The Mechanism Of CRF Modulating Mast Cells In The Pathogenesis Of Irritable Bowel Syndrome

IntrodutionIrritable bowel syndrome(IBS)is one of the most common functional gastrointestinal disorders,with the prevalence ranging from 1.1% to 29.2% in the general population diagnosed by the Rome III criteria.IBS patients can experience constipation,diarrhea,or both.IBS significantly reduces health-related quality of life and work productivity.Multiple factors contribute to the pathogenesis of IBS,such as brain-gut axis dysregulation,gut barrier dysfunction,visceral hypersensitivity and dysmotility,the food intolerance and psychological stress are considered as important triggers of IBS.Psychological stressor may predispose to the development of IBS.Patients with IBS are more likely to have experienced anxiety,tension,anger and fear,which can contribute to the development of the disease through brain-gut axis.Corticotropin releasing factor(CRF)is a 41 amino acid peptide,produced in the central nervous system and peripheral tissues in response to stress and has been shown to play a central role in stress-induced intestinal pathophysiology.The activity of CRF are mediated by activation of specific seven trans-membrance G-protein coupled recptors(GPCRs)known as CRF-R1 and CRF-R2.Some researchers found that the expression of CRF was increased in the intestinal of IBS patients.The water avoidance stress and restraint stress can lead dysmotility and visceral hypersensitivity in rodents,with the increased CRF in the gut.More and more researchers indicate low-grade intestinal inflammation as a key role in the pathophysiology of IBS,and attentions have been focused on the roles of mast cells(MCs)in the intestinal.Increases in the number of mucosal MCs have been observed in IBS patients in the rectum,colon,cecum,terminal ileum,jejunum,and duodenum.Moreover,it varies in different genders,in distinct bowel segments and in subgroups of IBS patients.Accounting for the close relations of MCs with major intestinal functions,the hyperplasia and activation of MCs can lead to symptoms of IBS--abdominal pain and/or discomfort,bloating,and abnormal bowel function.Commensal microflora contains a number of components able to activate innate and adaptive immunity,such as LPS,peptidoglycans,superantigens,bacterial DNA,heat shock protein.Unlimited immune activation in response to signals from commensal bacteria could pose the risk of inflammation.The mucosal immune system has developed precise regulatory for eliminating or tolerating non-dangerous,food and commensal microorganisms.One of such a protective mechanism is endotoxin tolerance(ET),a phenomenon in which cells exposed to LPS enter into a transient unresponsive state and are unable to respond to further challenges with endotoxin.This phenomenon has been observed both in vitro and in vivo in animal models as well as in humans.Toll-like receptor 4(TLR4)takes part in the progress of endotoxin tolerance.The suppressors of cytokine signaling(SOCS)proteins are also involved in tolerance phenomena as negative regulators of the pro-inflammatory response,via the TLR4-NFκB pathway.CRF-R1 and CRF-R2 can be detected on MCs,and TLR4 is also expressed on MCs.It is a question that whether CRF can break the endotoxin tolerance of MCs by regulating the TLR4 on MCs.The gut barrier is a functional entity separating the gut lumen from the inner host,and consisting of microbiota,mucus,epithelial layer,immune elements(lymphocytes,innate immune cells)and neurological elements.It prevents against the entry of antigens and microorganisms in while allowing absorption of nutrients from the diet.Abnormal intestinal permeability,which facilitates enhanced antigen exposure that may activate the intestinal immune system,has been implicated in the pathogenesis of IBS.Increases in the numbers of MCs in the gut of IBS patients were found to be related to changes in gut permeability.CRF produced in response to stress is a key factor in the pathogenesis of IBS.MCs are important innate immune cells in the intestinal barrier and they keep endotoxin tolerance to LPS under normal circumstance.In this study,we tested the hypothesis that CRF could modulate the MCs to breach the established endotoxin tolerance