Research On Effect And Mechanism Of T-lymphocyte Subsets And Related Molecules At Different Stages Of Immunopathogenesis By Schistosome Infection

As an infectious disease seriously endangering human health, schistosomiasis is widely epidemic in Asia, Africa and Latin America, and is listed as one of the Neglected Tropical Diseases(NTDs) by the World Health Organization. For schistosomiasis, the core pathogenic link is hepatic egg granulomas and secondary liver fibrosis. The cause of disease is the abnormal T-lymphocyte-mediated immune response, and even continuous immune response generated by CD4+T(Th) cells to hepatic egg antigen in special cases, but the specific mechanism remains to be investigated and discussed. It has been shown from previous researches that Th1/Th2 immune deviation is closely related to the schistosomiasis immunopathological injury and development of egg granuloma and its immune dysregulation. As Th-cell subsets, Th17 and Tfh, participating in schistosome infection immunity and its pathological process, have attracted much attention in recent years. Functional imbalance of Th-cell subsets(Th1/Th2/Th17/Tfh), induced after the host is infected with schistosome, is an important factor of schistosomiasis immunopathological mechanism. So far most researches on T-cell immune response during schistosome infection immune response have focused on CD4+T cell and its subsets, but very little on CD8+T cells. It was believed from some researches that CD8+T cells possibly play a role in suppressing the immune pathology in the process when schistosome Mansoni infects host, but no conclusive evidence has been established yet. Therefore it has great significance to study dysequilibrium of T-cell subsets at different pathological stages of schistosomiasis and search for the targets to regulate and control T-cell subgroup immune imbalance for controlling or preventing the onset and development of schistosomiasis immunopathological injury.It has been proved that effective activation and functional mediation of T-cells requires the synergy of double signals: one is antigen peptide- MHC complex, which is identified by T-cell Antigen Receptors(TCRs); and the other is co-stimulatory signal, which is provided by co-stimulatory molecular ligand and receptor pairs on the surface of antigen presenting cells(APCs) and T-cells. Inducible co-stimulatory molecules(ICOS) and programmed death-1(PD-1) are expressed on activated T-cells, costimulatory molecules and their ligands(ICOS/ICOSL and PD-1/PDL-1) interact to mediate the positive or negative synergy signals in the process of immune response of the host, which play an important role in regulating host immune response. Study relating to this, therefore, has become a hot spot for the research on tumor immunity, infection immunity and autoimmune diseases.This research topic was carried out with schistosome-infected mice as the experimental animal model and T-cell subgroup as the main line, so as to investigate the dynamic changes in the expression of T-cells and the related immune molecules at different stages of schistosome infection and re-infection, and study its correlation with host immunopathological changes caused by schistosome. Further research on in-vivo intervention with the ICOS/ICOSL and PD-1/PDL-1 signaling pathways over the schistosome-infected model in order to explore how to control egg granuloma reaction, the key immunopathological injury for schistosome-infected host, with positive costimulatory signal ICOS and negative one PD-1, and explore the key pathological injury-egg granuloma by schistosomiasis.Part I Expression of T-cell subset and related molecules in schistosomeinfected mouse models and the correlation between the expression and hepatic pathological changesObjective: To observe the expression changes in T-cell subgroup, Th subgroup(Th1/Th2/Th17/Tfh) as well as related molecules at different stages of schistosomeinfected mice, and pathological changes at different stages of schistosomiasis were parallel observed.Methods: Schistosome-infected animal model was established by adopting the abdominal patching method; the expression changes of T-cell subgroup, Th-cell subgroup(Th1/Th2/Th17/Tfh) as well as co-stimulatory molecules PD-1/PDL-1 and ICOS/ICOSL at different stages of schistosome-infected mice were detected through use of multi-parameter fluorescent labeled flow cytometry; fluorescence quantitative PCR method was applied to detect the expression level of Th subgroup(Th1/Th2/Th17/Tfh) transcription factor in mice at the same time; and immunohistochemical HE staining method was employed to observe the reaction process of main immunopathological injury, liver schistosomasis egg granuloma, and to analyze its correlation with the expression of T-cell subgroup, Th subgroup(Th1/Th2/Th17/Tfh) and co-stimulatory molecules PD-1/PDL-1 and ICOS/ICOSL.Results:(1) After liver immunohistochemical HE staining, the analysis of the area of hepatic egg granulomas in schistosome-infected mice at different stages showed that the host’s egg granuloma to schistosomiasis