Immuno-inflammatory response in patients after myocardial infarction and involvement in fibrosis in vitro

Abstract

Pathophysiology of acute myocardial infarction (AMI) has been described in mice and is based on a three-stage model involving temporal modifications of immune cells and fibroblasts. The acute inflammatory phase is followed by a reparative phase and fibrous scar maturation phase. Purinergic signaling, particularly P2Y11 receptor, has been reported to be involved in the regulation of inflammation after ischemia and could be an actor of resolution of inflammation after AMI. The aims of our study were: (1) to characterize the immune and P2Y11R profiles of AMI patients and (2) to analyze interactions between patients’ immune cells and cardiac fibroblasts in vitro. We collected peripheral blood mononuclear cells (PBMC) of 182 patients at various times (H0, H4, H24, H48, D3, M1, M6, M12) after reperfused ST-segment elevation AMI, and of 30 healthy donors. Expression level of genes involved in tolerogenicity profile of dendritic cells (HMOX1, STAT3, IDO1), in T cell polarization (CD4, FOXP3, TBX21/GATA3) as well as P2RY11 were evaluated by RT-PCR. P2Y11R expression was analyzed using flow cytometry. PBMC and human cardiac fibroblasts (HCF) were co-cultured during one day (5PBMC/1 HCF) and gene level expression (ACTA2/VIM, COL1A1), phenotype (α-SMA/vimentin) and secretory (soluble collagen) profiles were analyzed by RT-PCR, flow cytometry and Sircol assay. In the first 48 hours after AMI, the expression level of HMOX1 (fold-change = 8.478 at H48, P < 0.0001), STAT3 (2.856 at H0, P = 0.0027) and CD4 increased (2.451 at H48, P = 0.0052); IDO1 (0.2055 at H24, P < 0.0001) and TBX21/GATA3 ratio decreased (0.4498 at H48, P = 0.0026); FOXP3 did not vary significantly. In the same time, the expression level of P2RY11 increased (2.124 at H0, P = 0.0015) as well as protein expression in T cells (median MFI = 845.5 at H0 vs. 229.5 for healthy donors, P = 0.0375). In vitro, we observed a non-significant increase of ACTA2/VIM ratio in HCF co-cultured with H24, M1, M6 PBMC; of α-SMA/vimentin with H48, M12 PBMC; of COL1A1 with H24, H48, M12 PBMC; and of soluble collagen in coculture supernatant with M1 PBMC. Our results suggest that P2Y11R could be involved in evolution of immune response after AMI, and that, in the first 2 days, circulating immune cells have a reparative profile and could participate in the transformation of fibroblast into myofibroblasts.