Mast cell function and death in Trypanosoma cruzi infection

Abstract

Although the roles of mast cells (MCs) are essential in many inflammatory and fibrotic diseases, their role in Trypanosoma cruzi-induced cardiomyopathy is unexplored. In this study, we treated infected CBA mice with cromolyn, an MC stabilizer, and observed much greater parasitemia and interferon-γ levels, higher mortality, myocarditis, and cardiac damage. Although these data show that MCs are important in controlling acute infection, we observed MC apoptosis in the cardiac tissue and peritoneal cavity of untreated mice. In the heart, pericardial mucosal MC die, perhaps because of reduced amounts of local stem cell factor. Using RT-PCR in purified cardiac MCs, we observed that infection induced transcription of P2X(7) receptor and Fas, two molecules reportedly involved in cell death and inflammatory regulation. In gld/gld mice (FasL(-/-)), apoptosis of cardiac, but not peritoneal, MCs was decreased. Conversely, infection of P2X(7)(-/-) mice led to reduced peritoneal, but not cardiac, MC death. These data illustrate the immunomodulatory role played by MCs in T. cruzi infection and the complexity of molecular interactions that control inflammatory pathways in different tissues and compartments.

Figure 1

Cromolyn treatment. Mice were divided into T. cruzi–infected (G1-G3) and uninfected (G4-G6) groups, where G1 and G4 were cromolyn treated, G2 and G5 were PBS treated, and G3 and G6 were untreated. Parasitemia was counted in microscopic slides on dpi 17 (A), and mortality was checked daily (B). Plasma was collected for cytokine evaluation by flow cytometry (C) on dpi 20 and creatine kinas MB (CK-MB) activity (D) on dpi 15 from the indicated groups. MCP-1, monocyte chemotactic protein-1. Each bar represents the mean ± SD of three independent experiments with six mice per group. *P < 0.05 compared with the other groups. Representative cardiac tissue sections were collected from G4 (E), G5 (F), G1 (on dpi 15) (G), and G2 (on dpi 15) (H), all stained with H&E. Arrows indicate inflammatory foci. Scale bar = 100 μm.

Figure 2

MC identification. Cardiac tissue sections were collected from uninfected and untreated mice (G6) (A and inset) and from T. cruzi–infected and untreated mice (G3) on dpi 7 (B) and dpi 17 (C) and were stained with AB/S/TB (A–C). Blue arrows indicate MMC subpopulations; red arrows, CTMC subpopulations. Data are representative of at least 15 mice per group in three independent experiments. Scale bar = 100 μm.

Figure 3

Cardiac and peritoneal MC density. MCs from control (G6) and T. cruzi–infected (G3) mice were collected at the indicated time points and were stained with AB/S/TB or TB for total cardiac MC quantification (A) and phenotypic separation as MMCs, CTMC, and hybrid MCs, as described in Materials and Methods (B). Each bar represents the mean ± SD of three experiments with six animals. Total peritoneal MCs were collected from control and infected mice on dpi 7 and 17 and were also stained with AB/S/TB (C) to reveal different MC subpopulations (D). E: Gene expression of chymase and tryptase in the left ventriculum of reperfused hearts was evaluated by real-time quantitative PCR. Each bar represents the mean ± SD of three independent experiments with seven animals per time point. *P < 0.05 compared with control; ^†P < 0.05 comparing AB/S/TB with TB; ^‡P < 0.05 compared with dpi 7.

Figure 4

Cardiac MC apoptosis. All 16-μm-thick frozen sections were collected from control (G6) (A) and T. cruzi–infected (G3) mice on dpi 7 (B) and dpi 17 (C) for apoptosis detection by TUNEL and MC staining with AB/S/TB. Arrows indicate MCs; arrowheads, TUNEL⁺ pericardial cells; double arrows, TUNEL⁺ cells composing inflammatory foci. Two independent experiments were performed with six mice per group. Scale bars = 100 μm.

Figure 5

SCF and IL-3 production in cardiac tissue. SCF (A–D) and IL-3 (E–H) levels were evaluated by immunohistochemical analysis in cardiac tissue from control (G6) mice (A and E) and from T. cruzi–infected (G3) mice on dpi 7 (B and F) and dpi 17 (C and G). Original magnification, ×200. SCF (D) and IL-3 (H) stromal labeling were quantified using Image-Pro Plus 4 image analyzer software in cardiac tissue from control (G6) and T. cruzi–infected (G3) mice on dpi 7 and 17. Each bar represents the mean ± SD of three independent experiments with five animals per time point. *P < 0.05 compared with control.

Figure 6

MC transcription of P2X₇ receptor and Fas. Peritoneal (A and C) and cardiac (B and D) MCs were purified from control (G6) and T. cruzi–infected (G3) mice on dpi 7 and 17 using Percoll to evaluate the transcription of P2X₇ receptor (A and B) and Fas molecule (C and D) by RT-PCR. Each bar represents the mean ± SD of densitometric units (normalized for the GAPDH) of three experiments per time point. The samples enriched in MCs (≥98%) were obtained from 20 animals. *P < 0.05 compared with control; ^†P < 0.05 compared with dpi 7.

Figure 7

Fas-L and P2X₇ receptors in cardiac MCs. Tissue sections were collected from control and T. cruzi–infected (as indicated in the panels) BALB/c (A and B), gld/gld (Fas-L^−/−) (C and D), C57Bl/6 (E and F), and P2X₇^−/− (G and H) mice for total MC quantitation (left panels) and subpopulation identification (right panels). We used AB/S/TB staining solution. Each bar represents the mean ± SD of two independent experiments with six mice per group. *P < 0.05 compared with control.

Figure 8

General scheme of pathways of pericardial MC death after T. cruzi infection. The increase in glucocorticoid levels induced by the infection (1) and/or possible death of pericardial SCF-producing cells (2) could lead to the reduction in cardiac stromal SCF. This down-modulation can trigger the decrease in the number of MCs by reduced chemotaxis of MC precursors to the heart (3) and/or apoptosis of mature cardiac MCs (4). Mechanistic pathways that could be playing a role in MC apoptosis include induction of Fas expression on MCs and interaction with Fas-L⁺ cells in the tissue (4a); predominance of vacant receptors of SCF (CD117) directly leading to MC death (4b); and increased levels of MC cytotoxic cytokines, such as IFN-γ, produced by natural killer (NK) cells in the early acute phase of the infection and later by T lymphocytes (4c). Although we observed no MC death through P2X₇ receptor activation, using P2X₇^−/−-infected mice, the infection induced its transcription for still unexplored functions. Moreover, infected necrotic cells releasing extracellular ATP could be the source of receptor agonistic stimulus.