| Part Ⅰ:High-dose dexamethasone corrects impaired myeloid-derived suppressor cell function via Etsl in immune thrombocytopeniaBackground:Myeloid-derived suppressor cells(MDSCs)are heterogeneous cells characterized by a myeloid origin,immature state,and remarkable ability to suppress T-cell responses,which expands during cancer,inflammation and infection.These cells circulate in the blood,and accumulate in the lymphoid tissue,bone marrow,and especially tumor sites.T-cell inactivation can be mediated by MDSCs through IFN-γ-dependent nitric oxide production or the Th2-mediated IL-4/IL-13-dependent Arginase-1(Arg-1)pathway.L-arginine deprivation results in repressed expression of the T cell-signaling molecule,CD3ζ,and the arrest of T-cell cycle.Therefore,two enzymes that compete for L-arginine metabolism(Arg-1 and inducible nitric-oxide synthase[iNOS])are crucial for MDSC-induced T cell dysfunction.In mice,MDSCs are generally characterized by the expression of cell-surface antigens Ly-6C/G and CD 11b.and their human counterparts are typically CDllb+CD33+HLA-DR-.Reports have demonstrated that myeloid cells could intrinsically suppress T cell proliferation and maintain immune homeostasis in murine models of autoimmune diseases.However,the role of MDSCs in human autoimmunity remains obscure.Immune thrombocytopenia(ITP)is an autoimmune hemorrhagic disorder in which enhanced anti-platelet T helper cell activity and abnormal activation of CD8+ T cells are involved.High-dose dexamethasone(HD-DXM)has been widely used as the first-line therapy for ITP patients.Previous literature confirmed that a 4-day regimen of HD-DXM expanded both T regulatory cells(Tregs)and myeloid dendritic cells(mDCs)in ITP,suggesting that the immunosuppressive therapy with glucocorticoids(GCs)could restore regulatory cells in an autoimmune scenario.GCs have been found to induce a specific monocytic phenotype with anti-inflammatory properties in humans,and a corresponding subset of monocytes in mice has been proved to resemble tumor-derived MDSCs.Nonetheless,little has been defined about the effect of GCs on MDSCs in ITP and whether this effect contributes to remission.MDSCs have a gene expression profile distinct from that of normal monocytes and polymorphonuclear neutrophils,among which Etsl is regarded a potential factor responsible for various autoimmune diseases.In this study,by looking into the unique function of MDSCs,we investigated whether a comparative loss in number and suppressive activity of MDSCs had a place in the pathogenesis of ITP.Our data elucidated both in vitro and in vivo that HD-DXM restored the insufficiency of MDSCs in ITP and identified a role for DXM in correcting impaired MDSC function.We implicated that this effect might be achieved via a possible crosstalk between glucocorticoids receptor(GR)and the transcription factor Etsl.Our results provided novel insights into a possible mechanism of glucocorticoid strategy in the management of ITP.Objective:To explore the role of MDSCs in the pathogenesis of ITP,and investigate both in vitro and in vivo whether HD-DXM could correct the impaired function of MDSCs as well as the underlying mechanism.Methods:1)Patients and controls:Twenty-one patients with active primary ITP were enrolled and received HD-DXM 40 mg/d for 4 consecutive days.Spleen specimens from 5 patients with ITP and 5 patients with traumatic spleen rupture were analyzed by immunofluorescence.2)A murine model of ITP:Splenocytes from CD61 knockout mice immunized with CD61+ platelets were transferred into severe combined immunodeficient(SCID)mouse recipients(C57BL/6 background)to induce a murine model of severe ITP.3)Fluorescence-activated cell sorter analysis:The phenotype of MDSCs was analyzed for the cell surface markers CD11b,CD33,and HLA-DR.Intracellular expression of Arg-1 and iNOS was also determined.4)In vitro generation of human MDSCs:Both PBMCs(peripheral blood mononuclear cells)and splenocytes were cultured with recombinant human IL-6 and GM-CSF(10 ng/mL each)in the presence or absence of DXM.5)Quantitative real-time PCR:Total RNA was extracted and converted into cDNA to evaluate the expression of IL-10,TGF-β(transgenic growth factor-β),and VEGF(vascular endothelial growth factor).6)Suppression assay:CFSE-labeled CD4+ T cells were cocultured with CD33+MDSCs at 1:8,1:4,or 1:2 ratios and acquired for FACS analysis.7)Cytotoxicity assay:MDSC-modulated CTLs(105/mL)were incubated with autologous platelets(106/mL)and early apoptosis of platelets was detected using a mitochondrial membrane potential assay kit.8)Immunofluorescence:Frozen sections were stained with anti-CD 11b,anti-iNOS,and anti-CD33 antibodies and detected using confocal laser scanning microscopy.9)siRNA