CD86 Expression by Monocytes Influences an Immunomodulatory Profile in Asymptomatic Patients with Chronic Chagas Disease

Abstract

In the chronic phase of Chagas disease, 60% of the patients develop the asymptomatic form known as indeterminate (IND). The remaining 30% of the patients develop a life-threatening form in which digestive and/or cardiac (CARD) alterations take place. The mechanisms underlying the development of severe forms of Chagas disease remain poorly understood. It is well known that interactions between immune cells such as monocytes and lymphocytes drive immune responses. Further, the co-stimulatory molecules CD80 and CD86 expressed by monocytes and subsets induce lymphocyte activation, thereby triggering cellular immune response. Here, we revealed, for the first time, the functional-phenotypic profile of monocytes subsets in Chagas disease. Using flow cytometry, we evaluated the effect of in vitro stimulation with Trypanosoma cruzi antigens on the expression of the co-stimulatory molecules CD80 and CD86 in different monocyte subsets of patients with IND and CARD clinical forms of Chagas disease. We also assessed the expression of toll-like receptor (TLR)-2, TLR-4, TLR-9, HLA-DR, IL-10, and IL-12 in the monocyte subsets and of CTLA-4 and CD28, ligands of CD80 and CD86, in T lymphocytes. CD86 expression in all monocyte subsets was higher in IND patients when compared with non-infected (NI) individuals. After stimulation with T. cruzi, these patients also showed a higher frequency of CD4⁺CTLA-4⁺ T lymphocytes than NI individuals. We found an association between CD80 and CD28, and between CD86 and CTLA-4 expression, with a high frequency of regulatory T (Treg) cells in IND patients. We proposed that CD86 may be involved in immunoregulation by its association with CTLA-4 in asymptomatic patients. CD86 and CTLA-4 interaction may influence Treg activation, and this could represent a new strategy to control inflammation and tissue damage.

Keywords: chagas disease; immune response; immunoregulation; innate immunity; monocytes.

Figure 1

Analysis of total monocytes and subsets. Flow cytometry gate strategy of non-infected (NI) individuals and frequency of total monocytes (A), classical (CD14⁺⁺CD16⁻), intermediate (CD14⁺⁺CD16⁺), and non-classical (CD14⁺CD16⁺⁺) subsets in culture without stimulation (control) and after in vitro stimulation with T. cruzi antigens (TRIPO) (B). The groups evaluated were NI individuals (n = 6), indeterminate (IND, n = 10), and cardiac (CARD, n = 9) clinical forms of Chagas disease. Significant differences (p < 0.05) between groups are evidenced by lines and asterisks (*) according Kruskal–Wallis test, followed by Dunn’s post hoc test. The # sign indicates significant difference between control and TRIPO cultures using Wilcoxon signed-rank test. Boxes show the median and interquartile ranges, whiskers indicate the highest and lowest observation, and dots represent the outliers.

Figure 2

Expression of the recognition and activation molecules of total monocytes and subsets. Expression of toll-like receptors (TLR) (A), and HLA-DR (B) by total monocytes, classical (CD14⁺⁺CD16), intermediate (CD14⁺⁺CD16⁺), and non-classical (CD14⁺CD16⁺⁺) monocytes subsets in cultures without stimulation (control) and after in vitro stimulation with T. cruzi antigens (TRIPO). The groups evaluated were non-infected (NI) individuals (n = 6), indeterminate (IND, n = 10), and cardiac (CARD, n = 9) clinical forms of Chagas disease. Significant differences (p < 0.05) between groups are evidenced by lines and asterisks (*) according Kruskal–Wallis test, followed by Dunn’s post hoc test. The # sign indicates significant difference between control and TRIPO cultures using Wilcoxon signed-rank test. Boxes show the median and interquartile ranges, whiskers indicate the highest and lowest observation, and dots represent the outliers. MFI, mean fluorescence intensity.

