| Approximately 200 million people worldwide are infected with Schistosoma spp, making it a serious public health problem. S. japonicum is popular in our country. The mechanism of the host immune response to S. japonicum infection is complicated. However our lack of knowledge of the details of the mechanism represents a research bottleneck for the prevention and control of schistosomiasis and for the development of vaccines against S. japonicum. Most studies have focused on the role of T-helper (Th) cells, such as Thl, Th2, Th17 and T-regulatory (Treg) cells in S. japonicum in-fection; however, there has been a little research on T follicular helper (Tfh) cells and their involvement in the mechanism of S. japonicum infection is unclear. Tfh cells belong to a subpopulation of CD4+T cells that promote the generation of germinal center and the germinal center B cells to produce antibodies. The inducible T-cell costimulator (ICOS) molecule on Tfh cell surface can promote liver granuloma for-mation in mice infected with S. japonicum. In this study, the phenotype and functional changes in Tfh cells from patients infected with S. japonicum were explored. In addi-tion, C57BL/6 mice infected with S. japonicum were used as the model to analyze the phenotype and function of Tfh cells. Moreover, an Agilent microarray analyzed the differentially expressed genes in Tfh cells between infected mice and uninfected mice. Immune regulation molecules were screened which provided a basis for the study of the biology of the immune response mechanism in mice infected with S. japonicum. In addition, the responses of Tfh cells in mice infected with S. japonicum and treated with the effective drug praziquantel treatment were observed. The results of this study will contribute to our understanding of the mechanism of the host immune response and will provide a scientific basis for immunization and vaccine research of schisto-somiasis.1. T follicular helper cells and related molecules in patients infected with S. ja-ponicumTfh cells belong to a subgroup of CD4+T cells that promote formation of the germinal center and germinal center B cells to produce antibodies. In this study, the role of Tfh cells in schistosomal infections was assessed.In this study, we selected 12 patients with acute schistosomiasis,11 chronic schistosomiasis patients and 10 healthy controls in Hunan Province endemic areas. The proportion Tfh cells and the expression levels of Tfh surface markers were de-tected by flow cytometry in subjects’peripheral blood. The distribution of B cell sub-sets was also examined. The levels of serum interleukin 21 (IL-21) and IgG antibodies specific to S. japonicum soluble egg antigen (SEA) or soluble worm antigen (SWA) were detected by ELISA.The results showed that the expression of programmed cell death protein 1 (PD-1) molecule on Tfh cell surface in acute schistosomiasis patients (7.42%±3.15%) in-creased (P< 0.01); The proportion of Tfh cells in chronic schistosomiasis patients (23.67%±3.12%) also increased (P< 0.01) and the expression of the surface mole-cules ICOS (12.44%±2.84%) and PD-1 (5.38%±1.42%) in chronic schistosomiasis patients were also increased (P< 0.05). Furthermore, the proportion of PD-1+ CXCR5+CD4+Tfh cells in chronic schistosomiasis patients was positively correlated with serum IL-21 (rs=0.782,P=0.004), SEA specific IgG (rs=0.688, P=0.028), IgGl(rs=0.709, P=0.015) and IgG4 (rs=0.527, P=0.024) antibody. The results indicat-ed that Tfh cells participate in the immune response through their surface molecules and secreted cytokines.Our study indicated, for the first time, that PD-1highCXCR5+CD4+Tfh cells might play an important role in the production of specific antibodies that contribute to the immune response caused by S. japonicum.2. The phenotype and function of Tfh cells in mice infected with S. japonicumIn this study, splenic Tfh cells were observed using immunofluorescence in C57BL/6 mice infected with S. japonicum. The proportion of Tfh cells and other Th cells were observed after different infection times (3,5,8,13wks) using flow cytome-try. The proliferation of lymphocytes and Tfh cells stimulated by SEA were analyzed. In addition, the changes in germinal center B cells were also detectd.Dynamic observation showed that the proportion of Tfh cells among CD4+T cells increased gradually with the time of S. japonicum infection, reaching a peak at 8wks, after which it decreaed gradually. Both SEA (4.868%±0.650%,12.268%± 0.672%) and SWA (4.380%±0.444%,11.532%±1.967%) antigens can cause an in- crease in Tfh cells in vitro (P< 0.05) and in vivo (P< 0.01) studies. The proliferation ability of infected spleen lymphocytes decreased (P< 0.05) and the proliferation of Tfh cells in infected spleen did not significantly decrease in response to S. japonicum infection in vitro. In addition, germinal center B cells were activated. The levels of serum IL-21 secreted by Tfh in infected group mice (0.240±0.034) were higher than in normal group mice (0.143±0.015) (P< 0.05). The variation trend of germinal center B cells was as same as Tfh cells. Notwithstanding these observations, the role and molecular mechanism of Tfh cells in mice infected with S. japonicum remain to be further elucidated.3. Differential expression of genes in Tfh cells of mice infected with S. japoni-cumPrevious studies found that Tfh cells were significantly increased in the peripher-al blood and spleens of mice and patients infected with S. japonicum. PD-1+CXCR5+CD4+ Tfh cells were significantly positively correlated with IgG anti-bodies specific to SEA in chronic schistosomiasis patients; however, the exact mecha-nism is not clear. In this study, the immune regulatory molecules of spleen Tfh cells between mice infected with S. japonicum and normal mice were screened using