The Function And Mechanism Study Of Fgl2 In Inflammatory Bowel Diseases And Tumor Microenvironment

Part 1:The function and mechanism of Fgl2 in inflammatory bowel diseasesColorectal cancer is one of the most common malignant tumors of the digestive tract.It is the third most common malignant tumor in the world,with more than 1 million new cases diagnosed worldwide each year.Previous studies have shown that the degree of inflammatory bowel disease and the duration of the disease are positively correlated with the risk of cancer,and epidemiological studies have shown that about 20% of IBD will progress to cancer within a period of 30 years,and more than 50% of patients with colorectal cancer.The inflammatory bowel diseases(IBDs),including ulcerative colitis(UC)and Crohn's disease(CD),are characterized by chronic inflammation in the gastrointestinal tract.The incidence rate of IBD has risen greatly worldwide over the past few decades,and extensive attention has been focused on exploring the immunopathogenesis of IBD,which leads to immunotherapies against IBD such as anti-tumor necrosis factor alpha(anti-TNF-?)treatment.Nonetheless,considerable portions of CD and UC patients do not respond to anti-TNF-? treatment.Thus,further understanding of the immunopathogenesis of intestinal inflammation is required to help find more effective treatments for IBDs.Fgl2,a member of the fibrinogen super family,is a pleiotropic cytokine impacting diverse cellular functions.It possesses prothrombinase activity,acts as an effector molecule of Treg cells to suppress T cell proliferation,induce B cell apoptosis and inhibit maturation of dendritic cells.Fgl2 was found up-regulated both in the inflamed colon tissue and the plasma of IBD patients with active diseases.Nonetheless,the function of Fgl2 in IBD pathogenesis is still unclear.In the present study,we explored the contribution of Fgl2 in the development of IBD and colitis-associated carcinoma(CAC),by using well-established DSS-induced experimental colitis model and AOM/DSS-induced CAC model.Methods: 1.The function of Fgl2 in DSS-induced colitis.(1)Generate DSS-induced colitis model in WT and Fgl2-KO mice.(2)Compare the degrees of weight loss,rectal bleeding and diarrhea of Fgl2-KO and WT littermates during colitis induction.(3)Compare the degrees of colon shortening of Fgl2-KO and WT littermates after colitis induction.(4)Using histopathological analysis of the colonic tissues,compare the degrees of colon histological severity of Fgl2-KO and WT littermates after colitis induction.(5)Measure the amounts of TNF-?,IL-1?,IL-6,IL-17 A,IL-4,IL-10 and TGF-? in the colon tissue harvested at day 9 in the experimental cohorts by ELISA.2.The function of Fgl2 in AOM/DSS-induced CAC.(1)Generate AOM/DSS-induced CAC model in WT and Fgl2-KO mice.(2)Compare the degrees of weight loss of Fgl2-KO and WT littermates during CAC induction.(3)Count the tumor number and mesure the sum area of all tumors in each mouse.3.Identification of Fgl2 expressing cells during DSS-induced colitis.(1)Generate DSS-induced colitis model in C57BL/6 mice.(2)Harvest the colons of colitis mice at indicated time point(day 0,day 2,day 4,day7)and measure the length of the colons.(3)Using histopathological analysis of the colonic tissues,compare the degrees of colon histological severity of colons of different time points.(4)The expression of menbrance-bound Fgl2 in the colon of different time point was detected by Western Blot.(5)The level of soluble Fgl2 in the colon of different time point by was detected ELISA.(6)CD45-Ep CAM+,CD4+CD25high,CD11b+F4/80+,CD11b+CD11C+ and CD11b+Gr-1+ cells were sorted from colitic C57BL/6 mice colons by FACS.(7)The expression of soluble Fgl2 in CD45-Ep CAM+,CD4+CD25high,CD11b+F4/80+,CD11b+CD11c+ and CD11b+Gr-1+ cells was measured by ELISA.(8)Immunofluorescence was done to analyze the interaction between m Fgl2 and F4/80,CD11 c as well as CD31.4.The function of Fgl2 produced by hematopoietic cells in DSS-induced colitis.