Passive Immune Thrombocytopenia Research Articles

Lipoprotein lipids and apolipoproteins in primary immune thrombocytopenia: Results from a clinical characteristics and causal relationship verification, potential drug target identification by Mendelian randomization analyses.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease. Cellular and systemic lipid metabolism plays a significant role in the regulation of immune cell activities. However, the role of lipoprotein lipids and apolipoproteins in ITP remains elusive. The automatic biochemistry analyser was used to measure the levels of serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), apolipoprotein A-I (apoA-I), apoB, apoE and lipoprotein a [LP(a)]. Genetic variants strongly associated with circulating lipoprotein lipids and apolipoproteins (LDL-C, apoB, TG, HDL-C and apoA-I) were extracted to perform Mendelian randomization (MR) analyses. Finally, drug-target MR and passive ITP mice model was used to investigate the potential druggable targets of ITP. Levels of HDL-C, apoA-I, decreased and LP(a) increased in ITP patients compared with healthy controls. Low HDL-C was causally associated with ITP susceptibility. Through drug-target MR and animal modelling, ABCA1 was identified as a potential target to design drugs for ITP. Our study found that lipid metabolism is related to ITP. The causative association between HDL-C and the risk of ITP was also established. The study provided new evidence of the aetiology of ITP. ABCA1 might be a potential drug target for ITP.

Mesenchymal Stromal Cells Regulate M1/M2 Macrophage Polarization in Mice with Immune Thrombocytopenia.

Mesenchymal stromal cells have shown promising effects in the treatment of immune thrombocytopenia. However, the underlying mechanisms are not fully understood. In this study, we investigated the therapeutic effects of human bone marrow mesenchymal stromal cells (hBMSCs) and analyzed their unique role in regulating the M1/M2 macrophage ratio. We established a passive immune thrombocytopenia (ITP) mouse model and showed that there was a significant M1/M2 imbalance in ITP model mice by assessing the M1/M2 ratios in the liver, spleen, and bone marrow; we observed excessive activation of M1 cells and decreased M2 cell numbers in vivo. We have shown that systemic infusion of hBMSCs effectively elevated platelet levels after disease onset. Further analysis revealed that hBMSCs treatment significantly suppressed the number of proinflammatory M1 macrophages and enhanced the number of anti-inflammatory M2 macrophages; in addition, the levels of proinflammatory factors, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), were significantly decreased in vivo, while the levels of the anti-inflammatory factor interleukin-10 (IL-10) were increased. In conclusion, our data suggest that hBMSCs treatment can effectively increase platelet counts, and the mechanism is related to the induction of macrophage polarization toward the anti-inflammatory M2 phenotype and the decrease in proinflammatory cytokine production, which together ameliorate innate immune disorders.

MST4 kinase regulates immune thrombocytopenia by phosphorylating STAT1-mediated M1 polarization of macrophages.

Primary immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disorder in which macrophages play a critical role. Mammalian sterile-20-like kinase 4 (MST4), a member of the germinal-center kinase STE20 family, has been demonstrated to be a regulator of inflammation. Whether MST4 participates in the macrophage-dependent inflammation of ITP remains elusive. The expression and function of MST4 in macrophages of ITP patients and THP-1 cells, andof a macrophage-specific Mst4-/- (Mst4ΔM/ΔM) ITP mouse model were determined. Macrophage phagocytic assays, RNA sequencing (RNA-seq) analysis, immunofluorescence analysis, coimmunoprecipitation (co-IP), mass spectrometry (MS), bioinformatics analysis, and phosphoproteomics analysis were performed to reveal the underlying mechanisms. The expression levels of the MST4 gene were elevated in the expanded M1-like macrophages of ITP patients, and this elevated expression of MST4 was restored to basal levels in patients with remission after high-dose dexamethasone treatment. The expression of the MST4 gene was significantly elevated in THP-1-derived M1 macrophages. Silencing of MST4 decreased the expression of M1 macrophage markers and cytokines, and impaired phagocytosis, whichcould be increased by overexpression of MST4. In a passive ITP mouse model, macrophage-specific depletion of Mst4 reduced the numbers of M1 macrophages in the spleen and peritoneal lavage fluid, attenuated the expression of M1 cytokines, and promoted the predominance of FcγRIIb in splenic macrophages, which resulted in amelioration of thrombocytopenia. Downregulation of MST4 directly inhibited STAT1 phosphorylation, which is essential for M1 polarization of macrophages. Our study elucidates a critical role for MST4 kinase in the pathology of ITP and identifies MST4 kinase as a potential therapeutic target for refractory ITP.

