| Background and aimInflammatory bowel disease(IBD)is a chronic nonspecific inflammation of the intestinal,including Crohn’s disease(CD)together with ulcerative colitis(UC),the causes are not entirely clear.Recent studies have found that immune dysfunction is one of the important factors.The release of more inflammatory mediators from localized intestinal mucosal lesions,intestinal epithelial cells function changes,inflammatory cytokines and anti-inflammatory cytokines imbalance,leading to structural and functional changes in intestinal epithelial cells,seriously affect the normal intestinal digestion,absorption,material transport,and other functions.So patients are presented as diarrhea,weight loss and other symptoms,meanwhile quality of life is affected.The immune system is to identify,response and adapt numerous exogenous or self-molecules,so whether in a healthy or disease condition,the system is very important.The status of the intestinal mucosal immune system in the autoimmune diseases is very essential.As a part of the immune system of the organic,it is also known as gut-associated lymphoid tissue(GALT),and directly involved in the regulation of intestinal mucosal immune.The intestinal mucosal immune system consists of the following three lymphoid areas:the lamina propria(LP),lied underneath the basement membrane in the intestinal villi;the intraepithelial compartment,which contains the intraepithelial lymphocytes(IELs)and is located just above the basement membrane,between the columnar epithelial cells;and lymphoid nodules(akin to lymph nodes)embedded in the gut wall and Peyer’s patches(PPs),separated from the LP and IELs.The LP,IEL and PP lymphoid populations form a complex and interconnected network that responds to immunological diseases in the intestine.PP is the main place in the intestinal mucosa to induce specific immune responses;IEL and LP are the targets of the intestinal immune.The IELs of the intestine are considered derived from thymus-dependent and extrathymic or thymus-independent precursors in the intestinal wall.The thymus-dependent population consists of T cells that bear CD4 or heterodimeric CD8αβ molecules,Thy-1 and αβ γδ T cell receptor(TCR)of T cells,whereas the thymus-independent population expresses either αβ or γδ TCR,and the majority of these cells bear CD8 homodimeric αα molecules,however,a few are double negative and only some express Thy-1.IELs have many immunological functions:they secrete and respond to various cytokines and express molecules that directly interact with lymphocytes(such as MHC molecules).LP T cells mainly express αβ TCR,and possess a memory/activated phenotype characterized by the following surface marker molecules:CD45RO/45RBlo,CD44hi L-Sello and αβhi.In vitro environment,LP T cell respond to antigen or TCR stimuli but secrete large amonts of interferon(IFN)-γ,interleukin(IL)-4 and IL-5 upon stimulation.Compared to mice in GF environment,some indexes of mice raised in ordinary environment have increased:the numbers of PPs and cells in them;thickness and cells number of the lamina propria;the number of plasma cells in the original germinal centers;size and number of isolated lymphocytes;number and toxicity CD8+ T cells located at intestinal intraepithelial lymphocytes;number of CD4+ T and Th17 cells located in the small intestine lamina propria;the expression of FOXP3 in CD4+ CD25+ T cells located in mesenteric lymph node;expression of angiopoietin-4 and REG3y in Paneth cells;IgA secretion by B cells;ATP level in the small intestine;the expression of MHCⅡ molecules,TLR9 and IL-25 in mucosal epithelium.However,the number of colon Th17 cells reduced in mice kept in ordinary environment.In the development of IBD,the variety of number and function in CD4+ T lymphocytes involved in the disease process,particularly in gut lymphocytes.The reduction of CD4+ T lymphocytes caused by drugs or HIV infection can alleviate CD activity.Another significant treatment for IBD is TNF-α monoclonal antibody,which causes intestinal mucosal T cell apoptosis.T cells are divided into different subsets depends on the various inflammatory cytokines which they secrete,such as Th1,Th2,Treg and Th17 Most previous studies indicate that,the