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Molecular Immunology 48 (2011) 1505– 1511

Contents lists available at ScienceDirect

Molecular Immunology

jo u rn al hom epa ge: www.elsev ier .com/ locate /mol imm

he response to TLR ligation of human CD16+CD14− monocytes is weaklyodulated as a consequence of persistent infection with the hepatitis C virus

heng Penga,b,1, Bi-Sheng Liua,1, Robert J. de Knegta, Harry L.A. Janssena, André Boonstraa,∗

Department of Gastroenterology and Hepatology, Erasmus MC, University Medical Center, Rotterdam, The NetherlandsDepartment of Infectious Diseases, Xiehe Hospital, Tongji Medical College, Huazhong Science and Technology University, Wuhan, China

r t i c l e i n f o

rticle history:eceived 28 February 2011eceived in revised form 6 April 2011ccepted 9 April 2011vailable online 30 April 2011

a b s t r a c t

Little is known about the frequency and function of CD16+CD14− monocytes from chronic HCV patients.We observed that the absolute numbers and ratio of CD16+CD14− to CD14+CD16− monocytes were sim-ilar between chronic HCV patients and healthy individuals. Functionally, we found that CD16+CD14−

monocytes are more responsive to TLR8-ligation and only weakly responsive to LPS stimulation in pro-ducing TNF as compared to CD14+CD16− monocytes. We found no overt impairment of the function

+ −

eywords:onocyte subset

LRiral infectionepatitis C virus

of CD16 CD14 monocytes from patients, except for an augmented induction of MIP-1�-producingCD16+CD14− monocytes upon TLR4-ligation. However, the increased frequency of MIP-1�-producingCD16+CD14− monocytes was not associated with viral load, ALT or fibrosis level. Our findings indicatethat, different from other infectious diseases, the frequency and function of CD16+CD14− monocytesare only minimally altered as a consequence of the persistent state of HCV infections, and our findings

a role + −

therefore do not suggest

. Introduction

The hepatitis C virus (HCV) is a major cause of chronic liverisease that can result in cirrhosis of the liver and hepatocellu-

ar carcinoma. In the majority of infected individuals, the immuneesponse against HCV is insufficient to eradicate the virus. A weakCV-specific T cell response is generally observed in patientshronically infected HCV. In addition, numerical or functionalmpairment of dendritic cells (DC), natural killer (NK) cells, reg-latory T cells have been reported in patients with chronic HCVClaassen et al., 2010; Dustin and Rice, 2007; Liu et al., 2009;

anigold and Racanelli, 2007). Although monocytes are importantells in initiating and maintaining immune responses, and comprisebout 10% of circulating leukocytes, relatively little is known on theffect of chronic HCV infection on the functionality of monocytes.

Human blood contains two distinct subpopulations of cir-ulating monocytes, which can be distinguished on the basisf membrane expression of CD16 and CD14: CD16+CD14− and

D14+CD16− monocytes (Auffray et al., 2009; Grage-Griebenowt al., 2001; Passlick et al., 1989). Compared to CD14+CD16−

onocytes, CD16+CD14− monocytes are less frequent and

∗ Corresponding author at: Department of Gastroenterology and Hepatology,rasmus MC, University Medical Center Rotterdam, ‘s-Gravendijkwal 230, Room L-55, 3015 CE Rotterdam, The Netherlands. Tel.: +31 107035944; fax: +31 107032793.

E-mail address: p.a.boonstra@erasmusmc.nl (A. Boonstra).1 These authors contributed equally to this paper.

161-5890/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.molimm.2011.04.008

for CD16 CD14 monocytes in HCV pathogenesis.© 2011 Elsevier Ltd. All rights reserved.

