| Inflammatory bowel disease(IBD)is a chronic gastrointestinal inflammatory condition consisting of Crohn's disease(CD)and ulcerative colitis(UC).Environmental,genetic and microbial factors with the immune system result in dysregulated immune responses in IBD.However,the mechanism still remains unclear and the treatment is far from effective.Further research in pathogenesis of IBD should be conducted to provide new theraputic targets.Intestine mucosal dysimmunity plays a core role in the pathogenesis of IBD.CD4+T cells are the key effector cells in immune system.CD4+T cells are composed of six main subgroups: Th1 cells,Th2 cells,Th17 cells,Th22 cells,newly discovered Th9 cells,and Treg cells.Each group of cells secretes specific cytokines.PU.1,an ETS family transcription factor,also identified as the spleen focus forming virus proviral integration site-1(Sfpi1)is a key transcription factor in the process of Th9 cell activation.The CD4 Na?ve T cells simulated with IL-4,TGF-? and IL-2 differentiate into Th9 cells in vitro.Th9 cells can deteriorate a variety of autoimmune inflammation via secreting specific pro-inflammatory cytokine IL-9.Some clinical researches have shown that Th9 cells and IL-9 increased significantly in IBD patients.More importantly,the expression of IL9 mRNA and IL-9 protein in patients with active UC was higher than in control subject.It has been demonstrated that Th9 cells and IL-9 play a key role in IBD.Currently,TL1 A,a tumor necrosis factor superfamily member,has been identified as one of IBD susceptibility genes.TL1 A,lymphocyte costimulator,can induce T cells proliferation and activation.TL1 A plays a critical role in various diseases such as asthma,rheumatoid arthritis and IBD.Papadakis found that TL1 A synergizes with IL-12 and IL-18 enhanced IFN-? production in human T cells and NK cells.TL1A was found to enhance the Th1,Th2 and Th17 cells responses in chronic colitis model.However,the association between Th9 cells and IBD is still unknown.lymphocytes over-expressed TL1 A transgenic mice and WT mice were used in this study,DSS-induced chronic murine colitis model was established and splenic cells and mesenteric lymph node(MLN)were isolated,purified and differentiated into Th9 cells and treated by IL-9 antibody and TL1 A antibody after TL1 A stimulation for investigation of the relationship between TL1 A and Th9,which might provide a new evidence for prevention and treatment of IBD.The experiment mainly consists of the following three parts: Part one: The changes of Th9 cells and IL-9 expression in chronic experimental colitis of sustained TL1 A expressionObjective: To study the effects of Th9 cells and IL-9 on chronic experimental colitis of sustained TL1 A expression.Methods: 1)LCK-CD2-TL1A-GFP-transgenic mice were identified by real-time PCR.2)The WT and Tg mice were randomly divided as followed:(1)Control/WT group,(2)DSS/WT group,(3)Control/Tg group,(4)DSS/Tg group.Chronic colitis was induced by drinking 2.5% DSS from 1 to 5 days,8 to 12 days,15 to 19 days and 22 to 26 days,and distilled water was given during the remaining time.Animals were sacrificed at 29 th day.3)Severity of colitis was evaluated by body weight(BW)changes,disease activity index(DAI),colon length,colon histology changes and pathology score.4)The percentage of CD4+IL-9+ T cells in colon