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. 2014 Feb 17;11(3):294–304. doi: 10.1038/cmi.2013.70

An unbalanced PD-L1/CD86 ratio in CD14++CD16+ monocytes is correlated with HCV viremia during chronic HCV infection

Jiajia Zheng 1,2,5, Hua Liang 3,5, Chunhui Xu 1, Qiang Xu 1, Ting Zhang 1, Tao Shen 1,4, Fengmin Lu 1,4
PMCID: PMC4085489  PMID: 24531620

Abstract

Circulating monocyte subsets with distinct functions play important roles in hepatitis C virus (HCV) infection. However, the mechanisms have not been well studied. In this study, we analyzed the distributions and phenotypic characteristics of three circulating monocyte subsets—CD14++CD16−, CD14++CD16+ and CD14+/dimCD16+—in chronic HCV-infected patients, HCV spontaneous resolvers and healthy controls, and we evaluated the possible link between HCV viremia and disease progression. Our results indicated that the frequency of the CD14++CD16+ monocyte subset was decreased, and negatively correlated with HCV RNA and core antigen levels during chronic HCV infection. PD-L1 expression and the PD-L1/CD86 ratio in CD14++CD16+ monocytes were higher during chronic HCV infection than in spontaneous HCV resolvers and healthy controls. The PD-L1/CD86 ratio positively correlated with HCV viral load and core antigen levels. Finally, PD-L1 was significantly increased, while cytokine secretions were dramatically decreased upon Toll-like receptor (TLR) ligand binding and HCV JFH-1stimulation. These findings indicates the compromised immune status of the CD14++CD16+ monocytes during chronic HCV infection and provides new insights into the specific role of the CD14++CD16+ monocytes and their significance in chronic HCV infection.

Keywords: core antigen, hepatitis C virus, monocytes, PD-L1/CD86, viral load

Introduction

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease, with an estimated 170 million infected individuals worldwide.1 It has been estimated that 85% of HCV-infected individuals experience persistent infection, and about 20% of the persistently infected individuals develop liver cirrhosis and hepatocellular carcinoma.2,3 It has also been reported that about 20% of HCV-infected individuals had cleared the virus and were classified as spontaneous HCV resolvers.4 The persistence of HCV infection indicates that the virus has evolved mechanisms to evade host immunity, including both innate and adaptive immunity, of which monocytes have received little attention.3,5,6,7 The characteristics and roles of the different monocyte subsets in chronic HCV infection and spontaneous HCV resolvers remain largely unknown.

Recently, three subsets of monocytes have been identified according to the surface expression of CD14 and CD16: classical monocytes (CD14++CD16−), intermediate monocytes (CD14++CD16+) and non-classical monocytes (CD14+/dimCD16+); each subset has different phenotype marker expression and cytokine secretion patterns.8,9,10 Notably, the latter two monocyte subsets are usually summarized as CD14+CD16+ monocytes and account for only ∼10% of the total monocytes. It has been reported that, upon virus stimulation, monocytes initiate the adaptive immune response and affect Th1/Th2 polarization by producing inflammatory and immune-modulatory cytokines, such as IL-10, IL-12, and TNF-α. Accordingly, the abnormal expression of these cytokines may impair the capacity of the antigen presenting cells to prime the naive T cells and, thus, contribute to an insufficient immune response to HCV infection.11,12

PD-L1 is expressed in a variety of innate immune cells, including dendritic cells, macrophages, and monocytes. Upon interaction with PD-1, the PD-1/PD-L1 pathway delivers a co-inhibitory signal to activate the T cells and results in the resistance of immune responses in pathogenic organism infection.13,14,15 We have previously reported that PD-L1 expression and the PD-L1/CD86 ratio of dendritic cells (DCs) were increased in HCV-infected patients, and this increase was associated with the impaired allostimulatory capacity of DCs.16 Monocytes are the precursor cells that give rise to mature macrophages and myeloid DCs.17 Whether monocytes exhibited similar trends of co-stimulatory/inhibitory marker expression during HCV infection as the DCs remains undefined.

In this study, we aimed to examine the proportions of the three monocyte subsets and their correlations with virological indicators in HCV persistent infection and HCV spontaneous resolvers. As intermediate CD14++CD16+ monocytes were found to be negatively associated with HCV viremia in chronic HCV infection, the expression of co-stimulatory (CD86,CD80 and HLA-DR) and co-inhibitory markers (PD-L1) were examined in this monocyte subset. Additionally, the PD-L1 and CD86 ratio was evaluated to investigate their contribution to HCV disease progression.

Materials and methods

Subjects

The recruited subjects were composed of three population groups, and the overall characteristics of these groups are described in Table 1. Group 1 was composed of 51 chronic HCV-infected individuals with positive plasma HCV RNA, HCV core antigen, and had a reactive anti-HCV response. Group 2 was composed of 35 HCV spontaneous resolvers who had a reactive anti-HCV response but were negative for HCV RNA and HCV core antigen. None of these anti-HCV-positive patients received any HCV-specific antiviral therapy. All participants were negative for HIV-1 and HBV infection. Subjects in groups 1 and 2 were recruited from Wangying Village, Shangcai County of Henan Province in Central China, and had participated in unsanitary commercial blood donations in the early 1990s. All virological indicators were detected using standard protocols in two cross-sectional studies performed in August 2005 and August 2009. Group 3 was composed of 30 healthy individuals who were recruited from the same village as groups 1 and 2; these individuals were negative for HBV, HCV and HIV-1. All blood samples were collected after the patient gave their informed consent. The HCV genotypes included in group 1 were 1b (62.7%) and 2a (37.3%). This study was approved by the ethics committee of Peking University, and informed consent was obtained in all cases.

