Identification of miRNAs associated with dendritic cell dysfunction during acute and chronic hepatitis B virus infection

The uptake or expression of hepatitis B virus (HBV) proteins by dendritic cells (DCs) is considered important for disease outcome. Differential expression of microRNA (miRNA) may have a role in viral persistence and hepatocellular injury. The miRNA expression was investigated by microarray in DCs from different stages of HBV infection and liver disease namely, immune active (IA; n = 20); low replicative (LR; n = 20); HBeAg negative (n = 20); acute viral hepatitis (AVH, n = 20) and healthy controls (n = 20). miRNA levels were analyzed by unsupervised hierarchical clustering and principal component analyses and validated by quantitative polymerase Chain Reaction (qPCR). The miRNA‐messenger RNA (mRNA)regulatory networks identified 19 miRNAs and 12 target gene interactions in major histocompatibility complex and other immune pathways. miR‐2278, miR‐615‐3p, and miR‐3681‐3p were downregulated in the IA group compared to healthy control, miR‐152‐3p and miR‐3613‐3p in the LR group compared to IA group and miR‐152‐3p and miR‐503‐3p in HBe negative compared to LR group. However, miR‐7‐1‐1‐3p, miR‐192‐5p, miR‐195‐5p, and miR‐32‐5p in LR, miR‐342‐3p, and miR‐940 in HBe negative, and miR‐34a‐5p, miR‐130b‐3p, miR‐221‐3p, miR‐320a, miR‐324‐5p, and miR‐484 in AVH were upregulated. Further, qPCR confirmed changes in miRNA levels and their target genes associated with antigen processing and presentation. Thus, a deregulated network of miRNAs‐mRNAs in DCs seems responsible for an impaired immune response during HBV pathogenesis.


| INTRODUCTION
The hallmark of chronic hepatitis B (CHB) infection is the lack of a robust hepatitis B virus (HBV)-specific CD8+ and CD4+ T-cell response leading to the induction of virus-specific T and B cell responses. HBV can downregulate host immune responses by interfering with toll-like receptors (TLR) expression and signaling pathways or via inhibition of interferon response. 1 Besides, the expression of HBV proteins in/by dendritic cell (DC) can lead to impairment of their immune-stimulatory function, causing insufficient/ineffective priming of CD4+ T cells, possibly contributing to the persistence of viral hepatitis. 2 DCs do not support HBV replication but can take up HBV proteins which may affect DC activity. 3 Binding and uptake of HBV virions by DCs may also be responsible for the impaired function of DC in HBV-infected patients. Functional alterations in DC or its precursors from chronic HBV patients also contribute to the impaired HBV-specific immune response. 4,5 Patients with advanced fibrosis or cirrhosis show a decrease in DC numbers in peripheral blood of chronic HBV patients compared to healthy individuals. 5,6 Not surprisingly, DCs from CHB patients are less efficient in inducing T-cell proliferation in vitro than those isolated from healthy individuals 6,7 and show low expression of IFN-α and DC-specific intercellular adhesion molecule 3-grabbing non-integrin which may affect initial events of immune activation. 8 However, some clinical studies have shown that DCs in chronic hepatitis B carriers are functionally intact. 9 Thus, DCs appear to be a central player in initiating and controlling the magnitude and quality of adaptive immune responses. 4 However, the molecular mechanisms associated with DC dysfunction in CHB patients remain poorly understood.
Aberrant expression of certain micro RNAs (miRNAs) can lead to pathological consequences in many liver pathologies including viral hepatitis, acute and chronic liver diseases, and hepatocellular carcinoma. 10,11 Therefore, a detailed investigation on the expression of miRNAs in the DCs should provide an in-depth understanding of the molecular mechanisms associated with immune-compromised status in CHB patients and also useful in the management of liver diseases. 12,13 The natural history of chronic HBV infection is a dynamic process that can progress gradually or rapidly consisting of four phases of the underlying liver disease, of variable duration and outcome. All phases have been pathogenetically linked to the level of HBV replication and the strength and targets of the host immune reactivity against the replicating HBV. In childhood infection, the first phase is called as "immune-tolerant phase" characterized by normal serum levels of alanine aminotransferase (ALT) and very high levels of HBV DNA with minimal fibrosis and inflammation in the liver. The majority of chronically infected individuals can develop "immune active (IA) phase" (second phase) characterized by elevated ALT and high HBV DNA levels and persistent immune-mediated necroinflammatory activity. The third phase is called "HBeAgnegative chronic infection" characterized by low-level HBV DNA, raised ALT with significant liver disease on liver biopsy. The fourth phase is called a "LR phase," characterized by low HBV DNA level, persistent normal ALT with no significant liver disease on liver biopsy. Here, we have analyzed the aberrant expression of miRNA  Exclusion criteria included co-infection with hepatitis C, hepatitis D, or HIV; decompensated liver disease; chronic hepatitis B with Child-Pugh B and C; liver disease of unknown etiologies; serum creatinine more than 1.5 times upper limit of normal; hemoglobin <10 g/dl; platelet and WBC counts were <70,000 and <3000 per cubic millimeter respectively; no serious concurrent medical illnesses like malignancy, severe cardiopulmonary disease, uncontrolled diabetes mellitus, alcohol consumption more than 20 g/day.