was detected by immunefluorescence staining.The percentages of CD4+IL-9+ T cells in spleen,mesenteric lymph node(MLN)and lamina propria mononuclear cells were detected by Flow cytometry instrument(FCM).The expression of IL-9 mRNA was detected by Real-time PCR.The secretion level of IL-9 of serum,spleen,mesenteric lymph node(MLN)and lamina propria mononuclear cells(LPMCs)was detected by ELISA.5)The expression of TGF-? and IL-4 mRNA in colon was detected by Real-time PCR.The secretion level of TGF-? and IL-4 of spleen,MLN and lamina propria mononuclear cells mononuclear cells was detected by ELISA.6)The protein expression of TL1 A and PU.1 was detected by Western blot assay.The expression of TL1 A and PU.1 mRNA was detected by Real-time PCR.Results: 1)The TLlA DNA located at 192 bp in Tg mice and did not express at 192 bp in WT mice.2)Significantly increased DAI,reduced BW and colon length were observed in DSS/WT group and DSS/Tg group as compared to corresponding Control/WT group and Control/Tg group.More severe lesions in DSS/Tg as compared to DSS/WT group.3)Worsened inflammation characterized by increased cellular infiltrate,mucin depletion,crypt abscess,and architectural changes in DSS/WT group and DSS/Tg group as compared to corresponding Control/WT group and Control/Tg group(8.25±0.50 vs 0.00±0.00,P<0.05)and(12.50±0.58 vs 0.00±0.00,P<0.05).Significantly increased histological score was observed in DSS/Tg group as compared to DSS/WT group(12.50±0.58 vs 8.25±0.50,P<0.05).4)MPO activity: Significantly increased MPO activity was observed in DSS/WT group as compared to Control/WT group(1.09±0.21 vs 0.43±0.11,P<0.05).Significantly increased MPO activity was observed in DSS/Tg group as compared to Control/Tg group(1.80±0.12 vs 0.60±0.07,P <0.05).Significantly increased MPO activity was observed in DSS/Tg group as compared to DSS/WT group(1.80±0.12 vs 1.09±0.21,P <0.05).5)The percentage of CD4+IL-9+ T cells was detected by immunofluorescence staining.Significantly increased CD4+IL-9+ T cells were observed in DSS/WT group as compared to Control/WT group(0.24±0.03 vs 0.09±0.01,P<0.05).The expression of CD4+IL-9+ T cells in DSS/Tg group mice were higher than that in Control/Tg(0.44±0.03 vs 0.10±0.01,P<0.05).Significantly increased CD4+IL-9+ T cells was observed in DSS/Tg group as compared to DSS/WT group(0.44±0.03 vs 0.24±0.03,P<0.05).The results of between the percentages of CD4+IL-9+ T cells in spleen,mesenteric lymph node(MLN)and lamina propria mononuclear cells were detected by FCM,the secretion level of IL-9 of serum,spleen,mesenteric lymph node(MLN)and lamina propria mononuclear cells(LPMCs)were detected by ELISA,the expression of IL-9 mRNA in colon was detected by Real-time PCR and the result of immunofluorescence staining are consistent.6)The expression of TGF-? and IL-4 mRNA was detected by Real-time PCR.Significantly increased expression of TGF-? and IL-4 mRNA was observed in DSS/WT group(6.22±0.53,3.35±0.57)as compared to corresponding Control/WT group(1.02±0.20,1.01±0.17)(P<0.05).The expression of TGF-? and IL-4 mRNA in DSS/Tg group(15.06±1.63,4.80±0.89)was higher than that in Control/WT group(1.16±0.24,1.29±0.33)(P<0.05).Significantly increased expression of TGF-? and IL-4 mRNA was observed in DSS/Tg group(15.06±1.63,4.80±0.89)as compared to DSS/WT group(6.22±0.53,3.35±0.57)(P<0.05).The