Novel cystatin of Trichinella spiralis inhibits inflammation mediated by bone marrow-derived macrophages

Tue16  Apr12:48pm(3 mins)
Poster
98
Where:
Renold C16

Authors

P Kobpornchai1; R Flynn3; O Reamtong2; P Adisakwatana1
1 Mahidol University/Faculty of Tropical Medicine/ Department of Helminthology, Thailand;  2 Mahidol University/Faculty of Tropical Medicine/ Department of Molecular Tropical Medicine and Genetics, Thailand;  3 University of Liverpool/ Institute of Infection and Global Health/ Department of Infection Biology, UK

Discussion

To survive in their host, Trichinella spiralis exerts molecular machineries to regulate host environments and immune responses. Releasing of immunomodulatory molecules is one of an important strategy that can suppress host inflammation and may be used for treatment of unrelated inflammatory diseases in patients. In this study, we identified and characterized a novel immunonomodulatory protein derived from excretory-secretory (ES) product of muscle stage T. spiralis. ES products cultured from the larvae were fractionated by the anion exchange chromatography and evaluated an effect on cytokine responses by treatment with LPS-induced mouse bone marrow-derived macrophages (mBMDMs). Three fractions showing high immunomodulatory property by decreasing pro-inflammatory cytokines or increasing anti-inflammatory cytokines, were subjected to protein identification using nano-LC/MS/MS. In this regard, the conserved hypothetical protein (Tsp_04814) was presented the significant highest MS score and the structural homology comparison suggested that Tsp_04814 is closely similar to cysteine protease inhibitor (renamed as TsCstN). In silico 3D structure of TsCstN contains N-terminal region, four β-sheet and two α-helix, which the conformation among N-terminal region, loop1 and loop2 is a key inhibition of cathepsin L. The recombinant TsCstN (rTsCstN) was expressed in E. coli and used for production of mouse polyclonal antibody (pAb) and functional analysis. The pAb could detect native TsCstN in crude parasite and ES of muscle stage and predominantly localized in the stichosome. rTsCstN could inhibit cysteine proteases, especially cathepsin L. Incubation of rTsCstN with LPS-induced mBMDMs exhibited a downregulaton of MHC class II and CD40 expression. Moreover, the protein could suppress an inflammation by reduction of pro-inflammatory cytokines in both mRNA and protein level. In conclusion, TsCstN is a novel cysteine proteases inhibitor eliciting an anti-inflammatory property, which may be used as an alternative treatment for inflammatory diseases in the future Keywords: Trichinella spiralis; immunomodulation; cystatin; macrophages; inflammation

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