Influence of B-1 cell extracellular vesicles on the immune response of mice challenged with E. cuniculi

Abstract

The microsporidia of the Encephalitozoon species infect a wide variety of mammals and are opportunistic agents in immunosuppressed patients, such as HIV+ patients, as well as in other therapeutic or genetic conditions. In these cases, the disseminated infection determines death due to the lack of effective therapy. The adaptive immune response is essential for the elimination of these pathogens, especially due to the cytotoxic activity of CD8 T lymphocytes. B-1 cells are specialized B lymphocytes and act as antigen presenters, and phagocytes, producing and utilizing IL-10 as a potent negative regulator of cell-mediated immunity, among other functions. The form of participation of these cells in the dynamics of the inflammatory process of different etiologies has been studied. In a previous study, our group demonstrated that the presence of B-1 cells determined a predominance of macrophages with an M1 profile and of pro-inflammatory cytokines and increased phagocytic activity of adherent peritoneal cells obtained from peritoneal lavage, while the absence of B- 1, determines M2 profile, with delay in microbicidal activity and less cell death. In addition to the contact between B-1 cells, many extracellular vesicles were observed being released by them, suggesting the transfer of information between cells by them. The objective of this work will be to evaluate the influence of extracellular vesicles of B1 cells on the immune response of mice previously treated with extracellular vesicles and then challenged with Encephalitozoon cuniculi. B-1 cells will be collected from peritoneal washes of BALB/c mice, separated in magnetic columns, and challenged or not with E. cuniculi spores, previously cultured in RK13 cells. The EVs separated by ultracentrifugation will have their protein content quantified, size and concentration, morphology will be characterized, as well as their phenotype. Balb/c and Balb/c Xid mice will be treated or not with EVs and after 48h challenged with E. cuniculi intraperitoneally. The evolution of microsporidium infection will be carried out by quantifying the fungal load in the liver and the presence of histological lesions in the animals. Additionally, the immune response will be verified by the quantification of macrophages and definition of their profiles (M1 and M2), T and B lymphocytes, and for CD8 T lymphocytes the expressions of activation molecules (CD69, CD62L, CD107a, CD178). The pro- and anti-inflammatory cytokines in the serum of these animals will also be measured. One-way or two-way analysis of variance (ANOVA) will be used to compare groups. Values will be presented as experimental means ± the standard error. Values of p<0.05 will indicate statistical significance, with a confidence interval of 95%.(AU)