Table 1. Demographic features of the individuals recruited for this study in 2009 (median (IQR1–IQR3)).

  HCV-infected patients (Group 1, n=51) HCV spontaneous resolvers (Group 2, n=35) Healthy controls (Group 3, n=30)
Age, median years (IQRa) 51 (41–58) 46 (39–54) 37 (34–42)
Deduced infection time, median years (IQR) 20 (19–21) 20 (18–22) NAb
Gender (M/F) 24/27 17/18 15/15
HCV genotype (1b/2a) 32/19 NA NA
HCV RNA, median log10 IU/ml (IQR) 6.1 (5.5–6.6) NA NA
HCV core antigen, median log10 fmol/l (IQR) 3.1 (2.6–3.7) NA NA
Anti-HCV, median S/CO ratio (IQR) 14.6 (14.0–15.5) 7.5 (5.7–11.4) NA
CD4+ T cell, median count/µl (IQR) 882.0 (731.5–1174.0) 878.0 (616.5–950.0) NA
CD8+ T cell, median count/µl (IQR) 540.0 (455.5–901.5) 755.0 (437.5–1059.5) NA
ALTc, median IU/l (IQR) 39.0 (27.5–57.5) 15.0 (12.0–21.5) NA
ASTd, median IU/l (IQR) 39.0 (33.5–51.5) 24.0 (21.0–28.5) NA
γ-GTe, median IU/l (IQR) 21.0 (17.0–37.5) 17.0 (13.0–21.5) NA
APRIf, median (IQR) 0.19 (0.10–0.37) 0.13 (0.08–0.34) NA
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a

IQR, interquartile range.

b

NA, not applicable.

c

ALT, alanine aminotransferase.

d

AST, aspartate aminotransferase.

e

γ-GT, γ-glutamyltransferase.

f

APRI: aspartate aminotransferase to platelet ratio index.

Detection of clinical, virological and biochemical parameters

The plasma HCV viral load was determined using the Abbott Real-Time HCV Amplification Kit (Abbott Molecular Inc., Des Plaines, IL, USA). Values greater than 1.48 log10 IU/ml were considered positive according to the manufacturer's instructions. HCV core antigen was measured using the Abbott ARCHITECT HCV Ag assay (Abbott Diagnostics, Abbott Park, IL, USA) with a detection limit of 3 fmol/l. Serum HCV antibodies were measured using the ARCHITECT Anti-HCV System (Abbott Diagnostics), with values greater than or equal to 1.0 S/CO considered reactive. The CD4+ T/CD8+ T-cell counts and the levels of liver associated enzymes were measured according to the clinical standard diagnostic criteria established by the local Center for Disease Control and Prevention. The HCV genotype was determined by amplification and sequence analysis of a 573-bp fragment of the core gene, as described in a previous study.7

Monocytes surface markers analysis

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized whole blood within 2 h of collection using the density gradient centrifugation technique with Ficoll-Hypaque (Sigma Chemical Co., St Louis, MO, USA). Then, 1×106 PBMCs were resuspended in RPMI-1640 medium containing 10% fetal bovine serum and were stained with antibodies against surface markers at room temperature for 30 min. The following monoclonal antibodies were used for monocyte phenotypic characterization: CD14-PE-Cy7, CD16-APC-Cy7, HLA-DR-Percp, CD80-FITC, CD86-APC and PD-L1-FITC (BD Biosciences, San Jose, CA, USA). After staining, the PBMCs were washed twice with FACS buffer and analyzed using a BD FACSAria multiple-flow cytometer (BD Biosciences). The monocyte subsets were gated on the forward/side scatter and the CD14/CD16 expression patterns. The data were analyzed using the FlowJo software (Tree Star, Inc., San Carlos, CA, USA), and the mean fluorescence intensity (MFI) was adopted for the expression of the surface markers.

HCV JFH-1 culture and quantification

Huh7.5 cells were infected with 1×105 IU/ml HCV JFH-1 stocks. The cells were passaged every 3 days at a ratio of 1∶3, and the cell culture supernatants were harvested and ultracentrifuged to collect the HCV JFH-1virus. The concentration of HCV JFH-1 was measured using the Abbott Real-Time HCV Amplification Kit (Abbott Molecular Inc.); the final concentration of the culture was 1×107 IU/ml.

Toll-like receptor (TLR) ligands and HCV JFH-1 stimulation

The CD14++CD16+ monocytes were sorted using a BD FACSAria (BD Biosciences). Then, 1×105 cells were resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum and were seeded in 96-well plates. The cells were stimulated with TLR ligands, including 5 µg/ml LPS (Invivogen, San Diego, CA, USA) and 10 µg/ml R848 (Invivogen) or with HCV JFH-1 at a multiplicity of infection of 10 for 20 h. The culture supernatants were collected for cytokine detection, and the cells were harvested for surface marker staining.

Cytokine detection

The levels of IL-1α, IL-1β, IL-12p70, TNF-α and IL-10 in the plasma or cultured supernatants were analyzed using ELISA kits according to the manufacturer's instructions (eBioscience, San Diego, CA, USA). The sensitivities of the ELISAs were 1.6 pg/ml for IL-1α, 0.32 pg/ml for IL-1β, 2.1 pg/ml for IL-12p70, 5 pg/ml for TNF-α and 1 pg/ml for IL-10.

