HLA-C dysregulation as a possible mechanism of immune evasion in SARS-CoV-2 and other RNA-virus infections

Abstract

One of the mechanisms by which viruses can evade the host's immune system is to modify the host's DNA methylation pattern. This work aims to investigate the DNA methylation and gene expression profile of COVID-19 patients, divided into symptomatic and asymptomatic, and healthy controls, focusing on genes involved in the immune response. In this study, changes in the methylome of COVID-19 patients' upper airways cells, the first barrier against respiratory infections and the first cells presenting viral antigens, are shown for the first time. Our results showed alterations in the methylation pattern of genes encoding proteins implicated in the response against pathogens, in particular the HLA-C gene, also important for the T-cell mediated memory response. HLA-C expression significantly decreases in COVID-19 patients, especially in those with a more severe prognosis and without other possibly confounding co-morbidities. Moreover, our bionformatic analysis revealed that the identified methylation alteration overlaps with enhancers regulating HLA-C expression, suggesting an additional mechanism exploited by SARS-CoV-2 to inhibit this fundamental player in the host's immune response. HLA-C could therefore represent both a prognostic marker and an excellent therapeutic target, also suggesting a preventive intervention that conjugate a virus-specific antigenic stimulation with an adjuvant increasing the T-cell mediated memory response.

Keywords: COVID-19; DNA methylation; HLA-C; SARS-CoV-2; enhancer transcriptional regulation; gene expression; symptomatic and asymptomatic COVID-19; upper airways cells.

Figure 1

Schematic overview of the possible mechanism used by SARS-CoV-2 to downregulate HLA-C and evade the host’s immune response. Created with BioRender.com.

Figure 2

Study design, workflow and main results. The upper part describes the sample cohort used for the genome-wide methylation and HLA-C gene expression analyses. Below, workflow and main results are summarized. Created with BioRender.com.

Figure 3

Immune related gene categories affected by DNA methylation alterations. (A) Pie chart showing the percentage of DNA methylation alterations in COVID-19 patients vs controls. (B) Pie chart showing the percentage of DNA methylation alterations in COVID-19 asymptomatic vs symptomatic patients.

Figure 4

DNA methylation profile of HLA-C regions. The upper part shows HLA-C isoforms, CpG islands and their chromosomic localization. The arrow indicates direction of transcription (A) DNA methylation profile in the discovery dataset (upper airways) in COVID-19 patients (red line) and controls (blue line). (B) DNA methylation profile in the discovery dataset (upper airways) in COVID-19 symptomatic (red line) and asymptomatic patients (blue line). (C) DNA methylation profile in the validation dataset (blood) in COVID-19 patients (red line) and controls (blue line). Asterisks indicate the altered CpG sites identified in Castro de Moura et al. (13).

Figure 5

HLA-C expression in different sample groups. (A) Box plot showing HLA-C expression levels (as fold difference) in controls, COVID-19 and post-COVID-19 patients. Outliers are shown as dots outside the boxes. (B) Box plot showing HLA-C expression levels (as fold difference) in controls and the restricted group of COVID-19 patients (without potential confounding factors, see Materials and Methods). (C) Box plot showing HLA-C expression levels (as fold difference) in controls, asymptomatic, symptomatic and post-COVID-19 patients. (D) Bar plot showing HLA-C expression levels (as Delta Ct) in one subject at three time points. * indicates p-value <0.05, ** indicates p-value <0.01 and *** indicates p-value <0.001.

Figure 6

Potential altered mechanisms of HLA-C regulation in COVID-19 disease. (A) Integrated enhancers associated with HLA-C. (B) Integrated enhancer (chr6:31260493-31279454) associated with CGI chr6:31276241-31276526, found hypermethylated in COVID-19 patients. (C) Integrated enhancer (chr6:31240851-31241006) associated with S-shore region of CGI chr6:31238852-31240120, found hypomethylated in COVID-19 patients. (D) Schematic illustration of a possible mechanism of HLA-C downregulation observed in upper airway cells of COVID-19 patients. Lollipops are exemplificative representation of CpG sites, where empty and filled circles represent hypomethylated and hypermethylated CpG sites, respectively. Illustrations in panels A–C are from HACER database (http://bioinfo.vanderbilt.edu/AE/HACER/index.html); illustration in panel D was created with BioRender.com.