Oxytocin Regulates Dendritic Cells Function Via PI3K/AKT Signaling In The Intestine And Alleviates DSS-induced Colitis

Inflammatory bowel disease(IBD)includes ulcerative colitis(UC)and Crohn's disease(CD).The disease has a high incidence in Western countries and has developed rapidly in our country in the past decade.At present,the disease still lacks effective treatment.The disease is prone to recur,which causes a heavy burden on the patients mental and economic conditions.Although the pathological cause is still unclear,IBD is widely believed to be related to abnormal immune responses.Oxytocin(OXT)is a classic neuropeptide,mainly synthesized by the hypothalamus and released in the posterior pituitary gland.OXT via its receptor(Oxytocin Receptor,OXTR)plays an important role in mammalian childbirth,lactation,mother-infant connection and social communication.Ten years ago,our and others'laboratories confirmed that OXT and OXTR were expressed in enteric neurons,and played a regulatory role in visceral pain transmission.Recently,we found that OXT could alleviated DSS-induced colitis.On the contrary,colitis was more severe after systemic knockout of OXTR.However,the anti-inflammatory mechanism of OXT is not clear.In 2019,our team found that macrophages played an important role in OXT alleviating the development of colitis.Interestingly,we found that dendritic cells(DCs)also express OXTR.DCs are the most important antigen-presenting cells,which participant in the regulation of innate and adaptive immune responses,and can mediate inflammatory response or immune tolerance.In homeostasis,immature or semi-mature DCs present self-antigen to T cells,resulting in loss of T cells or inducing regulatory T cells(Treg),thereby mediating immune tolerance.Immature DCs begin to mature when expose to some stimuli,such as LPS,IL1? or TNF?.Mature DCs together with IL6,IL23,TNFa and other inflammatory factors prime CD4+T cells to Thl or Th17,which cause colon immune response,intestinal tissue damage and development of colitis.Here,we reveal for the first time that the OXT/OXTR signalling pathway mediated immune tolerance in DCs that participates in protection against colitis.This study may provide a new method and basis for the clinical therapy for IBD.Part ?Oxytocin regulates the Maturation of Dendritic Cells and the Underlying MechanismObjectives:Previous studies have demonstrated that OXT alleviates experimental colitis,and dendritic cells also play an important role in the occurrence and development of IBD.However,there are no research reports on the effect of OXT/OXTR on dendritic cells.Therefore,the purpose of this study is to explore whether the OXT/OXTR signalling system affects the function of dendritic cells and to explore its probable mechanism.Methods:1.Bone marrow-derived dendritic cells were induced by GM-CSF and IL-4.2.Lamina propria mononuclear cells(LPMCs)were separated by percoll density gradient centrifugation,and lamina propria dendritic cells(LPDCs)were obtained by CD11c+ magnetic bead sorting from LPMCs.3.The expression of OXTR on dendritic cells was detected by immunofluorescence and western blotting.4.CD 11c-Cre mice and OXTR-flox mice were used to generate specifically knocked-out OXTR on CD 11c+cells(OXTR?DC mice).5.BMDCs stimulation method:collect induced BMDCs on the 7th day,add OXT or LPS respectively,or add LPS stimulation after OXT pre-incubation for 40 minutes.In experiments using inhibitors,incubate BMDCs with inhibitors for 20 minutes first.BMDCs was cultured in the 37? 5%CO2 incubator.6.The expression of extracellular molecules of BMDCs was detected by flow cytometry.7.Cytokines produced by BMDCs were detected by using qPCR and ELISA.8.The phagocytic ability of dendritic cells was detected by using FITC-dextran.9.FITC-labelled Lactobacillus and Salmonella were used to detect the ability of dendritic cells to adhere to bacteria.10.The BMDCs was co-cultured with CD4+naive T cells isolated from the mesenteric lymph nodes of mice with experimental colitis induced by DSS,and the differentiation of CD4+T cells was detected by flow cytometry.11.The phosphorylation levels of AKT and I?B signalling were detected by western blotting.Results:1.Bone marrow-derived dendritic cells were induced successfullyThe typical dendritic cell morphology was observed under the microscope,and non-adherent cells were collected and analyzed for the percentage of CD1 lc positive cells by flow cytometry(>90%)by flow cytometry.2.DCs expressed OXTRThe immunofluorescence and western blot results indicated that OXTR were expressed on the mice BMDCs,lamina propria DCs(LPDCs)from mice and human.OXTR?DC mice were successfully reproduction3.OXTR?DC mice were successfully generatedThe generation of OXTRfl/fl mice and OXTRfl/fl/CD11c-Cre mice(OXTR?DC mice)by using Cre/flox system.The expression of OXTR of DCs from OXTRfl/fl