results of between the level of TGF-? and IL-4 of spleen,MLN and lamina propria mononuclear cells was detected by ELISA and the expression of TGF-? and IL-4 mRNA was detected by Real-time PCR are consistent.7)The expression of TL1 A and PU.1 mRNA was detected by Real-time PCR.Significantly increased expression of TL1 A and PU.1 mRNA was observed in DSS/WT group(9.19±1.24,2.68±0.46)as compared to corresponding Control/WT group(1.01±0.14,1.03±0.26)(P<0.05).The expression of TL1 A and PU.1 mRNA in DSS/Tg group(12.62±2.01,4.90±0.87)was higher than that in Control/WT group(1.21±0.16,1.20±0.18)(P<0.05).Significantly increased expression of TL1 A and PU.1 mRNA was observed in DSS/Tg group(12.62±2.01,4.90±0.87)as compared to DSS/WT group(9.19±1.24,2.68±0.46)(P<0.05).The results of between the expression of TL1 A and PU.1 protein in colon were detected by ELISA and the expression of TL1 A and PU.1 mRNA was detected by Real-time PCR are consistent.Part two: The role and mechanism of TL1 A in the differentiation of Th9 cellObjective: To research the differentiation of Th9 cell by TL1 A.Methods: 1)CD4 Na?ve T cells were isolated by magnetic cell sorting(MACS)negative sorting from spleen and mesenteric lymph node(MLN)mononuclear cells.The percentage of CD3~+CD4~+CD44-CD62L+ T cells(CD4 Na?ve T cells)was detected by FCM.2)CD4 Na?ve T cells were isolated from spleen and MLN mononuclear cells.Subsequently,CD4 Na?ve T cells were stimulated to Th9 cells.The percentage of CD4+IL-9+ T cells of before sorting cells and Th9 cells was detected by FCM.3)After stimulation of TL1 A with 100 ng/mL,the TL1 A antibody with 4 ug/mL or IL-9 antibody with 50 ng/mLwere further administrated in the process of Th9 cells differentiation.The percentage of CD4+IL-9+ T cells of Th9 cells from spleen and MLN mononuclear cells was detected by FCM.The level of CD4+IL-9+ T cells of Th9 cells from spleen and MLN mononuclear cells was detected by ELISA.The expression of PU.1 of Th9 cells from spleen and MLN mononuclear cells was detected by Western blot assay.Results: 1)The percentages of CD3~+CD4~+CD44-CD62L+ T cells(CD4 Na?ve T cells)were detected from spleen and MLN by FCM.The purity of the sorted cells from spleen and MLN indicated the isolated cells could used in the following experiment(90.18%±2.62%)and(92.12%±2.82%).2)The percentage of CD4+IL-9+ T cells of before sorting cells and Th9 cells from spleen was detected by FCM.Significantly increased percentage of IL-9 was observed in the both Th9 cells/WT and Th9 cells/Tg group(20.93%±3.60%,31.40%±3.66%)as compared to the corresponding before sorting cells/WT and before sorting cells/Tg group(0.28%±0.10%,0.42%±0.10%)(P<0.05).Meanwhile,significantly increased percentage of CD4+IL-9+ T cells was observed in the Th9 cells/Tg as compared to the Th9 cells/WT(31.40%±3.66% vs 20.93%±3.60%,P<0.05).The percentage of CD4+IL-9+ T cells from MLN and spleen before and after sorting was detected by FCM.3)After administration of TL1 A with 100 ng/mL,the percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 from spleen and MLN mononuclear cells were detected.The Th9 cells from spleen mononuclear cells: The percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 of the TL1A/WT group(CD4+IL-9+ T cells: 30.63%±2.32% vs 21.07%±2.35%,P<0.05;IL-9: 3867.63 pg/mL±88.11 pg/mL vs 