Statistical analysis

All statistical analyses were performed using the GraphPad Prism V5.0 software (GraphPad Software Inc., San Diego, CA, USA). The data were expressed as the mean±s.d., except for the demographic features of the recruited individuals, which were recorded as the median and the interquartile range. Comparisons between groups were performed using the Mann–Whitney U test. Pearson's correlation test was used to evaluate the correlations among the plasma HCV viral load, HCV core antigen, the proportions of the different monocyte subsets, and the monocyte surface markers. P values <0.05 were considered statistically significant.

Results

The proportion of CD14++CD16+ monocytes was significantly decreased in patients with chronic HCV infection

According to the CD14 and CD16 expression patterns, the peripheral blood monocytes were classified into three types: classical monocytes (CD14++CD16−), intermediate monocytes (CD14++CD16+) and non-classical monocytes (CD14+/dimCD16+). The gating strategy for the monocyte subsets is shown in Figure 1a. When compared to the healthy controls, the CD14++CD16− classical monocytes were decreased in the HCV-infected patients (P=0.005) and resolved individuals (P=0.026), while the CD14+CD16+ monocytes were increased in the HCV-infected group (P=0.002) and HCV-resolved group (P=0.019) (Figure 1b c). The increased CD14+CD16+ subset was attributed to the CD14+/dimCD16+ subset, as the proportion of CD14+/dimCD16+ monocytes was significantly increased in the HCV-infected group (P<0.001) and HCV-resolved group (P=0.001) (Figure 1d). The CD14++CD16+ intermediate monocytes accounted for the smallest proportion of monocytes, and this subset was significantly decreased in the chronic HCV infection patients as compared to the HCV-resolved group (P=0.002) and healthy controls (P<0.001) (Figure 1e). We also detected monocyte-associated cytokine secretion in the plasma of the chronic HCV-infected subjects, HCV resolvers, and healthy controls. The concentrations of IL-1α, IL-1β, IL-12p70, TNF-α and IL-10 cytokines, as detected by ELISA, were increased in the chronic HCV-infected group and HCV-resolved group compared to the healthy controls (P<0.001, Figure 1f), indicating the enhanced systemic activation state during chronic HCV infection.

Figure 1.

Figure 1

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Proportions of the monocyte subsets in HCV-infected patients, HCV spontaneous resolvers and healthy controls. (a) Gating strategies of the CD14++CD16−, CD14++CD16+ and CD14+/dimCD16+ monocyte subsets according to the CD14/CD16 expression pattern. Representative plot figures for chronic HCV infection, spontaneous HCV resolvers, and healthy controls are shown. (b–e) The percentages of the CD14++CD16− (b), CD14+CD16+ (c), CD14+/dimCD16+ (d) and CD14++CD16+ (e) monocytes in the chronic HCV-infected patients (n=57), HCV-resolved individuals (n=35) and healthy controls (n=30). (f) Levels of serum IL-1β, IL-1α, IL-12p70, TNF-α and IL-10 in the chronic HCV-infected patients (n=57), HCV-resolved individuals (n=35) and healthy controls (n=30). HCV, hepatitis C virus.

Decreased CD14++CD16+ monocytes were negatively correlated with HCV viremia in chronic HCV infection patients

Because the distribution of all three monocyte subsets significantly changed during HCV infection, we analyzed the relationships between the monocyte subset distributions and the virological indicators of chronic HCV infection. The proportion of CD14++CD16+ monocytes was negatively correlated with the plasma HCV viral load (P=0.007, r=−0.372) and the HCV core antigen (P=0.025, r=−0.313) (Figure 2a) in the chronic HCV-infected group. There was no association between the proportions of either the CD14+/dimCD16+ or CD14++CD16− monocyte subsets and the virological indicators of chronic HCV infection (Figure 2b and c). These data suggested that the CD14++CD16+ monocytes were inversely correlated with HCV replication status in the chronic HCV infection patients.

Figure 2.

Figure 2

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The proportion of CD14++CD16+ monocytes in the HCV-infected patients was negatively correlated with plasma viral load and HCV core antigen. (a–c) Correlations between the proportions of the CD14++CD16+ (a), CD14++CD16− (b) and CD14+/dimCD16+ (c) monocyte subsets and plasma viral load (left panel) and HCV core antigen (right panel). HCV, hepatitis C virus.

Immunophenotypic characteristics of the CD14++CD16+ monocytes during HCV infection

Considering that the proportion of CD14++CD16+ monocytes was negatively correlated with HCV viremia, we next investigated the expression patterns of the monocyte surface co-stimulatory markers HLA-DR, CD86, CD80, and the co-inhibitory marker PD-L1 in this monocyte subset. The frequency of HLA-DR-positive CD14++CD16+ monocytes and the MFI of HLA-DR on the CD14++CD16+ monocytes were increased during chronic HCV infection (P=0.005 for frequency, and P=0.009 for MFI), and only the HLA-DR MFI showed an increased expression in spontaneous HCV resolvers (P<0.001) (Figure 3a). Chronic HCV-infected individuals had a higher HLA-DR frequency and MFI compared to the HCV resolvers (P=0.014 for frequency and P=0.013 for MFI) (Figure 3a). The frequency of CD86-positive cells and the MFI of CD86 increased in both the chronic HCV infected group and the HCV resolvers (P<0.001 for all) (Figure 3b). However, increased frequency of CD80-positive cells and the MFI of CD80 was observed in the HCV resolvers (P<0.001), while only the increased MFI was observed in the chronic HCV infection group (P=0.031) (Figure 3c). The expression of the co-inhibitory marker PD-L1 was increased in the chronic HCV-infected group (P<0.001 for both frequency or MFI) (Figure 3d). Interestingly, PD-L1 expression in HCV resolvers appeared to decrease when compared to the healthy controls (P<0.001 for frequency and P=0.05 for MFI), and significantly decreased expression was observed when compared to the chronic HCV-infected group (P<0.001 for both frequency and MFI) (Figure 3d), indicating that lower PD-L1 expression in HCV resolvers might contribute to virus clearance. As we found no association between the frequencies of classical or non-classical monocytes and HCV viremia, decreased frequencies of PD-L1-positive cells were found in the chronic HCV infection group for both the CD14+/dimCD16+ and CD14++CD16− monocyte subgroups, as well as for the CD14++CD16− monocytes in the spontaneous resolvers (Supplementary Figure 1).