incidence and maintenance of IBD are related to the imbalance of Th1 and Th2 cells.CD mainly mediated by Th1 cells,meanwhile UC is mainly mediated by Th2 cells.Present studies show that Th17 and Treg cells are also participated in the occurrence of these diseases.Th17 cell,named by the secretion of IL-17 initially,is a subset of T cells.It is widely dispersed in the mucosa of gastrointestinal tract,especially the small intestine.It secretes IL-17A,IL-17F,IL-22,IL-21 and IFN-γ.Naive CD4+ T cells which are stimulated by antigen or presented by APC produce a variety of cytokines.These cytokines induce naive CD4+ T cells to differentiate into diverse directions.Th17,one subset of them,is mainly regulated by IL-6,IL-23 and TGF-β.The activation of Th17 cells effects both UC and CD.Studies suggested that Th17 cells were mostly located in the mucosa of patients with UC and submucosa in patients with UC,moreover,number of Th17 cells and levels of IL-17 in patients with UC and CD elevated.The level of IL-17 secreted by peripheral blood mononuclear cells of UC patients is interrelated with the degree of inflammation.The increase of Th17-induced cytokines could enhance IBD inflammation through the induction of Thl differentiation.Cdc42(cell division cycle 42)is a member of Rho GTP enzyme protein family.Activated Cdc42 binding with GTP can regulate cell polarity,an active form by overexpression or mutation-negative regulator of actin cytoskeleton rearrangement,cell migration,differentiation and survival.In T cells,over-expression mutation of Cdc42 influences the polarization,migration and differentiation of cytoskeleton of actin and tubulin.Cdc42 is also involved in positive selection and maturation of lymphocytes.Production of thymus T lymphocytes requires cell migration and adhesion.In our previous work,we made use of two-dimensional gel electrophoresis and immunoblotting to analyze colonic mucosa of patients with UC.The results showed that level of Cdc42 was significantly higher than that of health control,at the same time,both p38 and Cdc42 were upregulated.In detecting the relationship between them,lipopolysaccharide(LPS)activateed p38MAPK pathway of murine macrophages RAW264.7,as while as the Cdc42 expression;however,SB203580,a special inhibitor of p38,downregulated both p38 and Cdc42.The activity of Cdc42 had relationship with p38 degree.Based on the preliminary findings,we assume that Cdc42 participated in the regulation of T cells and intestinal mucosal immune cells,furthermore,and it will be altered as the improvement of the inflammation.According to the above research background and theoretical basis,we designed experiments in terms of human and experimental animal model in order to verify the assumption.We conformed that,in the process of inflammatory bowel disease,not only the ratio of Th17 cells,but also the expression of Cdc42 are affected.In addition,experiments were planed to observe whether the change of Cdc42 will affect the differentiation of Th17 cells.In experimental animal models,we also tested the changes in the intestinal mucosal lymphatic system and Th17-related proteins.Contents are as below:1.The changes of Th17 cell ratio,interrelated proteins and Cdc42 of the healthy controls,UC patients and CD patients;2.Interfering the process of Th17 cells induction with Cdc42 specific inhibitor in.human blood,and then we observed if the differentiation will be affected;3.Detect the inflammatory indexes and change of expressions of Cdc42 in intestinal mucosal lymphocyte and Thl7 cell-associated proteins.Methods1.Based on the recommended diagnostic criteria of Consensus of diagnosis and treatment of inflammatory bowel disease,published by the Inflammatory Bowel Disease Group of the Chinese Medical Association in 2012,we made the inclusive and exclusive criteria:Diagnosis of active UC as follows:1.clinical manifestations:persistent or recurrent diarrhea,mucus blood and pus with abdominal pain,tenesmus,and varying degrees of symptoms,duration of more than 4-6 weeks,you can have the skin,mucous membranes,jointS,eyes and liver and gallbladder and other intestinal manifestations;2.colonoscopy:(1)mucosal vascular texture,disorder or disappear,mucosal edema,crisp,spontaneous