Regulating The Monocyte/Macrophage System And CD4~+T-cell System In Restoration Of Immune Tolerance In ITP

Part Ⅰ:TNF-α blockade corrects monocyte/macrophage imbalance in primary immune thrombocytopenia(ITP)Background:ITP is an autoimmune hemorrhagic disease with heterogeneous pathogenesis,predominantly characterized by autoantibody-mediated platelet destruction and/or impaired platelet production.The loss of immune tolerance plays a crucial role in the pathogenesis of ITP,manifested with imbalance of Thl/Th2,Th17/Tregs,dysfunction of cytotoxic T cells and NK cells.Increased platelet destruction remains the main mechanism of ITP,in the reticulo-endothelial system,where monocytes/macrophages phagocytose autoantibody-bound platelets in an Fcγ receptor(FcyR)-dependent manner.Based on CD16 expression,monocytes are divided into three groups:classical(CD14+ CD16-),intermediate(CD14+ CD16+),and non-classical(CD14 CD16+).Classical monocytes account for approximately 70 to 90%of total monocytes,and function as scavenger cells under healthy conditions.By contrast,pro-inflammatory CD16+ monocytes(intermediate and non-classical subgroups)are associated with tumor necrosis factor-α(TNF-α)production after stimulation and expand under infectious or inflammatory conditions.Murine monocyte counterparts are defined as classical/intermediate monocytes(Ly6C+CD11b CD115+)and non-classical monocytes(Ly6C-CD11b+CD115+).Monocytes are precursors of macrophages,and become tissue resident macrophages upon recruitment.Macrophages consist of the classically activated M1 type and the alternatively activated M2 type.M1 macrophages facilitate inflammation,defend against intracellular pathogens and tumors in the context of Thl-driven responses,and produce more TNF-a.On the contrary,M2 macrophages are anti-inflammatory and tissue repairing in Th2-driven activities.Deviation of monocyte/macrophage subsets contributes to the loss of immune tolerance in ITP,manifesting as higher ratios of pro-inflammatory CD16+ monocytes and M1 macrophages.First-line treatments for ITP include corticosteroids and intravenous immunoglobulin G(IVIG).An estimated 15 to 25%of patients are inexplicably refractory to first-line therapies.Though various second-line therapies exist-such as thrombopoietic agents,rituximab,and splenectomy are available-a subset of patients does not respond to any available strategy.Novel therapeutic methods are still needed to improve management of ITP.TNF-a is produced primarily by activated monocytes/macrophages.Higher TNF-a production and over-activation of TNF-a receptor signaling were associated with the pathogenesis of autoimmune diseases such as rheumatoid arthritis,inflammatory bowel disease,and psoriasis.Elevated serum or plasma TNF-a expression has also been reported in ITP patients.Because of the crucial role of TNF-a in promoting inflammation,it has been proposed as a therapeutic target for several diseases.For example,blockade of TNF-a with infliximab or etanercept has been widely used for management of rheumatoid arthritis and inflammatory bowel disease.Infliximab was also effective in a few refractory cases of ITP.Zhong and co-workers demonstrated in vitro that TNF-a blockade could expand regulatory T cells(Tregs)in ITP.However,the effect of TNF-a blockade on the monocyte/macrophage system in ITP remains unknown.Our study aimed to investigate the therapeutic value and mechanisms of TNF-a blockade in treatment of ITP.Objectives:(1)To explore the role of monocyte/macrophage subsets in regulating the immune tolerance of ITP,further improving the pathogenesis of ITP.