Myocardial gene expression of T-bet, GATA-3, Ror-γt, FoxP3, and hallmark cytokines in chronic Chagas disease cardiomyopathy: an essentially unopposed TH1-type response

Abstract

Background: Chronic Chagas disease cardiomyopathy (CCC), a late consequence of Trypanosoma cruzi infection, is an inflammatory cardiomyopathy with prognosis worse than those of noninflammatory etiology (NIC). Although the T cell-rich myocarditis is known to play a pathogenetic role, the relative contribution of each of the functional T cell subsets has never been thoroughly investigated. We therefore assessed gene expression of cytokines and transcription factors involved in differentiation and effector function of each functional T cell subset (TH1/TH2/TH17/Treg) in CCC, NIC, and heart donor myocardial samples.

Methods and results: Quantitative PCR showed markedly upregulated expression of IFN-γ and transcription factor T-bet, and minor increases of GATA-3; FoxP3 and CTLA-4; IL-17 and IL-18 in CCC as compared with NIC samples. Conversely, cytokines expressed by TH2 cells (IL-4, IL-5, and IL-13) or associated with Treg (TGF-β and IL-10) were not upregulated in CCC myocardium. Expression of TH1-related genes such as T-bet, IFN-γ, and IL-18 correlated with ventricular dilation, FoxP3, and CTLA-4.

Conclusions: Results are consistent with a strong local TH1-mediated response in most samples, possibly associated with pathological myocardial remodeling, and a proportionally smaller FoxP3(+)CTLA4(+) Treg cell population, which is unable to completely curb IFN-γ production in CCC myocardium, therefore fueling inflammation.

Figure 1

Expression of mRNA encoding transcription factors T-bet, Gata-3, and Rorγ-T in myocardium. Real-time qPCR analysis of mRNA expression in CCC and NIC myocardium. After normalization to GAPDH mRNA, relative increase was plotted in comparison to N group and data were calculated with the 2^−ΔΔCt method, as described in Methods section. The horizontal bar stands for the median; dotted lines indicate twofold increase or decrease of expression as compared with the control group.

Figure 2

The ratio of mRNA encoding transcription factors T-bet and Gata-3 in myocardium. The ratio of relative expression of T-bet/GATA-3 in CCC and NIC group. The ratio of relative expression was plotted in comparison to N group and data were calculated with the 2^−ΔΔCt method, as described in Methods section.

Figure 3

Myocardial expression of cytokine mRNA. Real-time qPCR analysis of mRNA expression in CCC and NIC myocardium. After normalization to GAPDH mRNA, relative increase was plotted in comparison to N group and data were calculated with the 2^−ΔΔCt method, as described in Methods section. The horizontal bar stands for the median. Dotted lines indicate twofold increase or decrease of expression as compared with the control group.

Figure 4

Expression of Foxp3, CTLA-4, IL-10, and TGF-β in myocardium. Real-time qPCR analysis of mRNA expression in CCC and NIC myocardium. After normalization to GAPDH mRNA, relative increase was plotted in comparison to N group and data were calculated with the 2^−ΔΔCt method, as described in Methods section. The horizontal bar stands for the median. Dotted lines indicate twofold increase or decrease of expression as compared with the control group.