Regulatory Role of B Cells and Its Subsets in Hepatitis E Virus Infection

Antibodies as well as memory B cells are the potential correlates of a protective immune response against hepatitis E virus (HEV) infection. Literature on the role of B regulatory cells (Bregs) in acute viral infections is limited. We have evaluated the role of IL-10 expressing Bregs in HEV infection. A total of 108 acute hepatitis E patients, 55 hepatitis E recovered individuals and 128 HEV naïve healthy controls were enrolled. The percentages of peripheral CD19+, immature CD19+CD24hiCD38hi, mature CD19+CD24intCD38int and memory CD19+CD24hiCD38− B cells were analyzed by flowcytometry. Intracellular cytokine staining for IL-10 and TGF-β, HEV-rORF2p specific T cell response (IFN-γ expression) pre/post IL-10/IL-10R blocking and CD19+IL-10+ B cells-depletion based assays were carried out to assess the functionality of Bregs. The percentage of HEV-rORF2p specific immature B cell phenotype was significantly higher in acute hepatitis E patients compared to hepatitis E recovered individuals and controls. Significantly higher IL-10 expression on B and HEV-rORF2p stimulated immature B cells of acute hepatitis E patients compared to controls indicated that Bregs are functional and HEV-rORF2p specific. Enhanced IFN-γ expression on CD8+ T cells upon IL-10/IL-10R blocking and also post CD19+IL-10+ B cells depletion suggested that CD3+CD8+IFN-γ+ T cells corroborate the regulatory potential of Bregs via IL-10 dependent mechanism. We have identified HEV specific functional, immature CD19+CD24hiCD38hi B cells having IL-10 mediated regulatory activities and a potential to modulate IFN-γ mediated T cell response in Hepatitis E. The prognostic/pathogenic role of Bregs in recovery from severe hepatitis E needs evaluation.


Introduction
Hepatitis E, a liver disease caused by hepatitis E virus (HEV) has emerged as a global health challenge, contributing to over 50% of the acute viral hepatitis cases in the endemic areas [1]. Though majority of patients with HEV infection follow a self-limiting course, very few develop severe form of hepatitis that may progress to fulminant hepatic failure (FHF) with a mortality rate of 20-30% in the third trimester of pregnancy. Chronic HEV infection and associated complications are also reported in immunocompromised and organ transplant recipients [2]. A remarkable deficit in knowledge still remains with respect to hepatitis E disease pathogenesis and control.
Association of B cells with antibody production and humoral immunity is a classic phenomenon. Robust anti-body response to HEV-open reading frame 2 (HEV-ORF2) protein in the recovered individuals from HEV infection has been the basis for the development of vaccine against hepatitis E [3]. B cells are typically characterized as antibody producing cells and are known for their ability to function as secondary antigen presenting cells. However, one of their subsets defined as 'B regulatory cells' (Bregs), having regulatory potential have emerged in the recent years [4]. The population of Bregs is relatively small (~less than 10%) under physiological conditions, but show substantial expansion in both patients and murine models of chronic inflammatory diseases, autoimmune diseases, infection, transplantation and cancer [5][6][7][8]. Bregs studies in human immunodeficiency virus (HIV) [9] and hepatitis B virus (HBV) infections [4] have indicated that the regulatory activities of Bregs are majorly interleukin-10 (IL-10) cytokine mediated, however immune regulation by production of transforming growth factor-beta (TGF-β) [10] and interleukin-35 (IL-35) [11] cytokines have also been reported. Phenotypically, subsets of Bregs have been defined as CD19 + CD24 hi CD38 hi immature/transitional B cells, CD19 + CD24 int CD38 int as mature B cells and CD19 + CD24 int CD38as memory B cells [12][13][14].
Our previous studies have reported higher levels of regulatory T cells (Tregs) and enhanced IL-10 and TGF-β cytokine production in acute HEV infection [15]. Subsequently, Tregs were found to be functional and exhibited TGF-β mediated suppressive activity [16]. Bregs are actively involved in inhibition of Th1 cells activation, Th17 cells differentiation and promotion, and maintenance of the Tregs population [17,18]. A role of Bregs in hepatitis E virus infection has not been explored. The current study presents a comprehensive investigation of B regulatory cells in patients with hepatitis E infection. We have assessed (1) frequency and phenotypic markers of B regulatory cells and (2) functionality of B regulatory cells in acute hepatitis E patients, hepatitis E recovered individuals and healthy controls.

