Participation Of Myeloid-derived Suppressor Cells And Cytotoxic T Lymphocytes In The Immunopathogenesis Of ITP

Part Ⅰ:Low-dose decitabine modulates myeloid-derived suppressor cell fitness via LKB1 in immune thrombocytopeniaImmune thrombocytopenia(ITP)is a common and multifaceted autoimmune bleeding disorder characterized by persistent thrombocytopenia.Immune dysregulation,including over-activation of immune effector cells and impaired function of immune regulatory cells,is pivotal to its pathogenesis.Myeloid-derived suppressor cells(MDSCs)are immunosuppressive immature innate immune cells.They inhibit T cell proliferation and activation,promote Tregs recruitment and amplification,and inhibit B cell proliferation and function.Their immunosuppressive function is based on normal fatty acid oxidation.In our previous study,high-dose dexamethasone corrected MDSC dysfunction via Ets1 in ITP.However,the sustained response rate to corticosteroids in ITP appears to be relatively low,and some patients develope chronic/refractory ITP.Decitabine is a hypomethylating agent that promotes cell differentiation at low doses and has a favorable effect on platelet elevation in myelodysplastic syndrome and post-transplantation thrombocytopenia.DNA methylation is imbalance in ITP;disruption of methylation balance in immune cells leads to abnormal gene expression,contributing to the loss of immune regulation.Our previously found that low-dose decitabine significantly increased the number of mature megakaryocytes by reducing methylation of TRAIL promoter region,thus promoting platelet production and exhibiting long-term clinical efficacy.Decitabine can regulate immune reaction in ITP by modulating Treg cells and inhibiting STAT3 activation,enhancing their immunosuppressive function in ITP,as well as restricting the cytotoxicity of Cytotoxic T lymphocytes(CTLs)to autologous platelets.However,it remains unknown whether decitabine can regulate the metabolic and suppressive activity of MDSCs in ITP.Liver kinase B1(LKB1),a tumor suppressor gene,encodes the serine STK11/LKB1.It is located upstream of AMP-activated protein kinase(AMPK),an energy-sensing molecule that can promote oxidative metabolism.The LKB1 signaling pathway was important in the linking of metabolic balance and functional homeostasis in immune cells.LKB1 is a key regulator of lipid metabolism in T cells,and participates in the optimal programming of suppressive activity,immune homeostasis,and modulation.It inhibited the cytotoxic effect of interferon y-producing CTLs,reduced the differentiation of CD4+ T cells into the pro-inflammatory subsets Thl and Th17,and induced the regulatory function of macrophages and dendritic cells at the transcriptional level.LKB1 deficiency interferes with the normal survival of Treg cells and with mitochondrial metabolism and inhibits the tricarboxylic acid cycle and fatty acid oxidation,leading to insufficient intracellular ATP synthesis and even the development of fatal early onset autoimmune disease.LKB1 promoter-region hypermethylation can inhibit gene transcription and reduce LKB1 protein kinase expression.Our objective was to elucidate the therapeutic effect of low-dose decitabine in ITP patients,and the mechanism of long-term remission in ITP.Objective:(1)The effects of low-dose decitabine on the generation and function of MDSCs in ITP,and the homeostatic regulation of LKB1 on the metabolism and immunosuppressive function of MDSCs in ITP patients was explored.(2)To intervene in vitro by using shRNA transfection,to clarify the effect of intervening LKB1 molecules on the function of low-dose decitabine-induced MDSCs,and to verify it in an active chronic ITP mouse model.Methods:(1)Patients and controls:For in vitro studies,43 ITP patients and 27 age-and sex-matched healthy volunteers were recruited.For in vivo studies,13 ITP patients were enrolled and treated with decitabine.(2)Active ITP murine model:CD61-knockout mice were given 108 wildtype C57BL/6J mice platelets weekly.SCID mice were subjected to 200 cGy total body irradiation,and injected intraperitoneally with 2×104 splenocytes from CD61-knockout mice.