(2)To clarify the regulatory effect of TNF-α blockade on monocyte/macrophage system subsets,and to explore the therapeutic effect of TNF-α blockade on ITP by restoring the balance of monocyte/macrophage system subsets through in vitro and in vivo experiments.Methods:(1)ITP patients and control:36 ITP patients were included,including 19 male patients and 17 female patients,aged 15-77 years,with a median age of 47 years,as well as 28 age and sex-matched healthy controls.Peripheral blood and plasma of the ITP patients and healthy controls were collected.Spleens were collected from ITP patients and spleen rupture control patients undergone splenectomy.(2)A passive ITP mouse model was established by peritoneal injection of anti-CD41a antibody,and the therapeutic effect of anti-TNF-α antibody on ITP mice was observed.(3)Flow cytometry:Expression levels of CD14,CD16 and TNF-α in human monocytes were detected by flow cytometry.The phenotypes of mouse macrophages F4/80,CD80,CD86,TNF-α,and mouse monocyte subsets CD11b,Ly6c,CD115 were detected.(4)Immunofluorescence imaging:Immunofluorescence was used to detect the expression of CD68 and TNF-α in the spleens of ITP patients and of patients with traumatic splenic rupture.(5)Enzyme-linked immunosorbent assay(ELISA):Plasma TNF-α from ITP patients and healthy controls was measured using ELISA according to the manufacturer’s instructions.(6)Macrophage culturing:Adherent monocytes/macrophages were cultured with macrophage colony-stimulating factor(M-CSF)to differentiate into macrophages.Macrophages were further treated with lipopolysaccharides(100 ng/mL,LPS)and interferon-y(20 ng/mL IFN-y)to elicit M1 polarization.Anti-TNF-a antibody,recombinant human TNF-α,and IgG isotype control were used as treatment.The mechanism of TNF-α regulation was investigated by adding NF-κB inhibitor JSH-23(7)Quantitative real-time PCR(q-PCR)and Western blot were used to detect the phenotype changes and signaling pathway of monocyte-derived macrophages after TN F-α and anti-TNF-α treatment.(8)In vitro phagocytosis assays:Monocytes/macrophages after the respective treatments were incubated with opsonized and CMFDA-labeled platelets for 1 hour at 37℃ or at 4℃ as a negative control.Fluorescence intensity was determined by flow cytometry.(9)Antigen presentation function of macrophages:The modulated macrophages were co-cultured with CD4+ and CD8+T cells,respectively,and the proliferation of CD4+and CD8+ T cells was determined by flow cytometry analysis after 6 days.(10)Platelet retention detection:The DIR(1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindotricarbocyanine Iodide)-labeled platelets were injected into ITP mice via retrobulbar vein.Mice were euthanized 30 min after injection.Spleens and livers were removed,and platelet retention analysis was assessed by measuring fluorescence intensity with an in vivo imaging system.Results:(1)Elevated TNF-a expression in ITPHigher plasma TNF-a levels were found in ITP patients compared with healthy controls(P-0.0001).TNF-a mRNA expression was also upregulated in ITP patient PBMCs(P<0.0001)and monocytes(P= 0.039).We found that TNF-a levels in plasma(r =-0.5509,P= 0.011 8),and its mRNA expression in PBMCs(r =-0.6936,P = 0.0002;)and monocytes(r =-0.6038,P = 0.0023)were inversely correlated with platelet counts in ITP patients,suggesting an important role of TN F-a in ITP.Notably,the number of TNF-a-expressing macrophages in ITP patient spleens was higher than that in healthy individuals with traumatic spleen rupture(P = 0.0045).(2)Increased CD16+ monocytes with elevated TNF-a expression in ITPHigher percentages of CD16+ monocytes were detected in ITP patients compared to healthy controls(P = 0.0192).Moreover,percentages of CD16+ monocytes correlated negatively with platelet counts in ITP patients(r=-0.3995,P= 0.0174).TNF-αexpression in CD16+ monocytes was higher than that in classical monocytes in both ITP patients(P<0.0001)and healthy controls(P<0.0001).Frequencies of CD16+monocytes were positively correlated with TNF-α expression in monocytes from ITP patients(r = 0.7580,P = 0.0003).