Materials and Methods
2.1. Patients and Controls. The study was approved by the Institutional Ethical Committee for Research on Humans, based on the guidelines set by the Indian Council of Medical Research (ICMR), New Delhi. Informed written consent was obtained from all the participants in accordance with the Declaration of Helsinki.
The study population (n =291) included the following groups: acute hepatitis E patients (n =108), hepatitis E recovered individuals (n =55) and anti-HEV negative healthy controls (n =128). The study groups were classified based on standard clinical and biochemical criteria [19]. Patient demographics are shown in Table 1.
Inclusion criteria: Acute hepatitis E patients: The patients were presenting with symptoms like icterus, dark coloured urine, fever, elevated alanine aminotransferase (ALT) levels (normal range 4-40 IU/L) and/or bilirubin >1 mg/mL in serum and/or presence of bile salts and pigments in urine. These patients were diagnosed positive for IgM antibodies against hepatitis E virus (anti-HEV IgM) and positive/negative for IgG antibodies against hepatitis E virus (anti-HEV IgG) by ELISA. Acute patients were from various outbreaks reported in India. Hepatitis E recovered individuals: The recovered cat-egory constituted of individuals with past history of acute hepatitis E infection and were from previously investigated hepatitis E outbreaks. These individuals were positive for anti-HEV IgG antibodies and negative for anti-HEV IgM antibodies with normal plasma ALT levels. The post onset days of illness for this group ranged from 0.2-2 years. Healthy controls: The control group constituted of age and sex matched apparently healthy individuals with no recent or past history of hepatitis E infection i.e. they were negative for anti-HEV IgM and IgG antibodies with normal ALT levels. The time of collection of healthy controls were same as that of the other two groups.
Exclusion criteria: Individuals with other viral infections including hepatitis A virus (HAV), HBV, HCV and HIV infections were excluded.

Serological and Molecular
Assays. All samples were screened by in-house ELISA for anti-HEV IgM and IgG antibodies as described previously [20]. ALT levels were measured using GPT (ALT) test kit (Span Cogent Diagnostics, India) in all plasma samples as per manufacturer's protocol [21]. Plasma HEV viral load was determined by Taqman reverse transcription polymerase chain reaction as previously reported [22].

Antigen (Recombinant HEV-ORF2 Protein) Preparation.
Recombinant ORF2 protein (rORF2p) was expressed in Sf9 insect cells and purified by anion exchange high performance liquid chromatography using AKTA BASIC 100 system (Amersham Biosciences, UK) as described previously [23]. rORF2p was used as the antigen for coating in ELISA for detection of anti-HEV IgM and IgG antibodies and as a recall antigen in the functional assays.

Peripheral Blood Mononuclear Cells (PBMCs) Isolation.
PBMCs were isolated using Ficoll-Hypaque density gradient centrifugation method and counted in the trypan blue dye exclusion method and the cells with more than 98% of viability were immediately used for assays. Isolated plasma was used for serological and biochemical assays and the PBMCs were used for all assays. The number of samples from each category used for different assays is shown in Supplementary Figure S1.

Frequencies of HEV-rORF2p Stimulated B Cells and Its
Subsets. PBMCs from 40 acute hepatitis E patients, 29 hepatitis E recovered individuals and 50 healthy controls were cultured For each sample, 50,000 events were acquired in BD FACS Aria-II flow cytometer and data were analyzed using BD FACS Diva software (BD Biosciences, CA, USA). Data from stimulated cells were analyzed after normalization with unstimulated cells. The gating strategy is depicted in (Supplementary Figure S2).

