Ikzf2 Regulates The Development Of ICOS~+Th Cells To Mediate Immune Response In The Spleen Of S.japonicum Infected C57BL/6 Mice

Background:Schistosomiasis is a disease caused by schistosome parasites that invade the human body.The prevalent schistosomiasis in China is schistosomiasis japonica.Once the body was infected,the various stages of Schistosoma japonicum,especially the eggs it releases,will be deposited in multiple organs(liver,lungs,etc.)in the body.At the same time,the miracidia in the mature eggs can secrete soluble egg antigen(SEA).It can cause a violent immune response in multiple immune organs,and the formation of characteristic egg granulomas in the tissues,causing damage,leading to liver fibrosis and splenomegaly.The spleen is an important immune organ of the body.The enlargement of the spleen and the proliferation and activation of various immune cells caused by Schistosoma japonicum infection are the focus of the study of Schistosoma japonicum infection.Inducible costimulatory molecule(ICOS)is a member of the CD28 family,which are mainly expressed in lymphocytes,while the initial T cells show a state of low expression.When the initial T cells encounter the CD3+CD28 activation signal,they can quickly activate and highly express ICOS molecule.ICOS plays an important regulatory role in the proliferation and differentiation of CD4+T cells.ICOS molecules can not only regulate the function of T cells,but also promote T cell-dependent antibody response and antibody affinity maturation in GCs by inducing and maintaining the differentiation and maturation of Tfh cells.A large number of literature reports that the ICOS molecule has a powerful and complex regulatory effect on Th cells,and can play very different roles in different infection models.Based on the important regulatory role of ICOS on Th cells in the immune response,it is very important to find upstream genes that regulate the expression of ICOS.Looking for the upstream genes related to the regulation of ICOS,we found that a series of transcription factors of the Ikzfs play an important role in regulating the differentiation and development of early lymphocytes.Objective:This study explored the changes in the content of ICOS+Th cells and the changes in the activation phenotypes and cytokines production capacity of ICOS+Th cells in the spleen of mice infected with Schistosoma japonicum.Then,through mRNA sequencing of CD4+ICOS+and CD4+ICOS-cells infected with Schistosoma japonicum.The aim is to explore the important signaling pathways involved in ICOS molecules and find important upstream genes that regulate the expression of ICOS molecule.Methods:1.A mouse model of Schistosoma japonicum infection was constructed.2.Spleen lymphocytes were extracted.qRT-PCR and flow cytometry were used to detect the changes of ICOS.3.Flow cytometry was used to detect changes in the content of ICOS in a variety of immune cells.4.Flow cytometry was used to detect changes in the activation phenotypes and cytokines production levels of ICOS+Th cells.5.ICOS+Th cells and ICOS-Th cells were obtained by flow cytometric sorting.The two groups of cells were subjected to RNA sequencing to explore the signaling pathways that the ICOS molecule participates in after Schistosoma japonicum infection and to search the upstream genes that regulate the ICOS molecule in Th cells.6.qRT-PCR and flow cytometry were used to verify the differential expression of Ikzf2,a key transcription factor regulating ICOS molecules,in ICOS+Th cells and ICOS-Th cells.7.The nuclear and cytoplasmic protein separation experiments and western blot technology were used to explore the nucleus of Ikzf2 in ICOS+Th cells in the spleen after Schistosoma japonicum infection.8.Finally,ChIP-qPCR was used to verify whether Ikzf2 directly regulates the expression of ICOS molecule in Th cells.Results:1.A mouse model of Schistosoma japonicum infection was established.2.In the spleen of infected mice,the results of qRT-PCR and flow cytometry shown that content of ICOS increased significantly,and the increase of ICOS+cells in Th cells was the most significant.3.The content of ICOS+Th cells continued to increase after infection,and reached a peak after 4-6 weeks of infection.4.ICOS+Th cell activation phenotypes such as CD69,CXCR5,CD40L have a significant increase,compared with ICOS-Th cells in the infection group and ICOS+Th cells in the control group.5.On the other hand,the production levels of cytokines such as IL-4,IL-10 and IL-21 in ICOS+Th cells also increased significantly,compared with ICOS-Th cells in the infection group and ICOS+Th cells in the control group.6.The results of mRNA sequencing suggested that ICOS participates in a variety of signaling pathways related to the activation of immune cells after Schistosoma japonicum infection.Compared with ICOS-cells,a total of 1289 genes are differentially expressed in ICOS+cells,of which 508 genes are up-regulated and 781 genes are down-regulated.7.GO analysis results show that these genes are significantly enriched in a variety of biological processes,cell components and molecular functions.In GO analysis,there is significant gene enrichment in immune system process,immune response,positive regulation of B cell activation,inflammatory response,DNA binding and other pathways.This indicates that ICOS molecule participates in immune response,inflammatory response,and DNA binding signaling pathways.8.It was found in KEGG molecules that ICOS molecule participates in hematopoietic cell lineage signaling pathways,cytokine-cytokine receptor interactions,T cell receptor signaling pathways,and Thl and Th2 cell differentiation signaling pathways.9.This study found that a transcription factor called Ikzf2 was significantly highly expressed in ICOS+Th cells.10.The methods of qRT-PCR and flow cytometry suggested that Ikzf2 is highly expressed at both the mRNA and protein levels in ICOS+Th cells.11.It was confirmed by nuclear and cytoplasmic protein separation combined with Western Blot technology that after Schistosoma japonicum infection,Ikzf2 entered the nucleus of ICOS+Th cells.12.Finally,ChIP-qPCR was used to confirme that Ikzf2 can directly bind to the ICOS promoter in Th cells regulate the expression of ICOS molecule.Conclusion:1.In the spleen of mice infected with Schistosoma japonicum,the content of ICOS+Th cells increased significantly,while the activation phenotype of ICOS+Th cells and the production of various cytokines increased significantly.2.The results of mRNA sequencing shown that the ICOS molecule participated in a variety of signal pathways related to the differentiation and development of immune cells.3.Ikzf2 can directly regulate the expression of ICOS in the spleen of mice infected with Schistosoma japonicum by directly binding to the promoter of the ICOS gene in Th cells.