immune response was the strongest in 8W after schistosome infection;(2) The flow cytometry analysis showed that CD4+T cells of peripheral blood and spleen decreased in the mice infected with schistosome, and CD8+T cells increased and reached the highest until post-infection 8W;(3) Tfh in spleen and lymph nodes of the mice increased from post-infection 3W, and while that in peripheral blood increased from post-infection 8w; Proportions of specific antibody level Ig G in serum and Tfh cells in spleen in infected mice are positively correlated;(4) Th1 type cytokine IFN-γ increased in post-infection 1w, decreased from post-infection 5W(egg formation period), and then decreased gradually; Th2 type cytokine IL-4 and Th17 type cytokine IL-17 increased in post-infection 6W, and continue to maintain at a high level; Tfh type cytokine IL-21 increased in post-infection 3w, reached the peak in post-infection 10 W, and stayed at a higher level in the later infection period; dynamic changes of Th1/Th2/Th17/Tfh specific transcription factor(TBet/RORyt/GATA3/Bcl-6) were consistent with those of its corresponding cytokines;(5) The expression of costimulatory molecules ICOS and PD-1 on CD4+T-cells in mice up-regulated after schistosome infection.Conclusions: Schistosome infection results in the decrease of CD4+T cells and the increase of CD8+T cells in the host; Host T-cell immune response gives priority to Th1 response at early infection stage, and as the disease course gets into the egg antigen generation until host hepatic egg granuloma forms and develops, Th2, Th17, Tfh immune response enhances, and the expression of co-stimulating molecules PD-1 and ICOS on T-cell surface has a significant rise. The balancing and regulating effect of Th1/Th2/Th17/Tfh, PD-1 and ICOS on the host immune response is worth further research.Part II Expression of T-cell subset and related molecules in schistosome reinfected mouse model and correlation between the expression and hepatic pathological changesObjective: To analyze changes in in-vivo expression levels of T-cell subgroup, Thcell subgroup(Th1/Th2/Th17/Tfh) and co-stimulatory molecules after primary infection, re-infection, and chemotherapy of host, to clarify the characteristics of immune response from T-cell subgroup, Th-cells and co-stimulatory molecules ICOS and PD-1 in schistosome re-infected host, and to lay a foundation for finding intervention measures to prevent schistosome re-infection and inhibit host immune pathology.Methods: Mice models of primary infection, re-infection and treatment were established; flow cytometry was adopted to observe the in-vivo expression levels of Tcell subgroup, Th-cell subgroup and co-stimulatory molecules PD-1 and ICOS in schistosome-infected mice after primary infection, re-infection and chemotherapy; the pathological changes of schistosome egg granuloma in the infected mice were observed by adopting immunohistochemical method; and the correlation between T-cell subgroup, Th-cell subgroup as well as ICOS/ICOSL & PD-1/PD-L1 and immunopathological changes under re-infection status was discussed.Results:(1) For mice in chemotherapy group, compared to those in infection group, the CD4+T proportion in peripheral blood and spleen was higher. The expression of CD8+T was lower. The expressions of PD-1 and ICOS down-regulated. IL-4 and IL-17 reduced to different extents. However, no significant change existed in IFN-γ, suggesting that the Th2/Th17/Tfh type immune response of the mice in chemotherapy group tends to fall;(2) For the mice in re-infection group, compared to those in primary infection group, the CD4+T proportion in peripheral blood and spleen was higher. The CD8+T proportion was lower. The ICOS and PD-1 in peripheral blood had not significant difference. The expression of ICOS and PD-1 on CD4+T in spleen upregulated. And no significant change existed in IFN-γ and IL-4. Neverthless, IL-17 and IL-21 type cytokines were lower;(3) For the schistosome re-infected mice, compared to those in primary infection group, the fluke loading quantity was less because primary infection has a protection rate of 19.9% for re-infected host. On the other hand, the area of egg granuloma has no significant difference.Conclusions: T-cell subgroup and Th-cell subgroup of re-infected host present different characteristics from those after primary infection. For the re-infected host, compared to the primarily infected one, the proportion of CD4+T increases, that of CD8+T decreases, Th17 and Tfh immune response down-regulate, expressions of costimulatory molecules PD-1 and ICOS up-regulate, and their ligand has no difference with primary infection.Part III Research on functioning mechanism of in-vivo interference of ICOS/ICOSL and PD-1/PDL-1 signals and combined treatment with praziquantel in schistosome-infected miceObjective: To observe the functions of interference in vivo of ICOS/ICOSL and PD-1/PDL-1 signals and combined treatment with praziquantel to schistosomiasis immunopathological injury, discuss the possible acting mechanism of PD-1/PDL-1 signal simulation and