Molecular Mechanism Of PAD2 Regulation Of MO-MDSCs Differentiation And Its Role In Collagen-induced Arthritis Mice

The citrullination catalyzed by peptidylarginine deiminase(PAD)plays an important role in the pathogenesis of rheumatoid arthritis(RA).We aimed to detect the expression of PAD and citrullinated proteins in myeloid-derived suppressor cells(MDSCs)in the mouse model of collagen-induced arthritis(CIA).Then we investigated the molecular mechanism of PAD2 in regulating the function or differentiation of MDSCs and its clinical application value.Methods(1)Detection of PAD and citrullinated proteins in MDSCs and their subgroupsWe separated MDSCs and their subgroups from CIA mouse spleen by magnetic beads.MDSCs were identified by flow cytometry(FCM).Expression of PAD in MDSCs and their subgroups were detected by real-time fluorescence quantitative PCR(qRT-PCR),Western Blot and fluorescent antibody technics.Citrullinated proteins in MDSCs and their subgroups were detected by Western Blot.(2)Effect of PAD on the function of MDSCsWe separated CD4~+T cells by magnetic beads and stained them with CFSE.In the presence of anti-CD3 antibody and anti-CD28 antibody,CD4~+T cell proliferation system was established in vitro.The MDSCs subgroups from CIA mice were isolated by magnetic beads to detect the expression of effector molecules including Arg-1 and NO,surface molecules(CD40,CD80,CD86,MHC-?),and their inhibitory ability on CD4~+T cell proliferation during the course of disease(18,35,42 days after the first immunization).The expression of PAD2 and PAD4 were down-regulated by small interfering RNA(siRNA)before the effector molecules of MDSCs and their inhibitory ability were detected.(3)Effect of PAD on the differentiation of MDSCsWe examined the proportion and number of MDSCs during the course of CIA mice by FCM and cell hemocytometer.By transfection with siRNA of PAD,we detected the effects of PAD on the differentiation from bone marrow cells(BMCs)to MDSCs,monocytic MDSCs(MO-MDSCs)to macrophages(M?)and dendritic cells(DCs)in vitro.We also explored the effect of PAD2 on the differentiation of MO-MDSCs by adoptively transferring CFSE-stained MO-MDSCs to CIA mice.(4)To screen the substrate of PAD2-catalyzed citrullinationThe expression of the citrullinated protein in the cytoplasm and the nuclear in the MO-MDSC weas detected by Western Blot after inhibiting of PAD2,which can determine the position of the reaction catalyzed by PAD2.The proteins from the nucleus of MO-MDSCs were detected by mass spectrometry(MS).After transfection with siRNA of PAD2,Western Blot was used to detect the citrullinated proteins of candidate substrates.IP experiment was performed to verify whether PAD2 could bind to candidate substrates.Citrullination in vitro was used to confirm that whether PAD2 specially catalyzed H4R3 into H4Cit3.(5)To screen the target gene regulated by citrullination of H4R3After inhibiting of PAD2,RNA-seq of MO-MDSCs was performed to observe the change of mRNA.The percentages of M?or DCs differentiated from MO-MDSCs were detected after inhibiting of candidate gene.We determined the connection of H4Cit3 and the transcription initiation site of GMPR by CHIP assay.(6)Effect of purine metabolites on the differentiation of MO-MDSCsAfter inhibiting of PAD2 in d42-MO-MDSCs,the levels of GMP and key metabolic enzymes in MO-MDSCs were detected.The GMPR gene carried by the vector was transferred into MO-MDSCs by liposomes.The ratio of M?and DCs differentiated from MO-MDSC were detected by FCM under the treatment of exogenous guanosine.(7)To observe the effect of PAD2-catalysed citrullination in CIA miceMO-MDSCs were treated with siRNA-PAD2,siRNA-GMPR and PAD2-specific inhibitor AFM-30A before adoptive transferred into CIA mice.The effects of MO-MDSCs on CIA mice were evaluated by joint clinic score,analysis of joint section,serum IgG,anti-type?collagen IgG,IFN-?and IL-17 levels.