Figure 3.

Figure 3

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Phenotypic profiles of the CD14++CD16+ monocytes in chronic HCV-infected patients, spontaneous HCV resolvers and healthy controls. (a–d) The frequency and mean fluorescent intensity of HLA-DR (a), CD86 (b), CD80 (c) and PD-L1 (d) in CD14++CD16+monocytes were analyzed. The black line represents the mean frequency or MFI value for each marker in the three different groups. HCV, hepatitis C virus.

PD-L1/CD86 ratio in CD14++CD16+ monocytes was closely correlated with HCV viremia

We previously reported that increased PD-L1 expression and the PD-L1/CD86 ratio in dendritic cells were associated with an impaired dendritic cell function in HCV infection.16 To determine whether the balance of co-stimulatory/co-inhibitory marker expression in the CD14++CD16+ monocytes was destroyed during HCV infection, we examined the changes in the PD-L1/CD86 and PD-L1/CD80 ratios in the CD14++CD16+ monocytes in the chronic HCV-infected group, spontaneous HCV-resolved group, and the healthy controls. Interestingly, we found that the PD-L1/CD86 ratio of the CD14++CD16+ monocytes was increased in the chronic HCV-infected individuals (P=0.007), while it was downregulated in the spontaneous HCV resolvers (P<0.001) compared to the healthy controls (Figure 4a). Similar changes were found for the PD-L1/CD80 ratio in the CD14++CD16+ monocytes from the chronic HCV-infected individuals and spontaneous HCV resolvers (P<0.0001 for both, Figure 4b). These results indicate that the CD14++CD16+ monocytes in the HCV resolvers retained a better immune status, as determined by the decreased PD-L1/CD86 and PD-L1/CD80 ratios. In contrast, the functional impairment of the CD14++CD16+ monocytes could be speculated in the chronic HCV-infected individuals, as the highest PD-L1/CD86 and PD-L1/CD80 ratios were observed in these cells. Additionally, the ratio of PD-L1/CD86 in the CD14++CD16+ monocytes was positively correlated with the level of HCV viral load (P=0.030, r=0.313) and core antigen (P=0.005, r=0.394) during chronic HCV infection (Figure 4c). No correlations were found between the PD-L1/CD80 ratio in the CD14++CD16+ monocytes and the virological indicators (Figure 4d). Overall, these results indicated that the PD-L1/CD86 ratio in the CD14++CD16+ monocytes was closely correlated with HCV viremia during chronic HCV infection and could be regarded as a hallmark of disease progression during HCV persistent infection.

Figure 4.

Figure 4

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The PD-L1/CD86 ratio in CD14++CD16+monocytes was closely correlated with HCV viremia. (a, b) The PD-L1/CD86 (a) and PD-L1/CD80 (b) ratios in CD14++CD16+monocytes in the chronic HCV-infected patients (n=57), HCV spontaneous resolvers (n=35) and healthy controls (n=30). (c, d) Correlations between the PD-L1/CD86 (c) and PD-L1/CD80 (d) ratios in the CD14++CD16+ monocytes and HCV plasma viral load (upper panel) and HCV core antigen (lower panel) during chronic HCV infection (n=57). HCV, hepatitis C virus.

Deregulated costimulatory/inhibitory marker expression and cytokine production in the CD14++CD16+ monocytes after stimulation with TLR ligands and HCV JFH-1

To further investigate how the CD14++CD16+ monocytes respond to pathogen-derived products in chronic HCV-infected individuals and HCV-resolved individuals, CD14++CD16+monocytes isolated from HCV infected individuals, HCV resolvers and healthy controls (n=6 for each group) were stimulated with LPS/R848 or with HCV JFH-1 viruses. The expressions of the co-stimulatory molecules HLA-DR and CD86 and the co-inhibitory molecules PD-L1 were subsequently detected. Upon stimulation with the TLR ligand LPS/R848, HLA-DR and CD86 expression decreased in all three investigated groups, as shown by fold change compared to the unstimulated samples, which was less than 1; on the other hand, the expression of the co-inhibitory marker PD-L1 was dramatically increased (Figure 5a). Compared to the healthy controls, the chronic HCV-infected subjects displayed a significantly decreased expression of HLA-DR (P=0.032) and CD86 (P=0.015) and a significantly increased expression of PD-L1 (P<0.001) (Figure 5a). There were no statistic differences in the expression of HLA-DR, CD86 and PD-L1 in the CD14++CD16+ monocytes between the HCV resolvers and the healthy controls (Figure 5a). When the HCV JFH1 virus was used to stimulate the CD14++CD16+ monocytes instead of the TLR ligands, similar changes in HLA-DR, CD86 and PD-L1 expression were observed in the chronic HCV infection group, HCV resolved group, and the healthy controls (Figure 5a).

Figure 5.

