The Protective Role And Mechanism Of Interleukin IL-33 Activating Enteric Glial Cells In Inflammatory Bowel Disease

BACKGROUND:The incidence of inflammatory bowel diseases（IBD）is increasing year by year and its pathophysiology remains uncertain.IBD’s pathogenesis is complex with heredity,environment,and abnormal immunity,neural-immune is a new frontline in IBD’s pathophysiology.Enteric glia cell（EGC）are an important contributor to enteric neural-immune homeostasis,its abnormality represents a significant feature in recent IBD research.Interleukin 33,a new player of the IL1 family,modulates central glial cells,which EGCs are similar to,by ST2 receptor.IL-33’s role has not been clearly clarified in IBD.Therefore,it’s meaningful to investigate the EGC’s role regulated by IL-33 in IBD.METHODS:The GEO database was analyzed for changes in IL-33/ST2 signaling in clinical patients with IBD,and clinical samples from IBD patients were collected to verify IL-33/ST2 signaling changes by RT-q PCR.The role of IL-33 was explored by IL-33-treated C57BL/6 wild mouse dextran sulfate sodium（DSS）colitis model,and the role of IL-33was also verified by IL-33 receptor ST2 knockout mouse(ST2^-/-)and C57BL/6 wild mouse DSS-induced colitis model.Immunofluo-rescence assay was used to detect the expression of ST2 in EGC.Bioinformatics analysis of single-cell sequencing database combined with RT-q PCR elucidated the change of quantity and role of EGC in IBD.Further RNA-seq sequencing was used to identify the key cytokines for IL-33-induced EGC secretion,which was validated by RT-q PCR.The possible interaction between EGC derived matrix metalloproteinase-9（MMP-9）and glial derived neurotrophic factor（GDNF）were explored by protein docking and Deep Cleave platform,a deep learning substrate prediction model.Finally,it was further investigated the IL-33’s role through EGC derived MMP-9to promote GDNF enzymatic maturation,and the outcome by blocking MMP-9 combined with RT-q PCR and Western blot in DSS-induced colitis model and EGC cell line.RESULTS:Analysis of the GEO dataset showed that IL-33 m RNA was transiently elevated for 24 hours and decreased after 48 hours in the intestinal transcriptome of IBD patients and healthy controls,while the inflammatory signals interleukin1β（IL1β）,monocyte chemoattractant protein-1（MCP-1）were consistently elevated within 48 hours.IBD patients and healthy controls were recruited from hospital,and intestinal IL-33 and ST2 m RNA expression was found to be elevated in IBD compared with healthiers using RT-q PCR.A mouse model of DSS-induced enteritis was constructed,and IL-33 treated group displays reduced colonic inflammation,and recovered weight loss,colon shortening length,and disease activity index were significantly improved compared with the DSS-treated group alone.In contrast,ST2^-/-mice showed more severe DSS-induced colitis than wild mice,with heavier weight loss,higher disease activity index,and more pronounced colonic shortening,as well as increased colonic permeability by FITC-dextran permeability assay.These results support a protective role of IL-33/ST2 in IBD.Immunofluorescence showed that EGC cells expressed ST2 receptor,and ST2 receptor co-localized with EGC marker GFAP in colon tissue,suggesting that IL-33 acts on EGC.Analysis of single-cell sequencing data from IBD revealed a decreased number of EGCs in the inflammatory mucosa of IBD,and RT-q PCR of intestinal tissues from DSS colitis mice revealed an increase in the expression of EGC function-related factors GFAP and S100β,suggesting the abnormal EGCs in IBD.In this study,the cellular RNA-seq revealed cytokines such as matrix metalloproteinase-9（MMP-9）production from EGCs after IL33 stimulus,and cellular experiment verified the significant differential genes in RNAseq by RT-q PCR.Further IBD cohort’s transcriptomic correlation analysis were used to reveal that IL-33 and MMP-9 were significant positive correlated.These results suggest that IL-33 activation of EGC in IBD may exert a protective effect through MMP-9.GDNF is the major protective molecule of EGC.Molecular docking revealed that the binding potential energy between MMP-9 and GDNF decreased significantly after GDNF bind to GFRα,which means MMP-9,as a proteinase,might exert effect before GDNF bind to its receptor.while Deep Cleave,a deep learning substrate prediction model,predicted that MMP-9 most likely enzymatically cleaves pro GDNF at the middle of amino acid residues 43-50 of the propeptide,thus promoting mature GDNF production.In EGC cellular experiments,IL-33 intervention promoted increased ERK and MMP-9 m RNA expression and did not affect GDNF m RNA expression,and Western blot found that ERK,PI3K,MMP-9,and GDNF protein expression increased,and blocking MMP-9 inhibited GDNF protein expression.Using the C57BL/6 mouse DSS colitis model,RT-q PCR revealed that IL-33 promoted MMP-9 expression but did not affect GDNF expression,Western blot revealed that IL-33 promoted mature GDNF production while mice were protected from colitis injury.Blocking MMP-9 by specific inhibitor reversed the protective effect of IL-33 on DSS-induced colitis with no change in transcription,while reduced mature GDNF.It is thus hypothesized that IL-33 affects GDNF maturation via EGC derived MMP-9’s cleavage and thus protects against colitis injury.CONCLUSION:In IBD,IL-33 plays a protective role by activating EGC to produce MMP-9 through MAPK signaling,EGC derived MMP-9promote GDNF maturation,and targeting the IL33-EGC-MMP9 pathway will bring new options for the treatment of IBD.