Genetic Polymorphism at CCL5 Is Associated With Protection in Chagas' Heart Disease: Antagonistic Participation of CCR1+ and CCR5+ Cells in Chronic Chagasic Cardiomyopathy

Abstract

Chronic cardiomyopathy is the main clinical manifestation of Chagas disease (CD), a disease caused by Trypanosoma cruzi infection. A hallmark of chronic chagasic cardiomyopathy (CCC) is a fibrogenic inflammation mainly composed of CD8⁺ and CD4⁺ T cells and macrophages. CC-chemokine ligands and receptors have been proposed to drive cell migration toward the heart tissue of CD patients. Single nucleotide polymorphisms (SNPs) in CC-chemokine ligand and receptor genes may determine protein expression. Herein, we evaluated the association of SNPs in the CC-chemokines CCL2 (rs1024611) and CCL5 (rs2107538, rs2280788) and the CCL5/RANTES receptors CCR1 (rs3181077, rs1491961, rs3136672) and CCR5 (rs1799987) with risk and progression toward CCC. We performed a cross-sectional association study of 406 seropositive patients from endemic areas for CD in the State of Pernambuco, Northeast Brazil. The patients were classified as non-cardiopathic (A, n = 110) or cardiopathic (mild, B1, n = 163; severe, C, n = 133). Serum levels of CCL5 and CCL2/MCP-1 were elevated in CD patients but were neither associated with risk/severity of CCC nor with SNP genotypes. After logistic regression analysis with adjustment for the covariates gender and ethnicity, CCL5 -403 (rs2107538) CT heterozygotes (OR = 0.5, P-value = 0.04) and T carriers (OR = 0.5, P-value = 0.01) were associated with protection against CCC. To gain insight into the participation of the CCL5-CCR5/CCR1 axis in CCC, mice were infected with the Colombian T. cruzi strain. Increased CCL5 concentrations were detected in cardiac tissue. In spleen, frequencies of CCR1⁺ CD8⁺ T cells and CD14⁺ macrophages were decreased, while frequencies of CCR5⁺ cells were increased. Importantly, CCR1⁺CD14⁺ macrophages were mainly IL-10⁺, while CCR5⁺ cells were mostly TNF⁺. CCR5-deficient infected mice presented reduced TNF concentrations and injury in heart tissue. Selective blockade of CCR1 (Met-RANTES therapy) in infected Ccr5^-/- mice supported a protective role for CCR1 in CCC. Furthermore, parasite antigen stimulation of CD patient blood cells increased the frequency of CCR1⁺CD8⁺ T cells and CCL5 production. Collectively, our data support that a genetic variant of CCL5 and CCR1⁺ cells confer protection against Chagas heart disease, identifying the CCL5-CCR1 axis as a target for immunostimulation.

Keywords: CCL5; CCR1; CCR5; Chagas disease; Met-RANTES; Trypanosoma cruzi; cell migration; heart disease.

Figure 1

CCL5 levels and association with disease severity, genotypes and left ventricular ejection fraction (LVEF) in T. cruzi-infected patients. (A) CCL5 levels in the serum of seronegative noninfected (NI) individuals and individuals who were seropositive for Chagas disease (CD); ***P < 0.001 (Student’s t-test). (B) CCL5 concentrations in the serum of CD patients grouped as A (asymptomatic), B1 [mild chronic chagasic cardiomyopathy (CCC)], or C (severe CCC) (analysis of variance, Bonferroni posttest). (C) Comparison of CCL5 serum levels according to the absence (CC) or presence of rs2107538 allele T (Mann–Whitney test). (D) Correlation between the CCL5 serum concentrations and LVEF in patients; P = 0.8078, r² = 0.0006 (linear regression).

