Resistin reinforces interferon λ-3 to eliminate hepatitis C virus with fine-tuning from RETN single-nucleotide polymorphisms

Abstract

The effect of resistin (RETN) on the response to anti-HCV therapy remains unclear. A prospective cohort study was performed using 655 consecutive HCV patients, of whom 513 had completed a course of interferon-based therapy. Multivariate and GEE analyses revealed four RETN single-nucleotide polymorphisms (SNPs), rs34861192, rs3219175, rs3745367 and rs1423096, to be synergistically associated with resistin levels. After adjusting for co-factors such as interferon λ-3 (IFNL3)-rs12979860, the resistin level and the hyper-resistinemic genotype at the 4 RETN SNPs were positively and negatively associated with a sustained virological response (SVR), respectively. RETN-rs3745367 was in linkage disequilibrium with IFNL3-rs12979860. Compared to non-SVR patients, SVR patients had higher levels of pre-therapy resistin, primarily originating from intrahepatic lymphocytes, stellate cells, Kupffer cells, hepatic progenitor cells and hepatocytes. This difference diminished over the course of therapy, as only SVR patients exhibited a 24-week post-therapy decrease in resistin. Both resistin and IFNL3 mRNAs were upregulated, but only resistin mRNA was upregulated by recombinant resistin in peripheral blood mononuclear cells with and without hyper-resistinemic genotypes of the 4 RETN SNPs, respectively. Fine-tuned by RETN SNPs, intrahepatic, multi-cellular resistin reinforced IFNL3 in eliminating HCV via immunomodulation to counteract pro-inflammation. These results encourage the development of novel resistin-targeted anti-viral agents.

Figure 1. Resistin levels and receiver operating characteristic (ROC) analyses.

(a) Box-and-whisker plots of pre-therapy resistin levels in chronic hepatitis C (CHC) patients according to resistin (RETN) scores. The outliers are shown as circles or stars. (b–d) Longitudinal trend of mean resistin levels (+/−95% confidence interval) in CHC patients according to RETN scores. Red lines: resistin levels of CHC patients with a sustained virological response (SVR); black lines: resistin levels of CHC patients without SVR. Blood draw time points: 1, 2 weeks before therapy; 2, after 4 weeks of therapy; 3, after 12 weeks of therapy; 4, after 24 weeks of therapy; 5, after 36 weeks of therapy; 6, after 48 weeks of therapy; 7, after 60 weeks of therapy; and 8, after 72 weeks of therapy. (e,f) ROC analyses of pre-therapy resistin levels in predicting SVR of CHC patients with a RETN score = 0 (e) or RETN score >0 (f).

Figure 2. Immunohistochemical analyses of resistin and other proteins in the livers of chronic hepatitis C (CHC) patients before anti-hepatitis C viral therapy.

(a) Resistin labeling in the liver from a CHC patient without sustained virological response (SVR). (b–f) Serial-section staining for resistin and other proteins in the liver from a CHC patient with SVR. (a,b) resistin staining. Clumps of resistin-positive nonparenchymal cells are labeled with white arrows; resistin-positive hepatocytes are labeled with black arrows. (c) Cluster of differentiation for 43(CD43) staining. (d) α-Smooth muscle actin staining. (e) CD68 staining. CD-68-positive cells (Kupffer cells) are labeled with red arrowheads. (f) Cytokeratin-7 staining. Cytokeratin-7-positive cells (hepatic progenitor cells) are labeled with red arrows.

Figure 3. The association between resistin (RETN) and interferon λ-3 (IFNL3).

(a) Pairwise correlations between genotypes and clinical parameters are shown in a color map. Spearman’s rank correlation coefficients are presented using a spectrum of colors ranging from dark brown to deep purple (the color bar is shown at the right). The HCV genotypes include genotype type 1 (coded as 1) and non-genotype type 1 (coded as 2). SVR: sustained viral response. SNPs are ordered by their locations along chromosome 19. The genotypes of 4 RETN SNPs are highly correlated with each other, forming a mosaic red-brown color block near the center of the color map. IFNL3 genotype “CC” is strongly correlated with RETN-rs3745367 genotype “AA” (Spearman correlation coefficient = 0.172, p = 0.013). (b,c) RNA fold changes for IFNL3 (b) and resistin (c) after stimulation with recombinant resistin (green bars) or IFNL3 (red bars) in peripheral blood mononuclear cells (PBMCs). (d) Proposed mechanism for the pathways associated with resistin and IFNL3 in interferon-based therapy stratified by RETN score. Panel to the left of the dashed line: pathways associated with RETN = 0; panel to the right of the dashed line: pathways associated with RETN >0. IFN: interferon; LD: linkage disequilibrium; Rx: reactions; HSCs: hepatic stellate cells; KCs: Kupffer cells; HPCs: hepatic progenitor cells; red arrows (straight or circle): upregulation. Please see the text for detailed descriptions.