Rhesus negative males have an enhanced IFNγ-mediated immune response to influenza A virus

Abstract

The Rhesus D antigen (RhD) has been associated with susceptibility to several viral infections. Reports suggest that RhD-negative individuals are better protected against infectious diseases and have overall better health. However, potential mechanisms contributing to these associations have not yet been defined. Here, we used transcriptomic and genomic data from the Milieu Interieur cohort of 1000 healthy individuals to explore the effect of Rhesus status on the immune response. We used the rs590787 SNP in the RHD gene to classify the 1000 donors as either RhD-positive or -negative. Whole blood was stimulated with LPS, polyIC, and the live influenza A virus and the NanoString human immunology panel of 560 genes used to assess donor immune response and to investigate sex-specific effects. Using regression analysis, we observed no significant differences in responses to polyIC or LPS between RhD-positive and -negative individuals. However, upon sex-specific analysis, we observed over 40 differentially expressed genes (DEGs) between RhD-positive (n = 384) and RhD-negative males (n = 75) after influenza virus stimulation. Interestingly these Rhesus-associated differences were not seen in females. Further investigation, using gene set enrichment analysis, revealed enhanced IFNγ signalling in RhD-negative males. This amplified IFNγ signalling axis may explain the increased viral resistance previously described in RhD-negative individuals.

Fig. 1. Schematic overview of the study.

Genomic variability was assessed through a genome-wide SNP array. Rhesus status was determined based on the rs590787 SNP. Whole blood samples were stimulated with a panel of ligands (polyIC and LPS) and a live influenza A virus. The expression of 560 immune-related genes was quantified using NanoString transcriptomics. R and gene set enrichment analysis (GSEA) were used to compare the response between RhD-positive and -negative individuals.

Fig. 2. Rhesus phenotype distribution of all individuals in the Milieu Interieur cohort.

RhD status was determined based on rs590787, a SNP in the RHD gene, using the Human Exome Bead Chip. a The frequencies of RhD-positive and -negative individuals was similar between males and females. b, c The genotype frequencies for the European cohort from the 1000 Genome Project were similar to those recorded for the MI cohort. The numbers in brackets are percentages.

Fig. 3. Rhesus antigenicity does not affect immune gene expression at baseline or in response to stimulation with PRR ligands in whole blood.

Immune gene expression in unstimulated and LPS and polyIC stimulated whole blood was assessed using NanoString transcriptomics. Comparisons between RhD-positive and -negative individuals in all donors, females only and males only showed no significant differences (q > 0.1, regression analysis with FDR correction) for (i) Null, (ii) LPS or (iii) polyIC. Shown in the heatmap are 100 representative genes chosen at random.

Fig. 4. Following influenza A virus stimulation, 45 genes were differentially expressed between RhD-positive and -negative males.

Whole blood was stimulated with influenza A virus and gene expression assessed using NanoString. a Volcano plot showing no significant differences between RhD-positive and -negative females (q > 0.1, regression analysis with FDR adjustment). b Volcano plot of the differentially expressed genes between RhD-positive and -negative donors in the male group (q < 0.1, regression analysis with FDR adjustment). c Heatmap of differentially expressed genes between RhD-positive and negative males. d Enrichment plot indicating upregulation of the IFNγ pathway in the RhD-negative males (p < 0.01).