(3)TNF-α blockade inhibited Ml polarization by suppressing NF-κB signalingTNF-α and LPS+IFN-y induced macrophages to polarize into Ml with increased TNF-α,MCP-1,and iNOS expression,and activated MAPK and NF-κB signaling pathway.While TNF-κ blockade reduced Ml polarization and inhibited the activation of NF-κB signaling pathway(P<0.05).(4)Anti-TNF-α treatment dampened macrophage phagocytosisrhTNF-α enhanced the phagocytic capacity of macrophages,whereas anti-TNF-αtreatment reduced the FcγR-mediated phagocytosis compared to IgG-treated macrophages(P<0.05).(5)TNF-a blockade-modulated macrophages suppressed T-cell proliferationWe co-cultured MDMs with CFSE-labeled T cells for 6 days and analyzed CD4+ T cell and CD8+ T cell proliferation by flow cytometry.M1 macrophages or rhTNF-α-treated MDMs from ITP patients enhanced the proliferation of CD4+ T cells and CD8+ T cells compared to IgG-treated MDMs.TNF-α blockade significantly reduced the stimulation of T-cell proliferation by Ml macrophages or MDMs(P<0.05).(6)TNF-α blockade ameliorated thrombocytopenia in ITP mouse modelPlatelet counts increased in anti-TNF-α-treated ITP mice compared to IgG-treated ITP mice on day 4(P = 0.0324)and day 6(P = 0.0474).Tracing of DIR-labeled platelets showed anti-TNF-α treatment decreased the retention of platelets in livers and spleens.Compared to normal mice,ITP mice had remarkably increased levels of CD80,CD86,and TNF-α on their splenic macrophages,and anti-TNF-α therapy reduced these levels(P<0.05).Moreover,TNF-α levels on macrophages from the liver and abdomen of ITP mice were also increased compared to normal mice,and anti-TNF-αantibody reduced these levels,too(P<0.05,P<0.01 respectively).Compared to normal mice,the frequencies of Ly6C-CD11b+CD115+ non-classical monocytes were considerably increased in the spleen and peripheral blood of ITP mice(P<0.001,P<0.01 respectively),and anti-TNF-α treatment reduced the percentages of non-classical monocytes(P<0.01).Conclusions:(1)The expression of TNF-α and the proportion of CD16+ monocytes in ITP patients were higher than that in healthy controls,and were negatively correlated with platelet numbers.(2)TNF-α blockade reduced the polarization of M1 macrophages,dampened macrophage functions in platelet phagocytosis and T-cell stimulation in ITP.(3)Anti-TNF-α therapy significantly reduced platelet retention in liver and spleen,leading to remarkably improved platelet counts of ITP mice.Additionally,anti-TNF-αtreatment reduced Ml macrophages in the spleen,liver and abdomen,and decreased non-classical monocytes in the spleen and peripheral blood in ITP mice.This study revealed the important regulatory role of TNF-α in ITP,and proposed that TNF-αblockade might be a new therapeutic strategy for ITP.Part Ⅱ:Interleukin-37 reduces in:flammation and impairs phagocytosis of platelets in immune thrombocytopenia(ITP)Background:Immune thrombocytopenia(ITP)is an autoimmune hemorrhagic disease with a considerable reduction in platelet count.As with other autoimmune diseases,inflammation plays an important role in the development of ITP.The disorders of immune homeostasis lead to the auto-attack on platelets,and impaired production of platelets from megakaryocytes,contributing to an inflammatory milieu manifested with higher expression of several pro-inflammatory cytokines,such as IL-1β,IL-18,TNF-α,IL-21,and the correlated aberrant immune cell subsets.Human interleukin(IL)-37 is a member of IL-1 family and is primarily known as an anti-inflammatory cytokine.IL-37 can be activated by pro-inflammatory stimuli and participates in the process of several inflammatory diseases.Patients with systemic lupus erythematosus,rheumatoid arthritis,and primary Sjogren’s syndrome expressed higher plasma/serum IL-37 which was positively correlated with disease severity,while the baseline expression level on healthy controls is very low.IL-37 is expressed in circulating monocytes as well as tissue macrophages,dendritic cells,tonsillar B cells,and plasma cells.Monocytes and macrophages are