transfection:Transfection with Etsl siRNA,NC siRNA,and FAM siRNA were performed and functionally evaluated by rT-PCR etc.10)Generation of MDSCs from murine bone marrow(BM):BM cells were cultured for 3 days in the presence of rmGM-CSF,rmIL-6,rmG-CSF(10 ng/mL each),and DXM.SCIDs were injected intraperitoneally with 6 ×106 MDSCs.Results:1)MDSCs in patients with ITP and controlsThe percentage of MDSCs in circulation was significantly lower in patients with ITP than in healthy controls(P<.01).Significantly lowerArg-1 levels(median,7.0[range.6.0-9.7]vs.median,9.3[range,7.0-13.0])and higher iNOS levels(median,13.0[range,3.5-23.5]vs.median,2.1[range,1.5-3.0])were found in MDSCs of patients with ITP compared with healthy controls(P<.01).The number of MDSCs in the spleen was greater in non-ITP control patients than in patients with ITP(5.8 ± 2.3vs.2.6 ± 1.8).Splenic MDSCs of patients with ITP had higher levels of iNOS than control patients.2)HD-DXM expanded CD11b+CD33+HLA-DRlow cell populations and upregulated IL-10,TGF-β,and VEGF expressionHD-DXM significantly elevated the percentage of circulating MDSCs in patients with ITP(P<.01).DXM with concentration gradients from 0.02 to 2mM could stimulate the expansion of MDSCs in a dose-dependent manner.We adopted an effective concentration of 0.5 mM and the percentage of CD11b+CD33+double-positive cells showed a significant increase after addition of DXM compared with pretreatment baseline(62.77 ± 7.02 vs.84.02 ± 9.53;P<.01).DXM modulation significantly augmented the expression levels of IL-10,TGF-β,and VEGF in cytokine-induced MDSCs(P<.05).3)The effect of DXM treatment on suppressive functions of MDSCsThe CD33+ MDSCs induced in the presence of DXM significantly enhanced the suppression of T-cell proliferation at 1:4 and 1:2 ratios by 25%and 35%,respectively(P<.01).CTLs induced significant autologous platelet apoptosis in patients with ITP.In these patients,MDSC-primed CTLs showed weaker cytotoxic effects and the reduced cytotoxicity was further demonstrated in the presence of DXM with significantly lower levels of platelets apoptosis(P<.01).4)The association of Etsl with DXM modulationThe relative mRNA level of Etslwas significantly higher in DXM-modulated MDSCs than in non-modulated cells(P<.01).The siRNA transfection produced a successful silencing of the Ets1 gene.The suppression of CD4+ autologous T-cell proliferation by DXM-modulated MDSCs after Etsl siRNA transfection was significantly attenuated compared with that by mock-transfected MDSCs,which demonstrated an evidential function decline(P<.01).5)MDSC treatment alleviated thrombocytopenia in a murine model of ITPThe transferred SCID mice had significant levels of serum antiplatelet CD61-specific IgG antibodies to indicate model criteria by the second week and were separated into 4 groups.Group 1 received MDSCs injection plus splenocyte engraftment,which was administered on the day of and 2 days after irradiation.Group 2 received spenocytes only.Group 3 received the same amount of MDSCs as group 1 without splenocyte engraftment.Group 4 served as blank control.Group 1 demonstrated an overall higher survival rate than group 2.Moreover,mice in group 1 showed significantly higher platelet levels than mice in group 2 at the third and fourth weeks after irradiation.All mice in groups 3 and 4 recovered from irradiation-induced thrombocytopenia by day 14.Mice in group 1 had a significantly elevated level of splenic Etsl expression in comparison with mice in group 2(P<.01).Conclusion:In summary,impaired MDSCs are involved in the immunopathogenesis of ITP.HD-DXM promoted the expansion and enhanced the suppressive function of MDSCs in patients with ITP.The effect of DXM-modulation on MDSCs was authenticated to correlate with the transcription factor Ets1.Our study sheds new light on immune therapeutic avenues targeting MDSCs in the management of ITP and suggests an innovative mechanism underlying the action of traditional glucocorticoids.Part Ⅱ:Percentage of reticulated platelets as a diagnostic indicator for adults with primary immune thrombocytopenia:a prospective multicentre double-blind studyBackground:The diagnosis and treatment-related decisions of primary immune thrombocytopenia(ITP)still remain principally dependent on exclusion of other conditions and clinical expertise rather than the demonstration of a standardized laboratory finding or high-quality clinical trial evidence.Measurement of anti-platelet autoantibodies specifically against glycoprotein(GP)Ⅱb/Ⅲa and/or GP Ⅰb/Ⅸ(i.e.,monoclonal antibody immobilization of platelet antigens or MAIPA)is considered a screening tool,but was not included in the diagnostic algorism of ITP due to relatively low sensitivity.Therefore,a reliable,non-invasive