Inflammation Enhances the Risks of Stroke and Death in Chronic Chagas Disease Patients

Abstract

Ischemic strokes have been implicated as a cause of death in Chagas disease patients. Inflammation has been recognized as a key component in all ischemic processes, including the intravascular events triggered by vessel interruption, brain damage and repair. In this study, we evaluated the association between inflammatory markers and the death risk (DR) and stroke risk (SR) of patients with different clinical forms of chronic Chagas disease. The mRNA expression levels of cytokines, transcription factors expressed in the adaptive immune response (Th1, Th2, Th9, Th17, Th22 and regulatory T cell), and iNOS were analyzed by real-time PCR in peripheral blood mononuclear cells of chagasic patients who exhibited the indeterminate, cardiac, digestive and cardiodigestive clinical forms of the disease, and the levels of these transcripts were correlated with the DR and SR. Cardiac patients exhibited lower mRNA expression levels of GATA-3, FoxP3, AHR, IL-4, IL-9, IL-10 and IL-22 but exhibited higher expression of IFN-γ and TNF-α compared with indeterminate patients. Digestive patients showed similar levels of GATA-3, IL-4 and IL-10 than indeterminate patients. Cardiodigestive patients exhibited higher levels of TNF-α compared with indeterminate and digestive patients. Furthermore, we demonstrated that patients with high DR and SR exhibited lower GATA-3, FoxP3, and IL-10 expression and higher IFN-γ, TNF-α and iNOS mRNA expression than patients with low DR and SR. A negative correlation was observed between Foxp3 and IL-10 mRNA expression and the DR and SR. Moreover, TNF-α and iNOS expression was positively correlated with DR and SR. Our data suggest that an inflammatory imbalance in chronic Chagas disease patients is associated with a high DR and SR. This study provides a better understanding of the stroke pathobiology in the general population and might aid the development of therapeutic strategies for controlling the morbidity and mortality of Chagas disease.

Fig 1. Indeterminate patients exhibited higher GATA-3, Foxp3, AHR, IL-4, IL-9, IL-10, and IL-22 mRNA expression than did cardiac patients.

The mRNA expression levels of transcription factors (A) and cytokines (B) were determined by real-time PCR in peripheral blood mononuclear cells of patients with the indeterminate (n = 18), cardiac (n = 17), digestive (n = 15) and cardiodigestive (n = 15) clinical forms of Chagas disease. The expression levels were normalized to the expression level of β-actin. The results are expressed as the means ± standard errors. *P < 0.05.

Fig 2. TNF-α, IL-10, T-Bet and FoxP3 expression levels in patients with chronic chagasic cardiomyopathy are correlated with death risk.

The mRNA expression levels of transcription factors (A) and cytokines (B) were determined by real-time PCR in peripheral blood mononuclear cells of patients with the cardiac (n = 17) and cardiodigestive (n = 15) clinical forms of Chagas disease that were classified into high (n = 10), medium (n = 12), and low (n = 10) death risk groups. The expression levels were normalized to the expression level of β-actin. The results are expressed as the means ± standard errors. *P < 0.05.

Fig 3. Patients who exhibited low stroke risk also exhibited high GATA-3, Foxp3, PU.1, AHR, IL-9, IL-22 and IL-10 expression.

The mRNA expression levels of transcription factors (A) and cytokines (B) were determined by real-time PCR in peripheral blood mononuclear cells of chagasic patients with low (n = 40), medium (n = 18) and high (n = 7) risks of stroke. The expression levels were normalized to the expression level of β-actin. The results are expressed as the means ± standard errors. *P < 0.05.

Fig 4. Patients who exhibited high death and stroke risks exhibited high iNOS expression.

The mRNA expression levels of iNOS were determined by real-time PCR of peripheral blood mononuclear cells of chagasic patients with the indeterminate (n = 18), cardiac (n = 17), digestive (n = 15) and cardiodigestive (n = 15) forms of disease (A). Patients were classified as having a high (n = 10), medium (n = 12), or low (n = 10) death risk (B) and were also grouped as having a low (n = 40), medium (n = 18) or high (n = 7) stroke risk (C). The expression of iNOS was normalized to the expression level of β-actin. The results are expressed as the means ± standard errors. *P < 0.05.

Fig 5. High TNF-α, iNOS and low Foxp3 expression are correlated with the risks of death and stroke.

The mRNA expression levels of Foxp3 (A and B), IL-10 (C and D), TNF-α (E and F) and iNOS (G and H) were determined by real-time PCR of peripheral blood mononuclear cells from chagasic patients classified as having a high (n = 10), medium (n = 12), or low (n = 10) death risk. The patients were also grouped as having a low (n = 40), medium (n = 18) or high (n = 7) stroke risk. Expression levels were normalized to the expression level of β-actin. The results are expressed as the means ± standard errors.