2464.15 pg/mL±122.63 pg/mL,P<0.05;PU.1: 1.25±0.08 vs 0.95±0.06,P<0.05)and TL1A/Tg group(CD4+IL-9+ cells: 37.63%±2.26% vs 29.90%±2.33%,P<0.05;IL-9: 4221.90 pg/mL±81.11 pg/mL vs 2855.90 pg/mL±128.59 pg/mL,P<0.05;PU.1: 1.42±0.07 vs 1.18±0.06,P<0.05)were significantly increased as compared to the corresponding control/WT and control/Tg group.Meanwhile,further increase were observed in the TL1A/Tg groups as compared to the TL1A/WT groups(P<0.05).The Th9 cells from MLN mononuclear cells: The percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 of the TL1A/WT group(CD4+IL-9+ T cells: 29.80%±2.3% vs 24.60%±1.67%,P<0.05;IL-9: 3939.17 pg/mL±166.15 pg/mL vs 2603.82 pg/mL±98.20 pg/mL,P<0.05;PU.1: 1.23±0.06 vs 0.93±0.06,P<0.05)and TL1A/Tg group(CD4+IL-9+ T cells: 42.67%±2.56% vs 31.40%±2.75%,P<0.05;IL-9: 4337.73 pg/mL±161.69 pg/mL vs 3142.04 pg/mL±153.75 pg/mL,P<0.05;PU.1: 1.41±0.08 vs 1.22±0.08,P<0.05)were significantly increased as compared to the corresponding control/WT group and control/Tg group.Meanwhile,further increase were observed in the TL1A/Tg groups as compared to the TL1A/WT groups(P<0.05).4)After administration of TL1 A antibody with 4 ug/mL,the percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 from spleen and MLN mononuclear cells were detected.The Th9 cells from spleen mononuclear cells: The percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 of the TL1 A Ab/WT group(CD4+IL-9+ T cells: 18.30%±1.51% vs 30.63%±2.32%,P<0.05;IL-9: 2556.13 pg/mL±83.65 pg/mL;PU.1: 0.63±0.07 vs 1.25±0.08,P<0.05)and TL1 A Ab/Tg group(CD4+IL-9+ T cells: 23.67%±2.06% vs 37.63%±2.26%,P<0.05;IL-9: 2556.13 pg/mL±83.65 pg/mL vs 3867.63 pg/mL±88.11 pg/mL,P<0.05;PU.1: 0.85±0.07 vs 1.42±0.07,P<0.05)were significantlyly reduced as compared to the corresponding TL1A/WT and TL1A/Tg group.Meanwhile,further decrease were observed in the TL1 A Ab/WT groups as compared to the TL1 A Ab/Tg groups(P<0.05).The Th9 cells from MLN mononuclear cells: The percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 of the TL1 A Ab/WT group(CD4+IL-9+ T cells: 17.33%±1.96% vs 29.80%±2.30%,P<0.05;IL-9: 2576.57 pg/mL±53.10 pg/mL vs 3939.17 pg/mL±166.15 pg/mL,P<0.05;PU.1: 0.63±0.09 vs 1.23±0.06,P<0.05)and TL1 A Ab/Tg group(CD4+IL-9+ T cells: 29.50%±2.01% vs 42.27%±2.56%,P<0.05;IL-9: 3111.39 pg/mL±62.44 pg/mL vs 4337.73 pg/mL±161.69 pg/mL,P<0.05;PU.1: 0.92±0.10 vs 1.41±0.08,P<0.05)were significantly decreased as compared to the corresponding TL1A/WT and TL1A/Tg group.Meanwhile,further decrease were observed in the TL1 A Ab/WT groups as compared to the TL1 A Ab/Tg groups(P<0.05).5)After administration of IL-9 antibody with 50 ng/mL,the percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 from spleen and MLN mononuclear cells were detected.The Th9 cells from spleen mononuclear cells: The percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 of the IL-9 Ab/WT group(CD4+IL-9+ T cells: 23.50%±1.93% vs 30.63%±2.32%,P<0.05;IL-9: 2218.88 pg/mL±90.83 pg/mL vs 3867.63 pg/mL±88.11 pg/mL,P<0.05;PU.1: 0.76±0.08 vs 1.25±0.08,P<0.05)and IL-9 Ab/Tg group(CD4+IL-9+ T cells: 26.77%±1.90% vs 37.63%±2.26%,P<0.05;IL-9: 2669.20 pg/mL±100.65 pg/mL vs 4221.90 pg/mL±81.11 pg/mL,P<0.05;PU.1: 0.94±0.06 vs 1.42±0.07,P<0.05)were significantly reduced as compared to the corresponding TL1A/WT and TL1A/Tg group.Meanwhile,further decrease were