A helminth protease inhibitor modulates the lipopolysaccharide-induced proinflammatory phenotype of microglia in vitro

Abstract

Objective: The aim of this study was to examine whether the natural protease inhibitor Av-cystatin (rAv17) of the parasitic nematode Acanthocheilonema viteae exerts anti-inflammatory effects in an in vitro model of lipopolysaccharide (LPS)-activated microglia. Methods: Primary microglia were harvested from the brains of 2-day-old Wistar rats and cultured with or without rAv17 (250 nM). After 6 and 24 h the release of nitric oxide (Griess reagent) and TNF-α (ELISA) was measured in the supernatant. Real-time PCR was performed after 2, 6 and 24 h of culture to measure the mRNA expression of IL-1β, IL-6, TNF-α, COX-2, iNOS and IL-10. To address the involved signaling pathways, nuclear NF-B translocation was visualized by immunocytochemistry. Morphological changes of microglia were analyzed by Coomassie blue staining. Differences between groups were calculated using one-way ANOVA with Bonferroni's post hoc test. Results: Morphological analysis indicated that LPS-induced microglial transformation towards an amoeboid morphology is inhibited by rAv17. Av-cystatin caused a time-dependent downregulation of proinflammatory cytokines, iNOS and COX-2 mRNA expression, respectively. This was paralleled by an upregulated expression of IL-10 in resting as well as in LPS-stimulated microglia. Av-cystatin reduced the release of NO and TNF-α in the culture supernatant. Immunocytochemical staining demonstrated an attenuated translocation of NF-B by Av-cystatin in response to LPS. In addition, Western blot analysis revealed a rAv17-dependent reduction of the LPS-induced ERK1/2-pathway activation. Conclusion: The parasite-derived secretion product Av-cystatin inhibits proinflammatory mechanisms of LPS-induced microglia with IL-10, a potential key mediator.