The Role Of IL-21Signaling Pathways In Schistosomiasis Granuloma Formation And Development

Objective： To construct and express recombinant immunotoxin ofmIL-21-LuffinP1，and determine the biological activity in vitro.We also observe itsinhibition in the formation of schistosoma granuloma and find the possible mechanismthrough schistoma japonicum infection model，as well as futher research and identifythe role of IL-21/Th17signal pathway in the formation and development of schistosomagranuloma.Methods:Using genetic engineering technology，we reconstruct the recombinantgene mIL-21-LuffinP1and clone into prokaryotic expression vector Pet32（a+）.Throughrestriction analysis and sequencing proving that the recombinant plasmid wasconstructed correctly，we transformed the plasmid to E.coli BL21(DE3) and inducedwith IPTG. In vitro we assayed its targeting and toxic effct through MTT and Flowcytometry. In schistosoma japonicum infection mice model，we verified its inhibition inthe formation of schistosoma granuloma through Pathological analysis，and detected themRNA level of Th17related cytokine IL-17A、 transcription factor ROR-γt、inflammatory chemokines CXCL1、CXCL2using real-time fluorescent PCR method.Results: We successfully build recombinant immunotoxin mIL-21-LuffinP1andinduce protein expression. Through MTT method,mIL-21-LuffinP1can significantlyinhibit myeloma cell SP2/0proliferation, which inhibition effect depending on theconcentration. We also find that in Flow cytometry double staining,mIL-21-LuffinP1can induce apoptosis on SP2/0cell,which highly express IL-21R.In schistosomajaponicum infection mice model，we started tail injection of mIL-21-LuffinP1orLuffinP1in the fourth week and killed mice after six weeks.By HE staining andstatistical analysis of granuloma area，we find that mIL-21-LuffinP1could significantly inhibit the granuloma lesion in vivo （P=0.018＜0.05）.Comparing with control group，IL-17level significantly decreased in mice liver of IL-21-LuffinP1group，and thetranscription factor of ROR-γt just the opoosite，while CXCL1and CXCL2mRNAlevel had no obvious statistical difference.Conclusion:We successfully build and purify immunotoxin mIL-21-LuffinP1,andconfirm its cytotoxicity and targeting in vitro.Mice experimental results showed thatmIL-21-LuffinP1could significantly inhibit the formation of schistosoma granuloma Itsmechanism may be through the role of targeting killing of Th17cells， eventuallyblocking IL-21signal pathways and inhibiting the level of IL-17, finally suppressgranulomatous inflammation.