observed in the IL-9 A b/WT groups as compared to the IL-9 Ab/Tg groups(P<0.05).The Th9 cells from MLN mononuclear cells: The percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 of the IL-9 Ab/WT group(CD4+IL-9+ T cells: 24.70%±2.10% vs 29.80%±2.30%,P<0.05;IL-9: 2215.48 pg/mL±129.40 pg/mL vs 3939.17 pg/mL±166.15 pg/mL,P<0.05;PU.1: 0.66±0.07 vs 1.23±0.06,P<0.05)and IL-9 Ab/Tg group(CD4+IL-9+ T cells: 30.20%±2.40% vs 42.27%±2.56%,P<0.05;IL-9: 2719.64 pg/mL±61.32 pg/mL vs 4337.73 pg/mL±161.69 pg/mL,P<0.05;PU.1: 0.88±0.08 vs 1.41±0.08,P<0.05)were significantly decreased as compared to the corresponding TL1A/WT and TL1A/Tg group.Meanwhile,further decrease were observed in the IL-9 Ab/WT groups as compared to the IL-9 Ab/Tg groups(P<0.05).Part three: The effect of IL-9 antibody on chronic experimental colitis of sustained TL1 A expression.Objective: To explore the effects of IL-9 antibody on chronic experimental colitis of sustained TL1 A expression.Methods: 1)The WT and Tg mice were randomly divided as followed:(1)Control/WT group,(2)DSS/WT group,(3)Ab Isotype/WT group,(4)IL-9 Ab/WT group,(5)Control/Tg group,(6)DSS/Tg group,(7)Ab Isotype/Tg group,(8)IL-9 Ab/Tg group.Chronic colitis was induced by drinking 2.5% DSS from 1 to 5 days,8 to 12 days,15 to 19 days and 22 to 26 days,and distilled water was given during the remaining time.In IL-9 Ab group and IL-9 Ab Isotype group,100 ug IL-9 antibody or control immunoglobulin G was injected into mice intraperitoneally every other day from the day 14 through day 29.Animals were sacrificed at 29 th day.2)Severity of colitis was evaluated by body weight(BW)changes,disease activity index(DAI),colon length,colon histology changes and pathology score.3)The percentage of CD4+IL-9+ T cells in colon was detected by immunofluorescence staining.The percentages of CD4+IL-9+ T cells in spleen,mesenteric lymph node(MLN)and lamina propria mononuclear cells were detected by Flow cytometry instrument(FCM).The expression of IL-9 mRNA was detected by Real-time PCR.The secretion level of IL-9 of serum,spleen,MLN and LPMCs was detected by ELISA.4)The expression of PU.1 was detected by Western blot assay.The expression of PU.1 mRNA was detected by Real-time PCR.Results: 1)Body weight(BW)change and disease activity index(DAI).After administration of IL-9 antibody,significantly increased BW and reduced DAI were observed in the both IL-9 Ab/WT group and IL-9 Ab/Tg group as compared to the corresponding DSS/WT group and DSS/Tg group.Meanwhile,significantly increased BW and reduced DAI were observed in the IL-9 Ab/Tg group as compared to the IL-9 Ab/WT group.There was no differerce in BW and DAI between Ab Isotype/WT group and DSS/WT group or Ab Isotype/Tg group and DSS/Tg group(P>0.05).2)H?E staining(×100)and histological score.After administration of IL-9 antibody,the lymphocyte cellular infiltrate and mucosal structure damage were alleviated.Significantly reduced histological score were observed in the both IL-9 Ab/WT group and IL-9 Ab/Tg group(3.83±0.98,6.33±0.82)as compared to the corresponding DSS/WT group and DSS/Tg group(11.00±0.63,13.67±1.03)(P<0.05).Meanwhile,significantly reduced histological score were observed in the IL-9 Ab/WT group as compared to the IL-9 Ab/Tg group(3.83±0.98 vs 6.33±0.82,P<0.05).There was no differerce in histological score between Ab