Myocardial chemokine expression and intensity of myocarditis in Chagas cardiomyopathy are controlled by polymorphisms in CXCL9 and CXCL10

Abstract

Background: Chronic Chagas cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium.

Methods and results: Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2-6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes.

Conclusions: Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our results may suggest that CXCL9 and CXCL10 are master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to the life-threatening form of CCC.

Figure 1. Histopathological analysis and localization of CCR5⁺, CXCR3⁺, CCR4⁺, CCL5⁺ and CXCL9⁺ cells in heart tissue.

Severe myocarditis with intense mononuclear inflammatory infiltrate in the heart tissues of CCC patient, and absence of myocarditis in the heart tissues of NIC patients and individuals without cardiomyopathies (N). In (A), imagens representative of heart fragments from CCC, NIC and N individuals. Haematoxylin-eosin stain. Representative images of immunofluorescence confocal microscopy from heart tissue sections from CCC and NIC (B). Sections were stained with primary antibodies against CCR5, CXCR3, CCR4, CCL5 and CXCL9 stained with AF633-conjugated anti-mouse IgG (red) and counterstained with DAPI (blue), as described in Methods. CTR- IgG isotype controls.

Figure 2. Expression of chemokines and chemokine receptors in myocardium.

Expression of chemokine receptors (A) in CCC and NIC myocardium. Expression of chemokines (B) in CCC and NIC myocardium. Real-time qPCR analysis of mRNA levels expression in CCC and NIC myocardium. After normalization to GAPDH mRNA, the relative expression was plotted in comparison to N group and data were calculated with the 2^−ΔΔCt method, as described in Methods. The horizontal bar stands for the median. Dotted lines indicate two-fold increase or decrease expression as compared with the control group.

Figure 3. Correlation between histopathologically assessed intensity of myocarditis and mRNA expression of CXCL9 in CCC myocardium.

Two-tailed p value obtained with Spearman's nonparametric correlation. n = 14.

Figure 4. Chemokine and chemokine receptor expression in myocardium from patients bearing the CXCL9 and CXCL10 SNPs.

Expression of chemokines and chemokine receptors in myocardium samples from patients bearing the CXCL9 rs10336 SNP (A). Expression of chemokines and chemokine receptors in myocardium samples from patients bearing CXCL10 rs3921 SNP (B). Real-time qPCR analysis of mRNA levels expression in myocardium samples from patients bearing the CXCL9 rs10336 CC (n = 5) or CT (n = 9) genotypes (A) and CXCL10 rs3921 GG (n = 5) or CC or CG (n = 9). The bar stands for the median.