Elevated O-linked GlcNacylation Promotes Intestinal Inflammation In Crohn’s Disease

Objective Crohn’s disease(CD)is a chronic relapsing form of inflammatory bowel disease,whose treatment remains a challenge due to limited insights for its pathogenesis.We aimed to determine the role of O-linked β-N-acetylglucosamine(O-Glc NAc)in the development of CD and evaluate the therapeutic effects of O-Glc NAc inhibitors on CD.Methods 1.Immunohistochemical staining(IHC)was used to detect the expression of O-Glc NAc,OGT,and OGA in intestinal tissues from normal,inactive,and active CD individuals.2.Western blot was used to detect the protein expression of O-Glc NAc,OGT and OGA in colon cancer cells infected with AIEC LF82.3.C57BL/6 mice were treated with AIEC LF82 by gavage.The expression of O-Glc NAc in ileum tissue of mice was detected by IHC.4.Intracellular UDP-N-acetylglucosamine(UDP-Glc NAc)was quantified by UV detection after calibration with UDP-Glc NAc standard.5.O-Glc NAc of IKK / IκB / NF-κB was measured with s WGA pull-down assay.6.Immunofluorescence(IF)and western blot were used to detect the localization of NF-κB p65 subunit.The activation of p65 was detected by luciferase reporter assay.Inflammatory cytokines downstream of NF-κB signal were detected by enzyme-linked immunosorbent assay(ELISA).7.GFP-LC3,a vector for monitoring autophagy,was transfected into colon cancer cells.IF was used to detect the formation of autophagosome/autolysosome. Western blot was used to detect the expression of autophagy-related gene LC3,p62,and ATG16.8.Colony-forming units(CFU)assay was applied for determining intracellular AIEC.9.Established a mouse colitis model by co-treatment with AIEC LF82 and DSS,and monitor the improvement of mouse body weight,typical symptoms of colonic inflammation,and survival after the application of O-Glc NAc inhibitor DON in the mice.Results 1.O-Glc NAC in intestinal tissue of active CD was markedly increased as compared with that in human normal intestinal tissue.The intestinal tissue of inactive CD individuals exhibited a modest increase in O-Glc NAc.2.In normal and inactive/active CD intestinal tissues,the expression of OGT and OGA was only marginally altered.3.O-Glc NAc in intestinal epithelium of mice infected with AIEC LF82 was increased,whereas the changes of OGT and OGA in these tissues were not significant.4.Infection of AIEC LF82 up-regulated the level of UDP-Glc NAc,a donor glucosamine for glycosylation,and increased O-Glc NAc in human colon epithelial HCT116 and HT-29 cells.Bacterial UDP-Glc NAc inhibitor 2-phenylbenzofuran significantly inhibited UDP-Glc NAc levels and O-Glc NAc modification in host cells.5.IKKβ and NF-κB were markedly O-Glycosylated in AIEC LF82-treated cells.Mutations of IKKβ(S733A)and p65(T352A)abrogated the O-Glc NAc of IKKβ and NF-κB and inhibited the activation of NF-κB resulted from AIEC LF82 infection.6.Treatment of 6-diaz O-5-ox O-L-norleucine(DON),an agent that blocks the production of UDP-Glc NAc and inhibits O-Glc NAc,led to the inactivation of NF-κB in AIEC LF82-infected HCT116,and reduced the release of pro-inflammatory cytokines IL-1 / IL-6.7.AIEC LF82 infection inhibited the formation of autophagy in host cells.O-Glc NAc inhibitor DON promotes autophagy and the clearance of AIEC LF82 in cells.8.DSS and AIEC LF82 combined treatment induced mice to develop typical colitis symptoms,such as weight loss,diarrhea,hematochezia,and shortened survival time.9.Administration of DON significantly alleviated the intestinal epithelial inflammatory response induced by DSS and AIEC LF82 in colitis mice and improved the survival of infected mice.Conclusions 1.O-Glc NAc was increased in intestinal epithelial tissues of CD patients and AIEC LF82-infected mice.2.Infection of AIEC LF82 up-regulated the level of UDP-Glc NAc and increased O-Glc NAc in human colon epithelial HCT116 and HT-29 cells.3.We identified that IKKβ and NF-κB were strikingly O-Glycosylated in AIEC LF82-treated cells.Application of O-Glc NAc inhibitor DON inactivated NF-κB in AIEC LF82-infected cells,enhanced the formation of autophagy,promoted the removal of cell-associated AIEC LF82,alleviated intestinal epithelial inflammation,and improved the survival of the colitis mice.