Identification of characteristic hub genes in the immune-active phase of chronic hepatitis B

Chronic hepatitis B (CHB) caused by the hepatitis B virus (HBV) remains a major global health burden. The immune-active (IA) phase, characterized by positive Hepatitis B surface antigens (HBsAg), elevated alanine transaminase (ALT) levels, and detectable HBV DNA, is a pivotal period in the natural history of CHB. Understanding the biological process in this phase is critical for elucidating CHB pathogenesis. With the accumulation of multi-omics data, it has become feasible to identify key molecular targets regulating the host-virus interaction during the IA phase. Identifing these targets provides a critical foundation for developing novel strategies to modulate the host immune response as an important supplement to antiviral therapy.

We integrated bioinformatics and machine learning approaches with clinical sample validation to identify IA-phase-specific hub genes in peripheral monocytes from CHB patients. Combining differential expression analysis and weighted gene co-expression network analysis (WGCNA), we found gene modules significantly correlated with the IA phase from the transcriptomic dataset GSE230397. IA-specific hub genes were subsequently identified using machine learning algorithms. While the potential roles of the four identified hub genes were initially explored via immune infiltration analysis, the expression of the key hub gene, solute carrier family 24 member 4 (SLC24A4), in peripheral classical monocytes from CHB patients in IA phase were specifically validated by single-cell RNA sequencing (scRNA-seq) datasets (GSE182159 and GSE283471) and confirmed by western blot analysis of clinical samples.The function of the key hub gene SLC24A4 was further explored through in silico knockout analysis. Structure-based virtual screening was conducted to identify potential U.S. Food and Drug Administration (FDA)-approved compounds targeting SLC24A4.

Compared to healthy controls (HC), 36 differentially expressed genes (DEGs) were identified as specific to the IA phase in GSE230397. WGCNA highlighted two key modules associated with IA features, from which 22 module-specific DEGs were derived. Machine learning integration pinpointed four hub genes: C-C motif chemokine receptor 5 (CCR5), interleukin 10 receptor subunit alpha (IL10RA), potassium voltage-gated channel subfamily A member 3 (KCNA3), and SLC24A4. Immune infiltration analysis of liver tissue revealed that SLC24A4 expression was positively correlated with the infiltration proportion of pro-inflammatory M1 macrophages and negatively correlated with the infiltration proportion of immunosuppressive M2 macrophages, suggesting its connection with a pro-inflammatory microenvironment. Validation in two independent scRNA-seq datasets and western blot analysis of clinical samples confirmed that SLC24A4 expression in peripheral classical monocytes was significantly higher in IA phase compared with HC. In silico knockout of SLC24A4 in classical monocytes using scTenifoldKnk showed the upregulation of genes involved in histocompatibility antigens class II (MHC class II)-mediated antigen presentation and the regulation of T cell activation. This suggested that SLC24A4 may act as a negative regulator of monocyte activation, and its high expression during the IA phase could represent a compensatory mechanism to restrain excessive inflammation. The possible binding of a FDA-approved compound Rucaparib to SLA24A4 was predicted by virtual screening.

This study revealed distinct transcriptional features of the CHB IA phase and identified SLC24A4 in peripheral classical monocytes as a potential key regulator and a potential drug target for immune modulation in CHB, offering novel insights into both pathogenesis and therapeutic development.