bleeding and purulent secretions or contact attachment,also common mucosal rough,was finely granular;(2)shows diffuse lesions in a prominent place,multiple erosions or ulcers;(3)visible colon bags lighter,blunt or disappeared and false polyp mucosa and bridges;(3)biopsy histology Check:diffuse intrinsic membrane impatient,chronic inflammatory cell infiltration,including neutrophils,lymphocytes,plasma cells,and eosinophils cells,especially epithelial cells infiltration of neutrophils and pouchitis,even crypt abscess;change the structure of crypts,crypt size,irregular,disorganized,and reduction of goblet cells;visible mucosal surface erosion,superficial ulceration and granulation tissue.Diagnosis of active CD is as follows:1.clinical manifestations:abdominal pain,diarrhea,bloody stools may have;systemic manifestations of weight loss,fever,loss of appetite,fatigue,anemia;intestinal manifestations similar to UC;a common complication of fistula,abdominal abscess,intestinal stenosis and obstruction,perianal disease,a rare gastrointestinal bleeding,perforation,longer duration may be cancerous.2.endoscopy:colonoscopy:segmental,asymmetric mucosal inflammation,ulcers and longitudinal pebble-like appearance;capsule endoscopy,enteroscopy:intestinal mucosa approximate performance of colonoscopy;endoscopy:esophagus,stomach,duodenal mucosa approximate colonoscopy performance.3.Imaging:CTE or MRE visible bowel wall thickening(>4mm),intestinal mucosa with intestinal stratification change significantly enhanced mucosal inner and outer ginger significantly enhanced,was "target sign" or "double halo sign" mesenteric vascular increase,expansion,twisted,was" comb "sign,the corresponding density increased mesangial fat,fuzzy,mesenteric lymph node enlargement.4,the inherent meningitis cells showed focal infiltration discontinuous;slit-like ulcers;aphthous ulcers;crypt structural abnormalities,glands,individual crypt abscesses,mucus secretion decreased obvious,visible or pyloric metaplasia Paneth cell metaplasia;non-caseous necrotizing granulomas;lymphocytes and plasma cells mainly chronic inflammatory cell infiltration in the lamina propria and submucosa bottom heavy,common lymphoid follicles;lymphangiectasis submucosal;ganglion cell proliferation and(or)ganglionitis around.Exclusion of bacillary dysentery,amoebic dysentery and other infectious colitis foundation and ischemic colitis,radiation colitis and other diseases on,can be diagnosed.At the same time patients have to take endoscopic and pathologic examinations and be diagnosed as IBD.Patients with the following situations should be excluded:cancer;complicated with other autoimmune diseases;diagnosed IBD patients who are using infliximab.21 patients with active UC,36 patients with active CD,40 healthy controls were included,while excluding cancer,complication with other autoimmune diseases and person who has been diagnosed with IBD and treated with infliximab.Collect the anticoagulant peripheral blood,and obtain plasma by centrifuging.Levels of IL-17A in the plasma were detected by ELISA method.The remaining blood cells were then separated by Ficoll density gradient centrifugation to get peripheral blood mononuclear cells(PBMCs).Total protein was extracted and tested by western blotting,at the same time,total RNA was extracted,reverse transcribed and quantitatively tested Cdc42,IL-17A,IL-23R and RORyt mRNA levels by real-time PCR.After been stimulated by phorbol ester(PMA),ionomycin and(BFA)for 4 hours,some of the PBMCs were marked by surface antibody:Human Anti-CD3-FITC and Human Anti-CD8-APC,and then marked by Human Anti-IL-17A-PE antibody after permeabilization and fixation.Measure the proportion of CD3+ CD8’ IL-17+ cell by flow cytometry machine.After taking similar steps,some of the PBMCs from patients were labeled and sorted by flow cytometry.When collected about more than 8000 cells,we could obtain total RNA which was extracted by micro RNA kit.Then reverse transcription and quantitative PCR were used to determine Cdc42,IL-17A,IL-23R and RORyt mRNA levels;2.According to the above inclusive and exclusive criteria,5 active UC patients,5 active CD patients and 5 healthy controls from Nanfang Hospital were chosen,non-adherent