Interferon-lambda 3 and 4 Polymorphisms Increase Sustained Virological Responses and Regulate Innate Immunity in Antiviral Therapy With Pegylated Interferon-Alpha

Andréa Marques Vieira da Silva, Lucia Elena Alvarado-Arnez, Tamiris Azamor, Leonardo Ribeiro Batista-Silva, Thyago Leal-Calvo, Ohanna Cavalcanti de Lima Bezerra, Marcelo Ribeiro-Alves, Fernanda de Souza Gomes Kehdy, Patrícia Cristina da Costa Neves, Camilla Bayma, Jane da Silva, Alessandro Fonseca de Souza, Marcelo Muller, Elisabete Ferreira de Andrade, Ana Carolina Magalhães Andrade, Eliane Matos Dos Santos, Janaína Reis Xavier, Maria De Lourdes De Sousa Maia, Rolando Páez Meireles, Hugo Nodarse Cuni, Guilherme Becker Sander, Paulo Dornelles Picon, Denise C S Matos, Milton Ozório Moraes, Andréa Marques Vieira da Silva, Lucia Elena Alvarado-Arnez, Tamiris Azamor, Leonardo Ribeiro Batista-Silva, Thyago Leal-Calvo, Ohanna Cavalcanti de Lima Bezerra, Marcelo Ribeiro-Alves, Fernanda de Souza Gomes Kehdy, Patrícia Cristina da Costa Neves, Camilla Bayma, Jane da Silva, Alessandro Fonseca de Souza, Marcelo Muller, Elisabete Ferreira de Andrade, Ana Carolina Magalhães Andrade, Eliane Matos Dos Santos, Janaína Reis Xavier, Maria De Lourdes De Sousa Maia, Rolando Páez Meireles, Hugo Nodarse Cuni, Guilherme Becker Sander, Paulo Dornelles Picon, Denise C S Matos, Milton Ozório Moraes

Abstract

Sustained virologic response (SVR) in chronic hepatitis C (CHC) treatment denotes that the host genetics controls the immune response and unequivocally contribute to viral clearance or disease severity. In this context, single nucleotide polymorphisms (SNPs) in the locus of interferon lambda 3 and 4 genes (IFNL3/4) have been important genetic markers of responsiveness to CHC as prognostic markers for the pegylated-Interferon-alpha/ribavirin (Peg-IFN-α/RBV). Here, we analyzed 12 SNPs at the IFNL3/4 region in 740 treatment-naïve patients with CHC infected with hepatitis C virus (HCV) genotypes 1, 2, or 3 treated with Peg-IFN-α/RBV. Individually, rs12979860-CC, rs8109886-CC, or rs8099917-TT were predictive markers of SVR, while rs12979860-CC demonstrated the stronger effect. Besides, the genotypic combination of these three predictors' genotypes, CC/CC/TT, increased the rate of SVR. Serum levels of cytokines and gene expression analysis on the genes IFNL3, IFNL4, IFNA1, and some of the IFN-stimulated genes (ISGs) were measured in a subgroup of 24 treated patients and 24 healthy volunteers. An antagonist effect was highlighted between the expression of IFNL3/4 and IFNA1 mRNA among patients. Besides, a prominent production of the pro-inflammatory chemokines CCL4 and CXCL10 was observed at a 12-week treatment follow-up. Lower serum levels of these chemokines were detected in patients with an rs12979860-CC genotype associated with the better treatment outcome. Also, lower expression levels of the IFI6, IFI16, IRF9 genes were observed among rs12979860-CC individuals. In conclusion, a combination of the genotypes at the IFNL3/4 locus can act as a better marker for the prognosis for virological responses in an admixed Brazilian population presenting the modulating effect over innate immunity and inflammation that are controlling the outcome of the viral infection, but also other infectious diseases. This study is registered on the ClinicalTrials.gov platform (accession number NCT01889849 and NCT01623336).

