TLR Signaling Pathway Of Dendritic Cells In Inflammatory Bowel Disease (Crohn’s Disease) And Effects Of Triptolide On It

Crohn’s disease(CD),a kind of inflammatory bowel diseases(IBD),exhibits a gastrointestinal segmental inflammatory damage,which seriously impact the patients’ quality of life.CD is relatively common in European and American.In recent years dortors has found the incidence of CD is in a growing trend in our country due to the change of lifestyle or environment and improvements of diagnostic technology.The complicated clinical problems caused by CD have made the specialist think highly of the disease.The etiology and pathogenesis of CD are not very clear today.The current study focused on the genetic predisposition,immune disorder,infection and environmental factors.The progress of reatment research on CD is slow,because the pathogenesis is not fully understood.The therapeutic effect of common drugs is poor,and the drugs may cause many side effects.Further reveal pathogenesis of CD,exploring efficient,low-toxic treatment methods has been dedicated to the study of gastroenterologist.Clinical and animal experiments both showed loss of immune tolerance of intestinal mucosa to the symbiotic bacteria is the major pathogenesis of CD.Dendritic cells(DCs)is recognized as a functional involving T cell activation and immune response initiation cell,and play an important role in the pathogenesis of CD.DCs is known as the most efficient Antigen-presenting cells,which connect specific immune response and non-specific immune response,and determine the type of immune response.Toll-like receptor(TLR)is an important tool for DCs to recognize and distinguish pathogen associated molecular patterns(PAMP)of intestinal microorganisms.DCs in intestinal lamina propria express various TLR,and different subtypes of TLR has different exogenous ligands.Current research has shown that TLR2,TLR4,TLR5 and TLR9 are relevant to steady-state of intestinal mucosa and inflammatory bowel diseases,and different subtypes of TLR plays different role in intestinal inflammation.DCs play an important role in maintaining intestinal mucosa steady and loss of intestinal immune tolerance,and research of TLR has pay close attention to the role in the pathogenesis of CD,however the effects of TLR signaling pathways in DCs on CD are not fully understood.Tripterygium Wilfordii Polyglyeosidium,which shows anti-inflammatory and immunosuppressive activities,has been used to treat autoimmune diseases such as rheumatoid arthritis and glomerulonephritis with satisfactory effect.We found Tripterygium Wilfordii Polyglyeosidium is effective in induction and maintain of clinical response and remission in CD.Some cases even achieved unexpected good effect.Triptolide,a diterpene triepoxide,is an active component of extracts from a traditional Chinese herb Tripterygium Wilfordii Hook,which is the major effective ingredient of Tripterygium Wilfordii Polyglyeosidium.Our previous study found triptolide could ameliorate the intestinal inflammation and increase survival rate and living condition of CD animal model(IL-10 knockout mice).This may be relative to inhibiting the expression of Thl cytokines.The results indicate that triptolide could be potential therapeutic effect in CD,however the precise mechanism is not clear.In this study,we investigated TLR signaling pathway in DCs of IL-10-/-mice and colon tissue samples from CD patients and effect of triptolide on the signaling pathway.It may conduce to the application of Tripterygium Wilfordii Polyglyeosidium on CD,and provide an economic and effective treatment methods for CD.Part 1Expression of TLR signaling pathway in the intestinal tissue of animal model of Crohn’s disease and effects of triptolide on it in vivoObjective:To investigate expression of TLR signaling pathway in animal model of Crohn’s disease(IL-10-/-mice)and wild type,and the effects of triptolide on TLR signaling pathway in IL-10-/-mice.Materials and methods:Establish chronic colitis in IL-10-/-mice.5-6 weeks male IL-10-/-mice were divided randomized into two groups.The control group were adminstered intraperitoneal injection normal saline every other day for 8 weeks.The triptolide group were similarly given an equivalent quantity of triptolide(0.07 mg/kg).Wild type mice of the same background were added if nessary.The histopathology changes in the colon of mice were observed.The level of MPO was evaluated by ELISA.The distribution of TLR2 and TLR4 were measured by immunohistochemistry.mRNA expression of TLR2,TLR4,and downstream factor MyD88 were measured by real time PCR.The protein expression of TLR2 and TLR4 were evaluated by Western blot analysis.Results:Triptolide significantly reduced colonic chronic inflammation and histological score in IL-10-/-mice.The expression of TLR2 and TLR4 were increased in IL-10-/-mice compared to wild type.Triptolide significantly inhibited the expression of TLR2,TLR4 and MyD88 in IL-10-/-mice.Conclusion:TLR signaling pathway was activated in the colon of IL-10-/-mice.Triptolide