The Function Of Trichinella Pseudospiralis Serpin In Parasites Immune Evasion

Trichinellosis is a widespread foodborne zoonotic disease which poses a greatthreat to human health, and it has been estimated that more than10million people areinfected with Trichinella. There is still a high prevalence in China. In the reported12trichinella species, trichinella pseudospiralis can infect both mammals species andsome carnivorous birds which is different from others, also, because of its specialcharacteristics it’s difficult to prevent and control.Serine proteinase inhibitor (Serpin) is a kind of regulators of serine proteinaseand cysteine protease, playing key roles in a variety of physiological functions such ascoagulation, inflammation, and apoptosis. They are found widely in animals, plants,worms, insects, microorganisms, and even some viruses. Parasite serpins play a keyrole in inhibiting blood coagulation, resistancing host protease damage, and also serveas targets for escaping host immune attack. T.pseudospiralis which no capsules shouldbe more susceptible to the host immune attack. Therefore, T.pseudospiralis which cantrigger strong inflammatory must produce a stronger immunosuppression to live in themuscle cells. So far the studies related T.pseudospiralis immune escape genes ismysterious, research on the immune regulation of T.pseudospiralis serine proteaseinhibitor during the invasion period of T.pseudospiralis will be benefical for us toprevent and control the trichinosis and other parasitic disease.First of all, the cDNA sequence of Trichinella pseudospiralis serine proteaseinhibitor was cloned by RT-PCR from total RNA of muscle larvae with specialprimers derived from the GenBank database. The PCR product was purified, digestedand ligated into the prokaryotic expression vector pET-28a(+). ThepET-28a-Tp-Serpin expression plasmid was transformed into E. coli Rosettagami(DE3) and the expression condition was optimized in order to increase thesoluble protein. The inhibitory activity of purified recombinant protein (rTp-Serpin) against different host-secreted serine proteases using substrate specificity. The resultshowed that the rTp-Serpin did display inhibitory activity against the mouse mast cellprotease-1, porcine pancreatic elastase and human neutrophil elastase, but did noteffectively inhibit human neutrophil cathepsin G. So we have demonstrated thatTp-Serpin is secreted by the adult stage of the T.pseudospiralis in order to neutralizethe potentially damaging effects of these digestive proteases within its immediateenvironment. In addition, rTp-Serpin did inhibitory activity against human neutrophilelastase suggests that this molecule may also be involved in inflammation and hostimmune evasion.Secondly, we investigated the effect of rTp-Serpin on modulating J774A.1macrophage activities. The CCK-8results showed that the viability of J774A.1macrophage was not reduced when treated with rTp-Serpin at low concentrations.Real-time PCR and ELISA results indicated that rTp-Serpin reduced the capacity ofmacrophages to express pro-inflammatory cytokines (TNF-α, IL-12, IL-6, IL-1β andIFN-γ) and effector molecule inducible nitric oxide synthase (iNOS), in response tolipopolysaccharide (LPS) challenge. However, rTp-Serpin boosted the expression ofanti-inflammatory cytokines interleukin-10(IL-10), transforming growthfactor-β(TGF-β) and effector molecule arginase1(Arg1). Western blot analysisshowed that the phosphorylation of both JAK2and STAT3in the J774A.1macrophages was stimulated by rTp-Serpin treatment. Therefore, we hypothesize thatTp-Serpin least partly depend on stimulating these pathway to induce theM2-polarization of macrophages. These results suggest that rTp-Serpin can regulatehost immune response in macrophages level by suppressing pro-inflammatoryproducts and effector molecule in J774A.1macrophages, and inducing macrophagesto polarize into the AAM by JAK2/STAT3signaling pathway activation, which maybe important for worm survival.Finally, the transcription of Tp-Serpin gene in T.pseudospiralis developmentalstages was systematically identified by semi-quantitative RT-PCR. In addition,Paraffin sections from parasites were prepared, and the expression of the Tp-Serpin gene of developmental stages was systematically identified by the Western blot andimmunolocalisation with the specific polyclonal antibody. The study provided apossibility of this antigen gene as a diagnostic antigen of T.pseudospiralis and laid thefoundation for further study on invasion, evasion, and self-development ofT.pseudospiralis. The results showed that Tp-Serpin gene was transcripted in everydevelopmental stage (AD3, NBL and ML) in this assay. Western blot analysis showedthat the antibody of the Tp-Serpin protein recognised crude antigens but not ESantigens. In addition, the immunolocalisation analysis showed that Tp-Serpin proteinwas mainly localized in surface of the worms. It was indicated that Tp-Serpin secretedby the epidermal cells may be functional protein was related to action against thedigestive enzyme in the intestine, involve in inflammation and evade host immuneeffect.