mice and OXTR?DC mice was detected by western blot and qPCR.OXTR expression was reduced in OXTRWDC LPDCs and BMDCs.4.OXT prevented the maturation of BMDCs4.1 OXT inhibited the expression of MHCII and costimulatory molecules on the surface of BMDCsThe expression of MHCII,CD80 and CD86 on BMDCs was increased stimulated with LPS(100 ng/ml),while pre-incubated with OXT(10-8M,40 min)inhibited the expression of these molecules on BMDCs stimulated by LPS.4.2 OXT promoted the phagocytic ability of BMDCsIncubation of OXT for 24h enhanced the phagocytosis of FITC-Dextran by BMDCs.Pre-incubation with OXT(10-8M,40 minutes)enhances the phagocytic ability of LPS-stimulated BMDCs.4.3 OXT inhibited the expressions of inflammatory cytokines in LPS-stimulated BMDCsThe expressions of TNF?,IL12,IL23 and IL6 increased after stimulating OXTRfl/fl BMDCs by LPS.OXT pre-treatment decreased the elevation of these cytokines of BMDCs.In contrast,OXT pre-treatment to OXTR?DC BMDCs cannot prevent the function of LPS.5.OXT inhibited T cell differentiation via its receptorThe naive CD4+T cells were obtained from mesenteric lymph nodes(MLN)from DSS-induced colitis mice by magnetic bead sorting.The sorted naive CD4+T cells were cocultured with BMDCs for 3 days(1000:1)and were detected by flow cytometry.OXTRfl/fl BMDCs and OXTR?DC BMDCs were stimulated by LPS with or without OXT pre-treated.OXT-pretreated OXTRfl/fl BMDCs but not OXTR?DC BMDCs markedly reduced the differentiation of Thl and Th17 cells.6.OXT affected the adhesion of BMDCs to different bacteriaOXT-incubated BMDCs promoted the adherence to the probiotic Lactobacillus but inhibited their adherence to the pathogenic bacteria Salmonella.7.OXT inhibited DCs maturation by activating PI3K/AKT pathway7.1 OXT activated PI3K/AKT pathwayThe phosphorylation of AKT increased after incubated with OXT in BMDCs,and was blocked by PI3K inhibitor(AS605240,Wortmannin and Ly294002),AKT inhibitor MK2206 and mTOR inhibitor Rapamycin.7.2 OXT inhibited I?B phosphorylationAfter stimulated with LPS,the phosphorylation of I?B increased,which was impaired by OXT.Notably,either MK2206 or wortmannin prevented the suppressive effect of OXT after LPS treatment.7.3 OXT inhibited the expression of MHCII and costimulatory molecules on BMDCs by activating PI3K/AKTAfter incubated with PI3K inhibitors(AS605240 and Wortmannin),AKT inhibitors(MK2206)and mTOR inhibitors(Rapamycin),OXT no longer inhibits LPS to stimulate BMDCs to express MHCII and CD86.Summary:1.DCs expressed OXTR2.OXT inhibited DCs maturation by activating PI3K/AKT pathway,leading to the downregulation of NF?B.3.OXT inhibited the ability of antigen presentation of DCs and inhibits the differentiation of CD4+T cells into Thl and Th 17.Part ?The Role of Dendritic Cells in the Experimental Colitis through Oxytocin SignallingObjectives:Our current study has demonstrated that OXT inhibits the maturation of DCs and the differentiation of T cells.To clarify whether the OXT signalling system in DCs plays a role in IBD,we used DSS-induced acute and chronic colitis mouse models to explore the function of the OXT signalling system in DCs in the development of IBD.Methods:1.The content of OXT in the plasma from IBD patients and normal people(health control,HC)was detected by ELISA.2.The expression of OXTR in LPDCs from DSS mice and normal mice was detected by qPCR.3.The expression of TNFa in LPDCs of DSS mice and normal mice was detected by qPCR.4.The mouse model of colitis was established by DSSWild mice with C57BL/6 background,OXTR?DC mice and the littermates OXTRfl/fl mice were used.For acute colitis,the OXTR?DC and OXTRfl/fl mice were administrated with 2%DSS in drinking water for 7 days and sacrificed.Mice from the OXTRfl/fl-water group and the OXTR?DC-water group drink normal water.Mice were induced with 2.5%DSS in drinking water for 7 days in the DCs therapy experiment.BMDCs(106 cells/mouse/day)or normal saline were injected via the tail vein on days 4 and 6.The DC(control mice were injected with BMDCs stimulated by LPS.The DC(OXT)mice were injected with BMDCs pre-treated with OXT andstimulated by LPS.For chronic colitis,mice were induced by three cycles of a five-day treatment with 3%DSS,1-5%DSS and 2%DSS respectively and followed with a seven-day's recovery on normal water drink.The level of inflammation was evaluated based on colon length,body weight change,disease activity index(DAI)and histologic score.The entire colon was quickly excised and washed by pre-cooled PBS.The proximal colons were used for Western Blotting and the middle part was kept in RNA fixer(Aidlab,Beijing,China)for RNA isolation.The distal parts were fixed in 4%paraformaldehyde overnight,embedded in paraffin,sectioned,and stained by H&E(Hematoxylin-eosin staining).5.The changes of LPDCs and T cells in LPMC from DSS mice colon5.1.The changes of LPDCs from DSS mice colonLPMCs were seperated by percoll density gradient centrifugation from colon of DSS-induced OXTR?DC and OXTRfl/fl mice.The LPMCs were labelled with MHCII-FITC,CD64-PE,CD103-Percp/Cy5.5,CD11c-APC,CD45-APC/Fire750,CD11b-Pacific Blue to identify CD103+CD11b+,CD103+CD11b-and CD103-CD11b+LPDCs subsets.5.2.The changes of CD4+T cells from DSS mice colonLPMCs were separated by percoll density gradient centrifugation from the colon of DSS-induced OXTR?DC and OXTRfl/fl mice.The LPMCs were labelled with CD4-FITC,Foxp3-PE,IFNy-Percp/Cy5.5 and IL17A-APC to identify CD4+Foxp3+Treg,CD4+IFN ?