Figure 2

CCL5 levels and CCR1 and CCR5 expression in CD8⁺ T cells and CD14⁺ macrophages of T. cruzi-infected C57BL/6 mice. Mice were infected with 100 trypomastigote forms of the Colombian T. cruzi strain and analyzed at 120 days postinfection. The heart was removed, proteins were extracted, and CCL5 concentrations were estimated by enzyme-linked immunosorbent assay. Splenocytes were collected and stained for intracellular cytokines (TNF, IL-10) and surface cell markers (CD8, CD14, CCR1, CCR5). (A) CCL5 levels in the heart tissue extracts of T. cruzi-infected mice compared to noninfected (NI) controls. (B) Percentages of total CD8⁺ T cells and CD8⁺ T cells expressing CCR1 and/or CCR5 in the spleen of NI and T. cruzi-infected mice. (C) The table shows percentages of total CD14⁺ macrophages. Graphs show the frequencies of CD14⁺ macrophages expressing CCR1 or CCR5 and CD14⁺ macrophages expressing TNF, TNF/IL-10, or IL-10 in the spleen of NI and T. cruzi-infected mice. (D) Pie charts represent the fractions of CCR1⁺ or CCR5⁺ CD14⁺ macrophages carrying each of the intracellular cytokine phenotypes shown in the legend. Graphs show the frequencies of CCR1⁺ or CCR5⁺ CD14⁺ macrophages expressing TNF, TNF/IL-10 or IL-10. Data represent two independent experiments with three NI and five to seven infected mice [*P < 0.05, **P < 0.01, ***P < 0.001, T. cruzi-infected mice vs NI (Student’s t-test)].

Figure 3

Effects of CCR5 deficiency on survival and myocardial injury in T. cruzi-infected mice. Mice were infected with 1,000 trypomastigote forms of the Colombian T. cruzi strain and analyzed at 70 days postinfection. (A) Survival curves of T. cruzi-infected Ccr5⁺/⁺ and Ccr5^−/− mice. Deaths were registered weekly. (B) Sera were collected, and CK-MB activity was determined by biochemical assay. Data represent two independent experiments with three noninfected (NI) and five to seven infected mice [*P < 0.05, T. cruzi-infected Ccr5^+/+ mice vs NI; ^#P < 0.05, Ccr5^−/− vs Ccr5^+/+ T. cruzi-infected mice (analysis of variance, Bonferroni posttest)].

Figure 4

Effects of Met-RANTES treatment on splenomegaly, myocardial injury, and CCL5 and TNF concentrations in the heart tissue of T. cruzi-infected mice. (A) Mice were infected with 100 trypomastigote forms of the Colombian T. cruzi strain and treated with Met-RANTES (Met-R, 10 µg/mice) from 120 to 150 days postinfection (dpi). Sera were collected (at 120 and 150 dpi) for estimation of CK-MB activity. Hearts were collected (at 150 dpi) for evaluation of CCL5 and TNF concentrations by enzyme-linked immunosorbent assay (ELISA). (B) The relative weight of the spleen (spleen weight in milligrams/body weight in grams) at 150 dpi. (C) CK-MB activity in serum determined by biochemical assay at 120 (pretherapy) and 150 dpi (posttherapy, Met-R). (D) CCL5 concentrations in extracts of heart tissue evaluated by ELISA at 150 dpi. (E) TNF concentrations in extracts of heart tissue evaluated by ELISA at 150 dpi. Data represent two independent experiments with 3–5 noninfected (NI) and 7–10 infected mice.*P < 0.05, **P < 0.01, ***P < 0.001, T. cruzi-infected mice vs NI; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001, pretreated vs Met-R-treated; ^&&P < 0.01, ^&&&P < 0.001, Ccr5^−/− vs Ccr5^+/+ T. cruzi-infected mice pretreatment (120 dpi); ^ψP < 0.05, ^ψψP < 0.01, ^ψψψP < 0.001, Ccr5^−/− vs Ccr5^+/+ T. cruzi-infected mice posttreatment (150 dpi) with Met-R (analysis of variance, Bonferroni posttest).

Figure 5

Expression of CCR1⁺ and CCR5⁺ on CD8⁺ T cells and CCL5 production after antigen stimulation in association with the severity of Chagas heart disease. Peripheral blood cells of Chagas disease (CD) patients grouped as A (asymptomatic, n = 20), B1 [mild chronic chagasic cardiomyopathy (CCC), n = 20], or C (severe CCC, n = 16) were collected and stimulated with T. cruzi antigen for 24 h. Cells and supernatants were harvested and analyzed for cell markers by flow cytometry and CCL5 production by enzyme-linked immunosorbent assay. (A) Frequencies of CCR1⁺ and CCR5⁺ cells and mean fluorescence intensity (MFI) of CCR1 and CCR5 on CD8⁺ T cells; ^##P < 0.01, antigen-stimulated cells from patients in group C vs group A [analysis of variance (ANOVA), Bonferroni posttest]. (B) CCL5 concentrations in supernatants of cell cultures of CD patients grouped as A, B1, and C; **P < 0.01, antigen-stimulated vs nonstimulated cells from patients in group C (ANOVA, Bonferroni posttest); ^##P < 0.01, antigen-stimulated cells from patients of group C vs group A (ANOVA, Bonferroni posttest).