The Role Of MD-1 Inhibition With Antisense Oligonucleotide In Murine Experimental Colitis

Backgrounds and Objectives Inflammatory bowel disease(IBD),a chronic and relapsing inflammation of gastrointestinal tract,comprises two clinical formsulcerative colitis(UC)and Crohn’s disease(CD).Although the precise etiology of IBD remains elusive,it’s widely believed that the health of gastrointestinal tracts is sustained by the interplay of genetic susceptibility,microbiota and the immune system.TLR4,a member of Toll-like receptors(TLRs),represents one of the important mechanisms associated with the pathogenesis of IBD.TLR4 requires an accessory molecule,myeloid differentiation 1(MD-2),to capture its ligand and modulate immune response.Once stimulated,TLR4 interacts with universal myeloid differentiation factor 88(MyD88)and a TIR domain containing adaptor inducing interferon(TRIF),initiating MyD88-dependent or MyD88-independent pathways.The MyD88-dependent pathway is primarily intracellular and can initiate NF-κB dependent inflammation,leading to production of pro-inflammatory factors,such as IL-6、TNF-α and IL-1β.Growing evidences have suggested that TLR4/NF-κB signaling contributes to the pathogenesis of IBD.Myeloid differentiation-1(MD-1)is a glycoprotein which shares 20% sequence identity to MD-2.Similar to TLR4/MD-2,MD-1 functions with radioprotective105(RP105)which is homologous to TLR4.Interestingly,recent studies have shown that RP105/MD-1 serves as a negative regulatory role in TLR4 signaling,which may be ascribed to heterodimerization of TLR4/MD-2 and RP105/MD-1.Compelling research have revealed the contributions of RP105/MD-1 to inflammatory disorders for arthritis,atherosclerosis,obesity and insulin resistance.However,the role of MD-1 in IBD remains unclear.To further investigate the involvement of MD-1 in IBD,we detected the protein levels of MD-1 in UC patients and experimental colitis mice;meanwhile,we inhibited MD-1 in colon with antisense oligonucleotide(AS-ODN)and assessed the effect of MD-1 inhibition on DSS-induced colitis.Methods Normal and inflamed colonic mucosal biopsies were obtained endoscopically from healthy controls(HC,n=11)and UC(n=12)patients.Western blot and immunohistochemical(IHC)staining were applied to detect MD-1 protein level in colon biopsy.Besides,DSS induced colitis model was established and the MD-1 protein level in colon of mice was evaluated by Western blot.To evaluate the effectiveness,specificity and duration of MD-1 AS-ODN to inhibit MD-1 in colon,mice were treated with 200μg AS-ODN by oral gavage,and R-ODN was administrated as a control.To examine the effect of MD-1 on the course of ongoing intestinal inflammation,mice were randomly placed into four groups(n=10 mice per group): H2O+R-ODN,H2O+AS-ODN,DSS+R-ODN,and DSS+AS-ODN.Our previous work showed that the effect of MD-1 AS-ODN to suppress MD-1 protein levels could sustain 60 hours at least.As a result,MD-1 AS-ODN or R-ODN was given every 60 hours to the experimental endpoint.The weight of mice,stool consistency,occult blood and disease activity index(DAI)scores were calculated daily.At the endpoint,the colon length were calculated.HE and IHC were performed to access tissue damages and neutrophil infiltration respectively.The mRNA expressions and protein levels of cytokines were detected by qPCR and Western blot.To explore the mechanism,TLR4 and MyD88 mRNA expressions were evaluated by qPCR,while the NF-κB signaling activation was examined by Western blot.Results MD-1 protein expression was remarkably decreased in both patients with ulcerative colitis and mice with DSS-induced colitis.IHC analysis of a small cohort of UC patients also revealed decreased MD-1 expression in colon,and MD-1 staining was mainly observed in immunocytes rather than the epithelium.For the first time we showed that oral administration of MD-1 AS-OND to mice significantly suppressed the MD-1 protein level in colon rather than the liver,kidney and spleen tissues;meanwhile the effect of MD-1 AS-ODN to suppress MD-1 protein levels could sustain 60 hours at least.MD-1 AS-ODN treated mice were more susceptible to DSS-induced colitis based on loss of body weight,colon length and disease activity index.Mice with AS-ODN administration had significantly higher histological scores and myeloperoxidase(MPO)activity.What’s more,MD-1 inhibition significantly enhanced proinflammatory cytokines IL-6 、IL-1β mRNA and protein levels in colons of colitis.Finally,mice treated with AS-ODN exhibited increased mRNA levels of TLR4 and MyD88 after DSS exposure,and showed upregulated phosphorylated p65 and IκBα levels,which suggested the enhanced NF-κB activation.Conclusions MD-1 in mice with DSS-induced colitis and UC patients were dramatically reduced,suggesting the involvement of MD-1 in IBD.Inhibition of MD-1 by AS-ODN exacerbates DSS-induced experimental colitis in mice and positively regulates NF-κB signaling pathway.Taken together,specifically suppression of MD-1 in colon tissues with AS-ODN exacerbates DSS-induced experimental colitis in mice,which is possibly related to activation of TLR4/ NF-κB signaling.