Decreased cyclooxygenase-2 associated with impaired megakaryopoiesis and thrombopoiesis in primary immune thrombocytopenia

BackgroundCyclooxygenase (COX)-2 is a rate-limiting enzyme in the biosynthesis of prostanoids, which is mostly inducible by inflammatory cytokines. The participation of COX-2 in the maturation of megakaryocytes has been reported but barely studied in primary immune thrombocytopenia (ITP).MethodsThe expressions of COX-2 and Caspase-1, Caspase-3 and Caspase-3 p17 subunit in platelets from ITP patients and healthy controls (HC), and the expressions of COX-2 and CD41 in bone marrow (BM) of ITP patients were measured and analyzed for correlations. The effects of COX-2 inhibitor on megakaryopoiesis and thrombopoiesis were assessed by in vitro culture of Meg01 cells and murine BM-derived megakaryocytes and in vivo experiments of passive ITP mice.ResultsThe expression of COX-2 was decreased and Caspase-1 and Caspase-3 p17 were increased in platelets from ITP patients compared to HC. In platelets from ITP patients, the COX-2 expression was positively correlated with platelet count and negatively correlated to the expression of Caspase-1. In ITP patients BM, the expression of CD41 was positively correlated with the expression of COX-2. COX-2 inhibitor inhibited the count of megakaryocytes and impaired the maturation and platelet production in Meg01 cells and bone marrow-derived megakaryocytes. COX-2 inhibitor aggravated thrombocytopenia and damaged megakaryopoiesis in ITP murine model.ConclusionCOX-2 plays a vital role in the physiologic and pathologic conditions of ITP by intervening the survival of platelets and impairing the megakaryopoiesis and thrombopoiesis of megakaryocytes.

Low-dose decitabine promotes M2 macrophage polarization in patients with primary immune thrombocytopenia via enhancing KLF4 binding to PPARγ promoter.

The first-line therapy is effective for the treatment of primary immune thrombocytopenia (ITP); however, maintaining the long-term responses remains challenging. Low-dose decitabine (DAC) has been adopted to treat refractory ITP, while its role in macrophage polarization has not been fully understood. We aimed to investigate the mechanistic role of DAC in M2 macrophage polarization and evaluated its therapeutic effect in ITP. The M2 monocytes were identified by flow cytometry from peripheral blood mononuclear cells in healthy controls (HCs) and ITP patients. The expression of PPARγ, Arg-1, DNMT3b and NLRP3, together with IL-10 plasma levels was measured to examine its function. Bisulfite-sequencing PCR was used to evaluate the methylation status of PPARγ promoter, and the binding affinity of KLF4 was measured by Cut&Tag. A sh-PPARγ THP-1 cell line was created to verify if low-dose DAC-modulated M2 macrophage polarization was PPARγ-dependent. The passive ITP models were used to investigate the therapeutic effects of low-dose DAC and its role in modulating polarization and immunomodulatory function of macrophages. NLRP3 inflammasome and reactive oxygen species were also tested to understand the downstream of PPARγ. The M2 monocytes with impaired immunoregulation were observed in ITP. After high-dose dexamethasone (HD-DXM) treatment, M2 monocytes increased significantly with the elevated expression of PPARγ, Arg-1 and IL-10 in CR patients. Low-dose DAC promoted M2 macrophage polarization in a PPARγ-dependent way via demethylating the promoter of PPARγ, especially the KLF4 binding sites. Low-dose DAC alleviated ITP mice by restoring the M1/M2 balance and fine-tuning immunomodulatory function of macrophages. The downstream of the PPARγ modulation of M2 macrophage polarization might physiologically antagonize NLRP3 inflammasome. Low-dose DAC promoted M2 macrophage polarization due to the demethylation within the promoter of PPARγ, thus enhanced the KLF4 binding affinity in ITP.