Quantification of IL-10 Cytokine in Plasma Samples.
Plasma concentrations of IL-10 cytokine were determined in representative plasma samples from each category of study subjects (acute hepatitis E patients, n =20; hepatitis E recovered individuals, n =20 and healthy controls, n =27) using a Bio-plex Multiplex Immunoassay System (Bio-Rad, Hercules, CA, USA) using a Bioplex ProTM Human Cytokine 27-plex assay kit as reported previously [24,25] as per the manufacturer's instructions.
The gating strategy is shown in (Supplementary Figure S3).

CD19 + IL-10 + Cells Depletion-Based B Cell Functional
Assay. To determine functionality of IL-10 expressing B cells, PBMCs from 33 acute hepatitis E patients, 22 hepatitis E recovered individuals and 52 healthy controls were processed for isolation of B cells. B cells were positively enriched from PBMCs using human CD19 MicroBeads (Miltenyi Biotec, CA, USA) and were separated over MACS MS Column (Miltenyi Biotec, CA, USA). The purity of the sorted population was 95-99%, as determined by flowcytometry. Isolated CD19 + and CD19cells were stimulated with/without HEV-rORF2p (10 μg/mL) for 24 h at 37°C followed by detection and isolation of IL-10 expressing B cells using human IL-10 cytokine secretion assay (Miltenyi Biotec, CA, USA) as per manufacturer's protocol. Further, magnetically sorted CD19 + IL-10 + and CD19 -IL-10cells were stimulated with/ without HEV-rORF2p (10 μg/mL) and co-cultured with PBMCs derived from the same patients/controls at 1 : 1 ratio in round bottom 96-well plate (Nunc, Denmark) in the presence of recombinant IL-2 (50 IU/mL) for 5 days. This was followed by intracellular staining with IFN-γ Alexa Fluor 647 antibody (BD Biosciences, CA, USA). The frequencies of HEV-rORF2p specific IFN-γ expression on CD4 and CD8 T cells in the presence and absence of IL-10 + B cells was determined. Isotype and FMO controls were used in all sets of experiments. The gating strategy is shown in (Supplementary Figure S3).       (Figure 5(D)) were also found to be comparable among the study groups.
The above results collectively suggest that Bregs are functional and IL-10 was noted to be the predominant cytokine expressed on Bregs in acute HEV infection.

Lack of Correlation between HEV Viral Load, ALT Levels, anti-HEV IgM/IgG Titers and HEV-rORF2p
Stimulated B Cells and Its Subsets Frequencies in Acute Hepatitis E Patients. There was no correlation between HEV viral load, ALT levels, anti-HEV IgM/IgG titers and HEV-rORF2p stimulated B, immature B, mature B and memory B cells frequencies in acute hepatitis E patients as assessed by spearman correlation analysis. Upon assessing the role of viral replication with the IL-10 producing B cells, it was observed that only a non-significant proportion of viremic acute hepatitis E patients had IL-10 producing B cells (17/36, 47.22%), further suggesting no association of viral replication with Bregs functionality in the self-limiting acute hepatitis E patients.