comprise about 5–15% of the total monocytes. Furthermore,CD16+CD14− monocytes are thought to produce higher levels ofpro-inflammatory cytokines, such as TNF and IL-1� in responseto TLR stimulation (Belge et al., 2002), and lower levels ofanti-inflammatory cytokines, such as IL-10 (Belge et al., 2002;Frankenberger et al., 1996). Recently, it was demonstrated thatCD16+CD14− monocytes are able to sense viruses via TLR7 or TLR8,resulting in the initiation of a pro-inflammatory response (Croset al., 2010). Several studies have reported that patients with vari-ous infections have increased numbers of CD16+CD14− monocytesin blood (Capsoni et al., 1995; Emminger et al., 2001; Nockher et al.,1994; Nockher and Scherberich, 1998; Skrzeczynska et al., 2002).Also, in a heterogeneous patient cohort of individuals with chronicliver disease (as a result of autoimmunity, alcohol toxicity, viralinfections or unspecified etiology) increased numbers of circulat-ing CD16+CD14− monocytes were observed (Zimmermann et al.,2010). The importance of CD16+CD14− monocytes in the pathologyof liver diseases was suggested since increased numbers of CD16+

cells were observed in the liver at areas of inflammation (Aspinallet al., 2010; Zimmermann et al., 2010).

Previously, we showed that CD14+CD16− monocytes fromchronic HCV patients produce lower level of TNF and IL-12p40as compare to healthy individuals upon TLR4 ligation and aug-mented production of TNF, IL-12p40 and IL-12p70 was observed

upon stimulation via TLR8 (Liu et al., 2011). Since at presentno information is available whether the function of CD16+CD14−

monocytes is affected as a consequence of chronic infection withHCV, we examined in detail the frequency and function of cir-

1 munology 48 (2011) 1505– 1511

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Fig. 1. The numbers and composition of the circulating monocyte compartmentwere not affected by chronic HCV infection. (A) The absolute number of leukocytesand monocytes in peripheral blood of chronic HCV patients and healthy individuals

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506 C. Peng et al. / Molecular Im

ulating CD16+CD14− monocytes in patients with chronic HCVnfections.

. Materials and methods

.1. Patients

Peripheral blood was collected from patients with chronic HCVnfection visiting our outpatient clinic and from healthy individuals.atients co-infected with human immunodeficiency virus, hepati-is A virus, hepatitis B virus or hepatitis D viruses were excludedrom the study. All characteristics of HCV patients used for theunctional assays are presented in Table 1. In addition, blood from9 healthy individuals was examined in this study (age = 33 ± 8;ale/female: 11/8). For the phenotypical analysis, 23 healthy indi-

iduals (average age 49.3; male/female: 14/9) and 39 chronic HCVatients (average age 49.7; male/female: 32/7) were evaluated. The

nstitutional review board of the Erasmus MC approved the proto-ols, and informed consent was obtained from all individuals.

.2. Enumeration of monocytes and leukocytes in whole blood

Absolute numbers of monocytes and leukocytes in whole bloodere determined by an automated impedance hematology ana-

yzer (ABX Micros-60, Horiba Medical). To determine the frequencyf distinct monocyte subpopulations, whole blood was stainedith antibodies against CD14-PE-Cy7 (61D3, eBioscience) andD16-PerCP-Cy5.5 (3G8, BD Biosciences), and evaluated by flowytometry (Canto-II, BD). The data was analyzed using BD FACSiva software.

.3. Stimulation of CD16+CD14− and CD14+CD16− monocytes

PBMC of healthy volunteers or patients with chronic HCVnfections were isolated from fresh blood by Ficoll-Paque gradi-nt centrifugation. PBMC were incubated with CD3-PE (UCHT1,iolegend) and CD3+ cells were magnetically depleted with anti-E microbeads (Miltenyi Biotec) following the manufacturer’snstructions. The CD3-depleted PBMC were further incubated withntibodies against CD14-Pacific Blue (M5E2, BD Pharmingen),D16-PerCP-Cy5.5 (3G8, BD Biosciences) and CD56-APC (N901,eckman). The purity of sorted CD16+CD14− and CD14+CD16−

onocytes was 98.3 ± 0.6%, 97.2 ± 2.4%, respectively. CD16+CD14−

nd CD14+CD16− monocytes sorted from both HCV patients andealthy individuals were cultured with serum-free X-VIVO15edium (BioWhittaker) at 2.5 × 105 cells/ml in 96-well flat bot-

om plates (200 �l/well) for 24 h stimulated with ultra pure LPS S.innesota (100 ng/ml, TLR4 agonist) or R848 (1 �g/ml, TLR7/8 ago-ist; Alexis). In some experiments, CD16+CD14− and CD14+CD16−

onocytes sorted from healthy individuals were stimulated with

am3CSK4 (100 ng/ml, TLR2 agonist), polyIC (25 �g/ml, TLR3/Mda-

agonist), flagellin (50 ng/ml, TLR5 agonist), CL264 (2.5 �g/ml,LR7 agonist) and CpG-2216 (5 �g/ml, TLR9 agonist, type A; Coleyharma). All TLR agonists used for stimulations were from Invivo-

able 1haracteristics of chronic HCV patients.

