Study On The Immune Responses Of ICOS-ICOSL Signaling Pathway In Mice And Developmental Proteins Analysis Of Angiostrongylus Cantonensis

Angiostrongyliasis cantonesis is a human zoonotic parasitic disease, results frominfections with the nematodes that A.cantonensis accidentally infects humans andmigrates to the central nervous system.Specifically, infections with A.cantonensis cause eosinophilic meningitis andeosinophilic enteritis. T cells require two signals to become fully activated. A first signal,which is antigen-specific, is provided through the T cell receptor which produced byA.cantonensis with peptide-MHC molecules on the membrane of antigen presenting cells(APC). A second signal, the co-stimulatory signal, is antigen provided by theA.cantonensis between co-stimulatory molecules expressed on the membrane of APCand the T cell. One of the best costimulatory molecules expressed by T cells is CD28,which interacts with CD80(B7.1) and CD86(B7.2) on the membrane of APC. Anothercostimulatory receptor expressed by T cells is ICOS, which interacts with ICOSL.In addition, when parasites changed their hosts to finish their life cycle, theyrequire the ability to respond to the host environments and regulate differential geneexpression. In the life cycle of A.cantonensis, adult worms reside in normal host（rats），which discharging the L1after infection. Widely disseminated via water, L1enter theintermediate host (such as Pomacantonensisea canaliculata), and develop into L3. L3infects rats, penetrates blood-brain barrier, and then develop into L4and L5, anddevelop into adults in the blood vessels of heart and lungs.This study established ICOSL-KO and C57BL/6J mice as experimentalAngiostrongyliasis model and further explained ICOS-ICOSL interaction contributes tothe immune respond in mice infected with A.cantonensis. Secondly, the stage-specificproteins of L3, FL5，ML5, FA and MA of A.cantonensis were also analysed by2D-DIGE binding mass spectrometry. The finding provided a new way for controlingeffectively the progress and regression of inflammatory reaction and provided the newstrategy of treatment of Angiostrongyliasis cantonesis. Objective: Establish the model of ICOSL-KO mice (CD275knockout mice） andC57BL/6J mice infection with A.cantonensis, observe the dynamic change of IgG、IgG1、IgG2a in serum, the co-stimulatory factor expression of CD4+T lymphocytesand the expression of cytokine spleen lymphocytes in different period of infection, thenexplore the immunological mechanisms that may be related to ICOS-ICOSL signalingpathway and the related regulatory function of hosts.Methods: Establish the model of ICOSL-KO mice and C57BL/6J mice infectionwith A.cantonensis. Collect blood from the mice eye socket, and obtain serum.Determinate the expression levels of IgG, IgG1and IgG2by ELISA in different periodof infection. Separate and observe the co-stimulatory signal of cell from mice infectedA.cantonensise in CD4+T lymphocytes. Measure the fluorescence of cells by flowcytometry (FCM), analysis the positive expression ratio with EXP30ADC software.Furthermore, culture spleen CD4+T lymphocytes of mouse, add soluble antigen of theL5of A.cantonensis and ConA, then collect cell supernatant after72hours ofincubation. Determinate the expression levels of cytokine such as IFN-γ, IL-12, IL-4,IL-5and IL-13by ELISA. Statistically significant was determined by Student’s t testor F test with SPSS17.0.Results: The level of IgG increased in both groups of C57BL/6J mice and thegroup of ICOSL-KO mice. The subtypes IgG1increased at3weeks postinfection andIgG2a reduceded at3week postinfection in the group of C57BL/6J mice，however, thelevels of IgG1was stable postinfection and IgG2a increased at3weeks postinfectionin the group of ICOSL-KO, shows that the signaling pathways are blocked in theprocess of the generation of protective immunity, so it is unable to quickly organize theeffective dominant response of Th2. The produce of co-stimulatory molecule oflymphocytes has significant hysteresis in the group of ICOSL-KO and has significantdifference after3weeks post infection; however, those of lymphocytes have significantdifference after1week post infection in C57BL/6J. It indicates that it has a closerelationship between the immune response of CD4+T lymphocytes and ICOSco-stimulatory molecules in different period of infection of A.cantonensis. The IFN-γand IL-12were increased in the early period and then gradually decreased along with infection in C57BL/6J; it indicates that the immune response level of Th1takesadvantage within2weeks post infection, and then replaced by the immune responselevel of Th2in the group of C57BL/6J. However, the stable of Th1cytokines such asIFN-γ and IL-12in the early infection and the expression of Th2cytokines IL-13wererelatively lower at3weeks post infection in ICOSL-KO mice. Above all, ICOS-ICOSLsignaling pathway is an important co-stimulatory molecule for infection-inducedexpansion of Th1and Th2responses.Conclusion: It indicated that Th2responses are predominant in mice infectedwith A.cantonensis. The ICOS-ICOSL signaling pathway plays a key role on theoccurrence and development of infection-induced expansion of Th1and Th2responses. Objective: Identify the stage-specific proteins between L3, FL5, ML5, FA, andMA of A. cantonensis. The results might prove evidence on key parasite proteins thatare involved in the host regulation or host-parasite interaction.Methods: Collect parasites from the life cycle of A. cantonensis in laboratoryconditions, separate L3, FL5, ML5, FA, and MA respectively. Pyrolysis the sampleswith lysis buffer and sonication, and obtain supernatant after centrifugation. The totalprotein concentration was determined with Bio-Rad protein assay reagent. Sampleslabeled with Cy3or Cy5, and the internal standard labeled with Cy2. After2D-DIGE,the gels were scanned by UMax Powerlook2110XL and Typhoon FLA9000, obtainedfluorescent images. Analysis images with DeCyder7.0software. Then the proteins wereidentificated by MALDI-TOF-MS and searched database. Two genes and the internalcontrol18s were chosen for quantitative real-time PCR (qPCR).Results: Obtain clear protein expression profiles, after statistical analysis (t test)the differential expressed proteins, select183protein spots that appear in24proteinexpression profiles.5spots were up-regulated in L5compared with adults, but showno significantly different in gender difference;89spots such were up-regulated in L5and adults compared with L3, in contrast,13spots were up-regulated in L3.12spots were up-regulated and7spots were down-regulated in MA relative to FA.3spots wereup-regulated in FL5and3spots were up-regulated in ML5. Compared with female,spot1496increases in both MA and ML5, suggesting a difference between the sexes.37proteins were successfully identified with high confidence (>95%) scores byMS. Among the predicted proteins,29spots were cytoskeleton-associated proteins andfunctional proteins, such as actin, paramyosin, heat shock protein, and transformationdomain-associated protein (TRR-1).8spots represented unnamed proteins.12spotswere matched to the EST of different-stage larvae of A. cantonensis, they have not beenidentified. Two genes corresponding to protein spots designated609and1004and theinternal control18s were chosen for qPCR analysis to quantify their levels oftranscription. The qPCR results were consistent with those of the DIGE studies,suggesting that the proteins here identified as differentially expressed were regulated atthe transcriptional level.Conclusion: The findings will provide a new basis for understanding thecharacteristics of growth and development of A. cantonensis in environmentalregulation and the host–parasite relationship. They may also provide a new way forcontroling effectively the progress and regression of inflammatory reaction and as drugtargets for the control of eosinophilic meningitis induced by A. cantonensis.