Discussion
The present study is an in-depth assessment of the role of B regulatory cells in self-limiting HEV infection where the immunoregulatory role of Bregs is being presented.
The percentages of B cells remained unaltered among our study cohort which goes in parallel with a study from our group evaluating role of B and memory B cells in HEV infection [26]. Similar observation was noted by Das et al. in a cohort of chronic hepatitis B patients [4]. Significant augmentation of HEV-rORF2p specific immature CD19 +-CD24 hi CD38 hi B cells phenotype in self-limiting acute hepatitis E patients compared to hepatitis E recovered individuals and healthy controls of our study mirrors similar observations documented in HBV [4], HIV and hepatitis C virus   BioMed Research International (HCV) infections [27,28]. Freshly isolated human IL-10 + Bregs have predominantly been detected within the CD24 or CD27 B cell subpopulations; Bregs were subsequently characterized as memory CD19 + CD24 hi CD27 + B cells by Tedder [29]. The discrepancies observed in phenotypes of Bregs between studies could partially be due to absence of a perfect panel of markers for characterization of this subset of B cells. However, though different phenotypes of human Bregs have been reported in different disease settings, only certain B cells have been shown to produce IL-10 [14,30]. Enriched HEV-rORF2p stimulated immature B cells in acute hepatitis E patients compared to both recovered individuals and controls, in spite of having comparable HEV-rORF2p stimulated CD19 + B cells among the study population is suggestive of the theory that a subset of B cells could have expanded from the existing pool of B cells. This is also suggestive of second model of Bregs development and differentiation pointing towards differentiation and expansion of this subset in response to external stimulation, HEV-rORF2p antigen in this case [31]. Existence of enhanced HEV antigen specific immature CD19 + CD24 hi CD38 hi B cells and mature CD19 + CD24 int CD38 int B cells in acute hepatitis E patients' group compared to recovered group may support heterogeneous existence of phenotypes of Bregs [31].
We chose to study immature B cells as the putative regulatory subset in HEV infection. IL-10 production has remained the hallmark of Bregs regulatory function, though TGF-β, IL-35 and IL-17 mediated several regulatory mechanisms have also come to light in recent years [32]. Simultaneous expressions of regulatory cytokines like IL-10 and TGF-β on Bregs have been demonstrated [33]. IL-10 is known to have a prognostic/detrimental role in infections [34]. Significantly higher IL-10 expression on CD19 + B and HEV-rORF2p stimulated CD19 + immature B cells of acute hepatitis E patients of the current study indicated that Bregs are functional. Further, a positive correlation was also observed between plasma IL-10 concentrations and HEV-ORF2p stimulated immature B cells frequencies in acute hepatitis E patients. We next demonstrated that IL-10 + B cells were of immature B cells (CD19 + CD24 hi CD38 hi ) phenotype only. It is important to note that though frequencies Control Recovered Figure 8: Flow cytometric analysis showing the effect of depletion of CD19 + IL-10 + cells on HEV-rORF2p specific CD4 and CD8 T cells response in terms of IFN-γ expression. B cells were magnetically sorted from PBMCs from 33 acute hepatitis E patients, 22 hepatitis E recovered individuals and 52 healthy controls, and were stimulated with/without HEV-rORF2p followed by detection and isolation of IL-10 expressing B cells. Sorted CD19 + IL-10 + and CD19 -IL-10cells were co-cultured with PBMCs derived from the same patients/controls and were intracellularly stained to determine the expression of IFN-γ on CD4 + T cells (%CD3 + CD4 + IFN-γ + ) and CD8 + T cells (%CD3 + CD8 + IFN-γ + ). Expression of IFN-γ on HEV-rORF2p specific CD4 T cells (CD3 + CD4 + IFN-γ + ) in (a) acute hepatitis E patients, (b) hepatitis E recovered individuals, (c) healthy controls and CD8 T cells (CD3 + CD8 + IFN-γ + ) in (d) acute hepatitis E patients, (e) hepatitis E recovered individuals, (f) healthy controls were assessed pre vs. post CD19 + IL-10 + cells depletion. Pair wise comparisons were done on matched samples wherein dots represent individual values. 