Chronic HCV patients

Age (years) Gender ALT

48 (32–61)a Male = 11 125 (10–375)a

Female = 8

a The range is shown in brackets.

is shown. (B) Monocytes were identified on the basis of their FSC/SSC profile, and fur-ther characterized by flow cytometry using CD14 and CD16 specific antibodies. Thecontribution of the specific subpopulation within the total monocyte pool is shown.

Gen, unless indicated otherwise. The levels of TNF in supernatantwere determined using sandwich ELISA (eBioscience) according tothe manufacturer’s instructions. The detection limit for TNF was15 pg/ml.

2.4. Intracellular detection of cytokine production

PBMC from HCV infected patients or healthy individuals werestimulated with ultra pure LPS S. minnesota (100 ng/ml; Invivo-Gen) or R848 (1 �g/ml; Alexis) in serum-free X-VIVO15 medium

(BioWhittaker) for 5 h, with brefeldin-A (10 �g/ml; Sigma) presentfor the last 3 h. Samples were fixed with 2% formaldehyde,permeabilized with 0.5% saponin, and stained with antibodiesagainst CD14-Pacific Blue (M5E2, BD Phamingen), HLA-DR-

Genotype Viral loads (IU/ml) Fibrosis

Genotype 1: 15 2.0 × 106 F0: 3Genotype 3: 3 (3.3 × 104–7.0 × 106)a F1: 1Genotype 4: 1 F2: 5

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erCP-Cy5.5 (LN3, ebioscience), TNF-PE-Cy7 (MAb11, eBioscience),D3-AmCyan (SK7, BD Biosciences), CD19-APC-H7 (SJ25C1, BD Bio-ciences), IL-8-FITC (6217, R&D), MCP-1-APC (5D3-F7, eBioscience)nd MIP-1�-PE (D21-1351, BD Pharmingen). The frequency ofytokine producing cells was determined by flow cytometryCanto-II, BD).

.5. Statistics

Continuous variables were represented as mean ± standardeviation, unless indicated otherwise. Mann–Whitney t-test wassed to compare variables between two independent groups. In allnalyses, a two-tailed p-value of less than 0.05 (confidence internal5%) was considered statistically significant. GraphPad Prism V5.0GraphPad Software Inc, San Diego, CA, USA) was used to performll analyses.

. Results

.1. The frequency of total monocytes and the ratio ofD16+CD14− monocytes in total monocytes were not altered inhronic HCV patients

We first examined whether the contribution of monocytes ineripheral blood of chronic HCV patients differs from healthy

ndividuals. We observed that the absolute numbers of periph-ral leukocytes and monocytes were similar between patients and

ealthy individuals (Fig. 1A). Within the monocyte compartmentFig. 1B), no differences were observed in the ratio of CD16+CD14−,D14+CD16− and CD16+CD14+ subpopulations when comparinglood from chronic HCV patients with healthy individuals.

ig. 2. CD16+CD14− and CD14+CD16− monocytes sorted from healthy individuals differere first depleted from PBMC of healthy individuals, followed by identification of tot

ntibodies against CD14 and CD16. (B) Sorted monocytes were stimulated with various Tepresentative data of 2 independent experiments. (C) CD16+CD14− and CD14+CD16− mnd TNF production was determined.

logy 48 (2011) 1505– 1511 1507

3.2. In contrast to CD14+CD16− monocytes, CD16+CD14−

monocytes isolated from healthy individuals highly respond toR848 but only weakly respond to LPS in producing TNF

Having demonstrated that the number of CD16+CD14− mono-cytes was not affected in chronic HCV patients, we examined iffunctional differences exist between patients and healthy individ-uals.