Production of recombinant human IgG1 Fc with beneficial N-glycosylation pattern for anti-inflammatory activity using genome-edited chickens

Intravenous immunoglobulin (IVIG) is a plasma-derived polyclonal IgG used for treatment of autoimmune diseases. Studies show that α-2,6 sialylation of the Fc improves anti-inflammatory activity. Also, afucosylation of the Fc efficiently blocks FcγRIIIA by increasing monovalent affinity to this receptor, which can be beneficial for treatment of refractory immune thrombocytopenia (ITP). Here, we generated genome-edited chickens that synthesize human IgG1 Fc in the liver and secrete α-2,6 sialylated and low-fucosylated human IgG1 Fc (rhIgG1 Fc) into serum and egg yolk. Also, rhIgG1 Fc has higher affinity for FcγRIIIA than commercial IVIG. Thus, rhIgG1 Fc efficiently inhibits immune complex-mediated FcγRIIIA crosslinking and subsequent ADCC response. Furthermore, rhIgG1 Fc exerts anti-inflammatory activity in a passive ITP model, demonstrating chicken liver derived rhIgG1 Fc successfully recapitulated efficacy of IVIG. These results show that genome-edited chickens can be used as a production platform for rhIgG1 Fc with beneficial N-glycosylation pattern for anti-inflammatory activities.

Glucocorticoid receptor modulates myeloid-derived suppressor cell function via mitochondrial metabolism in immune thrombocytopenia.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells and natural inhibitors of adaptive immunity. Intracellular metabolic changes in MDSCs exert a direct immunological influence on their suppressive activity. Our previous study demonstrated that high-dose dexamethasone (HD-DXM) corrected the functional impairment of MDSCs in immune thrombocytopenia (ITP); however, the MDSC population was not restored in nonresponders, and the mechanism remained unclear. In this study, altered mitochondrial physiology and reduced mitochondrial gene transcription were detected in MDSCs from HD-DXM nonresponders, accompanied by decreased levels of carnitine palmitoyltransferase-1 (CPT-1), a rate-limiting enzyme in fatty acid oxidation (FAO). Blockade of FAO with a CPT-1 inhibitor abolished the immunosuppressive function of MDSCs in HD-DXM responders. We also report that MDSCs from ITP patients had lower expression of the glucocorticoid receptor (GR), which can translocate into mitochondria to regulate the transcription of mitochondrial DNA (mtDNA) as well as the level of oxidative phosphorylation. It was confirmed that the expression of CPT-1 and mtDNA-encoded genes was downregulated in GR-siRNA-treated murine MDSCs. Finally, by establishing murine models of active and passive ITP via adoptive transfer of DXM-modulated MDSCs, we confirmed that GR-silenced MDSCs failed to alleviate thrombocytopenia in mice with ITP. In conclusion, our study indicated that impaired aerobic metabolism in MDSCs participates in the pathogenesis of glucocorticoid resistance in ITP and that intact control of MDSC metabolism by GR contributes to the homeostatic regulation of immunosuppressive cell function.

