Identification of key genes and pathways associated with different immune statuses of hepatitis B virus infection

Abstract

We aimed to identify key genes and pathways associated with different immune statuses of hepatitis B virus (HBV) infection. The gene expression and DNA methylation profiles were analysed in different immune statuses of HBV infection. Differentially expressed genes (DEGs) and differentially methylated genes (DMGs) were identified, followed by their functional and integrative analyses. The differential expression of IgG Fc receptors (FcγRs) in chronic HBV-infected patients and immune cells during different stages of HBV infection was investigated. Toll-like receptor (TLR) signalling pathway (including TLR6) and leucocyte transendothelial migration pathway (including integrin subunit beta 1) were enriched during acute infection. Key DEGs, such as FcγR Ib and FcγR Ia, and interferon-alpha inducible protein 27 showed correlation with alanine aminotransferase levels, and they were differentially expressed between acute and immune-tolerant phases and between immune-tolerant and immune-clearance phases. The integrative analysis of DNA methylation profile showed that lowly methylated and highly expressed genes, including cytotoxic T lymphocyte-associated protein 4 and mitogen-activated protein kinase 3 were enriched in T cell receptor signalling pathway during acute infection. Highly methylated and lowly expressed genes, such as Ras association domain family member 1 and cyclin-dependent kinase inhibitor 2A were identified in chronic infection. Furthermore, differentially expressed FcγR Ia, FcγR IIa and FcγR IIb, CD3^- CD56⁺ CD16⁺ natural killer cells and CD14^high CD16⁺ monocytes were identified between immune-tolerant and immune-clearance phases by experimental validation. The above genes and pathways may be used to distinguish different immune statuses of HBV infection.

Keywords: DNA methylation profile; Gene expression profile; IgG Fc receptors; T cell receptor signalling pathway; TLR signalling pathway.

Figure 1

The workflow of this study (A) and different expressed genes between different immune state of HBV infection patients (B). (a) Integrative analysis of gene expression and DNA methylation profiles of HBV infection; (b) Analysis of FcγRs expression in different immune state of chronic HBV‐infected patients; (c) Differential expression of FcγRs on immune cells in different statuses of HBV infection

Figure 2

Leucocyte transendothelial migration pathway (A) and Toll‐like receptor signalling pathway (B) enriched by differentially expressed genes between acute and chronic hepatitis B virus (HBV) infection. Pink nodes indicate differentially expressed genes

Figure 3

Analysis of the correlation between clinical indicators and gene expression levels. A, The correlation between alanine aminotransferase (ALT) levels and gene expression levels. B, The correlation between HBV DNA and gene expression levels. C, Key differentially expressed genes associated with ALT levels

Figure 4

The expression of FcγRs in different immune state of chronic HBV infection and analysis of the correlation between clinical indicators and FcγR expression levels. A, The expression levels of FcγRs. B, The correlation between ALT levels and FcγR expression levels. C, The correlation between aspartate transaminase (AST) and FcγR expression levels. Data were expressed as mean ± standard deviation. *P < .05 and **P < .01

Figure 5

The expressions of FcγRs on immune cells in different state of HBV infection. A, Different expression of FcγRs on the subsets of CD3‐CD56+CD16+NK cells between four groups. B, Different expression of FcγRs on the subsets of CD3‐CD19+CD5‐ and CD3‐CD19+CD5+B cells between four groups. C, Different expression of FcγRs on the subsets of CD3‐CD19+CD32+B cells between four groups. D, Different expression of FcγRs on the subsets of CD3‐CD5+CD32+B cells between four groups. E, Different expression of FcγRs on the subsets of CD14highCD16+ monocytes between four groups. One or two columns on the left show a schematic diagram of cell subsets in a flow cytometry experiment

Figure 6

The correlation between FcγR expression on CD14^highCD16⁺ monocytes and different clinical indicators, including ALT, AST, serum HBsAg and serum HBV DNA

Figure 7

The levels of key cytokines (IL‐6, IL‐1β, IL‐10, TNF, MIP‐1β and IL‐12p70) in plasma of patients with different immune statuses of HBV infection. Data were expressed as mean ± standard deviation. *P < .05, **P < .01 and ***P < .001