A total of 65 chronically HBV-infected patients were categorised as IT (n=10), EP‐CHB (n=15), IC (n=22) and EN‐CHB (n=18) [Supplementary(S)Table S1]. In addition, 19 HC were included.
Distribution of monocyte-subsets in different phases of CHI
We first determined the frequency of total-monocytes and their subsets in different phases of CHI (Fig. 1a-b). The frequency of HLA-DR+CD14+-total-monocytes was comparable across all study groups. However, the proportion of HLA-DR+CD14++CD16-[classical] monocytes was significantly diminished in IT (83.6±3.1%), EP‐CHB (82.5±3.6%) and EN‐CHB (82.7±2.4 %) relative to IC (92.5±1.7%) and HC (93.6±1.6%), while a stark increase was observed in HLA-DR+CD14++CD16+[intermediate], and HLA-DR+CD14+CD16++[non-classical] monocytes in IT (intermediate/non-classical-monocytes;9.4±1.3/6.9±2.3%), EP-CHB (9.7±1.6/7.7±2.4%) and EN-CHB (9.7±1.5/7.4±1.7%) as compared to IC (3.8±1.0/3.6±0.9%) and HC (3.3±0.8/2.9±0.9%) (Fig. 1b).
TLR expression and cytokine production by monocytes
Recognition of PAMPs by TLRs of monocytes represent a critical step for the clearance of infecting microbes. We noted significant reduction in TLR-2+-, TLR-4+- and TLR-9+-monocytes in IT and EP-/EN-CHB than IC and HC while no difference could be perceived in the incidence of TLR-8+-monocytes across the groups (Fig. 1c). All monocyte-subsets of IT and EP-/EN-CHB exhibited declining trends in TLR-2/4/9 expression (Fig. S1a). Simultaneously, marked diminution was observed in the percentages of total-monocytes (Fig. 1d) as well as all three-subsets (Fig. S1b) that expressed TLR-regulated pro-inflammatory cytokines TNF-α, IL-12 and IL-6 in IT and EP-/EN-CHB in comparison to IC/HC. While intramonocytic TNF-α and IL-6 level was equivalent in IT and EP-/EN-CHB, IL-12 was significantly low in EP-/EN-CHB than IT. Further, the frequencies of IL-12+-monocytes were less in IC than HC (Fig. 1d).
Additionally, we evaluated the production of inhibitory cytokines, TGF-β and IL-10 by the monocytes in different disease phases. We assessed the expression of TGF-β bound to the latency-associated peptide (LAP)  whereby lower cell-surface LAP-expression correlates with higher TGF-β secretion. IT as well as EP-/EN-CHB patients displayed significant decline in proportion of LAP-TGF-β+-monocytes, indicative of raised functional TGF-β levels, as opposed to IC and HC (Fig. 1e, S1c). Furthermore, an analogous expansion in IL-10+-monocytes (Fig. 1e, S1c) was apparent in IT and EP-/EN-CHB relative to IC/HC. Notably, monocytes of EP-/EN-CHB showed enhanced IL-10 expression than IT (Fig. 1e).
Phagocytic activity and oxidative response of monocytes in CHI
We analyzed the expression of FcγRI/CD64, the primary receptor for opsonic uptake of antigens on monocytes. In comparison to IC/HC, a deficit in phagocytic function of monocytes was noted in IT and EP-/EN-CHB, as evident from significantly low expression of CD64 in total-monocytes, including all subsets (Fig. 2a, S2a). Consistent with decreased CD64 expression, there was also substantial decline in the percentage of monocytes associated with zymosan-reporter signal in case of IT and EP-/EN-CHB, suggestive of poor zymosan uptake by these cells than those of IC/HC (Fig. 2a, S2b-S2c).
Phagocytosis leads to the generation of reactive oxygen and nitrogen species (ROS/RNS) within monocytes. The capacity of the monocytes (including all subsets) to produce ROS was significantly attenuated in IT and EP-/EN-CHB as inferred from decreasing DCF-fluorescence in these patients relative to HC and IC (Fig. 2b, S3a-S3b). RNS production is dependent upon nitric oxide that is generated by inducible nitric oxide synthase (iNOS). Compared to IC/HC, iNOS+ total-/monocyte-subsets were reduced in numbers in IT and EP-/EN-CHB, suggesting a decrease in iNOS-mediated RNS production (Fig. 2b, S3c). Additionally, IC harboured significantly lower frequency of iNOS-expressing monocytes compared to HC.
Regulation of monocyte functions by HBsAg and cytokines
We next sought to identify the viral antigen and systemic cytokines that might contribute to the altered monocyte functions during CHI. HBsAg is the most abundant viral protein in the sera of HBV-infected patients and it was found to be markedly high in IT (5.2±0.9 log10IU/mL) and EP-/EN-CHB (5.3±0.9 log10IU/mL) than IC (3.2±0.5 log10IU/mL) (Fig. 3a). A positive correlation was observed between serum HBsAg levels and IL-10-expressing monocytes while HBsAg titers correlated inversely with frequencies of TLR2+/IL-12+/CD64+/iNOS+-monocytes, implying a potential role of HBsAg in monocyte dysfunction (Fig. 3a). Further, treatment of CD14+-monocytes, sorted from HC, with high concentration of rHBsAg resulted in reduced frequency of classical-monocytes and amplification of non-classical and intermediate-subsets, along with suppression of TLR-2/CD64/IL-12/iNOS and augmentation of IL-10 expression as compared to untreated and β‐gal‐treated cells (Fig. 3b, S4). On the other hand, even at low HBsAg concentration, the monocytes exhibited significant decrease in iNOS- and IL-12 expression over control setups (Fig. 3b).