(1)WT?WT and Fgl2-KO?WT chimeric mice were irradiated and generated by injection of WT or Fgl2-KO derived bone marrow cells into caudal vein.(2)8 weeks after bone marrow injection,mice were reconstituted and detected by FACS of peripheral blood.(3)Compare the degrees of weight loss,rectal bleeding and diarrhea of WT?WT and Fgl2-KO?WT chimeric mice during colitis induction.(4)Expression of m Fgl2 and s Fgl2 in the colons of chimeric mice was detected by Western Blot and ELISA.(5)Compare the degrees of colon shortening of WT?WT and Fgl2-KO?WT chimeric mice after colitis induction.(6)Using histopathological analysis of the colonic tissues,compare the degrees of colon histological severity of WT?WT and Fgl2-KO?WT chimeric mice after colitis induction.(7)Measure the amounts of TNF-?,IL-1?,IL-6,IL-17 A,IL-4,IL-10 and TGF-? in the colon tissue harvested at day 9 in the experimental cohorts by ELISA.5.Fgl2 contributes to macrophage polarization during colitis.(1)Generate DSS-induced colitis model in WT and Fgl2-KO mice.(2)Colon lamina propria(LP)cells were isolated from colitic WT and Fgl2-KO mice at day 6 and day 9 post DSS induction.(3)Frequencies of macrophages,DCs and neutrophils in the colons of WT and Fgl2-KO mice were determined by FACS.(4)Frequencies of M1 and M2 macrophages in the colons of WT and Fgl2-KO mice were determined by FACS.6.Fgl2 is required to regulate macrophage polarization in vivo and in vitro.(1)Generate DSS-induced colitis model in WT and Fgl2-KO mice.(2)LP-macrophages were isolated from colitic WT and Fgl2-KO mice at day 9 post 2.5% DSS colitis induction.Expression of M1-associated genes(IL-1?,IL-6,NOS2 and IL-12p40,A)and M2-associated genes(MR,IL-10,Arg1 and Fizz1,B)were examined by real-time PCR analysis.(3)Peritoneal macrophage from WT and Fgl2-KO mice were isolated and cultured in vitro;(4)Peritoneal macrophages isolated from WT and Fgl2-KO mice were stimulated with LPS for M1 polarization.M1-associated genes were examined by real-time PCR analysis and ELISA.(5)Peritoneal macrophages isolated from WT and Fgl2-KO mice were stimulated with IL-4 for M2 polarization.M2-associated genes were examined by ELISA and real-time PCR analysis.Results 1.Fgl2 is required to protect DSS-induced colitis.(1)Compared with WT mice,Fgl2-KO mice lost significantly more weight,and showed higher clinic scores,demonstrating increased intestinal bleeding and more severe diarrhea.(2)Fgl2-KO mice exposed to DSS showed significantly shorter colon length.(3)Fgl2-KO mice indeed showed higher histological scores.(4)Compared to WT mice,Fgl2-KO mice produced higher levels of pro-inflammatory cytokines including TNF-?,IL-1? and IL-17 A following DSS treatment.Conversely,Fgl2-KO mice produced lower levels of anti-inflammatory cytokine such as IL-10.2.Fgl2 is required to protect AOM/DSS-induced CAC.(1)Faster body weight loss was observed in Fgl2-KO mice during AOM/DSS administration.(2)Compared with WT mice,Fgl2-KO mice had heavier tumor burden,containing more tumor number and larger sum area of all tumors in each mouse.3.Identification of Fgl2 expressing cells during DSS-induced colitis.(1)With the extension of DSS induction,the colon length of colitis mice was shortened,and the damage of epithelial barrier structure was more significant.(2)m Fgl2 was increased in the colon post DSS administration.(3)s Fgl2 was increased in the colon post DSS administration.(4)Colonic macrophages,Treg cells and DCs are the sources of s Fgl2 in colon post DSS administration.(5)Colonic macrophages and DCs are the main sources of m Fgl2 in colon post DSS administration.4.Fgl2 produced by hematopoietic cells plays a dominant role in regulating intestinal inflammation.(1)8 weeks after bone marrow injection,mice were reconstituted and detected by FACS of peripheral blood.Above 90% of WBCs in the peripheral blood were CD45.2 positive.