Excretory-secretory products produced by Paragonimus westermani differentially regulate the nitric oxide production and viability of microglial cells

Background: Tissue-invading helminth parasites secrete a large amount of cysteine proteases that may play critical roles in tissue invasion and immune modulation. However, roles of excretory-secretory products (ESP) secreted by Paragonimus westermani in the activation and death of microglial cells in brain are poorly understood. Objectives: In the present study, we investigated whether ESP could regulate microglial nitric oxide (NO) production and viability. Methods: The NO production and cell viability were assessed by respectively measuring the formation of nitrite and the release of lactate dehyrogenase. Results: At a low (0.2 μg/ml) concentration, ESP significantly stimulated NO production with no apparent cell injury or death in cultured microglial cells. However, at high (≥2 μg/ml) concentrations, ESP induced severe cell death. Inhibition of inducible NO synthase significantly reduced the NO productivity, but not cytotoxicity, of ESP. Similarly, inhibitors of the extracellular signal-regulated kinase, p38 and nuclear factor kappa B also blocked only the NO productivity of ESP. Interestingly, heat inactivation did not hamper the ability of ESP to stimulate microglial NO production. Similarly, pretreatment with thiol-crosslinking reagents dramatically reduced both proteolytic activity and cytotoxicity of ESP, but did not alter NO production in microglial cells. Interestingly, although cysteine protease competitive inhibitors and thiol-alkylating reagents markedly reduced the proteolytic activity of ESP, they did not influence the NO productivity and cytotoxicity of ESP. Conclusion: The present results indicate that the NO production and cytotoxicity by ESP may be differentially regulated via unknown mechanisms, not related with cysteine protease activity.