9 BioMed Research International of HEV-rOFR2p stimulated mature B cells were more in acute hepatitis E patients compared to hepatitis E recovered individuals their functionality was comparable as indicated by the comparable frequencies of IL-10 + mature B cells. Interestingly, IL-10 was noted to be the predominant cytokine expressed on Bregs in acute HEV infection. Similarly, elevated levels of IL-10 producing B cells were observed in patients with HIV, HCV and lymphocytic choriomeningitis virus (LCMV) infections and the increased IL-10 levels were correlated with suppression of T cells [35]. Different methods of stimulation could impact the induction of IL-10 production by Bregs [9]. Upon stimulation with CpG-B, an agonist of TLR-9, we observed an induction of TGF-β expression on B cells and these TGF-β + B cells were enriched in acute hepatitis E patients compared to recovered individuals and healthy controls. However, there was no detection of HEV-rORF2p specific TGF-β expression on B cells or B cells subsets which explain that most of the primary functions of Bregs cells in HEV infection are dependent on expression and release of IL-10. It is quite reasonable that Bregs act as the main source of IL-10 and may be considered as an immune regulator in self-limiting acute HEV infection.
HEV RNA positivity in sera of hepatitis E patients lasts beyond normalization of transaminases suggesting that liver injury is independent of viral replication [36]. We attempted to associate viremia and liver damage with the functionality of Bregs in acute hepatitis E patients. No correlation of increased ALT levels and viral replication with IL-10 producing B cells indicated that functional Bregs have probably no impact on the liver damage of the acute hepatitis E patients.
Bregs mediated CD8 + T cells inhibition and suppression of inflammation has been extensively studied in infectious diseases and cancers [32]. Das et al. have documented that sorted CD19 + CD24 hi CD38 hi cells suppressed HBV-specific CD8 T cell responses in an IL-10 dependent manner in chronic HBV infection [4]. A significant increase in IFN-γ expression on CD8 + T cells and no effect on IFN-γ expression on CD4 + T cells upon blocking of both IL-10 and IL-10R and depletion of CD19 + IL-10 + B cells in our study is an important observation. Current data clearly indicate that Bregs in Hepatitis E mediate T cell modulation via CD8 + T cells.
It could be suggested that CD3 + CD8 + IFN-γ + T cells in acute hepatitis E patients corroborated the regulatory potential of Bregs via IL-10 dependent mechanism. These observations of our study are partly in concordance as reported in HIV infection where Bregs suppress HIV-1 specific CD8 + T-cell responses via IL-10 production and possibly PD-L1 expression [37]. In the past, we have proposed a beneficial role for Tregs in self-limiting acute HEV infection based on the higher percentage of HEV specific, functional Tregs in self-limiting HEV patients [15,16,38] and absence of the Tregs in the liver of fatal hepatitis E FHF patients [39]. We had further elucidated TGF-β1 as the regulatory molecule responsible for enhancement of Tregs in self-limiting HEV infection and use of TGF-β1 was suggested as a possible supplement for boosting Treg response towards recovery from severe hepatitis E [16]. IL-10 producing B cells are known to play a major role towards the induction and maintenance of Tregs [9]. Our current results definitely suggest participation of Bregs in the maintenance of immune homeostasis and may allude possible existence of Bregs-Tregs interactions in HEV disease pathogenesis. It is tempting to speculate the mechanism of interaction among these two groups of regulatory cells.
The limitation of this study was that the role of Bregs was not explored among FHF patients with differential outcomes. Indeed, sans Bregs data of FHF patients, the prognostic/pathogenic role of Bregs in HEV infection remains an avenue unexplored. In the absence of any information about the composition of B cells among PBMCs before stimulation, the answers to questions including the differences due to composition of B cells before and after stimulation, cell survival/cell proliferation/effect of Bregs on induction of B cell populations and antibody generation are beyond the scope of the current dataset and can be considered as a limitation of the study.
In a nutshell, we have identified HEV specific functional, immature CD19 + CD24 hi CD38 hi B cells having IL-10 mediated regulatory activities that have the potential to modulate IFN-γ mediated T cell response in Hepatitis E infection.

Data Availability
The data used to support the findings of this study are included within the supplementary information file.

Conflicts of Interest
The authors declare that they have no competing interests.