Mechanistic Study On Twist1 Involved In Corticosteroid Resistance In Patients With Ulcerative Colitis

Ulcerative colitis（UC）is a continuous,nonspecific chronic inflammatory bowel disease involving the colonic mucosa and submucosa.The pathogenesis is still undefined and many studies discovered that it is due to the immune damage caused by the overreaction of immune cells in intestinal mucosal tissues to intestinal microbial antigens in genetically susceptible people in a specific environment.The main pathological findings were infiltration of inflammatory cells such as eosinophils and neutrophils.There is no specific treatment at present due to the unclear pathogenesis.The main treatment was systemic anti-inflammatory with glucocorticoids.Most patients responded,but 1/3 of them did not respond to the corticosteroid,which was called corticosteroid resistance（CR）.Patients with steriod resistance may need to increase dosage or duration of steriod,leading to an increase in steriod side effects（hyperglycemia,hypertension,central obesity,acne,osteoporosis,etc）.Glucocorticoid plays an anti-inflammatory role by binding to glucocorticoid receptorα（GRα）in cells.GRαcan be transferred into the nucleus to activate anti-inflammatory genes,thus achieving anti-inflammatory effect.Twist1（TW1）is an apoptosis suppressor gene,which is overexpressed by many tumor cells and promotes tumor cell overgrowth and epithelial mesenchymal cell transition.TW1 also has immunomodulatory effects,which can regulate the activation of macrophages,promote the function of dendritic cells,and participate in vascular sclerosis.This study mainly elucidates the expression of TW1 in CR UC and its regulation mechanism of steroid resistance in UC.Part 1Objective:To explore the expression of neutrophil elastase（NE）in UC serum,analyze the expression of NE in neutrophil granulocytes in UC intestinal tissue,and further compare the number of neutrophil granulocytes in UC intestinal tissue.Methods:Patients with acute severe UC requiring surgery and patients with colorectal cancer were included in this study.All patients were treated with Dexamethasone injection（Dex,7.5-12.5mg,equivalent to methylprednisolone40-60mg）for 1 week before surgery and then the dosage was gradually reduced.Corticosteroid sensitivity（CS）was defined as the severity of the disease changing from severe to moderate or from moderate to mild after 1 week of steroid use.CR was defined as no significant improvement in clinical symptoms after 1 week of steroid use.A total of 34 CR and 12 CS intestinal mucosal specimens and serum before and after steroid treatment were collected.The surgical specimens of 8 cases of colon cancer were collected from the adjacent non-tumor sites（confirmed by pathologists）.The expressions of NE,Eosinophil cationic protein（ECP）,IFN-γ,IL-17 and IL-4were detected.Neutrophils,eosinophils,B cells and T cells in CR and CS groups were isolated and cultured with stimulants,respectively,±Dex.The expression of NE,ECP and IL-10 in supernatant and T cell proliferation were detected.The propotions of neutrophils and eosinophils in Lamina propria mononuclear cell（LPMC）were counted by Fluorescence activating cell sorting（FACS）in intestinal tissue.Results:After Dex treatment,compared with before treatment,the expression of NE in CS group was significantly decreased,but there was no significant change in CR group.Neutrophils isolated from intestinal mucosa were cultured in groups.In the CS group,the expression of NE in supernatant cultured with Dex decreased significantly,while in the CR group,the expression of NE in supernatant cultured with Dex did not change significantly.Neutrophils accounted for about 30%of LPMC in the CR group,but only about 10%in the CS group.Conclusion:Neutrophils are the main inflammatory cells in CR UC.Neutrophils may be responsible for CR in UC.Part 2Objective:To investigate the cause of CR in UC neutrophils.Methods:Neutrophils from surgically resected colon specimens of CR group（n=34）,CS group（n=12）and NC group（n=8）were sorted and purified by FACS and performed RNA sequencing.The target gene was identified and verified by RT-q PCR.Results:Compared with CS and NC group,TW1 showed higher activity among the 15 differentially expressed genes（DEGs）,and was related to Ras,MPO and ELANE.Compared with CS and NC groups,the high expression of TW1 in neutrophils in CR group was also confirmed by RT-q PCR.In eosinophils,B cells,T cells and epithelial cells,TW1 showed no significant difference between CR,CS and NC groups.Conclusion:The high expression of TW1 in neutrophil in colon tissue is associated with CR in UC.Part 3Objective:To investigate the mechanism of TW1 on intestinal neutrophil CRMethods:The proteins of CR group（n=19）were extracted and co-immunoprecipitation（co-IP）was performed with anti-TW1 antibody.The proteins combined with TW1 were analyzed by mass spectrometry（MS）.protein complex of CR group（n=19）,CS group（n=12）and NC group（n=8）were analyzed by WB after co-IP experiment.Neutrophils in CR and CS groups were purified by FACS and cultured with stimulants,±Dex,or CRISPR was used to delete TW1 gene and NE expression was detected by ELISA.The binding of TW1 to GRαwas detected by competitive ELISA.Results:In CR group,the co-IP protein complex of anti-TW1 antibody was found to