Figure 5

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Phenotypic profiles and cytokine secretion patterns of the CD14+CD16+ monocytes in chronic HCV-infected patients, spontaneous HCV resolvers and healthy controls (n=6 for each group) following stimulation with TLR ligands or HCV-JFH1 virus. (a) HLA-DR, CD86 and PD-L1 expression in CD14+CD16+ monocytes following stimulation with TLR ligands (5 µg/ml LPS and 10 µg/ml R848, upper panel) or with the HCV JFH-1 strain (10 MOI, lower panel). The data are shown as the fold change vs. the unstimulated controls. (b) IL-1β, IL-1α, IL-12p70, TNF-α and IL-10 production from the CD14+CD16+ monocytes following overnight culture with LPS/R848 or HCV JFH-1 virus. HCV, hepatitis C virus; MOI, multiplicity of infection; TLR, Toll-like receptor.

CD14++CD16+ monocytes produced less cytokines after stimulation with LPS/R848 (P=0.021 for IL-1β, P<0.001 for IL-1α, IL-12p70 and TNF-α) or HCV JFH-1 (P=0.046 for IL-1β, P<0.001 for IL-1α, P=0.005 for IL-12p70 and P=0.017 for TNF-α) when compared to the healthy controls, except that the level of IL-10 was increased in the chronic HCV infection group (P=0.036 for LPS/R848 stimulation, and P=0.027 for JFH-1 stimulation) (Figure 5b). These data suggested the CD14++CD16+ monocytes are impaired during chronic HCV infection, which in turn may limit the immune response of the host to the virus.

Discussion

In this study, we analyzed the distributions and phenotypes of three different monocyte subsets and their relationships with HCV viremia during HCV infection. The CD14+CD16+monocytes were increased in both the HCV persistent and self-limited infections compared to the healthy controls, which was in agreement with previous reports that showed that CD14+CD16+ monocytes were expanded during inflammatory diseases, including HIV, HCV and HBV infection.18,19,20,21 CD14+CD16+ monocytes were shown to produce more pro-inflammatory cytokines, such as TNF-α and IL-1β, and less anti-inflammatory cytokines, such as IL-10, upon TLR ligand stimulation.8,22,23,24 Here, we found that the systemic activation status was enhanced in both chronic HCV infection, as well as spontaneous HCV resolvers, as shown by the increased levels of IL-1β, IL-α, IL-12p70, TNF-α and IL-10. CD14+CD16+ monocytes are thought to be responsible, at least in part, for systemic immune activation. Sustained systemic immune activation in spontaneous HCV resolvers also indicated that the immune function of the monocytes in the spontaneous HCV resolvers did not returned to normal levels even though viral clearance had occurred.

CD14+CD16+ monocytes are a heterogeneous population that contains the CD14+/dimCD16+ and CD14++CD16+ subsets.10,22 We found that the increase in CD14+/dimCD16+ monocytes was responsible for the increase in CD14+CD16+ monocytes in the HCV-infected and HCV-resolved individuals, as the CD14++CD16+ subset decreased during HCV infection (Figure 1b). The CD14+/dimCD16+ non-classical monocytes are regarded as the mature and pro-inflammatory subset of monocytes.8,25,26 Increased numbers of CD14+/dimCD16+ monocytes were observed during viral infection and autoimmune disease and have been reported to perform a ‘patrolling' function in vivo; additionally, these monocytes have been shown to poorly respond to TLR ligands in vitro.8 CD14++CD16+ monocytes had been previously shown to play pivotal roles in the anti-viral immune response.16,17 In the current study, the proportion of CD14++CD16+ monocytes was significantly decreased and was reversely correlated with HCV viral load and HCV core antigen during chronic HCV infection. Intermediate CD14++CD16+ monocytes represent a transitory stage of monocyte differentiation and are considered anti-inflammatory monocytes that can produce large amounts of IL-10.27,28 Several studies have showed that CD14++CD16+ monocytes were increased during HIV infection and platelet activation,21,29 indicating that the proportion of CD14++CD16+ monocytes was linked to immune activation. However, immune activation is not a characteristic of HCV infection, which could partially explain why inverse changes in the CD14++CD16+ monocyte subset were observed during HCV infection and HIV infection. The negative correlation between CD14++CD16+ monocytes and HCV viremia suggested that HCV replication affected the non-classical and intermediate monocyte subsets differently and could be regarded as a surrogate for the evaluation of the severity of HCV replication.

Negative signaling molecules expressed by monocytes, such as PD-L1 and Tim-3, are involved in immune responses by delivering inhibitory signals to T cells.30,31,32 Specifically, the binding of PD-L1 on APCs to PD-1 on T cells delivers a co-inhibitory signal that blocks T-cell activation.30,31 Excessive PD-L1 expression leads to PD-L1/PD-1 overactivation and has been reported to play an important role in the pathogenesis of viral infection and the formation of the inflammatory environment.33,34,35 The upregulation of PD-L1 in monocytes is associated with defective HCV-specific T-cell responses, and the inhibition of monocyte-associated PD-L1 significantly enhanced the frequency of IFN-γ-producing HCV-specific T cells and the production of Th1 cytokines.36 Our data showed that PD-L1 expression was elevated in the CD14++CD16+ monocytes during chronic HCV infection, but not in HCV resolvers, suggesting a compromised anti-viral immune status of CD14++CD16+ monocytes in chronic HCV-infected individuals; however, the enhanced expressions of co-stimulatory markers HLA-DR, CD86, and CD80 were also identified during HCV infection. Additionally, CD14++CD16+ monocytes exhibited limited responses to LPS/R848 and HCV JFH-1 virus, as shown by the decreased HLA-DR and CD86 expression, increased PD-L1 expression, the impaired secretion of IL-1β, IL-1α, IL-12p70 and TNF-α and increased IL-10 secretion after treatment. The expression levels of HLA-DR and CD86 were higher in the CD14++CD16+ monocytes than in the CD14+CD16− monocytes and the CD14+/dimCD16+ monocytes.28,37 We supposed that, upon TLR ligand or virus stimulation, the intermediate CD14++CD16+ monocytes may develop into non-classical monocytes, and as a result, the expression of co-stimulatory markers would be decreased. This effect could be further enhanced by viral infection or in disease conditions. These results were consistent with some reports that the production of monocyte-derived cytokines was decreased following LPS stimulation, while contradictory to other reports that showed cytokine production was increased or remained unchanged following HCV infection.38,39,40,41 These discrepancies might be due to the cell types used in the different studies; we used purified CD14++CD16+ monocytes, while whole PBMCs or bulk monocytes were used in the reports mentioned above. The CD14++CD16+ monocytes accounted for the smallest proportion of monocytes, and the levels of cytokine production, including TNF-α, IL-10, IL-1β and IL-6, from these cells were at their lowest or intermediary levels when compared to classical and non-classical monocytes.28

