Perfil mitocondrial de eosinófilos no contexto da ativação celular e doenças eosinofílicas

Abstract

The roles played by mitochondria in cells of the immune system has increasingly drawing attention. Mitochondria mediating immune functions and inflammation have been associated with diverse biological processes such as cytokine secretion. However, our current understanding of the mitochondrial activity, architecture and morphological changes in eosinophils under pathological conditions is still poorly known. Here, we investigated the activity, permeability, and ultrastructure of mitochondria within human eosinophils in response to a pro-inflammatory cytokine stimulation [tumor necrosis factor-alpha (TNF-α); C-C motif chemokine ligand 5 (CCL- 5); C-C motif chemokine ligand 11 (CCL-11)], and in the context of inflammatory bowel diseases (IBD) and respiratory diseases [Crohn’s disease (CD); ulcerative colitis (UC) and eosinophilic rinosinusitis (ERS)], which are characterized by recruitment and activation of eosinophils. Analysis was obtained from eosinophils isolated the human peripheral blood from healthy donors stimulated with TNF-α, CCL-5, CCLL-11 and analyzed with MitoTracker and MitoSox assays by flow cytometry. In parallel, blood eosinophils and intestinal and sinus biopsy samples from patients with IBDs and ERS were prepared for transmission electron microscopy (TEM). Tissue eosinophils detected in inflammatory sites showing the entire cell profile, nucleus and intact plasma membrane were investigated with single cell imaging analysis. A total of X eosinophils and X mitochondria were counted and the following mitochondrial aspects were evaluated: i) Mitochondrial total área, number and sigle size; ii) Mitochondrial cristae volume; iii) Mitochondrial cristae pattern; and iv) Mitochondrial-organellar interactions represented by membrane contact sites or tethers. All quantitative analyses were performed using Fiji software. We observed that mitochondrial activity and permeability of TNF-α, CCL-5 and CCL11-stimulated eosinophils significantly increased compared to control cells. The ultrastructural signatures of activated eosinophils were observed in all experimental groups in parallel to significant changes in mitochondrial total área, numeber, single size, cristae volume and cristae patterns represented by different morphologies (lamellar, tubular, and mixed) Our findings demonstrated that human eosinophils undergo mitochondrial cristae reshaping after both in vitro stimulation and during inflammatory diseases . Furthermore, the number of inter-organelle interactions increased during all inflammatory conditions, with significant mitochondrial interactions established with endoplasmic reticulum (ER), other mitochondria, secretory granules, lipid droplets (LDs), and eosinophils sombrero vesicles (EoSVs). Finally, we identified that the pattern of mitochondrial interactions changed according to the microenvironment, with mitochondria-EoSVs and mitochondria-LD contact sites detected only in activated eosinophils. Altogether, our findings identified that mitochondrial remodeling and mitochondria inter-organelle interactions are closely associated with inflammation-induced eosinophil activation and immunomodulation of their responses during inflammatory diseases. These events demonstrate that mitochondria within eosinophils are responsive to the surrounding milieu and may participate in signaling pathways that regulate eosinophil immune functions.