| Background:Oxytocin(OT)is traditionally considered a nonapeptide hormone synthesized in the hypothalamus that is released from the posterior pituitary into circulation and is involved in milk let-down and uterine contraction.Studies in our laboratory and others have made clear that OT is an endogenous neuropeptide in the gastrointestinal tract and regulates the secretion,motility,and immunity of the digestive tract.The intestine is the largest immune organ in the body and contains various innate immune and adaptive immune cells.Macrophages are the most important innate immune cells.Muscularis macrophages(MMs)are densely distributed and adjacent to enteric neurons and nerve fibres.The interaction of MMs and enteric neurons together regulated intestinal motility,protected tissues and maintained intestinal immune homeostasis.There is a direct connection between MMs and neurons,we previously demonstrated that OT regulated the polarization of macrophages to relieve intestinal inflammation.This article aimed to explore whether the polarization of macrophages regulated the expression of the OT and OT receptor(OTR)in the ENS.OT in the ENS was very likely to be the signalling molecule for the dialogue between intestinal macrophages and neurons.This "feedback loop" between macrophages and enteric neurons could be a pivotal "controller" that determines the tendency toward homeostasis or disease.Methods:1.Enteric neurons isolation and cultureEnteric neurons were obtained from C57BL/6 mice as previously described.Briefly,the mice were euthanized with no pain,and the outer skin of the peritoneum was cut.Three to five centimetres of colon tissue was excised and immediately placed in a Sylgard-lined dissecting dish containing carbogen-gassed Krebs saline.The mesentery was cut with venus scissors,and the colonic segment was cut longitudinally along the mesentery.The colonic segment was fixed in a dissecting dish,and the contents were thoroughly washed.The mucosa,submucosa,serosal layer,and circular muscle layers were carefully removed with venus forceps to expose the longitudinal muscle myenteric plexus(LMMP).The LMMP was incubated in Krebs saline containing papain(10 mg/ml)at 37℃ for 50 min.The LMMP was stretched as much as possible under the microscope.Immediately thereafter,the LMMP was placed in Dulbecco’s modified Eagle’s medium containing collagenase Ⅱ(1 mg/ml),cut into pieces with scissors,and digested in a humidified incubator with 5%CO2 at 37℃ for 55 min.After digestion,the medium was added to terminate and centrifuged at 1000 rpm for 6min.To culture enteric neurons,the sediment was collected and suspended in Dulbecco’s modified Eagle’s medium supplemented with 10%heat-inactivated foetal bovine serum and 1%penicillin-streptomycin solution.The medium was replaced with fresh medium daily,and the cell growth status was carefully observed.After 4 h of cell culture,the medium was changed,and the cell morphology was observed with a microscope.2.Acute colitis modelAcute colitis was induced by 2.5%dextran sulfate sodium(DSS)in drinking water for 7 days.In the second week,standard drinking water was available to the mice.On days 7,14,21,28 and 49,the mice were sacrificed.The degree of colitis inflammation was evaluated by recording the body weight,colon length,the score of disease activity index(DAI),mucosal permeability,H&E staining and histological score of mice.3.The expression of OT on enteric neurons,the expression of F4-80/iNOS double-positive cells in colon section,the expression of Iba1/Argl double positive cells in colon section and CD206 positive cells in LMMP was detected by immunofluorescence.4.The mRNA levels of M1 and M2 macrophage markers,inflammatory factors and OT/OTR were detected in cultured enteric neurons and LMMP by qPCR.5.The concentrations of OT and cytokines in cultured enteric neurons were detected by ELISA.6.The protein levels of STAT3 pathway,NF-κB pathway,SMAD2/3 pathways,OT,OTR and PEG3 in cultured enteric neurons and LMMP were detected by western blot.Results:1.Effect of macrophages supernatant on the expression of OT and OTR in enteric neuronsWe used LPS to induce Raw264.7 cells to differentiate into M1 macrophages and collected the supernatant(M1 supernatant).We also induced Raw264.7 cells to differentiate into the M2 type with IL4 and