring would be critical. Further studies directed atdelineating mechanisms and optimising the radioprotectiveeffects of probiotics are indicated.

FundingThis work was supported in part by the National Institutes of Health grantsDK55753 and DK33165 (to WFS), DK089016 and L30 RR030244 (to MAC), DK071619and DK07161-90251 (to TSS) and P30-DK52574 (to the Washington UniversityDigestive Diseases Research Core). This work was also supported in part by theCrohns and Colitis Foundation of America and a Global Probiotics Council YoungInvestigator Award (both to MAC). We thank Lynne Foster and Ellen Bettonville foradditional technical assistance, and Dr Parag Parikh for useful discussions on thiswork.

Competing interests None.

ContributorsStudy concept and design: MAC, WFS. Acquisition of data: MAC, MSR,TER, XE, CM, MSW, JMM, GMN. Analysis and interpretation of data: MAC, WFS,TER, CM, TSS, XE, GMN. Drafting of the manuscript: MAC, WFS. Critical revision ofthe manuscript for important intellectual content: TSS, TER, MSR, MSW, CM, XE.Statistical analysis: MAC. Obtained funding: MAC, WFS, TSS. Approved final versionof manuscript: all authors.

Provenance and peer reviewNot commissioned; externally peer reviewed.

Data sharing statementWe are happy to share our data. All the reagents used arecommercially available.

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Matthew A Ciorba, Terrence E Riehl, M Suprada Rao, et al.mannerTLR-2/cyclo-oxygenase-2-dependentepithelium from radiation injury in aLactobacillus probiotic protects intestinal

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Lactobacillus Probiotic Protects Intestinal Epithelium