PD-L1 (B7-H1), CD86 (B7-2) and CD80 (B7-1) all belong to the B7 family and can deliver different signals to regulate T-cell activation.42 Because several studies suggested CD80 and CD86 play differential regulation roles in the inflammatory response in certain diseases,43,44 we analyzed the PD-L1/CD86 and PD-L1/CD80 ratios in the CD14++CD16+ monocyte subset and evaluated their relationship with disease progression. The PD-L1/CD86 and PD-L1/CD80 ratios were dramatically higher in the chronic HCV-infected group compared to the HCV resolvers and healthy controls. Furthermore, the ratio of PD-L1/CD86, but not PD-L1/CD80, showed a positive correlation with the HCV viral load and HCV core antigen in the chronic HCV-infected group. However, no correlation was found between the PD-L1/CD86 ratio in the CD14++CD16+ monocytes and the clinical markers of inflammatory activity, such as serum alanine aminotransferase (data not shown). We previously reported that the PD-L1/CD86 ratio in DCs was increased in HCV-infected patients and was associated with the impaired allostimulatory capacity of the DCs.16 It has also been reported that a higher PD-L1/CD86 ratio was accompanied by an increased number of regulatory T cells in liver transplant tolerance.45 Combined with our results, these findings suggested that the balance of inhibitory B7-H1 to co-stimulatory B7-1/B7-2 molecule in APCs might affect the outcomes of their interactions with T cells, and the PD-L1/CD86 ratio in monocytes could be employed as an important indicator to reflect the immune responses and disease state during viral infection, including HCV infection.

In summary, our results demonstrated that the distributions of the monocyte subsets were altered during HCV infection, and the decreased CD14++CD16+ monocytes were negatively correlated with HCV viral load and core antigen in chronic HCV infection, indicating that the CD14++CD16+ monocytes play a pivotal role in modulating the immune response to HCV infection. The PD-L1/CD86 ratio in the CD14++CD16+ monocytes can be regarded as a hallmark to assess the immune status and disease outcomes of HCV infection. The impaired expression of co-stimulatory markers and cytokine secretion following TLR ligand or HCV JFH-1 virus treatment indicated the compromised immune status of the CD14++CD16+ monocytes and may affect the systemic responses to invasive pathogens. These findings provided new insights into the characterization of the CD14++CD16+ monocytes and their significance in chronic hepatitis C infection.

Acknowledgments

This work was supported by the National Natural Science of China (81271826, 31100126), the National Science Foundation of Beijing (7122108), SKLID development grant (2011SKLID207) and grants from the National S&T Major Project for Infectious Diseases (2012ZX10002003 and 2012ZX10002005).

The authors declare that they have no competing financial interests.

Footnotes

Supplementary Information accompanies the paper on Cellular & Molecular Immunology's website. (http://www.nature.com/cmi).

Supplementary Information

Supplementary information
Click here for additional data file. (621.1KB, pdf)