and further induce the gut barrier dysfunction.This study may provide a better understanding of the precise mechanism of CRF and MCs in the pathogenesis in IBS and may help develop strategies for managing IBS patients.Part Ⅰ CRF modulates mast cells to breach endotoxin tolerance in vitro Aims:To verify if CRF can modulate HMC-1 MCs to breach the established endotoxin tolerance,and to elaborate the precise mechanism in this progress.Methods:1.HMC-1 MCs were pretreated with different concentrations of LPS(0,10,100ng/ mL)in vitro,and then all of the groups were stimulated by 100ng/ mL LPS.The cells were observed in transmission electron microscope to evaluate the situation of degranulation.The levels of TNF-α,MCP(tryptase),IL-1β,IL-6 and IL-13 in the supernatant were tested by ELISA.2.The tolerant HMC-1 MCs were treated by different concentrations of CRF(0,50,100 pg/ mL),and the mRNA and protein of TLR2 and TLR4 were tested by realtime-PCR and Western blotting.In order to confirm CRF mediated the effects by which receptor(CRF R1 and/or CRF R2),and the pathway through(Erk1/2 or p38 MAPK),different antagonist were added to pretreate the cells before CRF.3.To demonstrate the key role of TLR4 in the breaching of endotoxin tolerance,TLR4 gene of HMC-1 MCs was silenced with shRNA.4.MCs were treated by the methods in step2,and then these groups and TLR4 null cells were stimulated by 100ng/ mL LPS.The cells were observed in transmission electron microscope to evaluate the situation of degranulation.The levels of TNF-α,MCP(tryptase),IL-1β,IL-6 and IL-13 in the supernatant were determined by ELISA.5.The tolerant HMC-1 MCs were treated by different concentrations of CRF(0,100 pg/ mL)or pretreated by CRF antagonist before CRF,and then they were stimulated with LPS.The expressions of SOCS-1 and SOCS-3 were detected by realtime-PCR and Western blotting.Results:1.Compared with the na?ve cells,the MCs pretreated by 100ng/ mL LPS released less TNF-α、IL-1β、IL-6 and IL-13(P<0.05),but the level of MCP was not different from that of na?ve group.Degranulations were not observed in all of the groups.2.After treated with 100pg/ml CRF,the expressions of TLR4 mRNA and protein in tolerant MCs were significantly increased(P<0.05).But the phenomenon was reversed by CRF-R1 antagonist or Erk1/2 antagonist.The expressions of TLR2 were not different in all of the groups.3.The TLR4 gene was silenced in HMC-1 MCs with shRNA.TLR4 null cell line was successfully established.4.CRF activated MCs by two pathways: CRF lead MCs to degranulation,during this progress,TNF-α and MCP were released.This effect was abolished by CRF-R1 or CRF-R2 antagonist.Meanwhile,CRF breached the endotoxin tolerance of MCs,at the same time,IL-1β,IL-6,IL-13 and TNF-α were released.This effect was abolished by CRF-R1 or Erk1/2 antagonist.TLR4 null cells didn’t establish the endotoxin tolerance to LPS and didn’t break the tolerance by the induction of CRF.5.CRF suppressed the mRNA and protein of SCOS-1 and SCOS-3 in endotoxin tolerant MCs.The suppression can be abolished by CRF antagonist.Conclusions:The established endotoxin tolerance of MCs can be breached by CRF,and the followings are the pricise machnisms: CRF that combined with CRF-R1 on MCs can up-regulate the expression of TLR4 on the surface of mast cells by Erk1/2 intracellular signal transduction pathway.At the same time,CRF suppresses SCOS-1 and SCOS-3,and further abolish the inhibition of the LPS-TLR4-NFκB pathway.Part Ⅱ CRF modulates mast cells to breach the intestinal barrier function in water avoidance stress mice Aims:To investigate the mechanism of the intestinal barrier dysfunction induced by the cytokines and mediators from mast cells that activated by CRF.Methods:1.Forty Balb/c mice(6-8w)were divided into 5 groups: Control,Sham,CRF-anta and MC stabilizer.They were treated differently due to the protocol and then they were treated with water avoidance stress(WAS)daily for 10 days.The mice were sacrificed on day 10.2.The permeability of colon mucosa was tested in Ussing chambers.The serosas and muscles were slipped form fresh tissues and then the mucosas were fixed into the chambers.After 30 minutes,the Isc and G were recorded per 30 min for 2 hours.The permeability to horseradish