A Novel Allogeneic Cell Therapy That Targets Pathogenic Autoantibodies for the Treatment of Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet counts (<100x10 9/L) and increased risk of bleeding. ITP is predominantly driven by immunoglobulin G (IgG)-autoantibodies directed against platelet surface antigens resulting in platelet destruction by the spleen and/or impaired platelet production by bone marrow megakaryocytes. Approximately 60% of ITP patients have measurable levels of anti-platelet autoantibodies in plasma and about 70% of them have autoantibodies directed against the fibrinogen receptor glycoprotein (GP) IIbIIIa.Current treatment strategies for ITP include non-specific immunosuppression (steroids, rituximab), inhibition of platelet clearance (immunoglobulins, splenectomy, anti-D immunoglobulin, and the Syk inhibitor fostamatinib) and stimulation of platelet production (thrombopoietin receptor agonists). In addition, therapeutics targeting the neonatal Fc receptor, responsible for IgG recycling, are under clinical investigation for ITP. Despite these treatment options, some patients with ITP are refractory or have inadequate responses to existing therapy.Here we describe a novel allogeneic cellular therapy, entitled platelet-like cells (PLC), that specifically targets pathogenic IgG for the treatment of thrombocytopenia in ITP. PLC are produced by differentiating a human induced pluripotent stem cell (hiPSC) line into megakaryocyte-like cells (MLC) which are further processed in a proprietary bioreactor that mimics the bone marrow environment and induces the release of anucleate PLC which are then cryopreserved. Analyses of PLC demonstrate sizes ranging between 65 nm and 10 μm. Expression of GPIIbIIIa protein as measured by enzyme-linked immunosorbent assay (ELISA) is evident in all particle sizes. PLC were administered intravenously (IV) into NOD-scid IL2Rgamma null (NSG) mice to evaluate their pharmacokinetics and biodistribution. PLC were rapidly cleared from the mouse circulation and predominantly distributed to the mouse liver where they colocalized primarily with Kupffer cells. PLC clearance through the liver was significantly inhibited by pre-treating mice with liposomes expressing phosphatidylserine (PS), indicating that one mechanism of PLC clearance is dependent on PS exposure on the PLC outer plasma membrane.The ability of PLC to bind anti-GPIIbIIIa autoantibodies was evaluated in an in vitro assay where PLC were incubated with ITP patient plasmas and subsequently removed by centrifugation and filtration. The concentration of anti-GPIIbIIIa autoantibodies remaining in the plasmas decreased following PLC treatment (96.6% ± 2.4%).The ability of PLC to clear anti-human GPIIbIIIa antibodies in circulation was evaluated by using a passive mouse model of ITP. Here, a fluorescently labeled mouse anti-human GPIIbIIIa monoclonal antibody (PAB-1) was dosed intravenously (IV) into NSG mice to achieve a circulating level of antibody comparable to those observed in ITP patients. The GPIIbIIIa antibody persisted in circulation for over 24 hours following a single dose in untreated mice and did not induce clearance of mouse platelets. Following administration of PLC in the passive ITP model, a decrease in antibody fluorescence in blood was observed within 2 minutes and full antibody clearance was observed at 3 hours post dosing. Furthermore, clearance of the antibody was observed to be both dose responsive and specific since PLC treatment did not affect the antibody concentration of an isotype matched control antibody. PLC activity in clearing the anti-GPIIbIIIa antibody was observed in all particle sizes. Fluorescence quantification of liver and spleen tissue demonstrated that PLC driven anti-GPIIbIIIa antibody clearance occurred preferentially in the liver. Taken together, these data indicate that by specifically binding and rapidly removing anti-platelet antibodies from circulation, PLC may disrupt the platelet degradation mechanisms underlying ITP pathogenesis and restore platelet counts. DisclosuresPatel-Hett: PlateletBio: Current Employment. Giannini: Platelet Biogenesis: Current Employment. Peters: Platelet Biogenesis: Current Employment. Dykstra: Platelet Biogenesis: Current Employment. Lehmann: Platelet Biogenesis: Current Employment. Russo: Platelet Biogenesis: Current Employment. Kohnke: Platelet Biogenesis: Current Employment. Carpenter: Platelet Biogenesis: Current Employment. Tomczak: Platelet Biogenesis: Current Employment. Lazarus: Platelet Biogenesis: Consultancy. Masuko: Platelet Biogenesis: Current Employment. Leung: Platelet Biogenesis: Current Employment. Meredith: Platelet Biogenesis: Current Employment. Pete: Platelet Biogenesis: Current Employment. Lee: Platelet Biogenesis: Current Employment. Falb: Platelet Biogenesis: Current Employment.

