Phosphorus Modifies Macrophage—Encephalitozoon cuniculi Interaction in a Potency-Dependent Basis In Vitro

Microsporidiosis is an opportunistic infectious disease caused by the fungus Encephalitozoon cuniculi (E. cuniculi), an intracellular parasite that infects different cell types in vertebrate and invertebrate hosts. The aim of this work was to know the cellular basis of macrophage response against this parasite after treatment with Phosphorus, the epidemic genus of microsporidiosis in rabbits. RAW 264.7 cells were co-cultivated with E. cuniculi in RPMI medium and treated with 20% Phosphorus 6cH, 30cH and 200cH at the time of the cell infection. The controls consisted of untreated co-cultures and cultures treated with the vehicle (0.06% alcohol) and pure water. The phagocytosis and the lysosome activity were evaluated after 1 and 24 hours of incubation, using the calcofluor and acridine orange staining methods followed by automatic image analysis. The cytokine production was evaluated using the MAGPIX-LUMINEX system. Alcohol itself was able to increase IL-6, MCP-1, and MIP1 production (p < 0.05) and reduce the number of phagocyted parasites (p < 0.0001) just after 1 hour of incubation. No significant effect was seen after 24 hours. Phosphorus 6cH increased the lysosome activity (or acridine staining intensity) after 1 and 24 hours of incubation and reduced the number of phagocyted parasites after 24 hours (p < 0.05). Phosphorus 30cH increased lysosome activity after 1 hour of incubation, followed by reduction in parasite internalization (p < 0.001) and increase in MCP-1 production (p < 0.05) after 24 hours, even in relation to cells treated with the vehicle. The 200cH potency also increased the lysosome activity in the 1st and 24th hours (p < 0.05), together with reduction in internalized parasites (p < 0.01) and increase in MCP-1 (p < 0.05) in relation to untreated cells and cells treated with the vehicle. The results suggest a specific potency-dependent increase in parasite digestion in Phosphorus-treated cells after 24 hours of incubation, corroborating the clinical success of Phosphorus observed previously.

Keywords: Encephalitozoon cuniculi, macrophages, Phosphorus, in vitro models