the major cells involved in autoantibody-opsonized Fcγ receptor(FcyR)-dependent platelet clearance in ITP.In our previous studies,aberrant expression of FcyRs was identified,manifested as a significant decrease in the inhibitory FcγRⅡb expression and a remarkable increase in activating FcyRI,FcγRⅡa,and FcyRIII expression in active ITP patients compared with healthy controls,leading to higher phagocytic capacity in platelet clearance.It has been reported that IL-37 induced anti-inflammatory M2-like macrophages and inhibited the pro-inflammatory M1 macrophages.However,the specific mechanism has not been elucidated.We speculate that it may be related to the regulation of the downstream inflammatory pathway of macrophages,and the distribution of FcγRs.ITP is an autoimmune disease characterized by low platelet count with heterogeneous bleeding manifestations,accounting for approximately one third of clinical hemorrhagic diseases.Common bleeding symptoms include petechiae,ecchymoses,epistaxis,genitourinary hemorrhage,gingival hemorrhage,and gastrointestinal hemorrhage.Although almost all bleeding symptoms in ITP are relatively minor events,fatal intracranial hemorrhage and overmedication to prevent severe bleeding are worthy of attention.However,platelet count does not completely relate to bleeding severity.Although the number of platelets is very low in some ITP patients,there is no clinical bleeding manifestations.However,in some other ITP patients,active bleeding may occur despite the platelet count is in the safe range(>30× 109/L).The goal of treatment is to increase the number of platelets on one hand and stop bleeding on the other.Therefore,in addition to platelet count,patient’s bleeding symptoms should also be an important reference for evaluating the efficacy of drugs.Several bleeding scoring systems have been used to evaluate the bleeding risk of ITP patients,but few of them has been widely used.In addition,some bleeding scoring systems are rather complex,making it difficult to popularize them in clinical practice.Thus,there is a great need for reliable indicators of the susceptibility of bleeding in ITP patients.Objectives:(1)To detect the expression level of IL-37 and its correlation with disease severity of ITP patients(2)To elucidate the regulatory effects of IL-37 on macrophage phenotype and phagocytic function,and to explore the therapeutic value of IL-37 on ITP.Methods:(1)ITP patients and control:43 ITP patients were enrolled,including 14 male patients and 29 female patients,aged 18-91 years old,with the median age of 50 years old,as well as 14 age and sex-matched healthy controls.Peripheral blood and plasma of the ITP patients and healthy controls were collected.(2)Enzyme-linked immunosorbent assay(ELISA):Plasma IL-37 from ITP patients and healthy controls was measured using ELISA according to the manufacturer’s instructions.(3)Monocytes/macrophages culturing:Peripheral blood mononuclear cells(PBMCs)of ITP patients and healthy controls were cultured in vitro for 2 hours,and the adherent cells were monocytes/macrophages.Recombinant human(rh)-IL-37,anti-IL-37 antibody or IgG control were added in the culturing for 24 hours.The expression levels of CD16,CD32a,CD32b and CD64 were detected by flow cytometry.(4)Quantitative real-time PCR(q-PCR):The expression of IL-37 in PBMCs of ITP patients and healthy controls was detected by q-PCR.The mRNA expression of monocyte/macrophage FcyRIIa and FcyRIIb was detected after culturing.(5)Macrophage culturing:Adherent monocytes/macrophages were cultured with macrophage colony-stimulating factor(M-CSF)to differentiate into macrophages.Macrophages were further treated with lipopolysaccharides(100 ng/mL,LPS)and interferon-γ(20 ng/mL,IFN-γ)to elicit M1 polarization.IL-37,anti-IL-37 antibody,or IgG control were added for intervention.Western blot was performed for analysis of inflammatory signaling pathways downstream of macrophages.