Despite the similar HBsAg levels in IT and CHB, we noticed a decline in IL-12+ and heightened IL-10+-monocytes in CHB as compared to IT. We postulated that these functional variabilities could be related to the differences in local cytokines in these two phases. Significant increases were found for serum IL-4 and TNF-α exclusively in EP/EN-CHB phases than other groups (Fig. 3c). In addition, we observed that treatment of monocytes with high concentration of rIL-4 conferred significant enhancement in IL-10+- and diminution in IL-12+-monocytes relative to untreated cells, while no discernible change was noted upon rTNF-α-treatment (Fig. 3c, S5a-S5b).
HBsAg and IL-4 activated β-catenin in monocytes
We next investigated the mechanism underlying HBsAg- or IL-4-mediated alteration in the properties of monocytes. Given that β-catenin could suppress TLR-triggered pro-inflammatory responses and induce anti-inflammatory cytokines [12, 13], we speculated that HBsAg/IL-4 could promote the aberrant monocyte function through activation of β-catenin. Our in vitro assays demonstrated that both HBsAg and IL-4 resulted in significant accumulation of β-catenin+-monocytes compared to untreated cells (Fig. 4a, S6a-S6b). Moreover, addition of β-catenin/TCF inhibitor led to enhanced frequency of classical-monocytes as well as that of TLR-2+/CD64+/iNOS+/IL-12+-monocytes but caused reduction in intermediate-/non-classical-subsets along with IL-10-expressing monocytes, relative to that observed when no inhibitor was added (Fig. 4b-c, S4, S5a). In parallel, β-catenin+ monocytes were significantly elevated in all chronically HBV-infected patients than HC and HBsAg titres correlated positively with percentages of β-catenin+-monocytes (Fig. 4a). Collectively, these findings signify that the functions of monocytes were compromised by induction of β-catenin by HBsAg/IL-4.
Characterization of in vitro differentiated macrophages
Monocytes from study subjects were differentiated in vitro to M1-/M2-macrophages and their cytokine production abilities were compared. HLA-DR+CD14+CD68+ M1-macrophages, particularly in CHB, and also IT were characterized by marked decline in IL-12 and TNF-α production relative to HC/IC (Fig. 5a). In addition, a heightened frequency of IL-10-expressing M1-macrphages was perceived in CHB followed by IT, while it was much lower in IC and HC (Fig. 5b). HLA-DR+CD14+CD68+ M2-marcophages from CHB and IT displayed superior abilities to produce IL-10 than IC/HC, although CHB showed higher IL-10 expression than IT (Fig. 5b). Irrespective of the study groups, all M2-macrophages expressed little IL-12 and TNF-α.
Monocyte-mediated differentiation pattern of CD4+T-cells-
Monocytes are known to drive the differentiation of CD4+T-cells into distinct functional populations . We explored whether the monocytes promote any specific CD4+T-cell differentiation program in CHI. Co-culture of sorted CD14+-monocytes from CHB and IT with autologous anti-CD3/anti-CD28 stimulated monocyte-depleted PBMC resulted in enrichment of CD4+CD25+FOXP3+Treg by ~4.4- and ~4-folds respectively and an expansion of CD4+CCR4+CCR6-Th2-subset by ~2.6- and ~2-folds. However, an equivalent population of Treg or Th2-cells did not emerge in presence of monocytes sorted from HC/IC. Conversely, monocytes of HC and IC favor the amplification of CD4+CXCR3+Th1-cells by ~4 fold while in IT, ~2.4 fold rise in Th1-cells was also seen (Fig. 5c).
Mobility traits of monocytes in CHI
CCR2 plays a key role in recruitment of monocytes to the liver . CCR2+-monocytes, inclusive of all subsets, were found to be markedly elevated in both EP-/EN-CHB patients than other groups while IT showed a greater percentage of CCR2-expressing monocytes than IC/HC (Fig. 5d, S7). This suggests a higher potential of the monocytes in CHB as well as IT to home to the liver.
Assessment of intrahepatic β-catenin+CD14+-monocytes
We also studied the frequency of CD14 and β-catenin double-positive cells in liver biopsy sections of CHB patients and HC by immunohistochemical staining. Liver histology indicated prominent lymphocyte-predominant lobular and portal inflammation in CHB than HC. β-catenin+CD14+-cell density was found to be substantially high in the liver of CHB and such cells were barely perceptible in HC (Fig. 5e).
Frequency/phenotype/function of monocytes in Tenofovir-treated CHB patients
Tenofovir is recommended as first-line monotherapy for CHB patients  and we tested the effect of Tenofovir treatment on the frequency and expression of different functional markers of monocytes in 12 CHB patients that included 5 EP-CHB and 7 EN-CHB. We observed that all patients achieved <250 copies/ml of HBV-DNA and normalization of serum ALT after one-year of therapy (Fig. 6a) but no significant change was detected in the monocyte-subset distribution or the proportion of TLR2/IL12/IL-10/CD64/iNOS-expressing monocyte-subsets between pre- and post-treatment time-points (Fig. 6b). Moreover, the serum levels of HBsAg and IL-4 in these patients remained similar to baseline values (Fig. 6a).