(3)Isolation and culturing of PBMCs and MDSCs:PBMCs were obtained and cultured with recombinant human IL-6 and granulocyte macrophage colony-stimulating factor(GM-CSF),with or without decitabine,for 7days.MDSCs were separated using anti-CD33 magnetic microbeads.(4)Flow cytometry:to analyze apoptosis,The phenotype of MDSCs were stained with FITC-labeled mouse anti-human CD33,PE-labeled mouse anti-human CD11b,APC/Fire 750 anti-human HLA-DR.Intracellular expression of Arg-1 and iNOS was also determined.(5)Metabolic analysis:MDSCs metabolism was analyzed using a Seahorse Extracellular Flux Analyzer.Human cultured MDSCs or mouse bone marrow MDSCs were subjected to various treatments for oxygen consumption rate detection.(6)ATP production measurement:Cells were inoculated in opaque 96-well plates(Corning,NY)with the same number of cells per treatment group and cultured overnight.ATP production was then measured.(7)Immunosuppression assays of MDSCs:MDSCs and CFSE-labeled CD4+CD25-T cells(Teff cells)were co-cultured for 5 days.proliferation of Teff cells was measured by flow cytometry.(8)CTLs-induced platelet apoptosis:Cytotoxic T lymphocytes(CTLs)were co-cultured with MDSCs for 3 days.Platelets were co-cultured with CTLs to analyze platelet apoptosis.(9)Quantitative real-time PCR:The mRNA expression of LKB1 signaling pathway,inhibitory cytokine and FAO/OXPHOS related genes were measured by real-time reverse transcription polymerase chain reaction(RT-PCR).(10)Western blotting:Gene expression was normalized to that of GAPDH for relative quantification.MDSCs were extracted for Western blotting.(11)Quantitative detection of Mass ARRAY targeted methylation sites:Total DNA was extracted from MDSCs using the QIAamp DNA Mini Kit.We used Agena’s EpiDesigner software to design primers for the core DNA methylation region:the final fragment to be detected was at-488 to 11 bp.(12)shRNA transfection:A lentivirus encoding the human LKB1 noncoding(NC)short hairpin RNA(NC shRNA)was transduced into human MDSCs.After 48 h,we added puromycin(1 mg/mL)to the culture medium for 24 h to screen the successfully transduced cells,LKB1 downregulation was measured by qPCR.(13)Immunofluorescence:Paraffin sections were incubated at 4℃ overnight with primary anti-Ly-6G/Ly-6C(Gr-1)and anti-LKB1 antibodies,followed by incubation with secondary antibodies.The nuclei were counterstained with 5μg/mL DAPI.(14)Generation of MDSCs from murine bone marrow:MDSCs were derived from the femurs and tibias bone marrow of wildtype C57BL/6 mice and sorted using magnetic beads.MDSCs were cultured with rmIL-6,rmGM-CSF,and rmG-CSF.(15)Cell therapy to active ITP murine model:Active ITP murine model were divided into groups.Via intraperitoneal injection,all the group received 2×104 splenocytes;part of the groups additionally received 3×106 different treated-MDSCs.(16)Enzyme-linked immunosorbent assay(ELISA):Serum samples of active ITP mice in different groups were tested for the level of IL-10,TGF-β and VEGF.Results:(1)Low-dose decitabine expand CD11b+CD33+HLA-DRlow cell populations.The proportion of CD11b+CD33+HLA-DRlow cell(MDSCs)in ITP patients were significantly lower than that in healthy controls.There was a significant increase in PBMCs after decitabine treatment in vivo.Relative to levels in healthy controls,ITP patients had significantly lower Argl expression and significantly higher iNOS expression in MDSCs.After decitabine treatment,Argl expression was elevated and iNOS expression was reduced.(2)Low-dose decitabine enhanced aerobic metabolism of MDSCs in ITP patients.MDSCs from ITP patients were cultured with various concentrations of decitabine(0-10 μmol/L).Low doses of decitabine(50 and 100 nmol/L)significantly increased the proportion of MDSCs in the samples from ITP patients.We performed a mitochondrial stress test,comparing cells treated with or without decitabine.Relative to the healthy controls,OCR was lower in MDSCs from ITP patients,but was significantly improved after decitabine treatment.