collected the supernatant(M2 supernatant).We separately incubated enteric neurons with M1 supernatant,M2 supernatant or unstimulated macrophages supernatant(M0 supernatant).In cells treated with M1 supernatant,the mRNA and protein levels of OT and OTR were markedly decreased compared with those in the control group,whereas treatment with the M2 supernatant increased the mRNA and protein expression of OT and OTR.M0 supernatant did not affect the expression of OT and OTR in enteric neurons.In conclusion,M1 macrophages and M2 macrophages inhibited and promoted the expression of OT and OTR in enteric neurons,respectively.We used LPS or IL-4 to stimulate enteric neurons and observed no obvious changes in OT or OTR mRNA expression compared with the control group.In summary,LPS or IL-4 did not affect the expression of OT and OTR in enteric neurons.2.Effect of cytokines in M1 supernatant on the expression of OT and OTR in enteric neuronsTo investigate cytokines in M1 supernatant that regulated the expression of OT and OTR,we first measured the levels of different cytokines in the M1 supernatant.The levels of TNF-α,IL-6 and IL-1β were obviously increased in M1 supernatant.Second,we tested the effects of these cytokines on the mRNA expression of OT and OTR.Treatment with TNF-α,IL-6 and IL1β significantly decreased the mRNA expression of OT and OTR in cultured enteric neurons.We pretreated neurons with Tocilizumab(IL-6 receptor antagonist),R-7050(TNF-α receptor antagonist),or IL-1RA(IL-1 receptor antagonist).We found that Tocilizumab,R-7050 and IL1RA reversed the inhibitory(IL-6,IL-1β and TNF-α)effects of the respective agonists.In addition,treatment with Tocilizumab,IL-1RA and R-7050 completely reversed the excitatory effects of M1 supernatant on the expression of OT.In summary,the inhibitory effect of M1 supernatant on OT and OTR expression was mainly through IL-1β,IL-6 and TNF-α.3.Effect of cytokines in M2 supernatant on the expression of OT and OTR in enteric neuronsTo investigate cytokines in M2 supernatant that regulated the expression of OT and OTR,we first measured the level of TGF-β in the M2 supernatant,TGF-β level was obviously increased.Second,we tested the effect of TGF-β on the mRNA expression of OT and OTR.Treatment with TGF-β significantly increased the mRNA expression of OT and OTR in cultured enteric neurons.Next,we pretreated neurons with LY2109761(TGF-β receptor antagonist),we found that LY2109761 reversed the excitatory(TGF-β)effect of the agonist.In summary,the excitatory effect of M2 supernatant on OT and OTR expression was mainly through TGF-β.4.Inflammatory cytokines downregulated the expression of OT and OTR proteins in enteric neurons via the STAT3 or NF-κB pathwayThe degree of STAT3 phosphorylation was significantly higher following treatment with IL-6 or TNF-α than in the control group.In addition,the protein expression of OT and OTR decreased significantly,and this inhibitory effect was reversed by Stattic(STAT3 inhibitor).Treatment with IL-1β did not change the degree of STAT3 phosphorylation but significantly upregulated the protein expression of P-NF-κB in enteric neurons.The decreases in OT and OTR expression following IL-1β administration were significantly reversed by pretreatment with Bay 11-7082(NF-κB inhibitor).Therefore,we believed that the inflammatory cytokines in M1 macrophage supernatant inhibit the expression of OT and OTR via different pathways.TNF-α and IL-6 elicited their effects through STAT3 phosphorylation,whereas the influence of IL-1β was mainly related to NF-κB phosphorylation.5.The SMAD2/3 pathways were involved in the upregulation of OT and OTR expression following TGF-βWe found that SMAD2/3 pathways were activated following TGF-β treatment,the protein expression of OT and OTR significantly increased,pretreatment with SIS3 HCl(SMAD2/3 inhibitor)blocked this change.Therefore,we believed that TGF-β increased OT and OTR expression in enteric neurons via the SMAD2/3 pathways.6.PEG3 was involved in the upregulation of OT and OTR expression induced by M2 macrophagesPEG3(Paternally expressed gene 3)encodes a DNA binding protein that acts as an inhibitory transcriptional regulator of the expression of OTR.We found that the treatment with M0 and M1 supernatant and inflammatory cytokines,including IL-6,IL-1β and TNF-α,did not