Multifaceted Defects in Monocytes in Different Phases of Chronic HBV Infection: Persistence After Antiviral Therapy

Monocytes play an important role in the control of microbial infection but monocyte biology during chronic HBV infection (CHI) remains inadequately studied. We investigated the frequency/phenotype/functions of monocytes in different phases of CHI namely, Immune-tolerant (IT), HBeAg-positive/HBeAg-negative (EP/EN) chronic hepatitis B (CHB) and Inactive carriers (IC), identied factors responsible for their functional alterations and determined the impact of antiviral-therapy on these cells.


Introduction
The outcome of chronic HBV infection [CHI] and the pathogenesis of liver disease are largely determined by immune-mediated host-virus interactions [1]. Inability to achieve sustained viral control in CHI has been correlated with the incapacity of the host to evoke an effective immune response against the virus [1]. Monocytes represent a critical component of innate immunity that play a fundamental role in the control of microbial infection and also contribute to the pathogenesis of in ammatory diseases [2]. They recognise pathogen-associated molecular patterns (PAMPs) by a set of Toll-like receptors (TLRs), and trigger intracellular signalling cascades leading to the expression of pro-in ammatory cytokines, enhanced phagocytic activity and generation of reactive oxygen and nitrogen intermediates [2][3]. These events orchestrate the early host response to infection that promote the clearance of pathogen. Based on the expression of CD14 and CD16, human monocytes can be divided into three subsets, CD14 ++ /CD16 − [classical], CD14 ++ /CD16 + [intermediate] and CD14 + /CD16 ++ [non-classical], whose relative percentages, functional properties and disease association had been reported to vary when studied in vitro and in vivo [4]. Monocytes tra c to the site of infection/in ammation and depending on microenvironmental stimuli, they differentiate into either M1-macrophages with pronounced pro-in ammatory phenotype, or M2macrophages with anti-in ammatory attributes [2]. Monocytes can also drive the CD4 + T-cell differentiation into distinct effector cells that could impact the elimination of microbes and disease pathogenesis [5]. Relatively little is known about the effects of chronic viral infection on monocytes.
Monocytes/macrophages serve as an important reservoir of HIV and the disease progression is closely linked to the expansion of CD16 + -monocytes [6]. Altered TLR-signalling and cytokine production by monocytes had been described in HCV-infected patients [7]. In chronically HBV-infected patients, changes were observed in the frequencies of monocyte-subsets and regulation of TLR-expression by HBV precore and surface protein had been reported [8][9]. However, there is still a lack of comprehensive understanding of monocyte biology during CHI, whose natural history includes four dynamic phases namely, immune-tolerant (IT), hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (EP-CHB), inactive carrier (IC) and HBeAg-negative chronic hepatitis B (EN-CHB) [10]. Hence, the present study aimed to appraise the distinct phenotype and functions of monocytes in different phases of CHI, identify the viral and host factors that could modulate the properties of these cells and study the interaction of monocytes with CD4 + T-cells. We also studied the impact of Tenofovir therapy on monocytes in CHB patients. This integrated knowledge would be vital for designing more effective therapies for CHB.

Methods
Detailed information regarding study subjects and experimental methodology is available in Supplementary information (SI).

Study subjects and samples
Treatment-naïve chronically HBV-infected patients were recruited from I.P.G.M.E.&R, Kolkata, India and grouped into IT, EP-CHB, IC and EN-CHB. Additionally, HBV-uninfected healthy individuals (HC) were included. Blood samples and liver biopsy specimens were collected from study subjects with written informed consent. The study was approved by the Ethical Review Committee of I.P.G.M.E&R.
Serum Hepatitis B surface antigen (HBsAg) and cytokine quanti cation Serum HBsAg and different cytokines in study subjects were quanti ed using Abbott Architect i1000sr platform and BD CBA Human Th1/Th2/Th17 Cytokine Kit respectively.

Differentiation of monocytes to macrophages
Puri ed monocytes were differentiated into macrophages with M-CSF followed by LPS and IFN-γ treatment to generate M1-macrophages or IL-4 to generate M2-macrophages and their intracellular TNFα/IL-12/IL-10 levels were ascertained by ow-cytometry.
Assessment of virological/immunological parameters following Tenofovir-therapy Blood samples were collected from 12 CHB patients treated with Tenofovir before therapy (=baseline) and after 12 months and frequency/phenotype/function of monocyte-subsets and serum HBV-DNA/ALT/HBsAg/cytokine levels were determined.

Statistical analysis
Statistical analysis was performed using GraphPad Prism5 software as relevant. P< 0.05 was considered statistically signi cant.

