This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In previous studies, macrophages activated in vitro with LPS and IFN-gamma were shown to kill intracellular microsporidia (ie. Encephalitozoon cuniculi). Reactive nitrogen and oxygen intermediates contributed to the macrophage-mediated killing of E. cuniculi based on inhibitor studies. In vivo murine studies, however, indicated that additional mechanisms of resistance participate in controlling E. cuniculi infection. B6.129S6Cybbtm1 mice, which carry the recessive disorder for chronic granumlomatous disease (CGD) and are unable to generate a phagocytic oxidate respiratory burst, when treated with aminoguanidine to also inhibit the production of nitric oxide, survived infection with E. cuniculi. Studies conducted during the previous year were performed to assess the role of iron during macrophage-mediated killing of intracellular microsporidia using a murine macrophage cell line, RAW264.7?NO-/-. Addition of the iron chelator, desferrioxamine (DFO) to non-activated macrophages resulted in inhibited replication of E. cuniculi compared with medium-treated macrophages. Addition of ferric citrate to activated macrophages, which does not require receptor-mediated transport, resulted in a significant increase in parasite recovery compared with activated macrophages not treated with ferric citrate. Non-activated macrophages infected with E. cuniculi and treated with ferric citrate resulted in higher replication of microsporidia than infected macrophages not treated with ferric citrate. Addition of ferric transferrin was less effective in reversing macrophage-mediated killing after treatment with LPS and IFN?, presumably due to reduced transferrin receptor expression on the activated macrophages. These results support the requirement of iron for microsporidia replication and that iron sequestration may contribute to the destruction of microsporidia by activated macrophages.
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