IMMUNE RESPONSES TO MICROSPORIDIA

Tulane University Of Louisiana, New Orleans LA

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Abstract

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. Microsporidia are fungi-related, obligate intracellular parasites. Members of the genus, Encephalitozoon, are clinically important as causes of emerging and opportunistic infections associated with diarrhea and systemic disease. E. cuniculi replicates within macrophages and is believed to disseminate via trafficking monocytes/macrophages. Little is known about how microsporidia survive within macrophages. To address this, the affect of E. cuniculi infection on the ability of macrophages to undergo apoptosis was evaluated. Cultures of human THP-1 macrophages were inoculated with live or dead E. cuniculi (2:1 spores to cells). After 1, 2, 4, and 8 days, the cultures were induced for apoptosis with staurosporine (50 ng/ml) for 4 hours and assessed via TUNEL. Macrophages inoculated with live spores exhibited 1.8-, 3.1- and 3.2-fold less TUNEL staining than those inoculated with medium after 1, 2 and 4 days, respectively. After 8 days, macrophages inoculated with live spores exhibited signs of rupture due to infection and displayed only a 1.64-fold reduction in TUNEL-positive cells compared with control macrophages. Caspase 3 activity associated with apoptosis was measured on day 4 and was 4.3-fold lower in macrophages treated with live spores compared with those incubated in medium only. Furthermore, macrophages treated with dead microsporidia exhibited significantly higher caspase 3 activity. Confocal microscopy demonstrated that only uninfected macrophages could be observed to undergo apoptosis in cultures incubated with live microsporidia and treated with staurosporine (i.e. none of the infected macrophages demonstrated signs of apoptosis). These early results suggest that E. cuniculi may inhibit apoptosis of macrophages to prolong their survival within these cells.