Figure 3

Expression of the co-stimulatory molecules of total monocytes and subsets. Expression of CD80 (A), and CD86 (B) co-stimulatory molecules by total monocytes, classical (CD14⁺⁺CD16), intermediate (CD14⁺⁺CD16⁺), and non-classical (CD14⁺CD16⁺⁺) monocytes subsets in cultures without stimulation (control) and after in vitro stimulation with T. cruzi antigens (TRIPO). The groups evaluated were non-infected (NI) individuals (n = 6), indeterminate (IND, n = 10), and cardiac (CARD, n = 9) clinical forms of Chagas disease. Significant differences (p < 0.05) between groups are evidenced by lines and asterisks (*) according Kruskal–Wallis test, followed by Dunn’s post hoc test. The # sign indicates significant difference between control and TRIPO cultures using Wilcoxon signed-rank test. Boxes show the median and interquartile ranges, whiskers indicate the highest and lowest observation, and dots represent the outliers. MFI, mean fluorescence intensity.

Figure 4

Ratio/proportion of CD80 and CD86 expression by monocytes. Ratio of CD86/CD80 expression by total monocytes (A), and proportion of these co-stimulatory molecules by the monocyte subsets (B). The groups evaluated were non-infected (NI) individuals (n = 6), indeterminate (IND, n = 10), and cardiac (CARD, n = 9) clinical forms of Chagas disease. Significant differences (p < 0.05) between groups are evidenced by lines and asterisks (*) according Kruskal–Wallis test, followed by Dunn’s post hoc test. MFI, mean fluorescence intensity.

Figure 5

Expression of cytokines of total monocytes and subsets. Expression of IL-10 (A), and IL-12 (B) by total monocytes, classical (CD14⁺⁺CD16), intermediate (CD14⁺⁺CD16⁺), and non-classical (CD14⁺CD16⁺⁺) monocytes subsets in cultures without stimulation (control) and after in vitro stimulation with T. cruzi antigens (TRIPO). The groups evaluated were non-infected (NI) individuals (n = 6), indeterminate (IND, n = 10), and cardiac (CARD, n = 9) clinical forms of Chagas disease. Significant differences (p < 0.05) between groups are evidenced by lines and asterisks (*) according Kruskal–Wallis test, followed by Dunn’s post hoc test. The # sign indicates significant difference between control and TRIPO cultures using Wilcoxon signed-rank test. Boxes show the median and interquartile ranges, whiskers indicate the highest and lowest observation, and dots represent the outliers. MFI, mean fluorescence intensity.

Figure 6

Analysis of lymphocytes and subsets. Flow cytometry gate strategy of non-infected (NI) individuals is represented. Frequency of CD4⁺ lymphocytes expressing CD25, CD28, and CTLA-4 (A) in culture without stimulation (control) and after in vitro stimulation with T. cruzi antigens (TRIPO). Frequency of Th1, Th2, Th17, and T regulatory (Treg) lymphocytes subsets (B) and, proportion of lymphocyte subsets (C). The groups evaluated were NI individuals (n = 6), indeterminate (IND, n = 10), and cardiac (CARD, n = 9) clinical forms of Chagas disease. Significant differences (p < 0.05) between groups are evidenced by lines and asterisks (*) according Kruskal–Wallis test, followed by Dunn’s post hoc test. The # sign indicates the significant difference between control and TRIPO cultures using Wilcoxon signed-rank test. Boxes show the median and interquartile ranges, whiskers indicate the highest and lowest observation, and dots represent the outliers.

Figure 7

Frequency of cytokines⁺ lymphocytes subsets. Flow cytometry gate strategy of non-infected (NI) individuals is represented. Expression of IFN-γ, IL-4, IL-17, and IL-10 by CD4⁺ T lymphocytes that characterize Th1, Th2, Th17, and regulatory T subsets, respectively. The groups evaluated were NI individuals (n = 6), indeterminate (IND, n = 10), and cardiac (CARD, n = 9) clinical forms of Chagas disease. Significant differences (p < 0.05) between groups are evidenced by lines and asterisks (*) according Kruskal–Wallis test, followed by Dunn’s post hoc test. Boxes show the median and interquartile ranges, whiskers indicate the highest and lowest observation, and dots represent the outliers.