Characteristics Of IL-17Induction By Schistosoma Japonicum Infection In C57BL/6Mouse Liver And Lung

Disease symptoms are due predominantly to the host immune response toschistosome eggs (ova) and the granulomatous reaction evoked. Granulomas destroythe eggs and sequester or neutralize otherwise pathogenic egg antigens but also lead tofibrogenesis in host tissues. Infection by S. japonicum, a multi-cellular parasite withan extremely diverse repertoire of antigens, induces the production of multiplecytokines that mediate the immune response. These cytokines are therefore potentialtherapeutic targets for schistosomiasis treatment. In fact, several studies haveconcluded that IL-17is the most directly cytokine associated with the severity ofhepatic granulomatous inflammation. Multiple studies have demonstrated that Th17isnot the only IL-17-producing T-cell population; CD8+T cells, NKT cells and γδT cellsare also IL-17-producing T cells in many infections, such as Listeria monocytogenes,Nocardia asteroides and Salmonella enterica serovar Typhi. It is of interest that thesource of IL-17is various under different conditions or different organs.The aim of the current study was to characterize the role of IL-17in the pathogenicprocesses of the S. japonicum-infected liver and lung. We compared expression andsecretion of IL-17from different lymphocyte subsets, CD4+T, CD8+T, NKT and γδTcells in response to non-specific stimulation with PMA and ionomycin in the liver andlung, respectively. Moreover, we found that25%of γδT lymphocytes produced IL-17,which was the largest cell population in the mouse liver, while CD4+T cells weredominated in the lung. To further ascertain the role of IL-17in the development of egg-induced granulomas, we treated mice with IL-17-neutralizing mAb or isotypecontrol mAb starting on3week post infection, and continuing every third days until2days before killing. Our study included the following two parts: First, characteristicsof IL-17induction by Schistosoma japonicum infection in C57BL/6mouse liver;secondly, roles of Th17cells in pulmonary granulomas induced by Schistosomajaponicum in C57BL/6miceCharacteristics of IL-17induction by Schistosoma japonicum infection inC57BL/6mouse liverC57BL/6mice were infected with40cercariae of S. japonicum and sacrificed at6week after infection. Single liver cell suspensions of normal and infected mice wereprepared and then cultured in the presence of anti-CD3mAb plus anti-CD28mAb.IL-17and IFN-γ mRNA expressions in the livers were detected by real-time reversetranscription quantitave polymerase chain reaction (RT-qPCR). Moreover，the culturesupernatants were collected after72h of incubation for detection of interferon-γ(IFN-γ) and IL-17by ELISA. CD3+and CD3-cells producing IL-17were detected byflow cytometry. We then compared IL-17expression in three hepatic T-cell subsets,CD4+T help cells, natural killer T cells, and γδT cells, to determine the major sourceof IL-17during infection. We administered an anti-IL-17mAb to infected mice toevaluate the role of IL-17in the host protective responses against S. japonicuminfection. Neutralizing anti-mouse IL-17A mAb or an isotype-matched rat IgG2amAb was first administered intraperitoneally3weeks after S. japonicum infection(62.5μg per mouse) then at the same dose every third day until2days before killing.Hemotoxylin and eosin (H&E) staining and Masson staining were used forpathological examination. Moreover, pro-collagen type III (PC-III), type IV collagen(IV-C), immunoglobulin G and IgE antibodies to soluble egg antigen (SEA) weremeasured by ELISA.Our results showed that IL-17protein can be detected in the liver cell culturesupernatant and IL-17mRNA expression was increased after infection, suggestingthat the production of IL-17in the liver is markedly enhanced by infection. Moreover,non-specific stimulation by PMA and ionomycin induced a much greater increase inthe proportion of IL-17+cells in the CD3+cell population from infected livers than inthe CD3-cell population from uninfected livers. We concluded that all three cell typesare sources of IL-17during S. japonicum infection, but that of these cell subtypes, the largest proportion of γδT cells produced IL-17. The extent of hepatic granulomatousinflammation and collagen deposition around schistosome eggs was reduced inanti-IL-17A neutralizing mAb mice compared to the mice which were provided theisotype control IgG. Moreover, IL-17normally serves to suppress SEA-specific IgGexpression, but has little effect on SEA-specific IgE expressionRoles of Th17cells in pulmonary granulomas induced by Schistosoma japonicumin C57BL/6miceC57BL/6mice were infected with40cercariae of S. japonicum and sacrificed at6week after infection. The percentages and absolute numbers of the CD3+cells andCD4+T cells in the pulmonary mononuclear cells of normal and infected mice wereextermined by FACS analysis. The levels of IFN-γ, IL-4, IL-17in the singlemononuclear lung cell suspensions of were detected by ELISA. The proportions ofIFN-γ+, IL-4+and IL-17+in the CD3-cells and CD3+cells were detected. FACS wasperformed to investigate whether CD4+Th, CD8+Tc, γδT cells, and natural killer Tcells are the producing-IL-17cells during S. japonicum infected lung and which is themain producing-IL-17cells population. The change of Th17cells from lungs wereobserved at0,4,5,6,7weeks after infection. Moreover, the correlation of Th17cellsto IFN-γ, IL-4, IL-5, IL-9and IL-10secreted Th cells was explored. The immuneresponse and pathological changes in the lungs were observed by HE.The absolute numbers of mononuclear cells (MNC) in the lung showed an increaseafter infection and the percentages of the CD3+cells and CD4+T cells weresignificantly increased during the infection. Moreover, FACS analysis showed theproportions of IL-17, IFN-γ and IL-4were significantly increased after infection inboth CD3+cells and CD3-cells compared with the control group. However, whencompared with CD3+cells, the proportions of IFN-γ+CD3-, IL-4+CD3-andIL-17+CD3-cells were lower in both normal control and infected group. Th17cellswere shown to be the initial source of IL-17during pulmonary infections with S.japonicum, which made up about40%of all IL-17positive cells. It indicated that theTh17cells after S. japonicum infection could secrete high proportion IL-4and IL-5compared with other cytokines. However, Th17cell hardly secret IL-9and IL-10. Alltogether, these results suggested that generation of IL-17during S. japonicuminfection may enhance pulmonary granulomatous inflammation, implicating thatIL-17promoted the severity of S. japonicum infected lung pathogenesis.In conclusion, we investigated the characteristics of IL-17in the liver and lung during the S. japonicum infection. The secretion of IL-17by liver and lunglymphocytes was significantly enhanced by S. japonicum. Our reports concluded thatIL-17was might produced mainly by γδT cells in the liver after infection by S.japonicum, while in the lung is T helper type17(Th17) cells. It concluded that IL-17is most directly associated with the severity of hepatic granulomatous and fibrosinginflammation. Targeting Th17/IL-17may represent a effective new approach tocurtailing and treating egg-induced immunopathology. We expanded the law of Thelper cells (Th) cytokine in murine schistosomiasis, and also enriched themechanism of immune regulation in schistosome granuloma development.