Immunological Mechanism Of Trichinella Spiralis SPI On TNBS-induced Colitis In Mice

Inflammatory bowel disease?IBD?is a chronic autoimmune disease that not only affects patients'physical health but also negatively affects the quality of their lives.More and more experimental evidence supports that immune disorders is critical to IBD.Recently,researchers have found that intestinal worms have great potential in regulating the host's immune response,so intestinal worms may have potential therapeutic effects on the inflammatory response induced by excessive immune response.Although worms may be beneficial for the treatment of IBD,there are still potential side effects of therapeutic worm infections.Therefore,the identification of anti-colitis immunomodulatory molecules derived from worms is important for searching the new treatment of IBD.Proteases and protease inhibitors,as important signaling molecules,participate in many highly controlled physiological activities which contains inflammatory responses.Therefore,we speculate that worm-derived proteases and protease inhibitors may be involved in regulating immune-related inflammatory responses through a series of mechanisms.In this study,recombinant Trichinella spiralis?T.spiralis?Serine protease inhibition?SPI??Ts Ka SPI,Ts Ad SPI?were selected as object,to analyze their intervention effects on TNBS-induced experimental colitis in mice and their potential immunological mechanism,thus laying a foundation for developing new effective worm-derived proteins/drugs to treat IBD.The contents are as follows:?1?Effects of Ts Ka SPI and Ts Ad SPI on colitis in miceIn this study,96 male BALB/c mice were randomly grouped into the prevention group and the therapy group.Changes in various indicators of colitis were detected to prove whether Ts Ka SPI and Ts Ad SPI as the main components of T.spiralis Excretion/secretion?ES?antigen can reduce the severity of TNBS-induced colitis.First,we recorded the weight changes of the mice every day and calculated the DAI scores.The results showed that the weight of mice in the PBS+TNBS group and the TNBS+PBS group continued to decrease,the DAI score continued to increase,and both reached the peak on the 7th day.The mice were then sacrificed and the role of recombinant proteins in intestinal inflammation was further analyzed by observing changes in macroscopic and microscopic damage of colon tissue.The results showed that both before and after the establishment of the colitis model,immunizing Ts Ka SPI and Ts Ad SPI could reduce the changes in macroscopic and microscopic damage of intestinal tissue,and thus have a certain relieve effect on intestinal inflammation.As a marker of neutrophils activation,MPO is involved in many processes that regulate the inflammatory response.Therefore,the expression of MPO in colon tissue was measured.The results of ELISA showed that in the Ts Ka SPI+TNBS group and the Ts Ad SPI+TNBS group,the MPO activity were significantly reduced compared with the PBS+TNBS group,indicating that the intestinal inflammatory response has been alleviated to a certain extent.In the therapy group,compared the TNBS+PBS group with the TNBS+Ts Ka SPI group and the TNBS+Ts Ad SPI group,we found the same results.By testing the above indicators,we can preliminarily believe that Ts Ka SPI and Ts Ad SPI have a certain degree of prevention and treatment of colitis in mice.?2?Changes of T and B cells during the intervention of recombinant proteinsIt is known that Th1 and Th17 immune responses are closely related to inflammatory responses,and infection with T.spiralis usually results in a Th1/Th2 mixed immune response dominated by Th2 in the host.Immunization of Ts Ka SPI and Ts Ad SPI can also results in a Th2-dominated immune response.At the same time,researchers have found that the aggressive inflammatory response is often accompanied by an inadequate Treg immune response,and Tregs infiltration is also found in T.spiralis infected muscle tissue.Therefore,this study further analyzed whether helper T cells were potential targets of recombinant proteins to exert an intervention effect on colitis by detecting changes in the number of CD4+IFN-?+Th1,CD4+IL-4+Th2,CD4+IL-17A+Th17 and CD4+CD25+Foxp3+Treg in mesenteric lymph node?MLN?and splenic lymphocyte single cell suspension.The flow cytometry?FCM?results showed that the percentage of CD4+IFN-?