(6)In vitro phagocytosis assays:Monocytes/macrophages after IL-37,anti-IL-37 antibody,or IgG control treatments were incubated with opsonized and CMFDA-labeled platelets for 1 hour at 37℃C or at 4℃ as a negative control.Fluorescence intensity was determined by flow cytometry.Results:(1)IL-37 expression is elevated in active ITP patientsPlasma IL-37 was significantly increased in ITP patients with platelet count lower than 30 x 109/L(median 163.50,interquartile range 263.70 pg/mL,n = 37)compared to healthy controls(median 95.30,interquartile range 116.87 pg/mL,n = 14,P =0.0129),and ITP patients with platelet count above or equal to 30 x 109/L(median 63.26,interquartile range 37.89 pg/mL,n = 6,P = 0.0001).A negative correlation was observed between plasma IL-37 level and platelet count in ITP patients enrolled(r =-0.5502,n = 43,P = 0.0001).Elevated plasma IL-37 was found in ITP patients with the bleeding score equal to or above 5(median 219.21,interquartile range 454.23 pg/mL,n = 21),compared with those ITP patients with scores below 5(median 88.30,interquartile range 67.04 pg/mL,n = 22,P<0.0001).Furthermore,Elevated plasma IL-37 correlated positively with bleeding scores(r = 0.6321,n = 43,P<0.0001).Plasma IL-37 levels were associated with clinical and laboratory parameters of ITP patients.The mRNA expression of IL-37 was consistent with plasma IL-37.(2)IL-37 inhibits inflammatory signaling in monocytes/macrophages from ITP patientsPhosphorylation of MAPK p38,MAPK JNK,AKT and NF-κB p65 was significantly upregulated after polarized by LPS and IFN-γ.rh-IL-37 reduced the phosphorylation of MAPK p38,MAPK JNK,AKT both of IgG-and LPS + IFN-γ-treated monocytes/macrophages,and reduced the phosphorylation of NF-κB p65 in LPS +IFN-y-treated monocytes/macrophages.Meanwhile,the anti-inflammatory role of IL-37 could be neutralized by anti-IL-37 antibodies.These data suggest that IL-37 inhibits inflammation by regulating MAPK,AKT,and NF-κB signaling in monocytes/macrophages from ITP patients.(3)IL-37 regulates FcγRI,FcγRII,and FcγRⅢ expression in monocytes/macrophagesLevels of FcyRI(CD64)on monocytes/macrophages decreased considerably after rh-IL-37 administration(60.66± 4.80 vs.70.34 ± 6.56,n = 6,P= 0.0021).Total FcγRⅡ(Ⅱa + Ⅱb;CD32)expression was not apparently affected after the respective treatment.Inhibitory FcyRⅡb(CD32b)expression on monocytes/macrophages increased after rh-IL-37 treatment(14.24± 1.61 vs.8.75 ± 0.42,n = 4,P = 0.0485).No significant changes were observed in FcγⅢ(CD 16)expression on monocytes/macrophages.rh-IL-37 treatment decreased FcyRⅡa mRNA expression(0.23 ± 0.05 vs.0.33 ± 0.04,n = 4,P= 0.023)and increased FcγRⅡb expression(0.063 ⒈ 0.008 vs.0.043 ⒈ 0.005,n = 4,P=0.0040)compared to IgG-treated monocytes/macrophages.Moreover,the ratio of FcγRIIa/FcγRⅡb mRNA expression on monocytes/macrophages was lower after rh-IL-37 treatment(3.54 ± 0.33 vs.7.92±0.39,n = 4,P = 0.023).The regulatory effects of rh-IL-37 on FcyRs could be eliminated by anti-IL-37 antibody.(4)IL-37 treatment decreases phagocytic capacity of monocytes/macrophagesMonocytes/macrophages treated with rh-IL-37 exerted significantly lower phagocytic capacity compared with monocytes/macrophages treated with IgG controls(2.81 ±0.31 v4.4.4± 0.34,n = 4,P = 0.0031).Addition of anti-IL-37 antibodies ameliorated the effect of IL-37 in dampening the phagocytosis.Conclusions:(1)IL-37 expression is elevated in ITP patients,which is correlated with platelet count and the severity of bleeding in ITP,indicating that IL-37 can be a candidate in evaluating disease severity of ITP.(2)IL-37 initiates an anti-inflammatory effect on monocytes/macrophages from ITP patients by down-regulating the phosphorylation of MAPK,AKT,and NF-κB signaling pathways.