Influence Of Myeloid-derived Suppressor Cells-derived Arginase-1 To Th17 Cells And Mechanism Of Quercetin Treatment In The Mouse Inflammatory Bowel Disease Model

ObjectiveInflammatory bowel disease(IBD)is a chronic and relapsing disorder that involves the whole intestinal system,and generally indicates ulcerative colitis(UC)and Crohn’s Disease(CD)in human.In IBD,interaction between multiple immune cells and Intestinal microorganism lead to IBD progression.In recent years,the key role of Th17 cells in IBD has become a research hotspot in the field.Thl7 cells are critical in the regulation of the immune response in IBD progression,which are the primary source of IL-17A and IL-17F.Our previous findings showed that myeloid-derived suppressor cells(MDSC)promotes Thl7 cell differentiation by secreting arginase-1(Arg-1),thereby exacerbating the progression of systemic lupus erythematosus.However,the relationship between MDSC and Thl7 cells in IBD remains unclear.In this study,mice with a conditional deletion of arg-1 in myeloid cells were used to construct IBD model to study the effect of MDSC on Thl7 cell differentiation and function.Our data highlight the role of MDSC-derived arg-1 in regulating IL-17A and IL-17F expression in the autoimmune diseases,investigated the mechanism of quercetin in IBD treatment,and provides further support for therapeutic approaches for IBDMethods(1)DSS was dissolved at a final concentration of 3.5%in water and gave to mice orally for 9 days or more days to establish an enteritis model.Weight loss,stool consistency and rectal bleeding were monitored daily to evaluate the disease activity index(DAI)of enteritis in mice(2)Before suppression assays,96-well plates were incubated with 2ug/ml anti-mouse CD3 purified antibody overnight in 4℃ conditions.Cells from lymph nodes of mice were stained with 2.5 μM of CFSE(eBioscience)for 15 min,and then incubated for 72h in the presence of lug/ml anti mouse CD28 purified antibody for 72h.To purify MDSC,CDllb+Gr-1+cells were isolated from the spleen of colitic mice according to the manufacturer’s instruction.Then MDSC were sorted(purity>95%)and co cultured with T cells for 72h.The Proliferation of T cells was determined based on CFSE dilution by flow cytometry(3)Single cell suspensions of peripheral blood mononuclear cells(PBMC),lamina propria mononuclear cells(LPMC)and spleen cells were generated as described previously.Peyer’s patches(PP)were carefully detached from the intestine to collect immune cells.MDSC,Thl7cells,Treg cells and γδ T cells were detected by flow cytometry.Colorectum of colitic mice was excised for hematoxylin and eosin stain(4)Level of Arg-1 in MDSC,IL-17A and IL-17F in Thl7 cells and γδ T cells were detected by flow cytometry.Expression of ACT1,IL-22,IL-23,OCLN,and COX-2 in the colorectum was detected by quantitative reverse transcription PCR.(5)MDSC were isolated from the spleen of colitic mice and suspended in PBS.2 ×106 WT-derived MDSC were transferred intravenously into colitic Argmye KO mice on days 0 and 2 after DSS administration.Mice were monitored daily for clinical signs of disease.Thl7cells and γδ T cells in PP and mLN were detected by flow cytometry(6)Quercetin was dissolved in dimethylsulfoxide upon receipt.Stock solution(lmmol/L)was stored as aliquots at-80℃ under sterile conditions.Mice were treated with quercetin(0.5 μM/g.diluted with PBS)was described previously.Briefly,mice were intraperitoneally injected with quercetin or vehicle(300μl per mouse)at day 0,3,5 and 7.From day 0,mice were treated with 3.5%DSS for 8-12 days,then sacrificed for further studies.Results(1)Weight loss,stool consistency and rectal bleeding were aggravated in Argmye KO mice during colitis.Whereas survival rate of Argmye KO mice decreased significantly when compared that with mice in WT group.(2)Colitic Argmye KO mice expressed a lower level of IL-17A in the colorectum;while IL-17F level was increased when compared that with mice in WT group.And Thl7 cells in PP of colitic Argmye KO mice expressed more IL-17F,other than IL-17A.(3)Percentage of MDSC in PBMC of colitic Argmye KO mice was lower than mice in WT group.Among this,the percentage of M-MDSC were significantly lower thanWT group,while there was no statistical significance between Argmye KO mice and WT mice in G-MDSC.In addition,the level of Arg-1,iNOS and IDO were lower in colitic Argmye KO mice.The expression of ACT1 and COX-2 were elevated in the colorectum of colitic Argmye KO mice,while the expression of IL-22,IL-23,and OCLN decreased in col orectum of colitic Argmye KO mice when compared that with mice in WT group.(4)Transfer of MDSC reversed weight loss of colitic Argmye KO mice.And colitic mice without transfer of MDSC had shorter colorectum in length.In addition,transfer of MDSC significantly elevated IL17A level in Thl7 cells in PP and mLN of colitic Argmye KO mice,while IL-17F level was reduced and eventually prolonged survival of colitic Argmye KO mice.(5)Quercetin treatment significantly alleviated colitis in mice,as evidenced by lower DAI scores,reduced body weight loss,and longer colon length.The MDSC population in PBMC and spleen of colitis mice was highly increased after quercetin treatment.G-MDSC and M-MDSC populations were also significantly expanded upon quercetin treatment.Finally,MDSC levels in LPMC were also increased after quercetin treatment.(6)Increased expression of both ESR1 and ESR2 in the colorectum suggested that ESR expressed by MDSC may recognize and bind to quercetin(Figure 7A).STAT3 has multiple biological activities involved in MDSC survival,proliferation,differentiation and apoptosis.Flow-cytometric analysis revealed that pSTAT3 expressed in MDSC from PBMC was increased upon quercetin treatment.G-MDSC,but not M-MDSC,more strongly expressed pSTAT3 in PBMC upon quercetin treatment.Activation of STAT3 enhanced Arg-1 synthesis in MDSC,and subpopulations of MDSC also secreted more Arg-1 in PBMC after quercetin treatment In addition,quercetin also induced elevated Arg-1 secretion in splenic MDSC,mainly G-MDSC(7)After quercetin treatment,IL-17A expression in the colorectum was elevated after quercetin treatment,whereas that of IL-17F was significantly reduced.We observed significant increases in CD4+IL-17A+Th17 cells in the PP and mLN respectively Intriguingly,IL-17F production by Thl7 cells was decreased in the mLN,but not in the PP.ACT1 expression in the colorectum was increased during colitis.Activation of ACT1 led to overexpression of IL-22,IL-23 and OCLN.Expression of the COX-2 in the colorectum during colitis was suppressed by quercetin treatmentConclusionsWe have demonstrated distinct roles of IL-17A and IL-17F in DSS-induced colitis mice model which was dependent on increased Arg-1 secret by MDSC after ESR/STAT3 activation.Administration of quercetin significantly enhanced accumulation of MDSC and Arg-1 secretion.And arg-1 elevated level of IL-17A and restricted IL-17F secretion in Thl7 cells,promoted expression of intestinal mucosal healing associated factors,thus alleviated colitis in mice.Our data highlight the role of MDSC-derived Arg-1 in modulating IL-17A and IL-17F secretd by Thl7 cells in IBD model,revealed the mechanism of quercetin in relieving colitis,revealed the mechanism of quercetin in relieving colitis and provides further support for therapeutic approaches for IBD.