Effect Of Oxytocin On Macrophage Polarization In Alleviating Experimental Colitis And Underlying Mechanism

Inflammatory bowel disease(IBD)is a world-wide chronic intestinal inflammation,including two major subtypes,Crohn’s disease and ulcerative colitis.The global incidence of IBD has increased over the past few decades,especially in developing countries.The pathogenesis of IBD is still unclear.It might be a result of the uncontrolled immune-medicated inflammatory response combined with unknown environmental triggers,intestinal flora imbalance and specific genetic factors.It has become an enormous financial burden on the patients and governments because of its prolonged healing and repeated recurrence.Innate immunity especially the intestinal monocyte-macrophage has been reported to play a pivotal role in IBD.The intestinal macrophages are derived from the bone marrow monocytes and polarize into two major subsets depending on the local microenvironment.Namely classically activated macrophages and alternatively activated macrophages,also known as Ml-type and M2-type macrophages.The Ml-type macrophages are sensitive to bacterial endotoxin and produce significant numbers of proinflammatory cytokines and chemokines,like IL-1β,IL-6,TNF-α,and IL-23,recruiting inflammatory cells and exacerbating the inflammatory response and tissue injury.M2-type macrophages exhibit tolerogenic properties,producing less pro-inflammatory cytokines but more IL-10 and TGF-β,participating in tissue restitution after injury.Numerous studies have found that IBD patients have abnormal polarization of intestinal macrophages.Regulation of the balance between M1 and M2 macrophage subsets might become an intriguing target in the treatment of IBDOxytocin is traditionally recognized as a hormone produced by the supraoptic nucleus and paraventricular nucleus of the hypothalamus and released from the posterior pituitary into circulation.In mammals,it is involved in reproductive functions like uterine contraction and milk ejection.Our previous study found that OT,as an endogenous neuropeptide of the enteric nervous system(ENS),was involved in the regulation of the secretion,movement,immunity of the digestive tract.OT receptors(OTR)were expressed in immune cells and participated in immune homeostasis.Our previous studies indicated that oxytocin administration protected the intestine from DSS colitis in mice,although the mechanisms were still unclear.Because of the import roles of macrophages in the development,progression and remission of IBD,in this study,we further explored the effect and mechanisms of oxytocin on the regulation of macrophage polarization and IBD,providing new targets for the treatment of IBD.Part ⅠEffect of Oxytocin on the Polarization of Macrophages and the Underlying MechanismObjectives:It was reported that oxytocin decreased TNF-α mRNA expression in bone marrow-derived macrophages induced by Lipopolysaccharide(LPS),but there were few studies about the effects of oxytocin on M2 macrophages.The aim of this study was to explore the regulatory effect of OT on macrophage polarization in mice and its underlying mechanism.Methods:1.The expressions of OTR on the membranes from different source of macrophages were measured by immunofluorescence.2.The expression levels of M1 or M2 type markers were measured by qRT-PCR and ELISA.3.The phosphorylation levels of NF-κB and STAT6 signaling were measured by western blot.4.The expression of β-arrestin2 was decreased by RNA interference.5.Human colonic specimens were stimulated by LPS and human IFN-y for 5 h with or without 40-min oxytocin preincubation and then LPMCs were isolated from each group.The transcription of IL-6,iNOS,TNF-α and CD206 were measured by qRT-PCR.Results:1.Macrophages had the expression of oxytocin receptors Immunofluorescence indicated that oxytocin receptors were expressed on the membrane of Raw264.7 macrophage cell line,mice bone marrow-derived macrophages induced by MCSF for 7 days,and mice peritoneal macrophage.2.Oxytocin inhibited proinflammatory mediators release in LPS-activated macrophagesThe effect of OT on M1-type polarization induced by LPS was observed by subcultured and primary macrophages.2.1 Oxytocin inhibited proinflammatory mediators release in LPS-activated RAW264.7 macrophagesFirstly,we used LPS to promote an M1 phenotype and explored the role of oxytocin on macrophages polarization.We found that LPS promoted macrophages to M1 polarization and oxytocin suppressed the transcription of inflammatory cytokines in LPS-stimulated RAW264.7 cells,like IL-1β,TNF-α,iNOS,and chemokines CCL2,CXCL10.Oxytocin(10-6 M to 10-9 M)lessened IL-6 transcription induced by LPS in a concentration-independent manner.Consistently,the LPS-induced expressions of NO,TNF-α,and IL-6 were also significantly restrained by pretreatment of oxytocin.These effects were reversed by atosiban,the OTR antagonist.2.2 Oxytocin inhibited proinflammatory mediators release in