(3)Low-dose decitabine augmented immunosuppressive functions of MDSCs in patients with ITP.In vitro,MDSCs were treated with PBS or decitabine and co-cultured with carboxyfluorescein succinimidyl ester(CFSE)-labeled CD4+CD25-effector T(Teff)cells.MDSCs generated from ITP patients had weaker inhibitory effects on Teff cells than those generated from healthy controls.In addition,low-dose decitabine significantly enhanced the immunosuppressive function of MDSCs in ITP.We further investigated whether MDSCs treated with decitabine inhibited CTL cytotoxicity.CTL-induced platelet apoptosis was significantly suppressed after co-culture with decitabine-treated MDSCs generated from ITP patients.(4)Decitabine-modulated MDSCs treatment ameliorated thrombocytopenia in active ITP mice.Active ITP murine were divided into 3 groups to receive splenocyte transfer,two groups were given additional transfer of PBS and decitabine-treated MDSCs,the decitabine-treated MDSCs group had significantly higher platelet counts than the control and PBS-treated MDSCs groups.The OCR analysis revealed a significant higher curve in the decitabine-treated MDSCs group.Moreover,we found that the mRNA expression level of ACADM,HADHA and PGC1β in control group was significantly lower than that in decitabine-treated MDSCs group.We found a significantly increased serum levels of TGF-β,IL-10,IL-4 in decitabine-treated MDSCs group.(5)Low-dose decitabine up-regulated the expression of downstream regulators of LKB1 signaling pathway and inhibitory cytokine in MDSCs.Western blot analysis and flow cytometry of the MDSCs from ITP patients revealed lower LKB1 protein levels and lower LKB1 expression than in healthy controls;increased after decitabine treatment.Following decitabine treatment,part of methylated residues in the LKB1 promoter region were demethylated in ITP patients.mRNA expression of LKB1,AMPKαl,AMPKα2,AMPKβ1,AMPKβ2,AMPKyl,AMPKy2,ND-1,ND-3,ATP-6,IL-10,VEGF,and TGFβ were significantly lower in ITP patients than in healthy control subjects,and the mRNA expression were increased after decitabine treatment.(6)LKB1 shRNA interference offset the altered metabolic activity of MDSCs induced by decitabine in vitro.shRNA transfection successfully silenced LKB1 in the MDSCs of ITP patients.The LKB1-knockdown efficiency was≥69.26%,revealed by RT-PCR.The OCR of the LKB1 shRNA group was significantly lower than that of the NC shRNA group,with no significant difference in OCR compared with the LKB1 shRNA+decitabine group.The intracellular ATP level was significantly lower in the LKB1 shRNA group.(7)LKB1 shRNA interference counteracted the functional augmentation of MDSCs induced by decitabine in vitro.The suppression of CFSE-labeled CD4+CD25-Teff cell proliferation was significantly lower in the group treated with LKB1 shRNA-transfected MDSCs than in the group treated with NC shRNA,indicating that the suppressive function of the MDSCs was reduced.Therefore,silencing LKB1 cancelled the decitabine-induced enhancement of MDSCs function observed in an earlier assay.(8)LKB1 shRNA interference offset rescuing effect of decitabine-treated MDSCs in a murine model of ITP.ITP mice were divided into 5 groups.On day 35,mice in group 2 had significantly higher platelet levels than those in group 4,suggesting that thrombocytopenia amelioration was dependent on LKB1.LKB1 expression in MDSC-positive cells was analyzed via and the expression was lowest in groups 4 and 5.OCR was lowest in group 1,similfluorescence intensity using Image:LKB1 expression was significantly higher in group 3,arly,following LKB1-knockdown,OCR curve were significantly lower in group 4,even following decitabine treatment(group 4).ACADM and PGC1β mRNA expression was significantly lower in groups 4 and 5 than in groups 2 and 3 and was highest in group 3.Conclusions:(1)The number and function of MDSCs in PBMCs are abnormal in ITP patients,which can be corrected after low-dose decitabine treatment.(2)The impaired metabolic function and immunosuppressive function caused by the low expression of LKB1 in MDSCs are involved in the pathogenesis of ITP.