Antibodies Against Macrophage CD44 Protect Platelets from FcγR-Mediated Phagocytosis: A Potential Mechanism for CD44 Antibody Amelioration of Murine Immune Thrombocytopenia

CD44 is a transmembrane protein that is widely expressed on leukocytes and other cell types. CD44 is the major receptor for hyaluronan, and CD44-hyaluronan binding plays an important role in regulating immune cell migration, inflammation, and wound repair. Interestingly, targeting CD44 with monoclonal antibodies (anti-CD44) has demonstrated therapeutic efficacy in numerous murine autoimmune disease models including passive antibody-mediated immune thrombocytopenia (ITP), where it is effective at a three log-fold-lower dose compared to intravenous immunoglobulin (IVIg). However, the anti-inflammatory mechanisms behind anti-CD44 remain largely unsolved. In passive antibody-mediated ITP, anti-platelet antibody is administered to induce thrombocytopenia by FcγR-dependent and independent mechanisms, and macrophage depletion as well as FcγR blocking antibodies protect platelets from FcγR-dependent removal. The efficacy of anti-CD44 in this model suggest to us that anti-CD44 may be interfering with platelet removal by phagocytes. Here we show that anti-CD44 inhibits macrophage FcγR-mediated phagocytosis and protects platelets from macrophage uptake in vitro. Anti-CD44 inhibited RAW 264.7 macrophage phagocytosis of antibody-opsonized platelets in a dose-dependent manner, up to near complete (>90%) inhibition as compared to isotype controls. Multiple anti-CD44 clones tested were capable of inhibiting macrophage uptake of platelets opsonized with different anti-platelet antibodies. The binding of antibody-opsonized platelets to anti-CD44-treated macrophages was not blocked, suggesting that inhibition of phagocytosis occurs at a post-FcγR binding step. Because CD44 can regulate actin polymerization by recruiting the Rho-family GTPase-activating proteins Tiam1 and Vav2, we asked whether anti-CD44 may be interfering with actin polymerization required for platelet uptake. Live-cell microscopy of RAW 264.7 macrophages transfected with LifeAct (plasmid encoding a fluorescent non-cytotoxic actin binding protein) revealed that anti-CD44 did not affect actin dynamics and filopodia formation in the absence of antibody-opsonized platelets. However, anti-CD44-treated macrophages failed to initiate phagocytic cup formation upon antibody-opsonized-platelet binding, suggesting that inhibition is upstream of actin polymerization initiated by FcγRs. Finally, the ability of anti-CD44 to inhibit platelet uptake also showed complete concordance with a blocking antibody against FcγRIIb/III, implicating that anti-CD44 may be mediating inhibition of the FcγRIII pathway. In conclusion, our data suggests that antibodies against CD44 protect platelets from macrophage phagocytosis in vitro by inhibition of FcγRs, and that inhibition occurs at a step post-binding but before phagocytic cup formation. New therapies for ITP are needed to replace current donor-derived therapeutics and irreversible splenectomy. This research helps to shed light on how anti-CD44 functions to normalize platelet counts in murine passive ITP, and may pave the way for future novel therapeutics to replace IVIg in the treatment of ITP. DisclosuresLazarus:CSL pharmaceuticals: Research Funding; St.Michael's Hosp/The Canadian Blood Services: Other: Patents on monoclonal antibodies as IVIg and anti-D replacements; Rigel pharmaceuticals: Research Funding; Momenta pharmaceuticals: Honoraria.

