Characterization of inflammatory protein expression patterns and their association with viral DNA load in hepatitis B virus infection via Olink proteomics analysis

Abstract

Objectives: To investigate the differential expression of inflammatory proteins in the sera of patients with chronic hepatitis B (CHB) using Olink Targeted Proteomics and to explore their correlations with viral deoxyribonucleic acid (DNA) load.

Methods: This retrospective study included 66 CHB patients and 22 healthy controls, with medical records collected between January and December 2023 at Nanning Customs, China. Viral DNA loads were quantified using real-time polymerase chain reaction (PCR), and 92 inflammatory proteins were profiled using Olink Targeted Proteomics.

Results: A total of 38 proteins were differentially expressed between CHB patients and healthy controls, of which 7 were upregulated and 31 were downregulated. Correlation analysis revealed that viral DNA load was positively associated with the expression of osteoprotegerin (OPG) (P = 0.021) and chemokine (C-X-C motif) ligand 9 (CXCL9) (P = 0.002), and negatively associated with interleukin-10 (IL-10) (P = 0.007), cluster of differentiation 40 (CD40) (P = 0.004), and caspase-8 (CASP8) (P = 0.020). Functional enrichment analysis indicated that these proteins were mainly enriched in neutrophil chemotaxis, granulocyte migration, chemokine signaling pathways, cytokine activity, chemokine activity receptors, and tumor necrosis factor (TNF) receptors.

Conclusions: Specific inflammatory proteins, including OPG, CXCL9, IL-10, CD40, CASP8, are associated with viral DNA load in CHB patients. These findings enhance the proteomic understanding of HBV pathogenesis and may offer potential therapeutic targets and biomarkers.

Keywords: Hepatitis B virus; bioinformatics analysis; inflammatory factors; proteomics.

Figure 1

Flow chart. HBV: hepatitis B virus; HBsAg: hepatitis B surface antigen; ALT: alanine aminotransferase; AST: aspartate aminotransferase; TBIL: total bilirubin; PEA: proximity extension assay; NPX: Normalization Protein Expression; GO: gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 2

Inclusion criteria based on quality control outcomes of serum samples. The horizontal axis represented the sample names, while the vertical axis denoted the NPX values. The light blue color indicated samples flagged for QC alerts and the dark blue color represented samples that had successfully passed the QC criteria. QC: quality control; NPX: Normalization Protein Expression.

Figure 3

Differentially expressed inflammation-related proteins between the HBV and healthy control groups. The blue points represented downregulated differentially expressed proteins, while the orange points indicated upregulated differentially expressed proteins. CDCP1: cub domain-containing protein 1; LAP TGF-beta-1: latency-associated peptide transforming growth factor-beta-1; IL-10: interleukin-10; CXCL9: chemokine (C-X-C motif) ligand 9; OPG: osteoprotegerin; TNFRSF9: tumor necrosis factor receptor superfamily member 9; AXIN1: axis inhibition protein 1; OSM: oncostatin m; TGF-alpha: transforming growth factor-alpha; ST1A1: stanniocalcin-1; SIRT2: sirtuin 2; TNFSF14: tumor necrosis factor superfamily member 14; IL-8: interleukin-8; STAMBP: signal transducing adapter molecule-binding protein; IL-15RA: interleukin-15 receptor alpha.

Figure 4

Bubble plot of GO pathway enrichment analysis for differential protein-coding genes between the HBV and healthy control groups. The x-axis signified the Rich factor, representing the count of differential proteins within each GO term. The y-axis depicted the GO Term, indicating functional annotation. Point size scaled with protein quantity, while color intensity reflected enrichment significance (P < 0.05 indicating statistical significance). This figure highlighted the top 30 most significantly enriched functions. GO: gene ontology; CXCR: C-X-C motif chemokine receptor; CCR: C-C motif chemokine receptor.

Figure 5

KEGG pathway analysis of differential proteins between the HBV-infected group and the healthy control group. The x-axis represented the Rich factor, indicating the count of differentially expressed proteins within each KEGG pathway. The y-axis, labeled “Pathway”, annotated the respective pathways. Point size was scaled with the number of proteins, while color intensity reflected the significance of enrichment, with P < 0.05 indicating statistical significance. This plot highlighted the top 30 most significantly enriched functions. TNF: tumor necrosis factor; NF-kappa B: nuclear factor kappa-light-chain-enhancer of activated b cells; JAK-STAT: janus kinase-signal transducer and activator of transcription; IL-17: interleukin-17; DNA: deoxyribonucleic acid; EGFR: epidermal growth factor receptor.