CD16+CD14− and CD14+CD16− monocytes were sorted by flowcytometry from PBMC on the basis of FSC/SSC profile, and exclu-sion of CD3+ and CD56+ cells (Fig. 2A). Morphological evaluationof CD16+CD14− and CD14+CD16− monocytes after cell sortingdemonstrated that both subpopulations showed a typical mono-cytic morphology (data not shown). Functionally, however, theTLR7/8 agonist R848 induced about 8–10 times higher levels ofTNF by CD16+CD14− monocytes than by CD14+CD16− mono-cytes, whereas LPS-challenged CD16+CD14− monocytes producedaround 8–10 times lower levels of TNF when compared withCD14+CD16− monocytes in response to LPS (Fig. 2B and C).

Both CD16+CD14− and CD14+CD16− monocytes did not respondto the TLR7 agonist CL264 (Fig. 2B), indicating that in human mono-cytes R848 triggers TLR8, but not TLR7. At the concentrations tested,Pam3CSK4 (TLR2 agonist), polyIC (Mda5/TLR3 agonist), flagellin(TLR5 agonist) and CpG (TLR9 agonist) induced relatively low orundetectable cytokines by both CD16+CD14− and CD14+CD16−

monocytes (Fig. 2B). Together, CD16+CD14− and CD14+CD16−

monocytes differ in their response to TLR4 and TLR8 stimulation.

3.3. TLR-induced TNF production by CD16+CD14− monocytesfrom chronic HCV patients is not affected as compared to healthy

individuals

Previously, we showed that the function of CD14+ monocytesfrom chronic HCV patients was modulated in response to TLR stim-

in their response to LPS and R848 stimulation in producing TNF. (A) CD3+ T cellsal monocytes on the basis of their FSC/SSC profile. Monocytes were sorted usingLR agonists for 24 h and TNF production was evaluated. The values depicted showonocytes obtained from 14 healthy individuals were stimulated with LPS or R848

1 munology 48 (2011) 1505– 1511

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Fig. 3. TLR-induced TNF production is comparable between CD16+CD14− mono-cytes from chronic HCV patients and from healthy individuals. CD16+CD14− andCD14+CD16− monocytes purified from chronic HCV patients (n = 4) and healthy indi-

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508 C. Peng et al. / Molecular Im

lation as compared to healthy individuals. Yet, the response ofD16+CD14− monocytes from chronic HCV patients to TLR ago-ists is still not clear. To examine this we stimulated CD16+CD14−

onocytes from PBMC of chronic HCV patients and healthy indi-iduals. As shown in Fig. 3, no differences were observed in theevels of TNF produced by LPS- or R848-challenged CD16+CD14−

onocytes from chronic HCV patients versus healthy individuals.

.4. The percentage of MIP-1ˇ producing CD16+CD14−

onocytes induced by LPS is increased in chronic HCV patients asompared to healthy individuals

Next, we determined TLR-induced cytokine responses byD16+CD14− monocytes in a larger cohort of chronic patientsersus healthy individuals. For this, flow cytometric analysis ofytokine producing cells is preferred due to the low percent-ges of CD16+CD14− monocytes in peripheral blood. Antibodiesgainst HLA-DR and CD14 were used to identify CD16+CD14− andD14+CD16− monocytes, since the CD16 molecule on monocytes

s down-regulated upon stimulation (Supplementary Fig. S1). Ashown in Figs. 4 and 5, CD16+CD14− monocytes from healthy indi-iduals cultured without specific stimuli have lower percentages ofpontaneous MCP-1- and MIP-1�-producing cells as compared toD14+CD16− monocytes from healthy individuals. Without stim-lation, both monocyte subpopulations from healthy individualsxhibit a low percentage of TNF-producing cells (Figs. 4 and 5).omparison of CD16+CD14− monocytes from chronic HCV patientsnd healthy individuals showed similar percentages of cells pro-ucing TNF, MCP-1, or MIP-1� in the absence of stimuli (Fig. 5).

As expected, stimulation with LPS or R848 led to augmentedercentages of TNF-, MCP-1- or MIP-1�-producing CD16+CD14−

nd CD14+CD16− monocytes obtained from patients and healthyndividuals (Figs. 4 and 5). Upon LPS or R848 stimulation ofBMC from healthy individuals we observed that CD16+CD14− andD14+CD16− monocytes did not differ in the frequency of cells

ig. 4. The intracellular cytokine profiles by PBMC from healthy individuals. PBMC were sas determined by flow cytometry. Representative dot plots show CD16+CD14− and Croducing TNF and MIP-1� (left), or TNF and MCP-1 (right).

viduals (n = 5) were stimulated with medium, LPS or R848 for 24 h. TNF productionwas measured by ELISA.

producing TNF. However, the percentages of TLR-induced MCP-1- and MIP-1�-producing monocytes are significantly lower inCD16+CD14− monocytes as compared to CD14+CD16− populations(Fig. 5), which is in line with previous reports (Ancuta et al., 2003).