change the expression level of PEG3,by contrast,treatment with M2 supernatant and TGF-βsignificantly reduced the mRNA expression of PEG3 in cultured enteric neurons.Western blot confirmed the above results,after TGF-β incubated enteric neurons,the protein level of PEG3 was down-regulated in enteric neurons.Blocking the SMAD2/3 pathways with SIS3 HCl significantly attenuated the downregulation of PEG3 expression and upregulation of OT and OTR expression.In conclusion,M2 macrophages activated the SMAD2/3 pathways by releasing TGF-β,inhibited the expression of PEG3,and increased the expression of OT and OTR.7.Macrophage polarization at different periods of colitisExperimental colitis divided into inflammatory periods and repair periods,with different types of macrophage polarization in each period.Therefore,we constructed DSS-induced acute colitis model to explore macrophage polarization in different periods of colitis.In the first 7 days during which the mice were administered DSS in drinking water,the mice in the DSS treatment group had significantly reduced body weight,reduced colon length,and a higher DAI than those in the control group.These changes reached a maximum at the end of the first week after the beginning of DSS feeding and then began to decrease.At the third week,the weight of the mice and the colon length returned to normal,and the score of DAI decreased significantly.Histological evaluation at different periods showed that at 7 days following DSS treatment,the colon exhibited severe epithelial damage,crypt loss,submucosal oedema and inflammatory cell infiltration.However,these histological changes began to decrease at the second week and completely disappeared at the seventh week.Therefore,we divided DSS-induced colitis into the inflammatory periods(the first and second weeks)and the repair periods(the third week and later).In the inflammatory periods of DSS-induced colitis,on days 7and 14,the mRNA levels of IL-1β,IL-6 and TNF-α significantly increased.In addition,the mRNA levels of M1 macrophage marker CCL2 and iNOS significantly increased.In contrast,in the inflammatory periods,on day 7,the mRNA expression of anti-inflammatory factor TGF-β and M2 macrophage markers(Chil3,CD206)had no significant difference compared with the control group.On day 14,the mRNA expression of anti-inflammatory factor TGF-β and Chil3 increased,but the mRNA expression of CD206 had no significant difference compared with the control group.The results of immunofluorescence also showed that the number of M1 phenotypes(F4-80+/iNOS+)in the submucosal increased on days 7 and 14.During the repair periods,the opposite change occurred in macrophage polarization.On days 21 and 28,the mRNA expression of pro-inflammatory factors IL-6,TNF-α,CCL2 and iNOS returned to normal level.The mRNA levels of anti-inflammatory factor TGF-β and M2 macrophage markers(CD206,Chil3)increased.The results of immunofluorescence also showed that the number of M1 phenotypes(F4-80+/iNOS+)in the submucosal began to decrease.On day 49,all the above indicators returned to normal level.Therefore,we believed that macrophages polarized to the M1 type during the inflammatory periods and to the M2 type during the repair periods.8.The changes of OT and OTR in different periods of colitisIt has been demonstrated that macrophage polarization affected the expression of the OT and OTR.Whether the expression of the OT and OTR was affected has not been reported during colitis.Next,we explored the effect of macrophage polarization on the expression of OT and OTR in different periods of colitis.We assessed the expression of the OT and OTR in the two periods in DSS-treated mice.The mRNA expression of OT and OTR decreased significantly during the inflammatory periods and increased significantly during the repair periods.On day 7,the mRNA levels of OT and OTR decreased to the lowest level.On day 21 and 28,the mRNA levels of OT and OTR significantly increased.On day 49,the mRNA levels of OT and OTR had no significant difference compared with the control group.We believed that the expression of the OT/OTR and macrophage polarization in the colon were correlated in these two periods of DSS-induced colitis.9.The effect of D-mannose on macrophage polarization and expression of OT/OTRPrevious studies showed that macrophage