LPS-activated peritoneal macrophagesFor OTRmyel-WT peritoneal macrophages,LPS enhanced the expressions of inflammatory cytokines,like IL-1β,IL-6 and TNF-α.Oxytocin preincubation depressed the elevation of inflammatory cytokines induced by LPS.3.Oxytocin inhibited LPS-induced macrophages via its receptorsAtosiban is not entirely selective to OTR,so we established myeloid cell-specific deletion(OTRmyel-KO)mice to investigate the function of OT-OTR system on M1-type polarization exclusively3.1 Oxytocin receptors were deleted on peritoneal macrophages from OTRmyel-KO miceThe expression of OTR on OTRinyel-KO peritoneal macrophages were knocked down successfully compared to their wild littermates(OTRinyel-WT).3.2 Oxytocin displayed the anti-inflammatory function in LPS induced macrophages via its receptorsFor OTRmyel-WT peritoneal macrophages,LPS enhanced the expressions of inflammatory cytokines,like IL-1β,IL-6 and TNF-α.Oxytocin preincubation depressed the elevation of inflammatory cytokines induced by LPS.4.Oxytocin promoted the M2 macrophages polarization in coordination with IL-4The effect of OT on M2-type polarization induced by IL-4 was observed by subcultured and primary peritoneal macrophages.4.1 Oxytocin increased the RAW264.7 macrophages sensitivity to IL-4 stimulation in vitroWe used IL-4 to promote an M2 polarization and found that in cell line RAW264.7,oxytocin preincubation increased the sensitivity of macrophages to IL-4 stimulation with raised expressions of CD206 and Argl.4.2 Oxytocin increased the peritoneal macrophages sensitivity to IL-4 stimulation via its receptorsIn OXTRmyel-WT peritoneal macrophages,oxytocin preincubation increased the expression of CD206,Argl and Chil3 induced by IL-4.However,in OXTRmyel-KO peritoneal macrophages,oxytocin did not change the sensitivity to IL-4 stimulation.Oxytocin system is involved in promoting M2-type polarization.5.Oxytocin on the Human-derived macrophages 5.1 Oxytocin inhibited proinflammatory mediators release in LPS-activated THP-1 derived macrophages and promoted the M2macrophages polarization in coordination with IL-4LPS and human IFN-γ promote an M1 polarization with up-regulated transcription of inflammatory markers,like CCL2,TNF-α,CD86 and this effect was restrained by the pretreatment of oxytocin.Oxytocin preincubation enhanced the expression of CD206,F13A1 and PTGS2 induced by IL-4.5.2 Oxytocin ameliorated inflammation by inducing macrophages to a more anti-inflammatory phenotype Oxytocin effectively inhibited the expression of inflammatory cytokine induced by LPS and IFN-y in LPMC,such as IL-6,iNOS,and TNF-α.Besides,oxytocin also enhanced the transcription of CD206.6.Oxytocin modulated the polarization of macrophages through NF-κB and STAT6 pathway6.1 OT-OTR signaling inhibited NF-κB phosphorylationAfter stimulated by LPS for 1 hour,the degree of p65 phosphorylation in peritoneal macrophages was higher than that of the control group.Oxytocin preincubation blocked the activation of p65 induced by LPS partly.6.2 OT-OTR signaling promoted STAT6 phosphorylation Pretreatment of IL-4 enhanced the phosphorylation of STAT6 when peritoneal macrophages were treated with IL-4 and oxytocin potentiated this effect.7.Oxytocin modulated the polarization of macrophages through β-arrestin2We further studied the effect of β-arrestin2 on the polarization of macrophages and its interaction with NF-κB and STAT6 signaling.7.1 Oxytocin modulated the polarization of macrophages through OXTR-β-arrestin2 pathwayThe transcription and expression of β-arrestin2 both decreased after LPS treatment.Oxytocin preincubation abrogated the suppression on β-arrestin2 induced by LPS.IL-4 enhanced the expression of p-arrestin2,and oxytocin potentiated the effect of IL-4.These data indicate that β-arrestin2 might mediate the regulatory effect of OTR on macrophage polarization.7-2 β-arrestin2 of RAW264.7 cells were knocked down by RNA interferenceWe used RNA interference to knock down the expression of β-arrestin2 in RAW264.7 cells.After infected with β-arrestin2 shRNA(shp-arr2)lentivirus,the expression of β-arrestin2 in RAW264.7 cells significantly decreased compared with those infected with negative control lentivirus.7.3 Oxytocin modulated the polarization of macrophages via β-arrestin2After shβ-arr2 lentivirus interference,LPS could still induce the expression of inflammatory cytokines,like IL-1β,IL-6 and TNF-α mRNA in RAW264.7 cells,but oxytocin pretreatment did not influence the effect of LPS.Similarly,shβ-arr2 lentivirus infection did not influence the upregulation of IL-4 on the expression of Argl and CD206 but abrogated the potentiation of oxytocin.7.4 Oxytocin modulated NF-κB and STAT6 signaling via β-arrestin2In shβ-arr2 lentivirus infected cells,LPS could still increase the phosphorylation of p65,but oxytocin lost the inhibitory effect.At the meanwhile,when β-arrestin2 was knocked down,oxytocin did not enhance the phosphorylation of STAT6 induced by IL-4 