Compared to healthy individuals, TLR-challenged CD16+CD14−

monocytes from chronic HCV patients show a similar frequencyof cells producing TNF or MCP-1 (Fig. 5). However, the number ofLPS-induced MIP-1�-producing CD16+CD14− monocytes is higherin PBMC from chronic HCV patients; with an average of 70% inhealthy CD16+CD14− monocytes and 84% in CD16+CD14− mono-cytes from chronic HCV patients (Fig. 5). Importantly, no differenceswere observed in the level of LPS-induced MIP-1� production by

+ −

sorted CD16 CD14 monocytes from chronic HCV patients andhealthy individuals (data not shown), suggesting that on a per-cell basis the MIP-1� levels are reduced, whereas their frequencyis increased. Upon LPS stimulation, increased percentages of MIP-

timulated with medium, LPS or R848 for 5 h, and intracellular cytokine productionD14+CD16− monocytes (identified on the basis of HLA-DR and CD14 expression)

C. Peng et al. / Molecular Immunology 48 (2011) 1505– 1511 1509

Fig. 5. The frequency of MIP-1�-producing CD16+CD14− monocytes upon TLR4 stimulation is enhanced in chronic HCV patients as compared to healthy individuals. Thef resenH

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requencies of the monocyte subpopulations producing TNF, MCP-1 or MIP-1� are pCV patients and 14 healthy individuals.

�-producing cells are only observed for CD16+CD14− monocytesrom chronic HCV patients, but not for CD14+CD16− monocytesFig. 5). Also, there is no difference in the percentages of MIP-1�-roducing CD14+CD16− monocytes in response to R848 betweenhronic HCV patients and healthy individuals (Fig. 5), indicat-ng that the modulation of MIP-1� by CD16+CD14− monocytesrom chronic HCV patients is TLR4 pathway dependent. The ele-ated MIP-1� producing CD16+CD14− monocytes from chronicCV patients in response to LPS were not associated with age, viral

oad, ALT or fibrosis level of chronic HCV patients studied (dataot shown). Interestingly, TLR4-induced TNF-, MCP-1- and IL-8-roducing CD16+CD14− monocytes were not altered in chronicCV patients when compared with healthy individuals (Fig. 5 andata not shown). Together, our findings show CD16+CD14− mono-ytes from chronic HCV patients are affected in their ability tonduce MIP-1� upon TLR4 ligation, whereas the production of MCP-

and TNF is not affected. The mild but specific changes in theunctionality of CD16+CD14− monocytes from chronic HCV patients

ay play a role in HCV disease or pathogenesis.

. Discussion

In this study, we evaluated the responses of CD16+CD14−

onocytes to several TLR agonists as well as the function of

ted upon stimulation with LPS or R848. The data shows the results from 17 chronic

CD16+CD14− monocytes from chronic HCV patients. We reporthere that CD16+CD14− monocytes isolated from healthy individu-als are more responsive to TLR8 ligation by their production of TNFas compared to CD14+CD16− monocytes. In contrast, CD16+CD14−

monocytes are less responsive to TLR4 ligation than CD14+CD16−

monocytes. Comparison of chronic HCV patients and healthy indi-viduals showed that the absolute numbers of monocytes and theratio of CD16+CD14− cells to other monocyte populations in periph-eral blood were similar. A detailed analysis of the functionalityof CD16+CD14− monocytes in blood from chronic HCV patientsshowed no overt modulation as compared to healthy individ-uals, except for an augmented induction of MIP-1� producingCD16+CD14− monocytes upon TLR4 ligation in monocytes fromchronic HCV patients as compared to healthy individuals.