(3)Low-dose decitabine corrects the abnormal expression of LKB 1 in MDSCs of ITP patients,improves the aerobic metabolism and immunosuppressive function of MDSCs.Part Ⅱ:Rapamycin Inhibits Cytotoxic T Lymphocytes-Mediated Platelet Destruction via Modulating PI3K/Akt/mTOR signaling pathway in Immune ThrombocytopeniaBackground:Primary immune thrombocytopenia(ITP)is a hemorrhagic disease characterized by decreased platelet count.The main pathogenesis is autoimmune intolerance,which leads to increased platelet destruction and decreased production.CD8+cytotoxic T lymphocytes(CTLs)of ITP patients express high level of lethal molecules,leading to the lysis and apoptosis of their own platelets,which is an important mechanism of platelet destruction in ITP patients.Mouse models of ITP further confirmed that CTLs in ITP can cause increased platelet destruction.Phosphatidylinosine 3 kinases(PI3Ks),and the downstream mediators Akt and mammalian target of rapamycin(mTOR)constitute the PI3K/Akt/mTOR signaling cascade of rapamycin,Akt,is a direct downstream effector of phosphoinositol 3-kinase(PI3K).mTOR is an evolutionarily highly conserved filament/threonine protein kinase,which mainly exists in two different complex forms,mTORCl and mTORC2.When mTORC1 is activated,the downstream signaling targets are mainly 4E-BP1 and p70S6K,activated mTORC2 can play a role in cell function by phosphorylation of downstream targets Akt and SGK1.The PI3K/Akt/mTOR signaling pathway has abnormal changes in gene level,protein expression and kinase activity in a variety of autoimmune diseases,which is closely related to the abnormal differentiation and activation of T cells.Previous studies have confirmed the importance of the PI3K/Akt/mTOR signaling pathway in CD4+T cell differentiation,MTORC1 regulates the differentiation of Thl and Th17 subsets,while Th2 subsets are dependent on mTORC2,and complete inhibition of mTOR activity can promote the generation of regulatory T cells.The PI3K/Akt/mTOR signaling pathway involved in the differentiation and activation of CD8+T cells,accumulating evidence have highlighted that the activation of PI3K/Akt/mTOR signaling pathway promotes the differentiation of CD8+T cells to CTLs,which leads to the up-regulation of cell-mediated immunity,suggesting the important role of PI3K/Akt/mTOR signaling pathway in the pathogenesis of ITP.Rapamycin,mTORCl molecular inhibitor,plays a role in autoimmune diseases,tumors,diabetes,etc.A clinical study has showed that rapamycin combines with low-dose prednisone achieve rapid clinical response and good lasting efficacy in the ITP,without significantly compromising safety,and no serious infection or other side effects.However,the endogenous mechanism remains to be explored.Our objective was to elucidate mechanism of rapamycin inhibiting CTLs cytotoxicity through PI3K/Akt/mTOR signaling pathway in ITP.Objective:(1)To explore the activation levels of PI3K/Akt/mTOR signaling pathway-related molecules in CD8+T cells of ITP patients.(2)To explore the regulatory effect of rapamycin on the activation of PI3K/Akt/mTOR signaling pathway,and to clarify the effect of rapamycin on the destructing of platelets by CTLs.Methods:(1)ITP Patients and controls:There were active ITP and healthy individuals were fit into in this study.(2)Isolation and culturing of CD8+T cells:PBMCs were separated and sorted by magnetic beads.CD8+T cells were cultured and inoculated with rh-IL-2,anti-human CD3 antibodies and anti-human CD28 antibodies for 72 hours.DMSO or rapamycin were added to the incubation medium.(3)Flow cytometry:The activation of PI3K/Akt/mTOR signaling pathway in CD8+T cells and the proportion of CD8+ T cell subsets were detected.The CD8+ T cell subsequently stained by anti-human/anti-mouse conjugated antibodies.Cells were subsequently analyzed.(4)CD8+ T cytotoxic cytokine secretion:The expression of perforin and granzyme B in CD 8+T cells of ITP and healthy control were detected by flow cytometry and labeled with corresponding monoclonal antibody.(5)CTLs-induced platelet apoptosis:CD8+T cells were co-cultured with platelets for 4 hours.And then detected by staining with Mitochondria Staining Kit(JC-1)to detect cell apoptosis.