+Thl and CD4+IL17A+Th17.6.The cytokines and related transcriptional factors(TF)from DSS mice colon were detected by qPCR6.1.IL6,IL23a and TGF? from DSS mice colon were detected by qPCR6.2.Th1 related TF T-bet,Th2 related TF GATA3,Th17 related TF RORyt and Treg related TF Foxp3.Results:1.The content of OXT in the plasma from IBD patients was higher than the healthy control.2.The expression of OXTR was lower in LPDCs from DSS-induced colitis mice than the healthy mice.3.The expression of TNF? was higher in LPDCs from DSS-induced colitis mice than the healthy mice.4.OXTR?DC mice are more sensitive to DSS-induced acute colitis compared with OXTRfl/fl miceThere was no significant change in body weight change and colon length from mice of OXTRfl/fl-water group and OXTR?DC-water group.OXTR?DC-DSS mice had more serious weight loss,shorter colon,higher DAI and histological score than OXTRfl/fl-DSS mice.The expressions of 116 and 1123 mRNA were higher and Tgf? mRNA was lower in colon tissue from OXTR?DC-DSS mice than OXTRfl/fl-DSS mice.5.The frequency of LPDCs and CD4+T cells from DSS-induced acute colitis OXTR?DC mice were changed5.1 The frequency of LPDCs from DSS-induced acute colitis OXTR?DC mice was changedThe LPDCs were classified by CD11b and CD103.OXTR?DC mice exhibited a much higher ratio of the CD 103+CD11b+DC subpopulation than OXTRfl/fl mice,while the ratio of CD103+CD11b-DC or CD103-CD11b+DC showed little difference.5.2 The frequencies of CD4+T cells from DSS-induced acute colitis OXTR?DC mice were changedThe frequencies of CD4+IFN ?+Thl and CD4+IL17A+Th17 cells in the colon were high in OXTR?DC mice compared to OXTRfl/fl mice detected by flow cytometry.The expression of T-bet and Roryt but not Foxp3 or Gata3 was markedly increased in the colon tissue of OXTR?DC mice compared with WT mice.6.OXTR?DC mice are more sensitive to DSS-induced chronic colitis compared with OXTRfl/fl miceIn the chronic colitis model,the body weight of OXTRfl/fl mice fluctuated with whether they drink DSS and the weight recovered to the initial stage at the end of the experiment.In contrast,although the weight of OXTR?DC mice also fluctuated,they recovered much slowly after drinking normal water,and the body weight was still lower than the initial stage at the end of the experiment.OXTR?DC mice have shorter colons,higher DAI scores and histological scores than OXTRfl/fl mice.The mRNA level of pro-inflammatory factors IL6 and IL23 increased,while the expression of anti-inflammatory factor TGF? decreased.7.CD4+T cells in the colon of chronic colitis OXTR?DC mice tend to differentiate towards Th 17Compared with OXTRfl/fl mice,the mRNA expression of Th17-related transcription factor ROR?t in the intestine of OXTR?DC mice was high,while the expression of other transcription factors did not change significantly.8.Injection of BMDCs incubated with OXT alleviated DSS-induced acute colitisDC(OXT)treatment but not DC(control)treatment protected against weight loss,reduced colon lengths,high DAI scores and histological damage in the colon compared with those of the control group.Summary:1.OXT system was abnormally elevated in the plasma of IBD patients.2.In enteritis,OXT signalling in DC affects the proportion of DC subsets and CD4+T cells differentiation.When OXTR was knocked out in DCs(OXTR?DC),DSS-induced colitis was aggravated,and the proportion of intestinal mucosal CD103+CD11b+DC subsets is higher,and CD4+T tends to differentiate into Th1 and Th17 cells.3.Injection of BMDCs incubated with OXT relieved acute colitis induced by DSS may be a new method for clinical treatment of IBD.Conclusion and innovation:1.We have demonstrated that dendritic cells express oxytocin receptors for the first time.2.OXT inhibited the maturation of DCs by down-regulating NF?B activity via activating the AKT.3.The endogenous OXT signalling system played a protective role in DSS-induced acute chronic colitis by regulating the function of DCs.4.OXT system was abnormal in IBD patients and DSS-induced colitis mice.5.Injection of BMDCs incubated with OXT relieved acute colitis induced by DSS may provide a new method for clinical treatment of IBD.6.The results above showed that the neuropeptide OXT participated in immune regulation and provided new evidence for neuro-immune communication.