In our patient cohort, we observed no differences betweenthe absolute numbers of monocytes in peripheral blood fromchronic HCV patients and healthy individuals. Also, the relativecontribution of CD16+CD14− and CD14+CD16− monocytes in thecirculation was similar in chronic HCV patients as compared withhealthy individuals in our cohort. Our observations are in line

with a recent study in chronic HCV patients in which the ratioof CD16+CD14− and CD14+CD16− monocytes was comparablewith healthy subjects (Martin-Blondel et al., 2009). Although thecomposition of monocyte subpopulations does not change in as a

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onsequence of infection with HCV, CD16+CD14− monocytes haveeen reported to be recruited to diseased human liver mediatedia vascular adhesion protein-1 and CX3CL1 (Aspinall et al., 2010).he preferential recruitment of the CD16+ monocyte populationay induce differentiation into dendritic cells or Kupffer cells in

he liver, thereby leading to augmented intrahepatic inflammation.owever, in this study no livers from chronic HCV patients werexamined for infiltration of monocytes, and we now show thathe frequency of CD16+CD14− monocytes in blood of chronic HCVatients is not affected.

Although the frequency of CD16+CD14− monocytes is notffected as a consequence of chronic HCV infection, the functionf this populated may be altered. In this study, we first examinehe function of CD16+CD14− monocytes isolated from healthyubjects. We report here that CD16+CD14− monocytes stimulatedith the TLR7/8 agonist R848 produce significantly higher levels

f TNF as compared to CD14+CD16− monocytes from healthyndividuals. It has been reported that TLR8 mRNA expressionn CD16+CD14− and CD14+CD16− monocytes is similar (Ancutat al., 2009; Cros et al., 2010), which makes it plausible that theumber of R848-responsive monocytes is comparable betweenD16+CD14− and CD14+CD16− monocytes, This is confirmed byur findings that the percentages of TNF-producing CD16+CD14−

nd CD14+CD16− monocytes are similar (Fig. 5B). The fact thatD16+CD14− monocytes produced higher levels of TNF thanD14+CD16− monocytes on a per-cell basis suggests that theignaling pathways downstream of TLR8 may differ in these twoonocyte subpopulations. Indeed, in line with our findings, itas recently found that signaling cascades differ between bothonocyte populations (Cros et al., 2010).Using intracellular cytokine staining, we observed that the

requency of MIP-1� producing CD16+CD14− monocytes wasignificantly increased upon TLR4 ligation in chronic HCV patients.IP-1� is an important chemokine in the pathogenesis of some

nflammatory conditions and diseases (Bystry et al., 2001). Forxample, MIP-1� is one of the predictors for the severity of dengueatients (Bozza et al., 2008) and induces inflammatory responsesgainst pathogens such as influenza or parasites (Menten et al.,002). However, we find that the increased percentage of MIP-� producing CD16+CD14− monocytes is not associated withge, viral load, and ALT and fibrosis level (data not shown).he immunomodulatory effects of MIP-1� are currently notompletely known. Interestingly, it has been suggested thatD14+CD16− monocytes from HCV patients spontaneously secreteigher levels of MIP-1�, and that MIP-1� modulates the differen-iation of monocyte-derived dendritic cells, resulting in reducedFN-gamma production by allogeneic T cells (Perrin-Cocon et al.,008). Moreover, A number of studies have demonstrated higherIP-1� levels in serum of chronic HCV patients as compared to

ealthy individuals, and importantly, MIP-1� levels are reducedpon therapy-induced viral load reduction in chronic HCV patients.dditionally, liver tissue obtained from chronic HCV patients alsoxhibit enhanced MIP-1� mRNA expression as compared to controliver tissue (Larrubia et al., 2008).

In summary, our findings show that CD16+CD14− monocytesre highly responsive to TLR8 ligation. We demonstrate that the fre-uency and function of CD16+CD14− monocytes are only minimallyltered as a consequence of the persistent state of HCV infections,nd our findings therefore do not suggest a role for CD16+CD14−

onocytes in HCV pathogenesis.

onflict of interest

The authors declare no financial or commercial conflict of inter-st.

logy 48 (2011) 1505– 1511

Acknowledgements

We would like to thank Heleen van Santen for collecting bloodsamples from chronic HCV patients visiting our outpatient clinic,and Bettina Hanssen for statistical analyses. Furthermore, we aregrateful to Jin Liu and Wenda Schoordijk for their excellent assis-tance in obtaining sorted monocytes.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.molimm.2011.04.008.

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