Distribution of monocyte-subsets in different phases of CHI
We rst 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. 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 signi cant 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 TLRregulated pro-in ammatory 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 signi cantly 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) [11] whereby lower cell-surface LAP-expression correlates with higher TGF-β secretion. IT as well as EP-/EN-CHB patients displayed signi cant 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 de cit in phagocytic function of monocytes was noted in IT and EP-/EN-CHB, as evident from signi cantly 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 signi cantly attenuated in IT and EP-/EN-CHB as inferred from decreasing DCF-uorescence 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 signi cantly 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 log 10 IU/mL) and EP-/EN-CHB (5.3±0.9 log 10 IU/mL) than IC (3.2±0.5 log 10 IU/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 ampli cation 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 signi cant 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. Signi cant increases were found for serum IL-treatment of monocytes with high concentration of rIL-4 conferred signi cant 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-in ammatory responses and induce anti-in ammatory 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 signi cant 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 signi cantly elevated in all chronically HBV-infected patients than HC and HBsAg titres correlated positively with percentages of β-catenin + -monocytes (Fig. 4a). Collectively, these ndings 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 [5].
We explored whether the monocytes promote any speci c CD4 + T-cell differentiation program in CHI. Coculture 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 ampli cation 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 [14]. 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 lymphocytepredominant lobular and portal in ammation 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 rst-line monotherapy for CHB patients [10] 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 signi cant 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).

