Influence of B-1 cells or their extracellular vesicles on effector immune cell activity in microsporidiosis

Abstract

Microsporidia are recognized as opportunistic pathogens in individuals with immunodeficiency, especially T-cells deficient. Although CD8 T lymphocyte activity is essential for eliminating these pathogens, macrophages and others innate immunity cells play a critical role in activating adaptive immunity. B-1 cells are a key component in innate immune response against pathogens but their participation is still poorly understood. Previously, we demonstrated that B-1 cells-deficient mice (Xid) were less resistant to experimental encephalitozoonosis and showed a high fungal burden despite the increase in CD8 T lymphocyte population. In vitro, in the absence of B-1 cells, were identified E. cuniculi infected peritoneal macrophages of the M2 profile. In the presence of B-1 cells or B-1 cell-derivated phagocyte, macrophages were predominantly M1 and B-1 cells showed extreme physical contact with other immune cells such as lymphocytes and macrophages, and extensive releasing of extracellular vesicles. The aim of the present project is to evaluate the effects of B-1 cells or their extracellular vesicles (EVs) on CD8 T lymphocyte cytotoxic activity or macrophage phagocytic and microbicidal activity on microsporidiosis. Three experiments will be performed: a) evaluation of CD8 T lymphocyte activity from mice with and without B-1 cells co-cultured with E. cuniculi infected macrophages, b) characterization of extracellular vesicles as size, content and function of E. cuniculi infected B-1 cells and their effects on bone marrow macrophages, c) analysis of the in vivo effect of extracellular vesicles of E. cuniculi infected B-1 cells on CD8 T lymphocytes and macrophages. BALB/c, BALB/c Xid and Xid+B-1 mice (adoptively transferred with B-1 cells) will be infected or not with E. cuniculi spores and after 7 and 14 days CD8 T lymphocytes from their spleen will be purified and co-cultured with E. cuniculi infected macrophages. After 36 and 72h, proliferation, viability and presence of activation molecules (CD62L, CD69, CD107a, CD178, anti-perforin) in CD8 T lymphocytes and their cytokine production will be evaluated. E. cuniculi infected macrophages will be analyzed for viability and microbicidal activity. The activation molecules will be evaluated ex vivo in CD8 T lymphocytes from the peritoneum, mesenteric lymph nodes and spleen of E. cuniculi infected mice with and without B-1 cells. Purified B-1 cells from BALB/c mice will be infected or not with E. cuniculi. After incubation, EVs will be analyzed for size, quantity, phenotype and protein concentration. Subsequently, EVs will be used for in vitro treatment of bone marrow macrophages or in vivo BALB/c and Xid mice. Macrophages and mice treated with EVs will be challenged with E. cuniculi. Phagocytic and microbicidal functions, cytokine production and death of macrophages will be evaluated. The fungal burden into the liver and the presence of histological lesions in the animals will be analyzed. Activation molecules (CD62L, CD69, CD107a, CD178, anti-perforin) will also be measured ex vivo in the CD8 T lymphocytes from the peritoneum, mesenteric lymph nodes and spleen of mice treated with EVs. One-way or two-way analysis of variance (ANOVA) will be applied for comparison between groups. Values will be presented as sample means±standard error. Values of p<0.05 with a 95% confidence interval will be considered. (AU)