A Helminth Protease Inhibitor Modulates the Lipopolysaccharide-Induced Proinflammatory Phenotype of Microglia in vitro

Neuroimmunomodulation. 2016;23(2):109-21. doi: 10.1159/000444756. Epub 2016 Apr 19.


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-x0138;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-x0138;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.

MeSH terms

  • Acanthocheilonema*
  • Animals
  • Animals, Newborn
  • Cells, Cultured
  • Helminths
  • Inflammation Mediators / antagonists & inhibitors
  • Inflammation Mediators / metabolism*
  • Lipopolysaccharides / toxicity*
  • Microglia / drug effects
  • Microglia / metabolism*
  • Phenotype*
  • Protease Inhibitors / isolation & purification
  • Protease Inhibitors / pharmacology*
  • Rats
  • Rats, Wistar


  • Inflammation Mediators
  • Lipopolysaccharides
  • Protease Inhibitors