protects against chronic colitis of IL-10-/-mice by involving inhibition of TLR signaling pathway.Part 2Expression of TLR signaling pathway in intestinal dendritic cells of animal model of Crohn’s disease and effects of triptolide on it in vivoObjective:To isolate and purify colonic dendritic cells from IL-10-/-mice and investigate the effects of triptolide on TLR signaling pathway in colonic dendritic cells of IL-10-/-mice.Materials and methods:5-6 weeks male IL-10-/-mice were divided randomized into two groups.The control group were adminstered intraperitoneal injection normal saline every other day for 8 weeks.The triptolide group were similarly given an equivalent quantity of triptolide(0.07 mg/kg).Magnetic cell sorting technology was used for isolation and purification of colonic dendritic cells from IL-10-/-mice.Cells were used for further study after testing the purity and survival rate.mRNA expression of TLR2,TLR4,and downstream factor MyD88 were measured by real time PCR.The protein expression of TLR2,TLR4 and NFkB were evaluated by Western blot analysis.Results:The purity of dendritic cells(marked with CD11c)is more than 90%,the survival rate of cells is also more than 90%.Triptolide significantly inhibited the mRNA and protein expression of TLR2 and TLR4 in dendritic cells.Triptolide could also decrease the mRNA expression of MyD88 and nuclear protein expression of NFkB in dendritic cells from colon of IL-10-/-mice.Conclusion:Magnetic cell sorting technology was convenient and reliable for isolation and purification of colonic dendritic cells.Triptolide significantly inhibited TLR signaling pathway in colonic dendritic cells.Dendritic cells are the potential target of triptolide.Part 3Expression of TLR signaling pathway in intestinal dendritic cells of Crohn’s disease and effects of triptolide on it in vitroObjective:Colon tissues were obtained from surgical resection specimens from patients with active CD who accepted partial colon resection.To isolate and purify colonic DCs from colon tissues.To investigated the effects of triptolide on expression of TLR2,TLR4,MyD88 and NFkB in colon tissues or DCs,and explore the effects of triptolide on TLR signaling pathway in CD patients.Materials and methods:Colon tissues were obtained from surgical resection specimens from patients with active CD who accepted partial colon resection,and divided randomized into two groups.Explants were cultured in serum-free CMRL-1066 tissue culture medium on collagen I-coated plates at 37 ℃ in a 5%carbon dioxide atmosphere for 24 h.The medium was supplemented with 20 ng/ml triptolide and TLRs agonists:125 μg/ml of Zymosan and 50 μg/ml of Lipoteichoicacid(LTA)for TLR2 elicitation and lOug/ml of lipopolysaccharide for TLR4 elicitation.The controls were cultured in the same manner except for the triptolide supplementation.mRNA expression of TLR2 and TLR4 were measured by real time PCR.Magnetic cell sorting technology was used for isolation and purification of colonic DCs from tissues.Then the DCs were divided randomized into two groups.DCs were cultured in RPMI1640 culture medium similarly to the previous described.The level of TNF-α,INF-γ,IL-4 and IL-5 were evaluated by ELISA.MHCⅡand CD86 on DCs were analyzed by flow cytometry.mRNA expression of TLR2,TLR4,and downstream factor MyD88 were measured by real time PCR.The protein expression of TLR2,TLR4 and NFkB were evaluated by Western blot analysis.Results:Triptolide significantly inhibited the expression of TLR2 and TLR4 in colon tissue from CD patients.Triptolide decreased the level of TNF-a and INF-y,and did not affect the level of IL-4 and IL-5.Triptolide reduced the expression of costimulatory molecules MHCⅡ and CD86 on DCs.Triptolide significantly inhibited the expression of TLR2,TLR4,MyD88 and NFkB in dendritic cells from colon of CD patients.Conclusion:Triptolide decreased the level of Thl cytokines,and did not affect the level of Th2 cytokines.Crohn’s disease is Thl dominant inflammation.The results conduce to the application of triptolide on CD.Triptolide could reduce the expression of costimulatory molecules on DCs.This means triptolide could alter mature state of DCs,and induce immune tolerance.Triptolide inhibited TLR signaling pathway both in the colon tissue and DCs,which contribute to the application of triptolide on CD.Summary1.Triptolide could ameliorate the chronic colitis in IL-10-/-mice.TLR signaling pathway plays an important role in the pathogenesis of colitis in IL-10-/-mice.Triptolide could adjust the abnormal intestinal mucosa immune response and suppress intestinal inflammation by inhibiting TLR signaling pathway.2.Triptolide could inhibit TLR signaling pathway not only in IL-10-/-mice but also in colon tissues and DCs from CD patients,which may be contribute to the application of triptolide on CD patients.3.The study showed DCs are the major target of triptolide in vivo and in vitro.We conclude that targeting TLR signaling pathway in DCs may be a new strategy for the treatment of CD.It needs further study to certify.