U-bottomed 96-well plates were coated with 30μ1 10μg/ml Sterile Purified NA/LE mouse Anti-Human CD3 antibody each well.After incubation within 37℃ for 4h,the plate should be stored at 4 ℃ovemight until use.2ml anticoagulant venous blood of the subjects was extracted,gradient separated to obtain PBMCs in a sterile environment.The cells were marked by MACS beads,and then sorted Naive CD4+ T cells.We adjusted the cell concentration to 1×106/ml and seeded them in coated culture plates.Each specimens were divided into three groups:control group(Anti-Human CD28),induction group(Anti-Human CD28+ TGF-β +IL-6),an inhibitor group(Anti-Human CD28 + TGF-p +IL-6+ML-141).All of them were cultured in serum-free 1640 medium for 5 days.Cell suspension were collected,centrifuged to obtain the supernatant which was used to detect IL-17 content by ELISA.A portion of the cells were used for the extraction of total RNA to the measurement of IL-17,IL-23R and RORyt mRNA levels by quantitative PCR.The other part were marker and determined CD3+ CD8-IL-17+ ratio by the above method;3.Experiments were conducted in healthy Balb/c mice weighted 22-25g,6-8w.20 mice were divided into control group and DSS group.Normal diet in the control group.DSS group was treated with 4%dextran sulfate sodium in drinking water instead.All the mice were sacrificed after 1 week.Recorded body weight and general condition every day and conducted DAI score.Collect the anticoagulant peripheral blood,and obtain plasma by centrifuging.Levels of IL-17A in the plasma were detected by ELISA method.The remaining blood cells were then isolated by density gradient centrifugation to get peripheral blood mononuclear cells(PBMCs).After been stimulated by PMA,ionomycin and BFA for 4 hours,some of the PBMCs were marked by surface antibody,Rat Anti-CD4-FITC,and then marked by Rat Anti-IL-17A-PE antibody after permeabilization and fixation.The ratios of CD4+IL-17+ cells were determined by flow cytometry machine.The removed spleen should be ground through a 70μn nylon-net in order to obtain single cell suspensions,and conducted gradient centrifuging to separate lymphocytes.Since been labeled by flow cytometric antibodies as above,CD4+ IL-17A+ cells were sorted.Washed small intestine with cold ice PBS repeatedly,made frozen sections to carry out immunofluorescence staining with Cdc42 and GM-CSF;the paraffin sections were processed by HE stain and immunohisto-chemistric stain.Part of the tissue was digested by collagenase IV so as to receive the lamina propria lymphocytes.The total RNA of these cells was extracted by micro RNA kit.Then reverse transcription and quantitative PCR were used to determine Cdc42,IL-17A,IL-23R and RORyt mRNA levels.4.Statistics:data were statistically analyzed using SPSS 17.0 software:homogeneity of variance was conducted first.Measurement data were expressed as mean±standard deviation.Gray value of bar charts were analyzed by Gelpro analysis software.Measurement data were gathered and also expressed as mean±standard deviation.One-way ANOVA was used for parametric test whilst Welch or Kruskal-Wallis tests were used for non-parametric tests.Multiple comparisons between groups used LSD-t test.The body weight and DAI score needed sphericity test.If P>0.05,the data should be analyzed by One-way ANOVA;if P<0.05,a repeated measurement was required and we choose Greenhouse-Geisser to analyze.The indexes of induced-cells were analyzed by Two-way ANOVA.P<0.05 was considered as significant difference and there was statistically significant.Results1.plasma levels of IL-17A:healthy controls,patients with active UC and CD are different among the three groups was statistically significant(F= 260.13,P = 0.000);active UC and CD patients,plasma IL-17A levels were significantly higher than that in healthy controls(3.429 ± 0.258,P=0.000 vs control group 1.797 ± 0.142;3.103 ±0.205,P = 0.000 vs control group 1.797 ± 0.142).Compared between the two groups,the plasma levels of IL-17A is slightly higher than in patients with UC CD patients(3.429 ± 0.258 vs 3.103 ± 0.205,P = 0.000).PBMCs of Cdc42,IL-17A mRNA levels:Cdc42:statistical difference among the three groups(F = 3.663,P = 0.034).UC patients(n = 11)PBMCs in Cdc42 levels