(3)IL-37 restores the balance of activating and inhibitory FcyRs and decreases antibody-mediated platelet phagocytosis by monocytes/macrophages,suggesting that IL-37 may be a potential therapeutic agent in ITP.Part III:Indirubin modulates CD4+ T-cell homeostasis via PD1/PTEN/AKT signaling pathway in ITPBackground:Immune thrombocytopenia(ITP)is the most common hemolytic autoimmune disease.T cells play an important role in modulating the peripheral immune tolerance.Loss of peripheral immune tolerance through simultaneous decrease of CD4+CD25+Foxp3+regulatory T cells(Tregs)as well as unrestricted proliferation and activation of peripheral CD4+ effector T cells(Teffs)underpin the pathophysiology of ITP.Tregs play an important role in maintaining self-tolerance and are phenotypically defined as CD4+CD25+Foxp3+.Tregs inhibit the activation and proliferation of auto-reactive Teffs,identify autoantigenic peptides and secrete inhibitory cytokines.The number and function of Tregs in ITP patients were significantly decreased compared with normal controls.The excessive activation and proliferation of Teffs with decreased apoptosis,further aggravated the disturbance of immune tolerance in ITP.Indirubin is an active ingredient of a traditional Chinese herb called Indigofera tinctoria L which is clinically used for the treatment of thrombocytopenia patients.There are also reports of indirubin possessing anti-viral,anti-bacterial and anti-inflammatory properties,and is most well-known for its clinical use in the treatment of chronic myelogenous leukemia.In murine models of ITP and ulcerative colitis,indirubin was effective therapeutically through down-regulation of the immune response and increasing the percentage of Tregs.However,the efficacy of Indigofera tinctoria L in the treatment of ITP still needs to be confirmed by appropriate clinical trials and the endogenous mechanisms of action remains to be explored.Programed cell-death 1(PD1)belongs to the CD28 immunoglobulin superfamily,and is a co-receptor that is inducibly expressed on T cells,B cells,NK cells and monocytes.PD1 binds with its ligand PD-L1,initiating the programmed death of T cells and enabling tumor cells to obtain immune escape.Targeted therapy on PD1 and PD-L1 has achieved satisfactory clinical results in treatment of a variety of cancers.The PD1-PDL1 pathway regulates peripheral T-cell tolerance in several ways.On the one hand,this pathway impairs the initial phase of.activation,expansion,differentiation,survival and functions of self-reactive T cells.On the other hand,this pathway facilitates the development,maintenance,and function of induced Tregs.Tregs activation and expansion in parallel with PD1 signaling are critical for the maintenance of immune tolerance and elimination of either can result in its breakdown.The combination of PD1 and PD-L1 limits T-cell stimulation and promotes the differentiation and stabilization of Tregs by augmenting the expression of PTEN,a cellular phosphatase which inhibits the PI3K/AKT/mTOR pathway.Pten is a classical tumor suppressor gene,and tumor Tregs continuously express high level of PTEN to maintain the immune tolerance of the tumor.Dysregulation of the PD1/PTEN pathway can be an important contributor to autoimmune pathogenesis,as has been shown in rheumatoid arthritis,systemic lupus erythematosus,and multiple sclerosis.PD1 and its ligand PD-L1 were reported to be decreased in the PBMCs of ITP patients,which suggested the important role of PD1 pathway in the pathogenesis of ITP.We hypothesized that PD1/PTEN signaling pathway plays an important role in maintaining immune tolerance in ITP by regulating T-cell homeostasis.Indirubin may regulate the number and function of Tregs and Teffs,thus maintaining the immune tolerance of ITP,and have therapeutic potential in ITP.Objectives:(1)To investigate the effect of Indirubin on the number and function of Tregs and Teffs in ITP patients,and to clarify the regulatory effects of Indirubin on T-cell-mediated immune tolerance.