The Effects Of Taenia Solium Serine Protease Inhibitors On Host Immune Cell Functions

Taeniasis solium/Cysticercosis cellulose a serious foodborne parasitic disease of humans,which is harmful not only to human health but also the development of pig industry.The disease is distributed worldwide and human are its only definitive host,making it a threat to public health.At present,the vaccines and drugs used for the prevention and treatment of Taenia solium are not sufficient for control of the disease or transmission.Serine protease inhibitor,which could inhibit both serine protease and cysteine protease activity have been shown to participate in the regulation of physiological processes such as agglutination,inflammation and apoptosis.So far,Serpin has been isolated and identified from a variety of parasitic helminths,such as Filaria,Trichinella spiralis and Echinococcus multilocularis.However,there are few studies on T.solium(Ts)serine protease inhibitor(Ts serpin)and its role in host interaction is still unclear.In this study,the immunoregulatory effect of Ts serpin on host infection was investigated as follows:1.Expression of Taenia solium serine protease inhibitor at different developmental stages.The coding DNA sequences(CDS)of five Ts serpins(Ts serpin B6 is 1131 bp,Ts serpin 570 is 1206 bp,Ts serpin EP45 is 1350 bp,Ts serpin 4848 is 1065 bp and Ts serpin 12383 is 1149 bp)were amplified by RT-PCR with specific primers.qRT-PCR was used to analyze the expression of Ts serpin genes in adult and larval stages.2.Study on the protease inhibitor activity of Ts serpins.The purified recombinant protein were detected by chromogenic substrate method.The five Ts serpins listed above showed significant inhibitory activity against α-chymotrypsin,trypsin and pancreatic elastase.The results showed that the purified protein had enzyme activity and had obvious inhibitory effects on the activity of a number of proteases,suggesting that the Ts serpins possessed a broad spectrum of activity.Ts serpin 570 could also inhibit thrombin factor,better absorption of nutrients in the host circulation.3.The expression profiles of mRNA in THP-1 cells treated with recombinant Ts serpins for 24 h were determined.A total of 2957 mRNAs were significantly differentially expressed(fold change≥ 1,p<0.05)in THP-1 cell treated with Ts serpin 570.These included 1630 up-regulated and 1327 down-regulated mRNAs.Ts serpin B6 induced changes in the expression level of 2908 mRNAs,including 1514 up-regulated and 1394 down-regulated genes.The biological functional prediction indicated that these mRNAs were enriched in immune pathways to resist tapeworm infection,such as the NOD,TLR and TNF signaling pathways.These results suggest that Ts serpin may play an important role in parasite-host interaction.4.The effects of recombinant Ts serpins on LPS-activated THP-1 cells were studied in vitro.THP-1 cells treated with Ts serpins and LPS significantly inhibited the production of NO and the secretion of the pro-inflammatory cytokines TNF-α,IL-1β,IL-6,IL-12α,IFN-γ and iNOS2.The secretion of the anti-inflammatory cytokines IL-10 and TGF-β was elevated.It is suggested that Ts serpins,as immunomodulatory molecules,may promote immune-evasion by inhibiting the inflammatory response of the host macrophages,thus leading to long-term persistence in the host.5.The interaction of Ts serpin B6 and Ts serpin 570 with intermediate host proteins were determined.Proteins were studied by Co-Immunoprecipitation,His-pull down and liquid chromatography-mass spectrometry show that interactions occurred between Ts serpin B6 and Ts serpin 570.These proteins were identified and most of them were ribosomal proteins involved in translation,energy production and conversion and signal transduction.The host proteins binding to Ts serpins enriched the most significant pathway chemokines,PI3K-Akt and cell apoptosis related signaling pathways.The apoptosis level of THP-1 cells treated with Ts serpin B6 and Ts serpin 570 showed that both of them significantly increased the level of THP-1 apoptosis.It was suggested that these two serpins might modulate biological function through inflammatory pathways.