Murine microsporidiosis - the role of M1 and M2 macrophages, phagocytes derived from B-1 cells and efferocytossis

Abstract

The microsporidia infect vertebrates and invertebrates and are known as opportunistic agents in immunedeficiencies patients. Although T CD8 + lymphocyte activity is essential to eliminate the microsporidia, macrophages play a critical role in innate and acquired immunity activation. For some infectious agents, the polarization of M1 and M2 macrophages is crucial in setting the direction that the infection will take, as well as is considering the efferocytosis contribute as a facilitator mechanism and promoter of the infection. These phenomena were not analyzed for microsporidiosis, just as it is not entirely clear the role of B-1 cells and its relationship with macrophages. Thus, this project aims to evaluate the in vitro activity of phagocytic cells in infection Microsporidian Encephalitozoon cuniculi, particularly by phagocytes analysis derived from B-1 cells (B-1 CDP), polarized macrophages M1 and M2 and efferocytosis. Third experiment will be performed: 1) the assessment of B-1 cell activity of B-1 CDP will be made by inoculation of E. cuniculi obtained in cell cultures of peritoneal lavage from BALB/c, BALB/c XID and B-1 CDP cultures; 2) analysis of M1 and M2 macrophage activity by inoculating E. cuniculi in macrophages derived from bone marrow precursors (MOP) and differentiated in M1macrophages (LPS and recombinant IFN-³) and M2 (with the recombinant cytokines IL-4, IL-13 and IL-33); 3) efferocytosis and evaluation of antiinflammatory activity with MOP and treated or not with COX inhibitor (indomethacin), agonist and antagonist EP2 receptor, the MOPs are subsequently incubated with apoptotic cells and then be challenged with E. cuniculi. The adhesion and capturing of E. cuniculi will be evaluated with markers SCRI Renaissance Stain 2200 and PKH26 Fluorescent Cell Linker. The nitric oxide measurements and cytokines will be performed from the culture supernatants at different times used (0.5h, 1h, 48h, 96h and 144h). Additionally, cultures will be studied by transmission electron microscopy. The ANOVA with one or two ways will be used for comparison between groups. Values are presented as means (± standard error) of the experimental replicates, with significance set at p <0.05. (AU)