polarization was closely related to OT and OTR expression.It was proven that D-mannose inhibited LPS-induced macrophage activation by impairing IL-1β production.To further tested the hypothesis that M2 macrophages increased OT and OTR expression in LMMP,the mice were fed D-mannose for 7 days to activate macrophages,further observation of the expression of OT and OTR.Compared with the control group,after 7 days of D-mannose feeding,the mRNA expression of IL-6,IL-1β and TNF-α did not change,but the iNOS mRNA expression in LMMP was decreased.Surprisingly,the mRNA expression of surface markers of M2 macrophages,including CD206,Argl,Ym1 and Chil3 significantly increased.The mRNA levels of antiinflammatory factors TGF-β and IL-10 also significantly increased.The above results showed that D-mannose promoted the polarization of M2 macrophages and inhibited the polarization of M1 macrophages.Consistent with the change in macrophage polarization,after the mice drank D-mannose,the expression levels of OT and OTR in LMMP significantly increased.10.Macrophage depletion inhibited the role of D-mannose on the expression OT and OTRTo further verify that the expression of OT and OTR was associated with macrophage polarization in D-mannose model,we depleted systemic macrophages by intraperitoneal injection of clodronate liposomes.After clodronate liposome pretreatment,the number of M2 macrophages(Iba1/Argl double-positive cells,CD206-positive cells)were significantly reduced.Pretreatment with clodronate liposomes significantly reversed the upregulation of OT and OTR expression induced by systemic administration of D-mannose.11.PEG3 was involved in the upregulation of OT and OTR expression induced by DmannosePrevious studies showed that PEG3 was involved in the upregulation of OT and OTR expression induced by M2 macrophages.So,we further verified the role of PEG3 in D-mannose promoting the expression of OT and OTR.7 days following D-mannose treatment,the expression of PEG3 was inhibited,whereas the expression of OT was increased.We found that the injection of SIS3 HCl significantly abolished the effect of D-mannose on the expression of PEG3,OT and OTR.In conclusion,D-mannose promoted the expression of OT and OTR by promoting the polarization of colonic macrophages to M2 type,activating the SMAD2/3 pathways and inhibiting the expression of PEG3.Summary:1.M1 macrophages inhibited the expression of OT and OTR in enteric neurons through pro-inflammatory factors(IL-6,IL-1β,TNF-α),which were achieved by activating STAT3 and NF-κB pathways.M2 macrophages promoted the expression of OT and OTR in enteric neurons via TGF-β,which was achieved by activating SMAD2/3 pathways.2.Colonic macrophages were polarized to the M1 type during the inflammatory periods,the expression of OT and OTR decreased,while in the repair periods,colonic macrophages were polarized to the M2 type,the expression of OT and OTR increased,which indicating that the polarization of macrophages was closely related to the expression of OT and OTR.3.D-Mannose promoted the expression of OT and OTR by inhibiting the activation of M1 macrophages and promoting the activation of M2 macrophages.M2 macrophages activated the SMAD2/3 pathways by secreting TGF-β,thereby inhibited the expression of PEG3,which promoted the expression of OT and OTR.4.This was the first time to prove that the polarization of macrophages differentially regulated the expression of OT and OTR in enteric neurons.We found that the expression of OT and OTR increased in the repair periods,which indicated that OT signalling system was involved in the repair of inflammatory tissue.We have found that OT inhibited the polarization of macrophages to M1 type and promoted the polarization of macrophages to M2 type,in this experiment,we also found that the activation of M1 macrophages led to the reduction of OT and OTR expression.Therefore,for inflammatory bowel disease(IBD)patients,OT treatment not only inhibited inflammation in the inflammatory periods,but also promoted tissue repair during the repair periods by promoting the polarization of M2 macrophages and the expression of the OT signalling system itself.OT might be a potential drug target,which provided new ideas for the treatment of inflammatory diseases. |
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