be TW1-GRαprotein complex by MS analysis,and verified by the co-IP of anti-TW1 antibody and anti-GRαantibody.This protein complex existed in CR group,but was negative in CS group and NC group.NE expression in CR group and CS group was significantly increased after PMA or LPS stimulation culture.After adding Dex,NE expression in CS group decreased significantly,but remained unchanged in CR group.NE expression was significantly decreased after TW1 gene was deleted by CRISPR and cultured with Dex.These results indicated that neutrophils recovered corticosteroid sensitivity after deletion of TW1 gene.The binding of TW1 to GRαcan antagonize Dex bingding to TW1 and it was dose-dependent manner by competitive ELISA.Conclusion:TW1 antagonizes the binding of Dex to GRαby binding GRα,which leads to neutrophil resistance to corticosteroid.Part 4Objective:To explore the mechanism of TW1 in CR formation of neutrophil in miceMethods:BALB/C mice were divided into two groups,normoxic group（n=6）and hypoxic group（n=6）.Mice in normoxic group（21%O₂）and hypoxic group（10%O₂+90%N₂）were fed for one week and sacrificed on day 8.Neutrophils were purified from intestinal tissue of mice by FACS.The expression levels of TW1,GRαand GRβin the two groups were detected by WB.The TW1-GRαprotein complex in the two groups was detected by co-IP with anti-TW1 antibody and anti-GRαantibody.Purified neutrophils were cultured under the following conditions:with stimulants,±Dex,neutrophils TW1 gene knockout（TW1/KO/1）was performed before hypoxia treatment in mice,neutrophils TW1 gene knockout（TW1/KO/2）was performed after hypoxia treatment in mice,and NE expression was detected by ELISA.Results:Compared with normoxic group,hypoxia induced higher expression of TW1 m RNA and protein in neutrophil,but did not affect the expression of GRαand GRβprotein.TW1-GRαprotein complex was found in the hypoxic group by co-IP.The expression of NE was significantly increased after cultrue of purified neutrophil with PMA/LPS.After adding Dex,NE expression decreased in normoxic group,but no significant change in hypoxic group.However,NE expression in TW1/KO/1+Dex and TW1/KO/2+Dex groups decreased significantly.Conclusion:Hypoxia can induce TW1 expression and lead to CR.Conditional knockout of neutrophil TW1 restored steroid sensitivity.TW1 plays an important role in mouse neutrophil CR.Part 5Objective:To investigate the mechanism of maintenance of high expression of TW1 in colon neutrophilMethods:According to the results of RNAseq in part II,it was found that Ras expression of neutrophil in CR UC colon tissue was abnormally high,and a hypothesis was proposed that Ras expression was related to TW1 expression.Ras activity was detected by ELISA after neutrophil protein was extracted from CR（n=19）and CS（n=12）groups.The expression of TW1 m RNA（PCR）and protein（WB）,Ras activity（ELISA）,STAT3 and p STAT3 protein（WB）were detected after neutrophils was purified by FACS and cultured with stimulants.Co-IP and MS analysis were performed with anti-STAT3 antibody.Recombinant Ras,r RASAL1 and r STAT3 were used for competitive ELISA.Results:Compared with CS group,Ras activity in intestinal neutrophils was significantly increased in CR group.Purified neutrophils from peripheral blood of normal subjects cultured with IL-6,TGF-βand hypoxia,Ras was activated and the expression of TW1 m RNA and protein increased significantly.After FTS（a Ras inhibitor）was added in all three groups,Ras was deactivated and the expression of TW1 m RNA and protein were significantly decreased.These results suggested that Ras activation was associated with high TW1 expression.The expression of p STAT3protein was increased in all three groups,but decreased in groups cultured with FTS,suggesting that Ras activation can promote STAT3 phosphorylation.Co-IP and MS showed that STAT3 could interact with RASAL1.Meanwhile,competitive ELISA showed that STAT3 could antagonize RASAL1 binding with Ras by dose-dependent manner,thus maintaining Ras activity.Conclusion:Ras activation can maintain high expression of TW1.Part 6Objective:To explore the effect of inhibition of TW1 on CR colitis miceMethods:BALB/C mice were treated with DSS and hypoxia to construct CR colitis mouse model.The mice were divided into six groups,Naive（n=6）,DSS+hypoxia+saline（n=6）,DSS+hypoxia+Dex（n=6）,DSS+hypoxia+TW1KO（n=6）,DSS+hypoxia+TW1KO+Dex（n=6）,DSS+hypoxia+Harmine（TW1 inhibitor）+Dex（n=6）.The body weight of mice in 6 groups was measured,intestinal tissue was stained with HE,FACS was used to count CD45⁺,and the cytokines of IL-1β,TNFα,MPO,IFN-γ,IL-6 and IL-17 were detected by ELISA.Neutrophil which was purified by FACS was cultured with PMA/LPS and NE expression was detected by ELISA.LPMC was extracted from intestinal tissue of mice in 6 groups and neutrophil count was performed.Results:DSS+hypoxia can construct CR colitis mouse model.In DSS+hypoxic+TW1KO+Dex,DSS+hypoxic+Harmine（TW1 inhibitor）+Dex group,the number of CD45⁺cells decreased significantly;the expression of inflammatory cytokines decreased significantly;NE expression was significantly decreased;Neutrophil counts were significantly decreased in LPMC,compared with DSS+hypoxic+saline,DSS+hypoxic+Dex,DSS+hypoxic+TW1KO group.Conclusion:Inhibition of TW1 can restore corticosteroid sensitivity in mice with CR colitis.