(2)To explore the role of PD1/PTEN pathway in the pathogenesis of ITP,and the regulatory effect of Indirubin on PD1/PTEN pathway in ITP.Methods:(1)ITP patients and control:36 ITP patients were included,including 1 9 male patients and 17 female patients,aged 15-77 years,with a median age of 47 years,as well as 28 age and sex-matched healthy controls.Peripheral blood and plasma of the ITP patients and healthy controls were collected.(2)Establishment of active ITP mouse model:Platelets from wild-type C57/BL6 mice were injected into CD61 knockout(CD61 KO)mice for 4 weeks.Severe combined immunodeficient mice(SCID)were treated with 180 cGy radiation and then injected with spleen cells from immunized CD61 KO mice to establish the active ITP mouse model(3)Cell culturing:Peripheral blood mononuclear cells(PBMCs)and CD4+ T cells were isolated from ITP patients and healthy controls.Cultures of PBMCs or CD4+ T cells were treated with indirubin for 72 hours at a concentration of 0.01 μM,0.1 μM,0.2 μM,1 μM,2 μM,10 μM,whereas 1‰ DMSO was used as vehicle controls.(4)Flow cytometry:Flow cytometry was used to detect the number of CD4+CD25+Foxp3+Tregs,the proportion of activated CD25+ Teffs,and the apoptosis of PBMCs by Annexin-V and PI staining.(5)Tregs inhibitory function:Fresh CD4+CD25-T cells and CD4+CD25+ Tregs were magnetically isolated from PBMCs.CD4+CD25" T cells(Teffs)were labeled with CFSE.seeded at 2 × 105 cells/well,and then co-cultured with or without Tregs at a ratio of 4:1 on a 96 well-plate.Sample data was acquired for flow cytometry analysis 6 days after cells incubation with 1 μM indirubin or 1‰ DMSO.Data was analyzed using Flow Jo software.(6)The expression of PD1,PD-L1 and PTEN in ITP patients and healthy controls was detected by flow cytometry and quantitative fluorescence PCR(q-PCR).The effects of indirubin on PTEN/AKT/mTOR pathway of CD4+ T cells were analyzed by Western blot.(7)In vivo experiments:The active ITP mouse model was established(same as above),and treated with indirubin(intraperitoneal injection,40mg/kg,once a week)or corn oil as a control.Platelet numbers were monitored weekly.The SCID mice were euthanatized 5 weeks after irradiation.Spleens,thymuses and thigh bones were removed to detect the percentage of Tregs and expression of PD1 and PD-L1 by flow cytometry.Results:(1)Indirubin increased the number and suppressive function of Tregs among ITP patientsIndirubin could increase the number of Tregs in the culture of PBMCs from ITP patients at the dose of 0.2 μM and μM(P = 0.0335;P = 0.0224),and PBMCs from healthy controls were most responsive to indirubin in the upregulation of Tregs at the dose of 2 μM(P = 0.0488).The percentages of CD4+ T cells among the PBMCs cultured were unchanged after respective.treatments compared with DMSO controls.Dexamethasone was used as a positive control to enhance Tregs(ITP patients:P=0.0302;healthy controls:P = 0.0333).Tregs markedly suppressed the proliferation of effector T cells:in both groups(P = 0.0039;P = 0.0039).Furthermore,indirubin could significantly enhance the inhibitory function of Tregs compared with DMSO(P =0.0117).Indirubin itself had no apparent effect on the proliferation of effector T cells,suggesting that indirubin could enhance the function of Tregs in suppression of the proliferation of effector T cells.(2)Indirubin decreased fidelity of effector T cells in a dose-dependent mannerIn the culturing of PBMCs from ITP patients,we found the expression of CD25 in CD4+ T cells,CD4+CD45RA+ T cells and CD4+CD45RO+ T cells were significantly reduced after treated with indirubin in a dose-dependent manner,indicating that indirubin could decrease the activation of effector T cells in ITP.However,PBMCs from healthy controls did not show obvious changes in the activation of effector T cells after indirubin treatment.We further tested the apoptosis level of PBMCs after treated with indirubin at different doses.Indirubin could only induce the apoptosis of PMBCs from ITP patients at higher doses of 2 μM and 10 μM(P = 0.0493;P =0.0295),and the apoptosis level of PBMCs from healthy controls did not change much after indirubin treatment.We then selected 1 μM indirubin as the optimal dose as it could significantly induce the number and function of Tregs as well as dampen the activation of effector T cells without apparent induction of apoptosis.