(6)Western blots analysis:CD8+T cells were lysed in radio-immunoprecipitation assay(RIPA)buffer for western blot.Western blotting using monoclonal rabbit anti-human GAPDH,monoclonal rabbit anti-human mTOR,p-mTOR,Akt,p-Akt,4E-BP1,p-4E-BP1,P70S6K,and p-P70S6K antibodies.(7)Active ITP murine model:Splenocytes from CD61 knockout mice immunized with CD61+ platelets were transferred into severe combined immunodeficient(SCID)mouse to induce a murine model of ITP.Results:1.Abnormal activation of PI3K/Akt/mTOR signaling pathway in CD8+ T cells from ITP patients.The activation of PI3K/Akt/M-TOR in CD8+T cells of ITP patients were detected.It was found that the phosphorylation of p-Akt,p-MTOR,p-4E-BP1 and p-P70S6K were higher than that in healthy controls.2.CD8+T cell subsets are unbalanced in ITP patients.We found that CD8+T cell subsets were partially disbalanced.Naive T cell subsets were significantly lower than normal controls,while effector T cell subsets were significantly higher than normal controls,while central memory T cell subsets and terminal effector T cell subsets had no significant differences compared with normal controls.However,the proportion of CD8+CD28-inhibitory T cell subsets in ITP patients was no statistically significant difference compared with healthy controls.3.Increased expression of perforin and granzyme B in CD8+T Cells from ITP Patients.We found that the levels of perforin and granzyme B in ITP patients were significantly higher than that in healthy controls.4.The mTOR inhibitor rapamycin significantly inhibited the abnormal activation of PI3K/Akt/M-TOR signaling pathway in CD8+T cells.There were no significant difference in the expression levels of mTOR,Akt,4E-BP1,and P70S6K after rapamycin treatment,but the phosphorylation level of p-mTOR,p-Akt,p-4E-BP1,p-P70S6K was significantly inhibited by rapamycin treatment.Likewise,the ratios of p-mTOR/mTOR,p-AKT/AKT,p-4E-BP1/4E-BP1,p-P70S6K/P70S6K were also significantly down-regulated.5.The mTOR inhibitor rapamycin partially restored the imbalance of CD8+T cell subsets in ITP patients.After rapamycin treatment in vitro,flow cytometry analysis showed that rapamycin partially restored the imbalance of CD8+T cell subsets,increased expression of naive T cell subsets,decreased expression of effector T cell subsets and central memory T cell subsets.There were no significant differences in Terminal effector T cell subsets after rapamycin treatment.6.The mTOR inhibitor rapamycin inhibits the expression of perforin and granzyme B of CD8+T cells in ITP patients.CD8+T cells were selected and cultured with rapamycin in vitro.Flow cytometry analysis found that Rapamycin significantly inhibited the expression of perforin and granzyme B in CD8+T cells of ITP patients.7.MTOR inhibitor rapamycin reduced platelet apoptosis mediated by CD8+T cells.CD8+T cells were cultured in vitro.After 3 days,different groups of CTLs were incubated with platelets.CTLs-mediated platelet apoptosis was significantly reduced in the rapamycin-treated group.8.The mTOR inhibitor rapamycin inhibited the expression of PI3K/Akt/mTOR signaling pathway and ameliorated thrombocytopenia in ITP.To evaluate the previous in vitro experiments,we further established an active ITP mouse model.On days 28 and 35,platelet counts were significantly higher in ITP mice in the rapamycin group than in the control group.We detected the expression of PI3K/Akt/M-TOR signaling pathway in ITP mice and found that the activation levels of p-MTOR,p-Akt,p-4E-BP1 and p-P70S6K on thymus,spleen and CD8+T cells of ITP mice in rapamycin group were significantly reduced.Conclusions:(1)The activation of PI3K/Akt/mTOR signaling pathway-related molecules in CD8+ T cells is abnormal,and the CD8+T cells in patients with ITP have an imbalance of subsets.(2)Rapamycin inhibits the abnormal activation of signaling molecules,restores the imbalance of subpopulations to a certain extent,and reduces the secretion of toxic cellular molecules.(3)The inhibitory effect of rapamycin on CTLs cytotoxicity was partially contributed by suppression PI3K/Akt/mTOR signaling pathway.