| Isolation of dendritic cells and RNA
DCs were isolated from freshly separated PBMCs using a blood DC     | 3699 >90% purity was used for isolating total RNA and miRNA as described previously. 13

| Analysis of miRNA expression
The expression of miRNA was determined using Agilent´s human miRNA microarray version V16 as described earlier. 13 Unsupervised hierarchical clustering of differentially expressed genes between patient groups were done using Pearson uncentered algorithm with average linkage rule. The pathway enrichment analysis of miRNAs was done using DIANA-miRPath v3.0 while the miRNA:mRNA gene regulatory network was visualized using CytoScape V 2.8.2. 13

| Quantitation of miRNA and target gene expression
The levels of miRNAs and their target mRNAs isolated from DCs were measured by real time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) as described previously. 13 The details of miRNA and gene primers can be found in Tables S1 and S2 respectively. The results were analyzed by the ΔΔCt method. 14  Posthoc comparison revealed that in the AVH group serum bilirubin, ALT, aspartate aminotransferase (AST) and albumin levels was statically significant compare to all other groups (Table 1).

| Analysis of differentially expressed miRNAs and their clusters in DCs
The miRNA array data were analyzed by principal components analysis (PCA) as well as by unsupervised hierarchical clustering to distinguish (similarity vs. difference) between various patient groups. 15 Each patient group formed distinct clusters suggesting a high degree of reproducibility within replicate samples ( Figure 1A).
The hierarchical clustering analysis also showed distinct miRNA expression patterns as evident from greater branch length between healthy control, AVH, and chronic hepatitis B patients. Further, among chronic HBV patients, specific clustering was observed for HBeAg negative, IA and LR groups ( Figure 1B).
Next, we analyzed the distribution of differentially expressed miRNAs for specificity and commonality in various patient groups and healthy subjects (Table S3). In immune active group compared to healthy controls, just nine miRNAs were downregulated (Table S4A).
However, in low replicative patients, as compared to the immune active group, 44 miRNAs were upregulated and 4 miRNAs were downregulated (Table S4B). Between HBeAg negative and low replicative groups, 12 miRNAs were upregulated and 8 miRNA were downregulated (Table S4C). Importantly, in the AVH group compared to an immune active group, 58 miRNAs were upregulated and 27 miRNAs were specifically downregulated (Table S4D) Figure 2B).
The differentially expressed DC miRNAs were predicted to engage multiple cellular and signaling networks essential for antigen processing and presentation, MHC I signaling, type I interferon signaling, TLR signaling, T cell receptor signaling, MAP kinase, and PI3K-Akt signaling and apoptosis and so forth. (Figure 4).

| Pathway enrichment analysis for differentially expressed miRNAs in DCs
Gene pathway enrichment for differentially expressed miRNAs was de-  Figure S3E and Table S5).  Figure 5D).

| DISCUSSION
The phenotypic and functional impairment of DCs in CHB patients have been reported earlier by several research groups. 5,8,19 The impaired DC function is often correlated with ineffective antiviral response and thus, viral persistence in CHB patients 7. The degree of liver inflammation in CHB patients is found to be inversely proportional to DC numbers in peripheral blood owing perhaps to their migration from peripheral blood to the inflammatory site in the  as also corroborated by gene expression analysis ( Figure 5A). In fact, a low miR-2278 level is reported to correlate with enhanced calreticulin (CALR) gene essential for the production of MHC class I proteins. 23 Since expression of HLA antigens are critical for the cytotoxic T cell controlled immune response against viruses and other pathogens, 24 its dysregulation could be associated with increased risk, rapid progression, and severity of liver diseases. Importantly, there are no reports implicating miR-615-3p, and miR-3681-3p in the regulation of HLA, calnexin, caregulin, or heat shock protein.

CONFLICT OF INTERESTS
The authors of this article declare no relationships with any companies whose products or services may be related to the subject matter of the article.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
F I G U R E 5 Validation of expression of miRNAs and their target genes in DCs of hepatitis B patients. miRNAs and their target genes were analyzed by RT-qPCR using DC RNA samples and specific primers (Tables S3 and S4). U6 and 18S ribosomal RNAs were used as internal controls for miRNAs and mRNAs respectively. Bar charts represent results of miRNAs/mRNAs in immune active versus healthy control (A), Low replicative versus immune active (B), HBe negative compared to low replicative (C) and AVH versus immune active groups (D). All experiments were performed in triplicate. Data are shown as mean ± SD. ** and * represent significance with p values < .01 and < .05 respectively. AVH, acute viral hepatitis; DC, dendritic cell; HBV, hepatitis B virus; m RNA, messenger RNA; miRNA, micro RNAs; RT-qPCR, real time quantitative reverse transcriptase polymerase chain reaction