peroxidase(HRP)was also evaluated as an indicator of the intestinal permeability.3.The total RNA and protein were extracted respectively from the colon tissues.The expressions of claudin5,7,8,12,15 and Trek1 mRNA and proteins were detected by realtime-PCR and Western blotting.4.The expression of Trek1 on the colon was tested by immunofluorescence.5.T84 cells were exposed to TNF-α,MCP(tryptase),IL-1β,IL-6 and IL-13 in the culture respectively for 72 h.The cells were analyzed by realtime-PCR and Western blotting to test the mRNA and protein of Trek1.Results:1.Mice treated with WAS displayed increased permeability compared with other mice.But this effect was blocked by CRF antagonist or MCs stabilizer.2.HE staining showed that the epithelial cells of mice treated with WAS were irregular arranged,and the gaps between epithelial cells were bigger.The tissue sections from the other four groups performed similarly.3.The mRNA and protein of Claudin5,7,8,12,15 were not significantly different among the 5 groups.But the mRNA and protein of Trek1 in mice treated with WAS were significantly lower than those of the other 4 groups.4.The expression of Trek1 on the colon was significantly lower than that of other 4 groups showed by immunofluorescence.5.The exposure to either of the 5 cytokines markedly suppressed the expression of Trek1 in T84 cells,in contrast with the exposure to BSA,which did not affect the expression of Trek1.Because all these mediators are capable of activating p38 MAPK,which might be involved in the suppression of Trek1 by these cytokines.To test the inference,in the same experimental procedures above,an inhibitor of p38 was added to the culture,indeed,the suppression of Trek1 was abolishedConclusions:CRF modulates mast cells to breach the intestinal barrier function in water avoidance stress mice due to the mediators from activated MCs.These mediators inhibit Trek1 in the intestinal epithelia via the p38 MAPK pathway.Part Ⅲ The role of Trek1 in regulating the intestinal epithelial barrier function in vitro Aims:The disruption of epithelial barrier integrity is an important factor in the pathogenesis of IBS.It is refractory to restitute the compromised barrier function.This study aims to investigate the regulation of TWIK-related potassium channel-1(Trek1)to restitute intestinal epithelial barrier function.Methods:1.Human intestinal epithelial cell line,T84 cells were cultured in DMEM.Upon confluence,the cells were collected and seeded in the inserts of Transwells at 106 cells/ml.After the transepithelial resistance(TER)reached or was over 1000 ?/cm2(recorded with an ohmmeter)the cells were used for further experiments.2.The Trek1 gene was silinced in T84 monolayers in Transwells with shRNA.The effect of gene silencing was assessed by Western blotting.3.TER was evaluated as the indicators of the T84 monolayer barrier function.TER was recorded at time points 0h,8h,16 h,2d,3d,4d,5d,6d,7d.4.The permeability to horseradish peroxidase(HRP)was also evaluated as the indicators of the T84 monolayer barrier function.5.Trek1-null T84 cell monolayers were treated with rTrek1 in Transwells.TER was recorded and HRP flux was performed at 72 h after shRNA transduction.Results:1.T84 cells were cultured in Transwells.Upon reaching confluence,the Trek1 gene was knocked down by gene silencing in Transwells.Western blotting showed that the Trek1 gene was knocked down in T84 cells.2.The TER of T84 monolayers was recorded at 9 time points from 0 h to day 7.The results showed that,after the gene silence,TER was dropped significantly at 16 h and further dropped at 48 h.The TER was remained at low levels in the entire observation period.3.The permeability of the T84 monolayers was assessed by HRP flux assay.The results showed that the knockdown of the Trek1 gene also increased the permeability of T84 monolayers,which was inversely with the changes of TER.4.To corroborate the results,in separate experiments,we added recombinant Trek1(rTrek1)to the culture of Trek1-null T84 monolayers;then assessed the barrier function.The results showed that the presence of rTrek1 reversed the Trek1-deficiency-induced T84 monolayer barrier disruption.Conclusion:Trek1 is required in the maintenance of T84 monolayer barrier function.