(8)Effect of PAD2-mediated citrullination on differentiation of human MO-MDSCsWe isolated MO-MDSCs from peripheral blood mononuclear cells(PBMCs)-induced MDSCs by FCM.The effect of PAD2 or GMPR on the differentiation of MO-MDSCs into M?or DCs was detected by FCM after transfection with siRNA of PAD2 or GMPR.We analyzed the relationship between PAD2,H4Cit3,GMPR in MO-MDSCs and the pathological indexes of RA patients.The pathological indexes including erythrocyte sedimentation rate(ESR),C-reactive protein(CRP),rheumatoid factors(RF),anti-cyclic peptide containing citrulline(anti-CCP)and disease activity scores of 28 joints(DAS28)in RA patients.Results(1)The purity of MDSCs and their subgroups were higher than 90%.Both polymorphonuclear MDSCs(PMN-MDSCs)and MO-MDSCs expressed PAD2 and PAD4.Confocal microscopy showed that PAD2 and PAD4 were expressed in the nucleus.Both PMN-MDSCs and MO-MDSCs expressed citrullinated protein.(2)The CD4~+T cell proliferation system was interfered with d28-MDSCs,d35-MDSCs and d42-MDSCs,there was no significant change in the immunosuppressive ability of PMN-MDSCs.The CD4~+T cell proliferation system was interfered with d28-MO-MDSCs,d35-MO-MDSCs and d42-MO-MDSCs,respectively.The proportion of CD4~+T cell proliferation in the D35-MO-MDSCs treatment group was higher than that in the d28-MO-MDSCs treatment group(P<0.05).The proportion of CD4~+T cell proliferation in d42-MO-MDSCs treatment group was higher than that in d35-MO-MDSCs treatment group(P<0.05),indicating that the inhibitory ability of MO-MDSCs on CD4~+T cell proliferation was reduced(P<0.05).The levels of CD40,CD80,CD86 and MHC-?in MO-MDSCs were increased from d28 to d42.After inhibiting of PAD2 in MO-MDSCs,the immunosuppressive ability of MO-MDSCs increased,the levels of NO and Arg-1 in MO-MDSCs increased(P<0.05 or P<0.01).These results suggested that inhibiting the expression of PAD2 in MO-MDSCs enhanced the immunosuppressive function of MO-MDSCs.(3)The proportion and number of MDSCs and PMN-MDSCs in CIA mice were reduced(P<0.05 or P<0.01),the proportion and quantity of MO-MDSCs did not change significantly.There was no significant change in the proportion of MDSCs differentiated from BMCs after inhibiting of PAD2 or PAD4.The differentiation of MO-MDSCs into M?or DCs decreased after inhibiting of PAD2,(P<0.05 or P<0.01).The percentages of monocytes and DCS differentiated from MO-MDSCs were decreased in CIA mice after inhibiting of PAD2(P<0.01).(4)After inhibiting of PAD2,the citrullinated protein in the nucleus of MO-MDSCs was decreased,but no significant change was observed in the cytoplasm.The results of MS showed H4Cit3 in MO-MDSCs declined mostly.Moreover,inhibition of PAD2 significantly reduced the level of H4Cit3 in MO-MDSCs.IP experiment indicated that PAD2 could bind to H4Cit3.PAD2 could specifically catalyze H4R3 to H4Cit3.(5)Transcriptome-sequencing showed that the expression of GMPR in MO-MDSCs decreased significantly after inhibiting of PAD2.The inhibition of GMPR reduced the differentiation of MO-MDSCs into M?or DCs(P<0.05 or P<0.01).The results of CHIP assay showed that H4Cit3 could bind to the transcription initiation site(448?579 bp)of GMPR.(6)After inhibiting of PAD2,the expression of GMPR in d42-MO-MDSCs decreased,while the guanosine monophosphate(GMP)increased.With the increasing GMPR,the proportions of M?or DCs differentiated from MO-MDSCs were increased(P<0.05 or P<0.01),exogenous guanosine reduced the differentiation of MO-MDSCs into M?or DCs(P<0.01).(7)PAD2 and GMPR could be inhibited by siRNA or AFM-30a.Adoptive transfer of PAD2 or GMPR inhibited MO-MDSCs could reduce the clinical score,degree of joint destruction,serum IgG,anti-collagen type?IgG,IFN-?and IL-17 in CIA mice.(8)In response to inhibition of PAD2 or GMPR expression in human MO-MDSCs(h-MO-MDSCs),the percentages of M?and DCs decreased(P<0.05 or P<0.01).Compared with healthy volunteers and OA patients,PAD2,H4Cit3 and GMPR in MO-MDSCs were increased in RA patients(P<0.05 or P<0.01),they were positively correlated with DAS28(P<0.05).ConclusionsHistone H4R3 can be citrullinated by PAD2 in MO-MDSCs from CIA mice.H4Cit3 can promote the transcription of GMPR and contribute to the differentiation of MO-MDSCs into M?or DCs.After inhibiting of PAD2 or GMPR in MO-MDSCs,adoptive transfer of MO-MDSCs could alleviate the disease in CIA mice.The PAD2,H4Cit3 and GMPR in MO-MDSCs are expected to be indicators for the assessment of disease activity of RA.