Keywords: Interferon lambda 3 e 4; hepatitis C; immune response; pegylated interferon; sustained virologic response.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Silva, Alvarado-Arnez, Azamor, Batista-Silva, Leal-Calvo, Bezerra, Ribeiro-Alves, Kehdy, Neves, Bayma, Silva, Souza, Muller, Andrade, Andrade, Santos, Xavier, Maia, Meireles, Cuni, Sander, Picon, Matos and Moraes.

Figures

Figure 1
Figure 1
Association of the SNPs of the IFNL3/IFNL4 region with the outcome of Peg-IFN-α/RBV treatment. Logistic regression models included viral genotype, ethnicity, fibrosis, baseline viral load, and type of treatment as categorical covariates. P-values were adjusted by Bonferroni.
Figure 2
Figure 2
Distribution of the SVR rates from the patients with CHC treated with Peg-IFNα. The distribution is stratified by (A) the genotypes of the SNPs rs12979860, rs8109886, and rs8099917, (B) the genotypic combination of those SNPs, and (C) the genotypic combination of the opposite prognostic markers. The column numbers represent the available individuals in each group. The SVR percentage were compared among groups by the Fisher’s exact test (*P < 0.05, **P < 0.001, ***P < 0.0001).
Figure 3
Figure 3
IFNL3, IFNL4, and IFNA1 gene expression in patients with CHC during the treatment with Peg-IFNα and RBV. (A)IFNL3; (B)IFNL4; (C)IFNA1 and (D) Heat map showing the antagonistic effect between the expression of IFNL3/4 and IFNA1. Statistical analysis was performed by the Kruskal-Wallis test and Dunn’s multiple comparison test (*P < 0.01, **P < 0.001, ***P < 0.0001). ND, not detected; HV, healthy volunteer; w, week; wpt, week post-treatment.
Figure 4
Figure 4
Gene expression levels of the type I IFN signaling pathway and type III IFN of patients with CHC treated with Peg-IFNα/RBV stratified by the SNP genotype rs12979860. Quantification of the expression of the IFN type I signaling pathway genes from whole blood of patients with CHC (n = 24) treated with Peg-IFNα/RBV at times 0, 1, and 12 weeks of treatment. (A)IFI6; (B)IFI16; (C)IRF9;(D)IFNL3; (E)IFNL4 mRNA expression. Expression of other genes analyzed did not present statistically significant results. Statistical analysis was performed by Kruskal-Wallis test and Dunn’s multiple comparison test by each genotype in the same group and between the groups 0, 1w, and 12w (*P < 0.01, **P < 0.001, ***P < 0.0001). w, week; wpt, week post-treatment.
Figure 5
Figure 5
Serum levels of the CCL4 and CXCL10 of the patients with CHC treated with Peg-IFNα/RBV and healthy volunteers who received one dose of Peg-IFNα stratified by rs12979860 genotypes. (A) CCL4 in healthy volunteers; (B) CCL4 in patients; (C) CCL4 levels before treatment in patient responders and non-responders; (D) CXCL10 in healthy volunteers; (E) CXCL10 in patients; (F) CXCL10 levels of the first week of treatment in patient responders and non-responders. Number of the genotypes in patients CC (n = 3), CT (n = 17) and TT (n = 4) and in health volunteers CC (n = 9), CT (n = 13) and TT (n = 2). Data were compared by the two-way ANOVA, plus post-hoc comparisons adjusted by the Sidak procedure (*P < 0.05). Results are represented as medians. h, hours; w, week; wpt, week post-treatment. Serum levels of other cytokines analyzed did not present statistically significant results.
Figure 6
Figure 6
Schematic model showing the main findings of this study. Patients with CHC present high levels of cytokines before treatment and display an antagonistic effect between IFNL3/4 and IFNA1 expression levels. After treatment, patients with the genotypic combination of SNPs rs12979869-CC/rs8109886-CC/rs8099917-TT can induce a balanced immune response, presenting lower levels of ISGs, IFI6, IFI16, and IRF9, and low serum levels of the chemokines CCL4 and CXCL10. These patients can increase the SVR prediction, especially those infected with GT1, contrary to the patients with genotypes rs12979869-TT/rs8109886-AA/rs8099917-GG.

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