Mechanisms Of Myeloid-derived Suppressor Cells Promoting Th17 Differentiation Through Polyamine And Its Correlation With Disease Progression Of Systemic Lupus Erythematosus

BackgroundMyeloid-derived suppressor cells(MDSC)are a group of immature myeloid cells that play an important role in tumor,inflammation,infection and autoimmune diseases.In recent years,MDSC and T helper cell 17(Th17)are widely studied for they often coexist in a certain pathological condition.Our previous experiments showed that MDSC promoted Th17 differentiation and aggravated disease progression by producing arginase-1(Arg-1)to decompose arginine(Arg)in systemic lupus erythematosus(SLE)patients and mice.However,the exactly metabolites promote Th17 differentiation after Arg decomposition is still unclear.ObjectiveIn this study,we used in vitro induction,clinical SLE blood samples and humanized mice to research the relationship between downstream metabolites of MDSC and Th17 differentiation.We intended to prove the immunometabolic pathway and specific mechanism of MDSC promoting Th17 differentiation and aggravating SLE disease progression.Methods1.Prostate cancer cell line RM-1 was subcutaneously inoculated in specific pathogen free(SPF)C57BL/6 male mice.Magnetic beads were used to separate WT mice na?ve CD4+T cells,and flow cytometry(FCM)was used to separate MDSC from tumor bearing mice.Human Cord blood mononuclear cells(CBMC)were collected,magnetic beads were used to separate na?ve CD4+T cells,and FCM was used to separate MDSC.Under mice or human Th17 differentiation condition,the cells were divided into 5 groups:na?ve CD4~+T cells culture alone,+ORN,+MDSC,+MDSC+DFMO and+DFMO co-culture groups.Proportion of CD4+IL-17A+cells and CD4~+RORγt~+cells was detected by FCM,concentration of IL-17A in the culture supernatant was detected by enzyme linked immunosorbent assay(ELISA)and mRNA levels of IL-17a and RORγt were detected by quantitative real-time PCR(qPCR).2.Under human Th17 differentiation condition,cells were divided into three groups:control,co-culture group with MDSC,with MDSC and nor-NOHA(Arg-1 inhibitor)groups.RNA chip was performed to detect the differentially expressed miRNAs(miR-542-5p).miR-542-5p expression were verified by qPCR in previously purefied RNAs.After miR-542-5p overexpression or silence,human na?ve CD4~+T cells were polarized to Th17,proportion of CD4~+IL-17A~+cells and CD4~+RORγt~+cells were detected by FCM,mRNA expression level of IL-17A and RORγt was detected by qPCR.As an important pathway for Th17 differentiation,we used bioinformatics anaylysis to find the target genes of miR-542-5p in TGF-β/SMAD signaling.Luciferase assay was performed to detect the interaction between miR-542-5p and target genes.Then,overexpression or silence miR-542-5p in na?ve CD4+T cells,with or without Th17 differentiation,SMAD3,SMAD7 and SMURF1 RNA levels were detected by qPCR,expression levels of SMURF1,SMAD2/3 and pSMAD2/3 were detected by FCM.3.Expression levels of polyamine synthetase and miR-542-5p in PBMC of SLE patients were detected and correlation with SLE disease activity index(SLEDAI)was performed.4.Humanized SLE mice were constructed by transferring PBMC from SLE patients to NOD/SCIDIL-2Rγ~nullull mice.One week later,control and miR-542-5p were transfected in vivo.4～5 weeks later,proportion of CD4~+IL-17A~+cells and CD4~+RORγt~+cells in mice PBMC and spleen were detected by FCM.il-17a,rorγt and polyamine synthetases RNA expression in spleen and kidney were detected by qPCR.Results1.Mice and human na?ve CD4~+T cells were cultured under Th17 differentiation condition,MDSC or ORN both promoted Th17 differentiation examined by qPCR,FCM and ELISA;if added DFMO and MDSC at the same time,inhibiting the further decomposition of ORN(metabolite of MDSC),no promoting effect exhibited.2.3 polyamine synthetases,ODC1,SRM and SMS expression increased,while MDSC promoted na?ve CD4~+T cells differentiating to Th17 in both mice and human.3.In vitro,miR-542-5p expression was significantly higher in co-culture with MDSC group than control,while obviously lower in co-culture with MDSC plus nor-NOHA group than MDSC group under human Th17 differentiation condition.4.In vitro,miR-542-5p expression in ORN or MDSC co-culture group was significantly higher than control,whereas miR-542-5p expression in MDSC+DFMO group was significantly lower than MDSC group under human Th17 differentiation condition.If added 3 polyamines,miR-542-5p expression was significantly higher than control.5.Overexpression or silence miR-542-5p in human na?ve CD4~+T cells,then added human Th17 differentiation inducing system,IL-17A and RORγt change level were consistent with miR-542-5p.6.Overexpression of miR-542-5p in human na?ve CD4~+T cells,gene and protein level of SMURF1 were downregulated,activated SMAD2/3 phosphorylation and TGF-β/SMAD pathway was positively regulated in both na?ve CD4+T and Th17,while TGF-β/SMAD pathway was negatively regulated in miR-542-5p silencing.7.miR-542-5p expression was positively correlated with 3 polyamine synthetases in PBMC of SLE patients.8.miR-542-5p and 3 polyamine synthetases expression in PBMC of SLE patients were higher than healthy controls,also positively related to the severity of SLE patients.9.In humanized SLE mice,MDSC-deplete group showed a decline proportion of Th17 in both PBMC and spleen,while miR-542-5p and 3 polyamine synthetases mRNAs expression levels downregulated in PBMC,spleen and kidney.10.In humanized SLE mice,overexpression miR-542-5p reversed the proportion of Th17 in PBMC,spleen and kidney in MDSC-deplete group.ConclusionOur study reveals MDSC promotes Th17 differentiation through Arg-1/polyamine/miR-542-5p/TGF-βpathway.Our data highlight the positive relationship between polyamine/miR-542-5p and SLE disease progression through Th17 induction in vitro,and humanized mice model in vivo.Our findings not only explain theoretically the relationship and mechanisms between MDSC increasing and aggravating in SLE patients,but also provides novel ideas and therapeutic targets for SLE.