(3)Indirubin normalized decreased PD1 and PTEN expression on CD4+ T cells of ITP patientsWe found reduced PD1 expression on CD4+ T cells(P = 0.0063)and increased PD1 expression on CD 14+ monocytes(P = 0.0144)of ITP patients compared with healthy controls.The mRNA expression level of PD1 on PBMCs from ITP patients was higher than that from healthy controls(P = 0.0215).Furthermore,we also tested the PD1 ligand,PDL1 expression on CD14+ monocytes and CD4+ T cells,but no significant differences were observed.PTEN,a signaling modulator downstream of PD1 activation,was significantly suppressed in ITP patients compared to healthy controls as evidenced by decreased PTEN expression on CD4+ T cells and decreased PTEN mRNA expression on PBMCs(P = 0.0393;P = 0.0429).Indirubin(1 μM)was found to significantly increase the PD1 expression on CD4+ T cells and Tregs in PBMCs from ITP patients(P = 0.0391;P = 0.0061)with a concomitant increase in PD1,PTEN mRNA expression levels(PTEN:P = 0.0269),though the increasing of PD1 mRNA expression was not significant.The PBMCs from healthy controls did not show significant changes in the PD1,PTEN mRNA expression level after indirubin treatment.(4)Indirubin ameliorated thrombocytopenia in an active murine modelFollowing radiation and transfer of anti-CD61 immune-sensitized splenocytes into SCID mice,platelet counts dropped to nadir on day 7.Indirubin(40 mg/kg)or control treatment(corn coil)was administered via intraperitoneal injections on day 13.On day 21,significantly higher platelet counts were observed in the indirubin-treated group compared to control group(728.33 × 109/L± 215.26 vs.403.33× 109/L± 111.83;P = 0.001 5).To investigate further in-depth the molecular mechanisms of therapeutic efficacy of indirubin in vivo,we quantified the percentage of CD4 CD25+Foxp3+Tregs from the spleen and thymus as well as the expression of PD1,PDL1 in the ITP mice.Compared with the control group,indirubin treatment increased splenic Tregs 35 days after splenic transfer(23 days after indirubin treatment;P = 0.005)and reduced the thymic Tregs(P = 0.129).PD1 expression on CD4+ T cells was upregulated in the spleens of indirubin-treated group(P = 0.047).Moreover,PDL1 was increased on splenic CD4+ T cells(P = 0.045)and CD80+CD86+ antigen presenting cells(APCs)after indirubin therapy(P= 0.002).(5)Indirubin regulated the homeostasis of CD4+ T cells via a PTEN/AKT/mTOR signaling pathwayWestern blot analysis of cell lysates from CD4+ T cells cultured with 1 μM indirubin revealed increased PTEN phosphorylation(P = 0.025)and decreased AKT(P = 0.0452)and mTOR phosphorylation(P = 0.0497).These results suggested that indirubin could restore the homeostasis of CD4+ T cells in a PTEN/AKT/mTOR signaling pathway which is important in the maintenance of immune tolerance in ITP.Conclusions:(1)Indirubin could increase the number and function of Tregs in cultured PBMCs from ITP patients,and decrease the activation of Teffs in a dose-dependent manner.High concentration of indirubin can induce apoptosis of PBMCs.(2)The expression of PD1 and PTEN in CD4+ T cells of ITP patients was significantly lower than that of healthy controls.Indirubin could restore the expression of PD1 and PTEN in CD4+ T cells of ITP patients and maintain the homeostasis of CD4+ T-cell through PTEN/AKT/mTOR signaling pathway.(3)Indirubin ameliorated thrombocytopenia of ITP mice,regulate the percentages of splenic and thymic Tregs cells,and restore the expression of PD1 and PD-L1 on CD4+T cells and APCs.