+Th1,CD4+IL-17A+Th17 and CD4+CD25+Foxp3+Treg in the Ts Ka SPI+TNBS group and Ts Ad SPI+TNBS group increased significantly,while the percentage of CD4+IL-4+Th2 cell decreased significantly compared with the PBS+TNBS group.The same results were obtained in the therapy group.At the same time,the expression of IFN-?,IL-4 and IL-17 in colon tissue were detected by ELISA kit,and the results were consistent with the changes in the percentage of Th1,Th2 and Th17cell.Therefore,it is preliminarily believed that recombinant protein can alleviate colitis in mice by regulating the helper T cell response,accompanied by increasing the expression of anti-inflammatory cytokines and reducing the expression of pro-inflammatory cytokines.At the same time,we also detected the changes in the expression of CD8+CD28-T cell and CD19+CD5+CD1d^hi Breg which exert immunosuppressive effects.The results showed that the degree of proliferation and differentiation of the above cells in colitis mice were significantly reduced,and immune recombinant protein could inhibit the excessive immune response by inducing the proliferation of the above cells,thus alleviating the symptoms of intestinal inflammation.?3?Changes in dendritic cells during the intervention of recombinant proteinsRecently,many researchers have indicated that dendritic cells?DCs?also play an important role in the occurrence and development of IBD.During the disease activity phase of IBD patients,a significant increase in the number of activated DCs can be detected,resulting in a disorder of immune function and a strong anti-autoimmune response.T.spiralis can inhibit the host immune response by inducing DCs to develop into an immature phenotype.Therefore,the maturity of DCs extracted from MLN and spleen were examined.The results showed that in TNBS-induced colitis model mice,DCs surface markers CD40,CD80,CD86 and MHC-?were highly expressed,while immune recombinant protein caused surface markers in a low expression state.Therefore,we speculate that recombinant proteins can alleviate the severity of colitis by limiting the maturity of DCs.Similarly,Toll-like receptor?TLR?-dependent signaling pathway also plays a critical role in inflammatory response,so we detected m RNA transcription levels of key factors in the TLR4signaling pathway in colon by q PCR.The results showed that the recombinant proteins could inhibit the over-activation of the TLR4 signaling pathway in colitis mice by reducing the expression levels of TLR4,My D88,IRAK,TRAF6,TRAM,TRIF and NF-?B,thereby inhibiting the excessive immune response and playing a role in alleviating intestinal inflammation.Meanwhile,Western Blotting was used to detect the NF-?B activation pathway,the results showed that immune recombinant proteins could limit the over-expression of NF-?B by reducing the phosphorylation level of IKB-?.In addition,we found similar results in the therapy group.Therefore,it is preliminarily believed that Ts Ka SPI and Ts Ad SPI can alleviate TNBS-induced colitis by regulating the maturity of DCs and the activation of TLR4/NF-?B signaling pathway.?4?Changes of macrophages during the intervention of recombinant proteinsIt is well known that macrophages can take part in the course of intestinal inflammatory by releasing pro-inflammatory cytokines,and previous studies have found abnormal activation of M1-phenotype macrophages in peripheral blood and inflammatory sites of IBD patients.While helminth infection promotes the activation of M2-phenotype macrophages.Therefore,we examined the degree of proliferation and differentiation of M1-and M2-phenotype macrophages in MLN and splenic lymphocyte single cell suspension to analyze whether macrophages were potential targets of recombinant proteins to relieve colitis.The results of FCM indicated that M1-phenotype macrophages in MLN and spleen lymphocyte single cell suspensions were reduced after TNBS induction,while immune recombinant proteins significantly increased M2-phenotype macrophages.At the same time,i NOS and Arg1 were the recognition markers of M1-and M2-phenotype macrophages,respectively.Therefore,the concentration of i NOS and Arg1 in colon tissues were detected by ELISA,and the results were consistent with expectation.Therefore,we believe that recombinant proteins can inhibit colitis not only by changing the polarization direction of macrophages,but also by inhibiting the expression of the pro-inflammatory cytokine TGF-?and promoting the expression of the anti-inflammatory cytokine IL-23.The Janus kinase/signal transducer and transcriptional activator?JAK/STAT?signaling pathway is involved in activating many important cellular processes.Therefore,the changes in the expression of key factors in the IL-6/JAK2/STAT3 signaling pathway were detected by Western Blotting.The results clarified that the recombinant proteins reduced the degree of the excessive immune response by increasing phosphorylation levels of related proteins in IL-6/JAK2/STAT3 signaling pathway,thereby alleviating TNBS-induced colitis.In conclusion,this study has confirmed the preventive and therapeutic effects of Ts Ka SPI and Ts Ad SPI on TNBS-induced experimental colitis in mice,and many kinds of immune cells can be potential targets for Ts Ka SPI and Ts Ad SPI to relieve colitis.Therefore,the results of this study have important implications for the research and development of new effective worm-derived drugs.