whole genome profiling.The spleen cells were collected from normal mice and mice infected with 5. ja-ponicum 8 weeks after infection. CD4+T cells were prepared using magnetic activated cell sorting (MACS) separation and then Tfh cells were sorted by flow cytometry. To-tal RNA from PD-1+CXCR5+CD4+Tfh cells and PD-1-CXCR5+CD4+ non-Tfh cells were extracted and the gene expression profiles were analyzed. Bioinformatics analy-sis identified differentially expressed genes (DEGs) between PD-1+CXCR5+CD4+Tfh cells and PD-1-CXCR5+CD4+ Tfh cells. The DEGs were subjected to gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses. The expres-sions of selected DEGs were validated using fluorescence quantitative PCR.Gene expressions between the infected group and normal group were significant-ly different. There were 1506 DEGs, including 642 up-regulated genes and 864 down-regulated genes. Their functions included granulocyte migration, leukocyte mi-gration involved in inflammatory response, positive regulation of endothelial cell mi-gration, positive regulation of endothelial cell migration, cytokine secretion activity and cytokine receptor activity. Fifteen genes were selected randomly and 14 were ver-ified by fluorescent quantitative PCR.The results showed that the differentially expression genes between PD-1+CXCR5+CD4+Tfh cells and PD-1-CXCR5+CD4+non-Tfh cells in the infection group were also significantly different. There were 710 differentially expressed genes including 406 up-regulated genes and 304 down-regulated genes. Main functions in-cluded positive regulation of cell migration, positive regulation of immune response, positive regulation of MAPK cascade, B cell receptor signaling pathway, antigen processing and presentation of exogenous peptide antigen via MHC class II and cyto-kine receptor activity. We selected 12 genes and 10 of them verified by fluorescence quantitative PCR.The identification of DEGs in PD-1+CXCR5+CD4+Tfh cells and PD-1-CXCR5+ CD4+non-Tfh cells between infected and normal mice provide a scientific basis for further molecular study of the immune function and mechanism of Tfh cells during S. japonicum infection.4. Tfh cells and related molecules in S. japonicum-infected mice after praziquan-tel treatmentCurrently, praziquantel is the only effective drug treatment for S. japonica; how-ever, praziquantel can influence the host immune response. Previous studies showed that Tfh cells are involved in the immune response of the host to S. japonicum infec-tion. However, does praziquantel influence the host immune response via Tfh? Thus, we investigated the effect of Tfh cells in S. japonicum-infected mice after praziquantel treatment.Female C57BL/6 mice were randomly assigned to the S. japonicum infection and praziquantel treatment group (treatment group), the untreated infected group and the uninfected with S. japonicum group (control group). Mice of the treatment group were administered with intragastic 200 mg/(kg/d) praziquantel at 6 weeks post-infection for 3 days. Mice were sacrificed after 4 weeks of treatment, and liver and spleen le-sions were observed. The proportion of Tfh cells and expression of surface molecules ICOS and PD-1 were detected by flow cytometry in the peripheral blood and spleen. The results showed that liver and spleen lesions improved significantly after pra-ziquantel treatment and the numbers of S. japonicum adults and eggs were reduced significantly. The expression of ICOS in the Tfh cells in the peripheral blood and spleens of treated mice were significantly lower than those in the untreated group. ICOS could be used as a biomarker to evaluate the efficacy of praziquantel.ConclusionsThis study explored the role of Tfh cells and their immune mechanism in the host during S. japonicum infection. The main results were as follows.1. Surface molecules of Tfh cells in patients with acute schistosomiasis and chronic schistosomiasis changed significantly. The expression of PD-1 in acute schis-tosomiasis patients increased while percentage of Tfh cells and the expressions of ICOS and PD-1 also increased in patients with chronic schistosomiasis. There were positively correlations between PD-1+CXCR5+CD4+Tfh cells and the levels of IL-21and SEA specific IgG, IgGl and IgG4 antibodies in the sera of patients with chronic schistosomiasis. The immune function of Tfh cells might depend on changes of surface molecules at different stages of S. japonicum infection.2. Tfh cells were located in the spleen of mice by immunofluorescence and using markers for Tfh cells:Tfh cells were classified as PD-1+CXCR5+CD4+. S. japonicum infection induced a decrease in the proliferation of splenic lymphocytes and the pro-liferation ability of Tfh cells did not decrease obviously. Tfh cells promoted an in-crease in germinal center B cells, as assessed by the increasing levels of the secreted cytokine IL-21. Our analysis showed that the germinal center B cells and Tfh cells showed a similar change trend:increase at initial infection, a peak at 8 wks, followed by a subsequent decrease.3. Differentially expressed genes of PD-1+CXCR5+CD4+Tfh cells and PD-1-CXCR5+CD4+non-Tfh cells between infected and normal mice were identified using Agilent mouse microarray analysis. The related immune molecules were screened and provided clues to the molecular basis of Tfh cells’involvement in the host response to S. japonicum infection.4. Treatment with praziquantel improved liver and spleen lesions significantly and reduced significantly the number of adults and eggs. The expression of ICOS in Tfh cells in the peripheral blood and spleen of treated mice were significantly lower than those in the untreated group. ICOS could act as a biomarker to evaluate the effi-cacy of praziquantel. |
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