combined praziquantel treatment to mitigate schistosomiasis immunopathological injury, and provide experimental basis for immune intervention measures against liver pathological damage caused by schistosomiasis.Methods: Intervention measures of ICOS/ICOSL and PD-1/PD-L1 were taken to BALB/c mice with schisotosoma infection. Inhibiting positive co-stimulatory signal(Anti-ICOSL m Ab) or enhancing negative signal(PDL-1.Fc) were taken in schistosome-infected mice, and insecticidal therapy by combining praziquantel was carried out at the same time, so as to observe the effect of positive signal blocking and negative signal enhancement on schistosomiasis immunopathological injury; since negative signal enhancement(PDL-1.Fc) and combined praziquantel intervention can significantly reduce and restrain the hepatic pathological injury, PD-1/PDL-1 signal stimulation or blocking intervention was further conducted at different stages of schistosome infection(3W, 6W, 12 W after infection) in the mice, so as to observe the influence of changes of CD4+T, CD8+T, CD19+B on the pathological injury after intervention.Results:(1) For the mice in the group of positive signal blocking(Anti-ICOSL m Ab) and combining praziquantel, the area of liver granuloma had no significant difference from that in the group of only praziquantel treatment;(2) For the mice in the group of negative signal enhancement(PDL-1.Fc) and combining praziquantel, the area of liver granulomas was lower than that in the group of only praziquantel treatment, indicating that PDL-1.Fc intervention can reduce the pathological damage of egg granuloma in schistosome-infected mice.(3) For the mice infected with schistosome and treated with immune intervention of PD-1/PDL-1 signal blocking(Anti-PD-L1 m Ab) at different stages(post-infection 3W, 6W, and 12W), the expressions of PD-1 on CD4+T and CD8 +T cells down-regulated, and also PDL-1 down-regulated on CD19+B cells in peripheral blood and spleen. The intervention at different stage has no effects the pathological damage of schistosomiasis, and the proportion of CD8+T cells has no difference with that in the treatment group mice.(4) For the mice infected with schistosome and treated with immune intervention of PD-1/PDL-1 signal enhancement at different stages(post-infection 3W, 6W, and 12W), the expressions of PD-1 on CD4+T and CD8 +T cells up-regulated, and also PDL-1 up-regulated on CD19+B cells in peripheral blood and spleen; intervention in post-infection 3W and 6W could reduce immunopathological injury of schistosomiasis and the proportion of CD8+T cells was lower than that in the treatment of PD-L1.Fc; especially for the immune intervention group with PD-1/PDL-1 signal enhancement and combining praziquantel, the intervention can killing worm and also reduce pathological damage in mice significantly, and percentage of CD8+T cells decreased obviously; but intervention in post-infection 12 W could not relieve the pathological injury of hepatic egg granulomas.Conclusions: Invention with ICOS/ICOSLsignal blocking(Anti-ICOSL m Ab) and PD-1/PDL-1 signal blocking(Anti-PD-L1 m Ab) could not relieve the pathological injury of hepatic egg granulomas. PD-1/PDL-1 signal enhancement and combining praziquantel can obviously alleviate the pathological damage of egg granuloma and kllling worm in schistosome-infected mice, and CD8+T cells decrease. PDL-1 early intervention combined with praziquantel treatment can significantly reduce irreversible liver pathological damage caused by schistosome infection, indicating its potential application prospect.In conclusion, in the immune response process of the schistosome-infected host, Tcell subgroup changes significantly during the disease progression, and CD4+T cells continue to fall while CD8+T cells rise. With the disease conversion from acute to chronic reaction, the dynamic changes and regulation of Th-cells show that the characteristics consistent with those of chronic infectious disease, that is to say, Th1-cell immune response acts predominantly for induction during the early stage of infection, but Th2/Th17/Tfh immune response strengthens during the late stage of infection and plays an important role in regulating the immunopathological injury of host. For the first time this study demonstrates that PD/PDL-1 signal enhancement can obviously reduce the immunopathological injury caused by schistosomiasis, and result in the increase of CD8+T inside the host. Therefore, PDL-1 combined with praziquantel, schistosomiasis chemotherapeutic drugs can significantly improve the schistosomiasis immune pathology. In summary, this study perfects the change rules of T-cell subgroup of schistosome-infected mouse model, enriches the regulating mechanism of co-stimulatory molecules in the immune pathology of schistosomiasis, and puts forward the intervention measure of combining targeted co-stimulatory signal PD-1/ PDL-1 combining with chemotherapy with praziquantel, so as to provide new therapeutic ideas and plans for the prevention and treatment of the pathological damage caused by schistosomiasis.