Follistatin-Like Protein 1 Promotes Intestinal Inflammation By Mechanisms Involving The Polarization And Related Signaling Pathways In Macrophages

Inflammatory bowel disease consists of Crohn’s disease(CD)and ulcerative colitis(UC),whose etiology remains largely unknown.It is now believed that multiple factors may be involved,including genetic susceptibility,environmental stimulus and abnormal immunological reactions.The gastrointestinal tract is one of the most macrophage dense organs.Macrophages are part of the innate immune system,providing the first-line defense to foreign pathogens.These cells function by phagocytosis,antigen presentation and secretion of multiple cytokines,and play a role in the clearance of pathogens and aging cells,modulation of inflammatory response,acquired immunity and tissue repair.Macrophages are a heterogeneous immune cell population,with diverse origins and functions.They can differentiate into the M1 proinflammatory macrophages or adopt the M2 anti-inflammatory phenotype depending on the stimulus,and this process is called polarization.STAT signaling pathways are activated by interferon-y(IFN-y)and lipopolysaccharide(LPS)to skew macrophage function toward the M1 phenotype(via STAT1)or by interleukin-4(IL-4)to skew toward the M2 phenotype(via STAT6).Normal intestinal macrophages exhibit an anti-inflammatory and pro-tolerogenic phenotype,while macrophages infiltrates in tissues from IBD shown to be proinflammatory in nature.M1 proinflammatory macrophages also accumulate during inflammation in models of IBD,and transfer of M2 macrophages helps ameliorate inflammation.TNF-αantibodies induce an M2-like phenotype in macrophages,and M2 macrophages were increased in the mucosa of patients that responded to infliximab.These results collectively suggested that macrophage polarization may be a potential therapeutic target for IBD.Follistatin-like protein 1(FSTL1)is a secreted glycoprotein produced mainly by cells of mesenchymal origin,and plays roles in the regulation of cell proliferation,differentiation,migration,organ development,carcinogenesis,and metastasis.Recent studies showed increased levels of FSTL1 in a variety of inflammatory diseases including rheumatoid arthritis,systemic lupus erythematosus,sicca syndrome,systemic scleroderma and polymyositis/dermatomyositis.The mechanism by which FSTL1 exerts its proinflammatory effects is poorly characterized and remains to be elucidated.Results from in vitro studies indicated that monocytes overexpressing FSTL1 secreted higher levels of interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and monocyte chemoattractant protein-1(MCP-1)after stimulation by phorbol-12-myristate-13-acetate(PMA)and LPS,and the transfected macrophages treated by LPS produced more IL-1β.Considering that no studies have been conducted to reveal the effects of FSTL1 on macrophage phenotypes and IBD colitis,we designed this research topic.This study aims to explore the effects of FSTL1 on IBD and colitis,and preliminarily reveal the possible mechanisms around macrophages.The expression levels of FSTL1 in the inflamed tissues of IBD and in the serum will be determined.The effects of FSTL1 overexpression on the proliferation,apoptosis,migration,phagocytosis and polarization of macrophages,together with the direct stimulatory effect of FSTL1 on cytokine secretion,will be investigated with emphasis on macrophage polarization and related signal pathways.The effects of FSTL1 knock-down on the development of DSS colitis will be studied,and the role of FSTL1 in the macrophage polarization will be validated in vivo to reveal one of the possible mechanisms.The research consists of three parts:PART 1 The expression of follistatin-like protein-1 in the intestinal tissue and serum of patients with inflammatory bowel diseaseAims:To investigate the expression levels of FSTL1 in the inflamed tissues and serum of IBD.Methods:The specimen database of pathology department was used to retrieve the surgical specimens of CD,UC and normal tissues at the edge of colon tumor for FSTL1 immumohistochemical staining.The fresh inflamed mucosa of IBD was taken during coloscopy for immunofluorescence staining of FSTL1 and vimentin(fibroblast marker).Active CD,nonactive CD,active UC,nonactive UC patients and normal controls were prospectively enrolled and the serum samples were obtained to determine the FSTL1 levels via ELISA.ROC curves were drawn to analyze the value of FSTL1 in IBD diagnosis and disease activity.Results:Immumohistochemical staining showed the upregulated levels of FSTL1 in the inflamed tissues of CD and UC patients.Colocalization of FSTL1 and vimentin was observed both in the CD and UC.Compared with that of the nonactive IBD and normal controls,the serum FSTL1 levels in the active patients were increased significantly.The area under the curve(AUC)of serum FSTL1 to diagnose IBD from normal controls or discriminate active IBD from nonactive ones was 0.626 and 0.734.Conclusion:The expression of FSTL1 was upregulated in the inflamed tissues of IBD which may partly derive from the fibroblasts.The serum FSTL1 was also increased