Discussion
In this study we enumerated the broad spectrum of phenotypic and functional alterations in monocytes of chronically HBV-infected patients and studied the mechanisms underpinning the changes, which would add to the ongoing efforts of de ning the process behind immune dysregulation in CHI and provide targets for development of new therapies aimed at reversing the defects.
An expansion of CD16 + -compartment (intermediate-and non-classical-monocytes) along with concomitant decrease in classical-monocytes was perceived in IT and CHB patients relative to IC/HC. Similar shifts in monocyte-subset distribution had been previously reported in Chinese HBeAg-positive CHB patients [15] and also in other infectious diseases [6,7]. The interaction of monocytes with the pathogen or pathogen-derived factors had been suggested to be the causal factor for this increase in CD16 + -monocytes [16]. The secretory HBsAg is known to have an immunomodulatory effect and circulating CD14 + -monocytes were found to harbour a detectable depot of HBsAg in CHB patients [17]. We demonstrated that at high concentration, HBsAg could stimulate the preferential generation of intermediate-and non-classical-monocytes and this concurred with the greater frequency of these two subsets in IT and CHB patients, who unlike IC, carried high serum HBsAg.
TLR-2 and TLR-4 of the monocytes sense the presence of virus via their proteins, whereas, TLR-7/8 binds single-stranded viral RNA and TLR-9 recognizes viral CpG DNA [3]. Previous studies on the TLRexpression on monocytes of CHB patients had revealed in many cases divergent data. Overexpression of TLR-2 and TLR-4 had been noted on CD14 + -monocytes in HBV-infected Chinese patients [15] while a study conducted in Australia reported a marked reduction in TLR-2 but not TLR-4 expression on monocytes of HBeAg-positive CHB patients relative to HBeAg-negative CHB and controls [8]. In contrast, we observed that both TLR-2 and TLR-4 along with TLR-9 were downregulated in both EP-and EN-CHB and also in IT in comparison to IC/HC. These discrepancies in TLR-expression pro le might partially result from investigating ethnically different patient populations or might be impacted by high exposure of a population to other pathogens causing tuberculosis, malaria, leprosy or to parasitic worms. TLRsignalling usually induce the expression of an array of in ammatory cytokines. We noted that the attenuation of TLR-expression in CHB/IT were reciprocated by greatly impaired production of TNF-α/IL-12/IL-6 by the monocytes while the levels of IL-10 and TGF-β were signi cantly enhanced. Intriguingly, higher expression of both TNF-α and IL-10 transcripts had been documented in the monocytes of Chinese CHB patients than healthy donors [18]. Different studies have established that monocyte-derived IL-12 could induce IFN-γ production from T-cells and also skew the naive CD4 + T-cells toward the Th1phenotype [5] that help in eliminating viral infection. Conversely, upregulation of IL-10 in monocytes coincides with impaired T-cell responses [3]. Hence, it appears that the inhibition of IL-12 and augmentation of IL-10 by monocytes in CHB/IT would limit the antiviral activities of T-cells and favour viral persistence.
A salient feature of the monocytes is their capability to phagocytose foreign organisms and generate ROS and RNS by specialized enzymes, NADPH oxidase and iNOS that can irreversibly oxidize and damage the cellular structures of the pathogens. Signi cant reduction in phagocytic activity, intracellular ROS production and suppression of iNOS was manifested by the monocytes of EP-/EN-CHB and IT when compared with IC/HC. Our nding was in congruity to that of Prieto et al. who had reported diminished phagocytosis by monocytes of CHB patients relative to HBeAg-negative/anti-HBe positive patients and HC [19]. This overall inadequate antimicrobial activity of monocytes in CHB/IT contribute to the chronicity of infection and high viral load in these phases.
Monocytes give rise to macrophages in tissues and the heterogeneity in monocytes underlie that of macrophages. To determine if MDM-subsets are functionally altered in CHI, we investigated the cytokine pro les of these macrophages, which can confer a better appreciation of functional polarization than that of cell-surface receptor expression. Our results revealed that M1-macrophages from CHB/IT acquired an M2-like anti-in ammatory state characterized by decreased production of TNF-α and IL-12 and increased IL-10. Similar cytokine secretion features of M1-cells had been reported in chronic HCV patients [7]. However, unlike M2-subsets from HCV-infected individuals, which secrete more pro-in ammatory cytokines than controls [7], the M2-macrophages in all phases of CHI produced very low concentrations of IL-12 and TNF-α. Moreover, M2-cells generated from CHB and IT displayed remarkable enhancement in IL-10 production in comparison to IC/HC. Thus, the aberrant functions of MDM in CHB/IT and their shifts towards M2-phenotype are likely to in uence T-cell activation and function that would contribute towards viral perpetuation and pathogenicity in CHI.
We next identi ed the HBV antigen and host cytokines that dictate the functional modi cation of monocytes during CHI. We demonstrated that high concentration of HBsAg encountered in IT and EP/EN-CHB, potentiated the reduced expression of TLR-2/CD64/iNOS/IL-12 and heightened expression of IL-10 by the monocytes. Moreover, HBsAg, even at low concentration, could inhibit IL-12 and iNOS expression and thus could explain the observed decreased production of these molecules by monocytes in IC relative to HC. The role of HBsAg in inhibiting TLR-2 ligand-induced IL-12 production in monocytes/macrophages had also been previously highlighted by Wang et al. [9]. In addition, in vitro assays depicted that IL-4, detected at high levels exclusively in EP-/EN-CHB phase, could constrain IL-12 but stimulate IL-10 expression in monocytes and thus could account for the greater immunosuppressor trait of monocytes in CHB patients. Similar IL-4-mediated inhibition of IL-12 production had been observed in murine peritoneal macrophages [20].
We dissected the molecular mechanisms through which HBsAg and IL-4 could affect the phenotype/function of monocytes and showed that both pathways impinge on the activation of βcatenin. An increased accumulation of β-catenin was seen in HBsAg or IL-4 treated monocytes whereas, pharmacological inhibition of β-catenin could potentially normalize the immune functions in these cells, signifying a crucial regulatory role of β-catenin in monocyte function during CHI. We also observed a positive correlation between HBsAg level and frequency of β-catenin + -monocytes in chronically HBVinfected patients. It seems plausible that high or low β-catenin concentration exert distinct effects on the expression of different functional markers of monocytes and thus could account for the difference in monocyte properties in CHB and IC.
The monocytes in CHB/IT displayed enhanced CCR2-expression and thus appeared to have high propensity to migrate to in amed liver. Interestingly, in regorafenib-resistant cancer cells, β-catenin was found to be a direct transcriptional activator of CCR2-expression [14] and it is conceivable that the upregulation of CCR2 in the monocytes of CHB/IT is related to β-catenin activation. In support to this conclusion, we also noticed a greater prevalence of β-catenin-expressing monocytes in liver tissues of CHB patients compared to HC.
Apart from the innate effector functions, monocytes can also act as a bridge to the adaptive immune system. Colocalization of CD14 + -cells with clusters of CD4 + T-cells had been reported at sites of in ammation [5], suggesting an interaction between these cells in vivo. We showed that monocytes from CHB/IT cause preferential skewing of CD4 + T-cell compartment towards Treg-and Th2-dominated phenotype and this together with profound suppression of Th1-polarization facilitate the chronicity of infection.
Finally, we tested whether Tenofovir therapy could modulate the monocyte phenotype/function in CHB patients. Our results highlighted that the monocyte-subsets continued to retain their baseline characteristics after one-year therapy and no reduction in HBsAg or IL-4 level was perceived. The persistence of immune suppressive cascade in CHB patients even after Tenofovir treatment may represent a pivotal risk factor for the advancement of liver diseases.
Taken together, this study has illustrated a multitude of functional defects in monocytes in different phases of CHI. It thus appears that therapeutic targeting of intramonocytic β-catenin or reducing the circulating HBsAg levels or modulation of the cytokine milieu in chronically HBV-infected patients might be successful in restoring the monocyte function, clearance of HBV and cure of HBV-induced liver disease.     TLR-2, IL-12, IL-10, CD64, iNOS in sorted CD14 + monocytes of healthy control (HC) treated with or without β-galactosidase (β-gal) (20μg/ml), recombinant Hepatitis B surface antigen (rHBsAg) (10μg/ml and 20μg/ml) for 48 hours. Statistical signi cance was assessed by one way ANOVA followed by Tukey's Multiple Comparison Test (**P < 0.005 and ***P < 0.0001). (c) Concentration of serum IL-4 and TNF-α in HC, IT, CHB and IC and grouped bar diagram of relative percentages of HLADR + CD14 + monocytes expressing IL-12 and IL-10 in PBMC of HC treated with or without rIL4 (5ng/ml or 25ng/ml) and TNF-α (50ng/ml). Statistical signi cance was assessed by one way ANOVA followed by Tukey's Multiple Comparison Test and two way ANOVA (**P < 0.005 and ***P < 0.0001).