laboratory-based method to differentiate patients with ITP is of urgent need in the clinical setting.Reticulated platelets(RP)represent newly released platelets in the circulation and are indicative of megakaryopoiesis in response to peripheral platelet destruction.Immature platelets or RP can be distinguished by their contents of messenger RNA residual,which is gradually lost within 24 hours as they mature and circulate.Thus,the employment of RNA-binding fluorochromes such as thiazole orange(TO),allows a real-time,direct assessment of thrombopoiesis to be made using flow cytometry.A high percent immature platelet fraction(IPF)is indicative of consumptive or recovering thrombocytopenic disorders in contrast to a low percent IPF seen in aplastic or hypoplastic states.As an idiopathic bleeding disorder,ITP often encounters overtreatment or empirical treatment,and sensitive laboratory approaches are lacking in its definitive diagnosis.Here,we explored a predicting assay of the percentage of RP(%RP)for patients with suspected and untreated ITP through a prospective,multicentre study.Objectives:To investigate the diagnostic value of FCM-measured%RP in the identification of adults with primary ITP.Methods:1)Study design:We designed the study to derive and validate a cutoff value(range)of%RP for the diagnosis of primary ITP in a two-phase approach(development and validation).Samples were collected from six medical centres in Shandong Province.Blinding was set between investigators who collected information and those who analysed data.A central,blinded expert panel provided the reference standard for diagnostic verification.Results of bone marrow examination and modified MAIPA were checked from patient records to confirm the diagnosis.2)Participants:Both admitted and out patients eligible for enrollment were aged 18 years or older of both genders.Each participant had a baseline peripheral platelet count<60 × 109/L,with or without the presence of bleeding symptoms upon enrollment.Any previous ITP-specific therapy within three months was considered an exclusion criterion.Patients with secondary ITP,seropositive detection of HIV,hepatitis B virus or hepatitis C virus infections,and severe kidney or liver function impairment were also excluded.3)Assessment of blood samples:Samples were collected from leftover of blood routine tests.Determination of%RP:flow cytometric measurement using anti-CD42b monoclonal antibody and TO.Plasma TPO concentration:using an enzyme-linked immunosorbent assay(ELISA)kit.Peripheral smear review:brilliant cresyl blue-stained.4)Statistical analysis:Predictive performance was assessed in the validation cohort by estimating negative and positive predictive values(NPV,PPV),sensitivity and specificity,and the area under curve(AUC)using established ROC.Results:1)Baseline characteristics:Between November 16,2016 and December 5,2017,we successfully processed 1542 samples resulting in valid analytical reports.Age,gender,baseline platelet counts,and median bleeding scores did not differ significantly between the development cohort and the validation cohort.2)Performance of the FCM-measured%RP:Panel review confirmed the classification of enrolled patients as follows:18.7%(289/1542)primary ITP;9.7%(149/1542)aplastic anemia(AA);48.9%(754/1542)chemotherapy-induced thrombocytopenia(CIT,suppressed phase);and 22.7%(350/1542)myelodysplastic syndromes(MDS),splenomegaly,or paroxysmal nocturnal hemoglobinuria(PNH),etc.Patients with primary ITP demonstrated a%RP of(14.4 ± 5.8)%,as compared with(5.8 ± 2.5)%,(4.8 ±2.0)%,and(5.7 ± 2.4)%among participants identified with AA,CIT,and MDS,respectively.Taken together,significant difference of%RP exists between ITP and non-ITP patients in the validation cohort(P<.0001).3)Development phase:The percentage of reticulated platelets obtained from 80 control subjects was(3.9± 1.6)%.Here with implementation of a cutoff optimization model,we derived a%RP cutoff value of 8.6%from the development cohort.4)Validation phase:In the validation cohort,the NPV of 8.6%or lower for the flow cytometric measurements of%RP was 96.4%(95%CI,94.7-97.6),and the PPV of%RP above 8.6%was 64.5%(95%CI,58.4-70.1)to rule in ITP.ROC curves for MAIPA and%RP models showed AUC of 0.776 and 0.923,respectively.The%RP cutoff model revealed a sensitivity of 86.7%(95%CI,81.1-90.9)and a specificity of 88.1%(95%CI,85.6-90.2).Conclusion:1)These data prospectively validated the flow cytometric measurement of%RP as a predictive tool to facilitate more definitive diagnosis of ITP.2)The cutoff value of 8.6%would be potentially applicable as routine laboratory screening for adults with primary ITP in a wide range of clinical settings. |
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