References

  1. Bowen DG, Walker CM. The origin of quasispecies: cause or consequence of chronic hepatitis C viral infection. J Hepatol. 2005;42:408–417. doi: 10.1016/j.jhep.2004.12.013. [DOI] [PubMed] [Google Scholar]
  2. Bowen DG, Walker CM. Adaptive immune responses in acute and chronic hepatitis C virus infection. Nature. 2005;436:946–952. doi: 10.1038/nature04079. [DOI] [PubMed] [Google Scholar]
  3. Hahn YS. Subversion of immune responses by hepatitis C virus: immunomodulatory strategies beyond evasion. Curr Opin Immunol. 2003;15:443–449. doi: 10.1016/s0952-7915(03)00076-1. [DOI] [PubMed] [Google Scholar]
  4. Hoofnagle JH. Hepatitis C: the clinical spectrum of disease. Hepatology. 1997;26:15S–20S. doi: 10.1002/hep.510260703. [DOI] [PubMed] [Google Scholar]
  5. Claassen MA, Janssen HL, Boonstra A. Role of T cell immunity in hepatitis C virus infections. Curr Opin Virol. 2013;3:461–467. doi: 10.1016/j.coviro.2013.05.006. [DOI] [PubMed] [Google Scholar]
  6. Liu B, Woltman AM, Janssen HL, Boonstra A. Modulation of dendritic cell function by persistent viruses. J Leukoc Biol. 2009;85:205–214. doi: 10.1189/jlb.0408241. [DOI] [PubMed] [Google Scholar]
  7. Tacke RS, Tosello-Trampont A, Nguyen V, Mullins DW, Hahn YS. Extracellular hepatitis C virus core protein activates STAT3 in human monocytes/macrophages/dendritic cells via an IL-6 autocrine pathway. J Biol Chem. 2011;286:10847–10855. doi: 10.1074/jbc.M110.217653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cros J, Cagnard N, Woollard K, Patey N, Zhang SY, Senechal B, et al. Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors. Immunity. 2010;33:375–386. doi: 10.1016/j.immuni.2010.08.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. van de Veerdonk FL, Netea MG. Diversity: a hallmark of monocyte society. Immunity. 2010;33:289–291. doi: 10.1016/j.immuni.2010.09.007. [DOI] [PubMed] [Google Scholar]
  10. Ziegler-Heitbrock L, Ancuta P, Crowe S, Dalod M, Grau V, Hart DN, et al. Nomenclature of monocytes and dendritic cells in blood. Blood. 2010;116:e74–e80. doi: 10.1182/blood-2010-02-258558. [DOI] [PubMed] [Google Scholar]
  11. Wedemeyer H, He XS, Nascimbeni M, Davis AR, Greenberg HB, Hoofnagle JH, et al. Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection. J Immunol. 2002;169:3447–3458. doi: 10.4049/jimmunol.169.6.3447. [DOI] [PubMed] [Google Scholar]
  12. Gruener NH, Lechner F, Jung MC, Diepolder H, Gerlach T, Lauer G, et al. Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus. J Virol. 2001;75:5550–5558. doi: 10.1128/JVI.75.12.5550-5558.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huang X, Venet F, Wang YL, Lepape A, Yuan Z, Chen Y, et al. PD-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis. Proc Natl Acad Sci USA. 2009;106:6303–6308. doi: 10.1073/pnas.0809422106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Muhlbauer M, Fleck M, Schutz C, Weiss T, Froh M, Blank C, et al. PD-L1 is induced in hepatocytes by viral infection and by interferon-alpha and -gamma and mediates T cell apoptosis. J Hepatol. 2006;45:520–528. doi: 10.1016/j.jhep.2006.05.007. [DOI] [PubMed] [Google Scholar]
  15. Yao S, Wang S, Zhu Y, Luo L, Zhu G, Flies S, et al. PD-1 on dendritic cells impedes innate immunity against bacterial infection. Blood. 2009;113:5811–5818. doi: 10.1182/blood-2009-02-203141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Shen T, Chen X, Chen Y, Xu Q, Lu F, Liu S. Increased PD-L1 expression and PD-L1/CD86 ratio on dendritic cells were associated with impaired dendritic cells function in HCV infection. J Med Virol. 2010;82:1152–1159. doi: 10.1002/jmv.21809. [DOI] [PubMed] [Google Scholar]
  17. Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, Ley K. Development of monocytes, macrophages, and dendritic cells. Science. 2010;327:656–661. doi: 10.1126/science.1178331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ziegler-Heitbrock L. The CD14+ CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol. 2007;81:584–592. doi: 10.1189/jlb.0806510. [DOI] [PubMed] [Google Scholar]
  19. Moniuszko M, Bodzenta-Lukaszyk A, Kowal K, Lenczewska D, Dabrowska M. Enhanced frequencies of CD14++CD16+, but not CD14+CD16+, peripheral blood monocytes in severe asthmatic patients. Clin Immunol. 2009;130:338–346. doi: 10.1016/j.clim.2008.09.011. [DOI] [PubMed] [Google Scholar]
  20. Zhang JY, Zou ZS, Huang A, Zhang Z, Fu JL, Xu XS, et al. Hyper-activated pro-inflammatory CD16 monocytes correlate with the severity of liver injury and fibrosis in patients with chronic hepatitis B. PLoS ONE. 2011;6:e17484. doi: 10.1371/journal.pone.0017484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Han J, Wang B, Han N, Zhao Y, Song C, Feng X, et al. CD14highCD16+ rather than CD14lowCD16+ monocytes correlate with disease progression in chronic HIV-infected patients. J Acquir Immune Defic Syndr. 2009;52:553–559. doi: 10.1097/qai.0b013e3181c1d4fe. [DOI] [PubMed] [Google Scholar]
  22. Auffray C, Sieweke MH, Geissmann F. Blood monocytes: development, heterogeneity, and relationship with dendritic cells. Annu Rev Immunol. 2009;27:669–692. doi: 10.1146/annurev.immunol.021908.132557. [DOI] [PubMed] [Google Scholar]
  23. Belge KU, Dayyani F, Horelt A, Siedlar M, Frankenberger M, Frankenberger B, et al. The proinflammatory CD14+CD16+DR++ monocytes are a major source of TNF. J Immunol. 2002;168:3536–3542. doi: 10.4049/jimmunol.168.7.3536. [DOI] [PubMed] [Google Scholar]
