Study On The Roles Of Trichinella Spiralis Extracellular Vesicles On Macrophage Polarization

Trichinellosis is a serious food-borne zoonosis caused by Trichinella spiralis.Trichinella spiralis invasion of the host usually induces a strong Th2 type immune response and an increase in Treg cells,as well as alternative activation of macrophages(M2 type macrophages)and incomplete matutation of dendritic cells accompanied by elevated expression of immunosuppressive fectors(IL-10 and TGF-β),creating an effective anti-inflammatory environment that not only ensures its successful parasitization and long-term survival in the host.During helminth infection,M2 subpopulation of macrophages protects the host by limiting the intense inflammatory response and repairing post-infection tissue,or promotes the Th2 response and exerts anthelmintic effects by controlling the Th1 response.Previous experiments have shown that infection with Trichinella spiralis,ES product and immunomodulatory factor induced M2-type macrophages,thus suggesting that Trichinella spiralis can alter macrophage activity to achieve long-term parasitism.Extracellular vesicles(EVs),as a new mode of parasite-host interaction,can regulate the function of receptor cells through their cargo.However,the mechanism by which Trichinella spiralis EVs(Ts-EVs)regulate macrophage activity remains unclear.The purpose of this study is to investigate the effects of Ts-EVs on macrophage polarization in vitro and in vivo,so as to provide a new idea for the study of the immune regulation mechanism of Trichinella spiralis.First,Ts-EVs were isolated and purified by ultracentrifugation.Bilayer vesicles were observed by transmission electron microscopy(TEM).Nanoparticle tracking analysis(NTA)identification showed that the main peak of particle size was 162 nm and the particle size was mainly distributed between 90-210 nm.Western blot analysis showed that Ts-EVs expressed EVs-specific marler proteins CD63,Enolase and heat shock protein70(Hsp70).The results showed that Ts-EVs were successfully obtained,and the isolated Ts-EVs can be used for further functional identification.Secondly,in order to determine which immune cell Ts-EVs mainly affected,we injected 15 μg and 30 μg Ts-EVs into C57BL/6 mice through tail vein,and peripheral blood immune cells were isolated after 12 h.We found that Ts-EVs significantly reduced the number of monocyte macrophages.Therefore,we investigate the effect of Ts-EVs on macrophage polarization in this study.Mouse bone marrow derived macrophages(BMDMs)were isolated and cultured with RPMI-1640 containing 10%FBS.BMDMs were identified by flow cytometry(F4/80)with a purity of over 90%.Then BMDMs were treated with Ts-EVs(5μg/m L)for 24 h.Western blot showed that Ts-EVs could significantly promote the expression of Arg-1,while inhibit NO production was detected by Giress reagent.q RT-PCR results showed that Ts-EVs group can significantly inhibit the expression of TNF-α,IL-1β,IL-17 A gene levels,promote IL-10,TGF-β,IL-4 gene levels,at the same time induce the expression of Arg-1,FIZZ1 and Ym1,reduce the expression of i NOS.In addition,phosphorylation of the JAK1/STAT3/STAT6 signaling pathway was detected by Western blot.The results showed that the expression of p-JAK1,p-STAT3 and p-STAT6 were significantly increased in Ts-EVs group compared to Control group,indicating that Ts-EVs inhibited BMDMs polarization to M1 through the JAK1/STAT3/STAT6 signaling pathways.We speculated that Ts-EVs could inhibit the production of pro-inflammatory cytokines,induce expression of M2 markers(Arg-1,FIZZ1 and Ym1),thereby inhibiting M1 macrophages polarization and promoting M2 macrophages polarization,regulating host immune response at the macrophage level,thus facilitating the parasitism of Trichinella spiralis.Finally,a mouse colitis model induced by DSS(DSS-IBD)was established to study the effect of Ts-EVs on macrophage polarization in vivo.The pretreatment procedure of Ts-EVs was as follows: the obtained Ts-EVs(50 μg/mice)was intraperitoneally injected into C57BL/6 mice,and then the mice were given water containing 3%DSS.Every day.the body weight and faces of mice were observed and recorded.On the 7th day,the mice were sacrificed.Body weight,DAI,colonic length and colonic macroscopic score results showed that Ts-EVs can prevent the clinical symptoms of DSS-IBD.H&E staining,PAS staining and MPO activity analysis showed that Ts-EVs could prevent the hhistopathological injury of DSS-IBD.The expression of various cytokines in the colon was determined by q RT-PCR and ELISA.The results showed that DSS induced the expression of inflammatory factors increased,while the early injection of Ts-EVs inhibited the expression of DSS induced pro-inflammatory factors.These results suggested that Ts-EVs can effectively interfere with the development of DSS-IBD by regulating the expression of cytokines in colon.Immunohistochemical analysis was performed to study the effect of Ts-EVs on macrophage polarization in DSS-induced colitis.The results showed that F4/80(macrophage marker)was expressed in the colon tissue of all groups,and CD11c(M1 macrophage marker)expression level in the DSS+Ts-EVs group was significantly lower than that in the DSS group.CD206(M2 macrophage marker)expression level in the Ts-EVs group was significantly higher than that in the Control group,indicating that DSS induced increased infiltration of M1-type macrophages in colon tissues,while the injection of Ts-EVs in advance significantly reduced the infiltration of M1-type macrophages.The effect of Ts-EVs on macrophage polarization was further observed by flow cytometry,and the results showed that Ts-EVs could inhibit macrophage polarization toward M1.This study showed that there were F4/80 positive cells in the mesenteric lymph nodes of all groups,and the number of CD16/32 positive cells in the DSS+Ts-EVs group was significantly lower than that in the DSS group.The number of CD206 positive cells in the Ts-EVs group were significantly higher than that in the Control group,which indicated that Ts-EVs could inhibit the polarization of macrophages toward M1.This study showed that Ts-EVs inhibited M1-type macrophage polarization,induced macrophage conversion to M2-type,and affect the pathological process of DSS-induced colitis by regulating macrophage polarization,which provided an experimental basis and theoretical rationale for studying the interaction between Trichinella spiralis and the host immune system.