in the active IBD patients,and the value of serum FSTL1 in the discrimination of active disease was better than that in the diagnosis.PART 2 Transfection of RAW264.7 macrophages by lentiviral vector encoding the follistatin-like protein-1 gene and the effects on the RAW264.7 macrophagesAims:RAW264.7 macrophages overexpressing FSTL1 were established,and the effects on the cell proliferation,apoptosis,migration,phagocytosis and polarization were investigated.The direct stimulatory effect of FSTL1 on the cytokine secretion was also studied.Methods:A lentiviral vector carrying the LV4-FSTL1 plasmid was created to transfect the RAW264.7 macrophages.FSTL1 expression was verified by both PCR and WB.Three experimental groups were set consisting of the blank group,the control group that used a control lentivirus,and the FSTL1 overexpressing group.The CCK8 assay was used to evaluate the cell proliferation.The cells were pretreated with H2O2 to induce apoptosis,and Annexin V/PI staining was conducted to determine the apoptotic cells.The cell migration of macrophages was determined via Transwell chamber.LPS was added and neutral red staining was used to investigate the phagocytic ability.The polarization toward M1 or M2 phenotype was induced by LPS&IFN-y or IL-4.The flow cytometry to detect CD11c and CD206 positive cells was performed,and the mRNA levels of IL-1β,IL-6,TNF-α,Fizzl and Ym1 were determined by PCR.The phosphorylation levels of STAT1/STAT6 proteins were measured by WB.Various concentrations of recombinant FSTL1 protein(100ng/mL and 300ng/mL)were used to directly stimulate the macrophages for different time periods(6h,12h,24h,48h,72h).The levels of IL-1β,IL-6 and TNF-α in the supernatant were measured by ELIS A.Results:The overexpression of FSTL1 in the transfected macrophages was confirmed by PCR and WB.The macrophages overexpressing FSTL1 had higher activities of proliferation,anti-apoptosis and migration while the phagocytic ability remained unchanged.As for the polarization,the differentiation toward M1 macrophages was enhanced while that toward M2 phenotype was inhibited in the macrophages overexpressing FSTL1.The phosphorylation of STAT1 was upregulated and that of STAT6 was downregulated.The secretion of IL-6 and TNF-α following recombinant FSTL1 stimulation was enhanced,and this effect was dose-dependent and weakened as time went on.There was no statistical difference regarding the levels of IL-1β.Conclusion:Overexpression of FSTL1 exerted the macrophages higher activities of proliferation,anti-apoptosis and migration,induced the polarization toward M1 phenotype while inhibiting the M2 transdifferentiation by upregulating the STAT1 signals and downregulating the STAT6 signals.Stimulation of recombinant FSTL1 increased the secretion of IL-6 and TNF-α but not IL-1β in the macrophages.PART 3 The effects of follistatin-like protein-1 on the DSS colitis and the intestinal macrophagesAims:To explore the role of FSTL1 in the DSS colitis,and confirm the effects on intestinal macrophages in vivo.Methods:2%DSS drinking water was used for 7 days to induce the colitis,while normal water was used in the control mice.All animals were sacrificed after 10 days and the tissues of middle colons were obtained.Four experimental groups were set consisting of FSTL1+/-colitic mice(n=7),wild-type colitic littermates(n=7),normal FSTL1+/-mice(n=5),and normal wild-type littermates(n=5).The changes in the weight,disease activity index(DAI),colon length and histological scores were monitored.CD86 and CD206 was measured by immunofluorescence staining.PCR was used to determine the mRNA levels of TNF-α,iNOS,Ym1 and Arg-1.The expression of FSTL1 and phosphorylation of STAT1/STAT6 were detected by WB.Results:The levels of FSTL1 increased markedly after induction of colitis by DSS in the FSTL1+/-mice and wild-type littermates.The FSTL1+/-mice produced less FSTL1 protein than the wild-type littermates.Compared with the wild-type colitic littermates,the FSTL1+/-colitic mice had lower weight loss,DAI and histological scores,and longer lengths of colon.Immunofluorescence staining showed that the number of M1 macrophages(CD86 positive cells)decreased while that of M2 macrophages(CD206 positive cells)increased in the FSTL1 knock-down mice.PCR confirmed that the mRNA levels of TNF-α and iNOS were lower in the FSTL1+/-colitic mice while the mRNA levels of Ym1 and Arg-1 were lower in the wild-type colitic littermates.WB found that the STAT1 signals were activated while STAT6 signals were inhibited in the DSS colitis.The phosphorylation of STAT1 was decreased in the FSTL1+/-mice while that of STAT6 was upregulated in the FSTL1+/-mice when compared with the wild-type littermates.Conclusion:Downregulation of FSTL1 helped alleviate DSS colitis,and promoted the polarization of M2 phenotype while inhibiting the M1 transdifferentiation.Considering the proinflammatory nature of M1 macrophages and the anti-inflammatory and pro-healing effects of M2 macrophages,we inferred that modulation of macrophage polarization may be at least a part of the mechanisms underlying the effects of FSTL1 on the colitis.