  24. Hijdra D, Vorselaars AD, Grutters JC, Claessen AM, Rijkers GT. Phenotypic characterization of human intermediate monocytes. Front Immunol. 2013;4:339. doi: 10.3389/fimmu.2013.00339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tacke F, Randolph GJ. Migratory fate and differentiation of blood monocyte subsets. Immunobiology. 2006;211:609–618. doi: 10.1016/j.imbio.2006.05.025. [DOI] [PubMed] [Google Scholar]
  26. Liaskou E, Zimmermann HW, Li KK, Oo YH, Suresh S, Stamataki Z, et al. Monocyte subsets in human liver disease show distinct phenotypic and functional characteristics. Hepatology. 2012;57:385–398. doi: 10.1002/hep.26016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Skrzeczynska-Moncznik J, Bzowska M, Loseke S, Grage-Griebenow E, Zembala M, Pryjma J. Peripheral blood CD14high CD16+ monocytes are main producers of IL-10. Scand J Immunol. 2008;67:152–159. doi: 10.1111/j.1365-3083.2007.02051.x. [DOI] [PubMed] [Google Scholar]
  28. Wong KL, Tai JJ, Wong WC, Han H, Sem X, Yeap WH, et al. Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets. Blood. 2011;118:e16–e31. doi: 10.1182/blood-2010-12-326355. [DOI] [PubMed] [Google Scholar]
  29. Passacquale G, Vamadevan P, Pereira L, Hamid C, Corrigall V, Ferro A. Monocyte-platelet interaction induces a pro-inflammatory phenotype in circulating monocytes. PLoS ONE. 2011;6:e25595. doi: 10.1371/journal.pone.0025595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000;192:1027–1034. doi: 10.1084/jem.192.7.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Okazaki T, Honjo T. The PD-1–PD-L pathway in immunological tolerance. Trends Immunol. 2006;27:195–201. doi: 10.1016/j.it.2006.02.001. [DOI] [PubMed] [Google Scholar]
  32. Wang JM, Shi L, Ma CJ, Ji XJ, Ying RS, Wu XY, et al. Differential regulation of interleukin-12 (IL-12)/IL-23 by Tim-3 drives TH17 cell development during hepatitis C virus infection. J Virol. 2013;87:4372–4383. doi: 10.1128/JVI.03376-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Watanabe T, Bertoletti A, Tanoto TA. PD-1/PD-L1 pathway and T-cell exhaustion in chronic hepatitis virus infection. J Viral Hepat. 2010;17:453–458. doi: 10.1111/j.1365-2893.2010.01313.x. [DOI] [PubMed] [Google Scholar]
  34. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, et al. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature. 2006;443:350–354. doi: 10.1038/nature05115. [DOI] [PubMed] [Google Scholar]
  35. Rodig N, Ryan T, Allen JA, Pang H, Grabie N, Chernova T, et al. Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T cell activation and cytolysis. Eur J Immunol. 2003;33:3117–3126. doi: 10.1002/eji.200324270. [DOI] [PubMed] [Google Scholar]
  36. Jeong HY, Lee YJ, Seo SK, Lee SW, Park SJ, Lee JN, et al. Blocking of monocyte-associated B7-H1 (CD274) enhances HCV-specific T cell immunity in chronic hepatitis C infection. J Leukoc Biol. 2008;83:755–764. doi: 10.1189/jlb.0307168. [DOI] [PubMed] [Google Scholar]
  37. Abeles RD, McPhail MJ, Sowter D, Antoniades CG, Vergis N, Vijay GK, et al. CD14, CD16 and HLA-DR reliably identifies human monocytes and their subsets in the context of pathologically reduced HLA-DR expression by CD14hi/CD16neg monocytes: expansion of CD14hi/CD16pos and contraction of CD14lo/CD16pos monocytes in acute liver failure. Cytometry A. 2012;81:823–834. doi: 10.1002/cyto.a.22104. [DOI] [PubMed] [Google Scholar]
  38. Villacres MC, Literat O, DeGiacomo M, Du W, Frederick T, Kovacs A. Defective response to Toll-like receptor 3 and 4 ligands by activated monocytes in chronic hepatitis C virus infection. J Viral Hepat. 2008;15:137–144. doi: 10.1111/j.1365-2893.2007.00904.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Liu BS, Groothuismink ZM, Janssen HL, Boonstra A. Role for IL-10 in inducing functional impairment of monocytes upon TLR4 ligation in patients with chronic HCV infections. J Leukoc Biol. 2011;89:981–988. doi: 10.1189/jlb.1210680. [DOI] [PubMed] [Google Scholar]
  40. Martin-Blondel G, Gales A, Bernad J, Cuzin L, Delobel P, Barange K, et al. Low interleukin-10 production by monocytes of patients with a self-limiting hepatitis C virus infection. J Viral Hepat. 2009;16:485–491. doi: 10.1111/j.1365-2893.2009.01094.x. [DOI] [PubMed] [Google Scholar]
  41. Peng C, Liu BS, de Knegt RJ, Janssen HL, Boonstra A. The response to TLR ligation of human CD16+CD14− monocytes is weakly modulated as a consequence of persistent infection with the hepatitis C virus. Mol Immunol. 2011;48:1505–1511. doi: 10.1016/j.molimm.2011.04.008. [DOI] [PubMed] [Google Scholar]
  42. Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med. 1999;5:1365–1369. doi: 10.1038/70932. [DOI] [PubMed] [Google Scholar]
  43. Hosiawa KA, Wang H, DeVries ME, Garcia B, Liu W, Zhou D, et al. CD80/CD86 costimulation regulates acute vascular rejection. J Immunol. 2005;175:6197–6204. doi: 10.4049/jimmunol.175.9.6197. [DOI] [PubMed] [Google Scholar]
  44. Nolan A, Kobayashi H, Naveed B, Kelly A, Hoshino Y, Hoshino S, et al. Differential role for CD80 and CD86 in the regulation of the innate immune response in murine polymicrobial sepsis. PLoS ONE. 2009;4:e6600. doi: 10.1371/journal.pone.0006600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tokita D, Mazariegos GV, Zahorchak AF, Chien N, Abe M, Raimondi G, et al. High PD-L1/CD86 ratio on plasmacytoid dendritic cells correlates with elevated T-regulatory cells in liver transplant tolerance. Transplantation. 2008;85:369–377. doi: 10.1097/TP.0b013e3181612ded. [DOI] [PubMed] [Google Scholar]

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