(2)The chimeric mice reconstituted with Fgl2-KO bone marrow cells developed more severe colitis than those reconstituted with WT bone marrow cells,displaying faster body weight loss and increased clinic scores.(3)The chimeric mice reconstituted with Fgl2-KO bone marrow cells developed more severe colitis than those reconstituted with WT bone marrow cells,displaying shorter colon length and invreased pathological scores.(4)The chimeric mice reconstituted with Fgl2-KO bone marrow cells expressed higher levels of pro-inflammatory cytokines.5.Fgl2 balances M1/M2 cell distribution by favoring M2 polarization during colitis.(1)The frequency of macrophage was decreased in the colonic LP of Fgl2-KO mice when compared with those of WT mice,while the percentage of DCs and neutrophils appeared comparable between WT and Fgl2-KO mice.(2)An increased percentage of M1 macrophages and a decreased frequency of M2 macrophages in Fgl2-KO colonic LP than those in WT mice.6.Fgl2 regulates the macrophage polarization in vivo and in vitro.(1)Macrophages isolated from Fgl2-KO colitic mice expressed increased M1 markers and effector molecules including but decreased M2 markers and effector molecules compared with WT colitic mice.(2)Fgl2 regulates macrophage polarization by favor M2 macrophages in vitro.Conclusions 1.Fgl2 is required to protect DSS-induced colitis.2.Fgl2 is required to protect AOM/DSS-induced CAC.3.Fgl2 is up-regulated in the DSS-induced colitic colons,which mainly expressed by macrophages,DCs and Treg cells.4.Fgl2 produced by hematopoietic cells plays a protective role in regulating intestinal inflammation.5.Fgl2 balances M1/M2 cell distribution by favoring M2 polarization during colitis.6.Fgl2 regulates the macrophage polarization directly.Part2: The function of stroma-derived Fgl2 in tumor microenvironmentTumor microenvironment is made up of tumor cells,stromal cells,extracellular matrix and cytokines secreted by these cells.A large number of studies have shown that tumor microenvironment plays an important role in tumorigenesis and tumor growth.Fgl2,a member of the fibrinogen super family,possesses prothrombinase activity expressed by macrophages and endothelial cells in a variety of diseases.It promotes the formation of coagulation cascades by catalyzing the transformation of thrombinogen to thrombin.In addition,Fgl2 acts as an effector molecule of regulatory T cells(Treg cells),which inhibits T cell proliferation,promotes B cell apoptosis,inhibits the maturation of DC cells,and exerts an immunosuppressive effect.Previous studies have shown that Fgl2 expression in a variety of solid tumors,such as liver cancer,kidney cancer,colon cancer,breast cancer,gastric cancer and ovarian cancer,and by affecting the biological function of tumor cells to promote tumor development.Fgl2 secreted by glioma cells can up-regulate the expression of PD-1 and CD39 and promote immunosuppressive cell recruitment in tumor microenvironment,thereby promoting tumor progression.However,we analyzed the m RNA levels of Fgl2 in various tumor cell lines using the Cancer Cell Line Encyclopedia(CCLE)database.It was found that Fgl2 in epithelial cell-derived tumor cell lines was lowly expressed,and in combination with the literature,Fgl2 is expressed mainly in immune cells in a variety of diseases,suggesting that Fgl2 from tumor stromal cells may play an important role in the development of tumors.Therefore,we established the mouse Lewis lung carcinoma(LLC)subcutaneous transplanted tumor model in Fgl2-KO mice to investigate the regulation of stroma-derived Fgl2 on the microenvironment of lung cancer and its role in the tumorigenesis and tumor growth.Methods 1.The function of stroma-derived Fgl2 in LLC subcutaneous transplanted tumor model.(1)Establish LLC subcutaneous transplanted tumor model in WT and Fgl2-KO mice.(2)The tumor size was measured every 3 days and the tumor growth curve was drawn.(3)Mice were anesthetized and sacrificed 21 days after tumor cell injection.The tumors were obtained and weighed.The weight of tumors from the WT and Fgl2-KO mice was compared.