Isotype/WT group and DSS/WT group(10.33±0.52 vs 11.00±0.63,P>0.05)or Ab Isotype/Tg group and DSS/Tg group(12.83±1.17 vs 13.67±1.03,P>0.05).3)MPO activity: After administration of IL-9 antibody.Significantly reduced MPO activity were observed in the both IL-9 Ab/WT group and IL-9 Ab/Tg group(0.13±0.02,0.42±0.07)as compared to the corresponding DSS/WT group and DSS/Tg group(0.97±0.13,1.94±0.12)(P<0.05).Meanwhile,significantly reduced MPO activity were observed in the IL-9 Ab/WT group as compared to the IL-9 Ab/Tg group(0.13±0.02 vs 0.42±0.07,P<0.05).There was no differerce in MPO activity between Ab Isotype/WT group and DSS/WT group(0.80±0.26 vs 0.97±0.13,P>0.05)or Ab Isotype/Tg group and DSS/Tg group(1.89±0.36 vs 1.94±0.12,P>0.05).4)The percentage of CD4+IL-9+ T cells in colon was detected by immunofluorescence staining.After administration of IL-9 antibody.Significantly reduced CD4+IL-9+ T cells were observed in the both IL-9 Ab/WT group and IL-9 Ab/Tg group(0.20±0.04,0.29±0.02)as compared to the corresponding DSS/WT group and DSS/Tg group(0.48±0.02,0.32±0.03)(P<0.05).Meanwhile,significantly reduced CD4+IL-9+ T cells were observed in the IL-9 Ab/WT group as compared to the IL-9 Ab/Tg group(0.20±0.04 vs 0.29±0.02,P<0.05).There was no differerce in CD4+IL-9+ T cells between Ab Isotype/WT group and DSS/WT group(0.33±0.02 vs 0.32±0.03,P>0.05)or Ab Isotype/Tg group and DSS/Tg group(0.44±0.04 vs 0.48±0.02,P>0.05).The percentages of CD4+IL-9+ T cells in spleen,mesenteric lymph node(MLN)and lamina propria mononuclear cells were detected by FCM,the level of IL-9 of serum,spleen,MLN and LPMCs was detected by ELISA,the expression of IL-9 mRNA in colon was detected by Real-time PCR and the result of immunofluorescence staining are consistent.5)The expression of PU.1 mRNA was detected by Real-time PCR.After administration of IL-9 antibody,significantly reduced expression of PU.1 mRNA were observed in the both IL-9 Ab/WT group and IL-9 Ab/Tg group(1.98±0.34,2.81±0.59)as compared to the corresponding DSS/WT group and DSS/Tg group(2.66±0.43,4.75±0.86)(P<0.05).Meanwhile,significantly reduced expression of PU.1 mRNA were observed in the IL-9 Ab/WT group as compared to the IL-9 Ab/Tg group(1.98 ±0.34 vs 2.81±0.59,P<0.05).There was no differerce in expression of PU.1 mRNA between Ab Isotype/WT group and DSS/WT group(2.69±0.44 vs 2.66±0.43,P>0.05)or Ab Isotype/Tg group and DSS/Tg group(4.80±0.86 vs 4.75±0.86,P>0.05).The results of between the expression of PU.1 protein in colon were detected by ELISA,the expression of PU.1 mRNA was detected by Real-time PCR and the expression of TL1 A mRNA was detected by Real-time PCR are consistent.Conclusions:1 Significantly increased of Th9 cells number and IL-9 production was observed in chronic experimental colitis of sustained TL1 A expression.TL1 A induced Th9 cells differentiation and IL-9 production in chronic experimental colitis.2 TL1 A induced Th9 cells differentiation and IL-9 production by upregulating PU.1 expression.On the contrary,TL1 A antibody inhibited Th9 cells differentiation and decreased IL-9 production by downregulating the expression of PU.1.3 IL-9 antibody significantly alleviated colonic inflammation by suppressing PU.1 expression and reducing Th9 cells differentiation and IL-9 production. |
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