Immunologic changes in the peripheral blood transcriptome of individuals with early-stage chronic Chagas cardiomyopathy: a cross-sectional study

Abstract

Background: Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a neglected disease that affects approximately 6 million individuals worldwide. Of those infected, 20-30% will go on to develop chronic Chagas cardiomyopathy (CCC), and many ultimately to advanced heart failure. The mechanisms by which this progression occurs are poorly understood. In this exploratory study, we sought to provide insight into the physiologic changes associated with the development of early CCC.

Methods: We used RNA sequencing to analyse the gene expression changes in the peripheral blood of six patients with Chagas disease with early structural heart disease, four patients with Chagas disease without any signs or symptoms of disease, thirteen patients without Chagas disease with early structural heart disease, and ten patients without Chagas disease or signs of heart disease. Pathway analyses and immune cell deconvolution were employed to further elucidate the biological processes underlying early CCC development.

Findings: Our analysis suggests that early CCC is associated with a downregulation of various peripheral immune response genes, including changes suggestive of reduced antigen presentation and T cell activation. Notably, these genes and processes appear to be distinct from those of non-Chagas cardiomyopathies.

Interpretation: This work highlights the potential importance of the immune response in early CCC, providing insight into the early pathogenesis of this disease and how it may differ from other cardiomyopathies. The changes we have identified may serve as biomarkers of early CCC and could inform future longitudinal cohort studies of markers of disease progression and strategies for the treatment of CCC in its early stages.

Funding: NIH, FONDECYT, IDSA, NSF.

Keywords: Biomarkers; Cardiomyopathy; Chagas; Gene expression; Immune.

Fig. 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 early cardiomyopathy (non-Chagas early-CARD or early-CCC) (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. Abbreviations: CARD, cardiomyopathy; CCC, chronic Chagas cardiomyopathy. Image created with BioRender.com.

Fig. 2

Distinct gene expression changes characterize early CCC. (a,c) Volcano plot of patients with early cardiomyopathy (CCC or CARD) vs those with no evidence of cardiomyopathy (IND or non-CARD) for a) chagas seropositive and c) seronegative individuals. Upregulated DEGs (FDR <0.1 and log₂FC > 0.585) are shown in red, downregulated DEGs (FDR <0.1 and log₂FC < −0.585) are in blue, genes with FDR <0.1 or |log₂FC| > 0.585 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 <0.1 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 early 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. Abbreviations: CARD, cardiomyopathy; CCC, chronic Chagas cardiomyopathy; IND, indeterminate.

Fig. 3

Early-CCC may be associated with 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 early 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 grey 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. Cluster color represents the comparison with predominant enrichment with red reflecting predominantly pathways enriched for Chagas positive, blue for Chagas negative, and grey when there is an near even enrichment for both groups. Abbreviations: MAIT, Mucosal-associated invariant T cell; NK, Natural Killer; Pos, Chagas seropositive; Neg, Chagas seronegative; CARD, cardiomyopathy; IND, indeterminate.