anymore.Conclusions:OT-OTR system participated in the modulation of macrophage polarization mainly by enhancing the expression of p-arrestin2,leading to the downregulation of NF-κB and upregulation of STAT6 signaling.Part ⅡEffect of Macrophagic Oxytocin Receptors on the Experimental ColitisObjectives:Our current study demonstrated that in vitro,oxytocin reduced LPS-induced M1 polarization with impaired expression of inflammatory cytokines and chemokines,but promoted IL-4-induced M2 polarization with a significant increase of expressions of M2 hallmarks.If oxytocin and its receptor could alleviate the progression of IBD by modulating the macrophage polarization is still unclear.Hence,we established DSS-induced OTRmyel-WT and OTRmyel-KO mice to explore the role of macrophagic oxytocin system in the progression of experimental colitis.Methods:Colitis was induced by administration of 2.5%DSS in OTRmyel-WT and OTRmyel-KO mice for 7 days.The degree of inflammation was assessed based on body weight loss,colon length,DAI,colonic mucosal permeability,and histologic score;LPMCs was isolated by Percoll gradient centrifugation and the hallmarks of Ml-or M2-like macrophages were measured by qRT-PCR;Immunohistochemistry was used to label submucosal F4/80+macrophages and CD206+M2-like macrophages;The level of p65 and phospho-p65 were measured by western blot.Results:1.OTRmyel-KO mice were more susceptible to DSS-induced acute colitisCompared with OXTRmyel-WT mice,the OXTRmyel-KO mice had more weight loss,shorter colon,greater macromolecular permeability,higher scores of DAI and histological assessment.1.1 The body weight change and colon lengthThe control mice had no significant body weight change and colon shorten.Mice treated with 2.5%DSS had more weight loss and shorter colon.Compared with OXTRmyel-WT mice,the OXTRmyel-KO mice had more weight loss and shorter colon.1.2 The disease activity index and mucosal permeabilityMice treated with 2.5%DSS had higher disease activity index and greater FITC fluorescence intensity in serum than the control groups.Compared with OXTRmyel-WT mice,the OXTRmyel-KO mice had higher disease activity index and greater FITC fluorescence intensity suggesting that OXTRmyel-KO mice had more severe mucosal damage and greater macromolecular permeability.1.3 The histological assessmentNo obvious histological abnormality was found in control groups.DSS-induced mice had varying degrees of tissue damage,glandular structure disorders,inflammatory cell infiltration.OXTRmyel-KO mice showed more severe epithelial destruction,crypt loss,submucosal edema and inflammatory cell infiltration in the colon than those of their wild littermates.The histological index of colitis in OXTRmyel’KO mice was also significantly higher than that of OXTRmyel-WT mice.2.The colonic macrophages from DSS-induced OXTRmyel-KO mice tended to polarize to M1-like macrophages2.1 Increased M1-like macrophages in the LPMCs from OXTRmyel-KO mice treated by DSSThe expression of IL-1β,IL-6 and iNOS mRNA were distinctly higher in OXTRmyel-KO than those in OXTRmyel-WT mice after the DSS administration.2.2 Decreased M2-like macrophages in the LPMCs from OXTRmyel-KO mice treated by DSSThe levels of CD206 and Argl,hallmarks of M2 macrophages,were apparently lower in the LPMCs from OXTRmyel-KO mice after the DSS administration.2.3 M2-type macrophages infiltrating into colonic submucosal tissue during colitis detected by immunohistochemistryThe numbers and staining intensity of F4/80-positive cells were significantly higher in the OXTRmyel-KO mice than those in OXTRmyel-WT mice after DSS treatment.The OXTRmyel-KO mice had lower CD206+/F4/80+ratio.3.OTRmyel-KO mice were more susceptible to DSS-induced acute colitis.3.1 The body weight change,colon length and DAI of control and chronic DSS-induced miceCompared with OXTRmyel-MT mice,the body weight of OXTRmyel-KO mice fluctuated more obviously,and the growth rate increased less.The OXTRmyel-KO mice had shorter colon lengths higher disease activity index after 41-day DSS induction.3.2 Increased Ml-like macrophages in the LPMCs from OXTRmyel-KO mice treated by chronic DSSCompared with OXTRmyel-WT mice,LPMCs from OXTRmyel-KO mice had higher expression of IL-1β,IL-6,TNF-α and lower expression of CD206.4.Activated NF-κB signaling in the colon of OXTRmyel--KO mice treated by DSSLevels of phosphorylation of p65 in DSS-induced OXTRmyel-KO and OXTRmyel-WT colonic tissue were measured by western blot.The expression of phosphorylated p65 in OXTRmyel--KO mice was higher than that in OXTRmyel-WT mice.Conclusions:Compared with OXTRmyel-WT mice,the OXTRmyel-KO mice were more susceptible to DSS-induced colitis.The colonic macrophages in OTR conditioned knockout mice stimulated by DSS exhibited enhanced proinflammatory properties with higher levels of IL-1β,IL-6 and iNOS,as well as lower levels of CD206 and Argl.The anti-inflammatory effect of oxytocin functioned mainly by inhibiting NF-κB signaling.