Immunologic changes are detectable in the peripheral blood transcriptome of clinically asymptomatic Chagas cardiomyopathy patients

Abstract

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a neglected parasitic disease that affects approximately 6 million individuals worldwide. Of those infected, 20-30% will go on to develop chronic Chagas cardiomyopathy (CCC), and ultimately many of these individuals will progress to advanced heart failure. The mechanism by which this progression occurs is poorly understood, as few studies have focused on early CCC. In this study, we sought to understand the physiologic changes associated with T. cruzi infection and the development of CCC. We analyzed gene expression in the peripheral blood of asymptomatic Chagas patients with early structural heart disease, Chagas patients without any signs or symptoms of disease, and Chagas-negative patients with and without early structural heart disease. Our analysis shows that early CCC was associated with a downregulation of various peripheral immune response genes, with gene expression changes suggestive of reduced antigen presentation and T cell activation. Notably, these genes and processes were distinct from those of early cardiomyopathy in Chagas-negative patients, suggesting that the processes mediating CCC may be unique from those mediating progression to other cardiomyopathies. This work highlights the importance of the immune response in early CCC, providing insight into the early pathogenesis of this disease. The changes we have identified may serve as biomarkers of progression and could inform strategies for the treatment of CCC in its early stages, before significant cardiac damage has occurred.

Figure 1:. Study design schematic.

10 Chagas-positive patients were randomly selected, and 23 Chagas-negative patients were matched by age, sex, region of origin, and cardiac disease stage. These patients were stratified by Chagas status to compare cardiac patients with asymptomatic early cardiomyopathy (abnormal EKG, normal echocardiography, no symptoms of heart failure) to those with no signs or symptoms of cardiomyopathy using differential gene expression, gene ontology and immune cell deconvolution. Early CARD – asymptomatic early cardiomyopathy. Image created with BioRender.com

Figure 2.. There are distinct gene expression changes that characterize early asymptomatic CCC.

(a,c) Volcano plot of patients with asymptomatic cardiomyopathy vs those with no evidence of cardiomyopathy for a) chagas seropositive and c) seronegative individuals. Upregulated DEGs (FDR <0.1 and log2FC > 1.5) are shown in red, downregulated DEGs (FDR <0.1 and FC < − 1.5) are in blue, genes with FDR < 0.1 or |FC| > 1.5 but not both are in black, and non-significant DEGs are in grey. (b,d) Heatmap of relative vst-transformed values for the most significant DEGs with FDR < 0.05 in chagas b) seropositive and d) seronegative individuals. Color represents the sample’s difference from the mean for that gene. e) Venn diagram of significant DEGs of asymptomatic cardiomyopathy vs non-cardiomyopathy for chagas seropositive and seronegative individuals. f) scatter plot of log2FC of seropositive DEGs vs the log2FC of these same genes in seronegative individuals with Pearson correlation test. Trendline shown in blue. CARD- cardiomyopathy, CCC- chronic Chagas cardiomyopathy

Figure 3.. Asymptomatic stage B CCC demonstrates reduced antigen presentation and T cell activation.

a) top 15 over-represented (ORA) GO biologic process (BP) terms (FDR <0.05) for downregulated (Down) and upregulated (Up) DEGs for asymptomatic cardiomyopathy vs non-cardiomyopathy patients that are Chagas seronegative (Neg) or seropositive (Pos). Circle size represents number of genes in the given pathway, color represents the FDR. b) box plot of antigen presenting cell deconvolution from bulk RNAseq data using ABsolute Immune Signal (ABIS) c) box plot of T cell deconvolution from bulk RNAseq data using ABIS d) enrichment map of gene set enriched analysis (GSEA) GO BP for seronegative and seropositive patients with lines connecting overlapping gene sets, and showing 15 clusters with > 2 biologic processes. Node size represents number of genes enriched in the pathway and line length represents pairwise similarity. Abbreviations: MAIT - Mucosal-associated invariant T cell, NK – Natural Killer, Pos – Chagas seropositive, Neg – Chagas seronegative, CARD- cardiomyopathy