This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Macrophages and dendritic cells (DCs) play major roles in the innate immune responses to microsporidia. Encephalitozoon speces of microsporidia replicate within 'resting' macrophages, but are killed by IFN(-activated macrophages. Macrophage-mediated killing of these microsporidia ex vivo occurred through generation of reactive oxygen and nitrogen intermediates (ROI and RNI, respectively) which was reversed by addition of RNI or ROI inhibitors to the cultures. Mice deficient in their ability to generate ROI and RNI, however, survived infection with E. cuniculi, although they carried statistically significantly higher parasite burdens during the first three weeks of infection compared with wild-type infected mice. Iron sequestration was subsequently found to contribute to macrophage-mediated killing, as well, and addition of ferric citrate to activated macrophage cultures reversed microsporidia killing in a dose-dependant manner to reach parasite levels of approximately 50% of the non-activated infected macrophages. While the activated macrophages increased expression of RNI, non-activated macrophages infected with E. cuniculi also expressed increased levels of RNI, while activated and infected macrophages produced the highest levels of RNI. Infected resting macrophages displayed significantly lower levels of apoptosis than non-infected macrophages ex vivo. These results support further studies to explore the various roles of nitric oxide in regulating microsporidia replication and apoptosis in infected macrophages.Early studies on the role of murine DCs in microsporidiosis revealed that the inability of aged DCs to prime either young or old T cells ex vivo could be reversed by addition of IL-15 to the cultures. Continued ex vivo studies are planned using murine, non-human primate, and human models to better understand the role of DCs in priming cytotoxic T cells and macrophages to control microsporidia.
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