than the control group(n = 17 patients)was significantly decreased(15.010 ± 5.908 vs 22.560 ± 11.974,P＝0.031),PBMCs of Cdc42 mRNA levels of CD patients(n = 18)are lower than that in healthy controls(n＝17)(15.557 ± 6.131 vs 22.560 ± 11.974,P = 0.022).No significant differences(P=0.871)between UC and CD group;IL-17A:there is a significant difference(F =3.356,P = 0.044)before the three groups.UC patients(n = 11)PBMCs in IL-17A than in healthy control subjects(n = 17)(0.0138 ± 0.0179 vs 0.0316 ± 0.0270,P=0.036),CD patients(n = 18)PBMCs in IL-17AmRNA is also higher than in healthy controls(0.0155 ± 0.0162 vs 0.0316 ± 0.0270,P=0.030).But between the two groups were compared,the difference was not statistically significant(P = 0.835);Cdc42 protein levels in PBMCs of:UC patients(n = 14)lower than healthy controls(n = 13)(0.7626 ± 0.3843 vs healthy controls 1.0620 ± 0.3175,F = 4.828,P=0.037).With active CD patients(n = 18)expression levels were lower than in healthy controls(n = 9)(0.9582 ± 0.9079 vs healthy controls 1.7543 ± 0.9635,F =4.434,P = 0.045).Sorting peripheral blood Th17 cells,Cdc42 mRNA levels:There were significant differences(F = 4.075,P = 0.039)in the three groups,UC group(n=6)lower than the healthy control group(16.888 ± 6.513 vs 26.617 ± 7.777,P =0.023).CD group(n = 6)is also lower than the healthy control group(17.379 ± 5.457 vs 26.617 ± 7.777,P = 0.029).No significant differences(P = 0.871)expression between UC and CD set of Cdc42.Three patients in the proportion of Th17 cells:between IBD patients and healthy control subjects was statistically significant(F =40.490,P = 0.000)differences.UC group(n = 6)was significantly lower than the proportion of the healthy control group(n = 6)(2.915 ± 0,528 vs 0.711 ± 0.238,P ＝0.000).CD group(n = 6)is also higher than the proportion of the healthy control group(2.813 ± 0.594 vs 0.711 ± 0.238,P = 0.000).No significant differences(P=0.718)between UC and CD groups.2.After iTh17 system induced IL-17A medium content:the healthy control group(n = 5,3.475 ± 0.353 vs 1.689 ± 0.232,P = 0.000),UC group(n = 5,3.750 ±0.287 vs 1.641 ± 0.431,P＝0.000),were significantly higher than the baseline water CD group(n = 5,3.485 ± 0.348 vs 1.894 ± 0.220,P =0.000);after joining the ML141,IL-17A content to rise further:the healthy control group(n = 5,6.104 ±0.415 vs 3.475 ± 0.353,P = 0.000).UC group(n = 5,5.319 ± 0.341 vs 3.750 ± 0.287,P＝0.000),CD group(n = 5,6.133 ± 0.229 vs 3.485 ± 0.348,P = 0.000).iTh17 system after induction,Th17 cell ratio:healthy controls(n = 5,6.006 ± 1.385 vs 3.066 ± 1.345,P = 0.033),UC group(n = 5,7.096 ± 1.226 vs 4.198 ± 0.871,P＝0.002),were significantly higher than the baseline water CD group(n = 5,11.996 ±1.200 vs 4.026 ± 0.981,P=0.000)were significantly higher;after joining the ML141,the ratio in healthy control subjects(n = 5,17.258 ± 2.737 vs 6.006 ± 1.385,P =0.000),UC group(n = 5,11.874 ± 1.387 vs 7.096 ± 1.226,P＝0.000),CD group(n =5,19.158 ± 1.738 vs 11.996 ± 1.200,P = 0.000)were significantly increased.3.After feeding mice with DSS for 7 days,compared to the control group,body weight decreased significantly(23.446 ± 0.931 vs 29.268 ± 0.743,F= 238.717,P=0.000),DAI scores were significantly higher(P = 0.000).Colon HE score significantly higher degree of inflammation apparent.Immunohistochemistry shows high expression of Cdc42 in the DSS group.There was no significant difference in the proportion of Thl7 cells in peripheral blood and spleen.Increased colonic lamina propria lymphocytes Cdc42 mRNA levels.Conclusions1.Compared to healthy controls,in active IBD patients,plasma levels of IL-17A and ratio of Th17 cells were increased,expression of Cdc42 protein and mRNA levels of IL-17A were declined;2.Cdc42 mRNA level of sorted Th17 cells of human peripheral blood in active IBD patients was significantly higher than that in healthy controls;3.Cdc42 specific inhibitor ML141 promoted Naive CD4+ T cells differentiate into Th17 cells;4.Mice in DSS group compared with them in the control group,the proportion of Th17 cells in peripheral blood and spleen were not significantly different,but the colonic lamina propria lymphocytes increase in the expression of Cdc42. |
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