(4)CCK-8 was used to detect the function of recombinant Fgl2 on the proliferation of LLC cells.(5)Annexin V / 7AAD staining was used to detect the function of recombinant Fgl2 on the apoptosis of LLC cells.2.The function of Fgl2 in LLC tumor microenvironment.(1)Establish LLC subcutaneous transplanted tumor model in WT and Fgl2-KO mice.(2)Mice were anesthetized and sacrificed 15 days after tumor cell injection.The frequency of CD45+ cells,TAMs(F4/80+CD206+),DCs(CD11c+MHCII+),MDSCs(CD11b+Gr-1+),Treg cells(CD4+Foxp3+)and CD8+T cells(CD3+CD8a+)in tumor microenvironment was analyzed by flow cytometry(FACS).(3)The expression of NOS2,ARG1,VEGF and TGF-? in the tumors of WT and Fgl2-KO mice was detected by q RT-PCR.(4)The percentage of MDSC cells in bone marrow and spleen of Fgl2-KO and WT mice was analyzed by FACS.(5)The expression of CXCL12 and CXCL5 in the tumors of WT and Fgl2-KO mice was detected by q RT-PCR.(6)The expression of CXCL12,CD33 and CD14 was obtained in human NSCLC using gene expression data from The Cancer Genome Atlas(TCGA)portal.3.Fgl2 regulates the activation and function of CAF cells.(1)Establish LLC subcutaneous transplanted tumor model in WT and Fgl2-KO mice.(2)Mice were anesthetized and sacrificed 15 days after tumor cell injection.The frequency of CAF(PDGFR?+F4/80-)in tumor microenvironment was analyzed by FACS.(3)CAF cells were sorted from LLC tumors by FACS and treated with recombinant Fgl2 in vitro.The expression of ?-SMA,FAP and PDGFR? was detected by q RT-PCR and Western Blot.(4)CAF cells were sorted from LLC tumors by FACS and treated with recombinant Fgl2 in vitro.The expression of CXCL12,HGF,TGF-?,VEGF and IL-6 was detected by q RT-PCR.Results 1.Stoma-derived Fgl2 promotes tumor growth in LLC subcutaneous transplanted tumor model.(1)The rate of tumor growth in the Fgl2-KO mice was slowed compared with WT mice.(2)The weights of excised tumors at termination(day 21)were also markedly smaller in Fgl2-KO mice.(3)Fgl2 is derived from tumor stromal cells in LLC subcutaneous transplanted tumor model.(4)The proliferation rate of LLC cells was not affected by recombinant Fgl2.(5)The apoptosis rate of LLC cells was not affected by recombinant Fgl2.2.Fgl2 is required for the infiltration of MDSCs in tumor microenvironment.(1)Mice were anesthetized and sacrificed 15 days after tumor cell injection.The percentage of MDSCs(CD11b+Gr-1+)was significantly reduced in Fgl2-KO mice compared with WT mice.(2)The level of NOS2,ARG1,VEGF and TGF-? was decreased in Fgl2-KO tumors.(3)The frequency of MDSCs both in the bone marrow and spleen was similar at baseline in the two genetic backgrounds.The frequency of MDSCs in the spleen and bone marrow of WT and Fgl2-KO tumor-bearing mice was also similar.(4)CXCL12 was decreased in the tumors of Fgl2-KO mice compared with tumors of WT mice.(5)CXCL12 gene expression was indeed correlated with MDSC markers(CD33 and CD14)in the tumors of NSCLC patients.3.Fgl2 promotes the activation and function of CAF cells.(1)Mice were anesthetized and sacrificed 15 days after tumor cell injection.The percentage of CAFs was similar in Fgl2-KO mice compared with WT mice.(2)The expression of ?-SMA,FAP and PDGFR? was up-reguated in CAF cells treated with recombinant Fgl2.(3)The expression of CXCL12,HGF,TGF-? and VEGF was up-reguated in CAF cells treated with recombinant Fgl2.(4)Fgl2 expression was positively correlated with the expression of with CAF markers and effectors,such as FAP,PDGFR?,CXCL12 in the tumors of NSCLC patients.MDSC markers and effectors,including CD33,CD14 and IL-10 were found to be positively correlated with the expression of Fgl2 in the tumors of NSCLC patients.Conclusions 1.Stoma-derived Fgl2 promotes tumor growth in LLC subcutaneous transplanted tumor model.2.Fgl2 promotes the activation and secretion of CXCL12 in CAF cells.3.Fgl2 is required for the infiltration of MDSCs by up-regulating the level of CXCL12 in the tumor microenvironment.