Mesenchymal Stem Cells Improve Platelet Counts in Mice with ITP

INTRODUCTION: Patients with chronic refractory immune thrombocytopenia (ITP) have the high risk of death and complications, which urge us to seek for new treatment alternatives. Currently, bone marrow-derived mesenchymal stem cells (BMSCs) have been extensively utilized for autoimmune diseases. However, it is still lack of evidence in vivo about the therapeutic benefits and the possible underlying mechanisms of BMSCs in ITP, which is also known as an acquired autoimmune disease. In this study, we transplanted BMSCs into a passive ITP mouse model to explore the therapeutic effects and immunosuppressive mechanisms of BMSCs in ITP disease.METHODS: The passive ITP mouse model was induced by dose-escalation injection of monoclonal antibody MWReg30. Mice were randomly divided into two groups: experimental group (2-4×106 BMSCs were injected into each ITP mouse through the tail vein on Day 3) and control group (ITP mice). PLT counts were enumerated by an automatic blood cell counter. The serum levels of cytokines (IL-10 and TGF-β1) were examined by ELISAs and the frequency of Treg cells by flow cytometry. The statistical analysis was processed by SPSS software version 19.0.RESULTS: We successfully induced a passive ITP mouse model with very low levels of PLT counts for 8 days (PLT counts were reduced to half of the initial values by 24 hours and were relatively constant throughout the study). The PLT counts in experimental group on Day 4 to Day 8 were (263±17)×109/L, (482±35)×109/L, (367±38)×109/L, (385±107)×109/L, and (317±48)×109/L, which were significantly higher than those in control group [ (159±19)×109/L, (238±20)×109/L, (175±32))×109/L, (129±7)×109/L, and (170±16)×109/L, respectively ]. The levels of serum TGF-β1 and IL-10 were significantly higher in experimental group compared with the control group [ (116.61±12.59)pg/ml and (348.89±44.49)pg/ml vs. (72.78±15.70) pg/ml and (134.61±27.59)pg/ml; p <0.05]. The ratios of Treg cells both in splenocytes and peripheral blood were significantly higher after transplantation of BMSCs [ (15.79±0.55)% and (6.37±0.45)% vs. (10.25±0.55)% and (4.80±0.26)%; p<0.05].CONCLUSIONS: BMSCs transplantation is an effective treatment for ITP in mice. The key mechanisms underlying the positive therapeutic impact may due to the BMSCs secreting suppressive cytokines TGF-β1 and IL-10 and increasing the proportion of Treg cells, which play key roles in both the maintenance of immune tolerance and the inactivation of macrophage. DisclosuresNo relevant conflicts of interest to declare.

Erythrocyte-Specific Antibodies As a Potential New Therapeutic Modality in Autoimmune and Inflammatory Disorders Beyond Immune Thrombocytopenia (ITP)

ITP is an autoimmune bleeding disorder characterized by platelet destruction and suppression of platelet production. In the majority of patients, platelet destruction due to IgG antibodies reactive with platelet GPIIbIIIa results in platelet phagocytosis by phagocytic cells in the mononuclear phagocytic system. Anti-D is an effective first-line therapy for patients with ITP and was originally brought into practice based on the ability of anti-D to competitively inhibit opsonized-platelet clearance by phagocytic cells. Similar to anti-D, an RBC-specific monoclonal antibody (TER-119) is also able to inhibit platelet clearance in a murine model of passive ITP, also ostensibly due to RBC clearance and competitive inhibition of phagocytic cells. Here, we show that TER-119 only caused measurable anemia after 3 hours post-injection while its ability to ameliorate thrombocytopenia in the passive-murine ITP model was clearly evident by 30 minutes post-injection. Further, TER-119 mediated maximal anemia 4 days after its injection, yet displayed no measurable ameliorative effects in passive-ITP at this time point. Based on these observations, we hypothesized that anti-inflammatory activity, rather than simple competitive inhibition of opsonized-platelet clearance, might be involved in this mechanism. To test this hypothesis, the ability of TER-119 to successfully treat inflammatory diseases not involving antibody-mediated splenic cellular sequestration were examined. In the K/BxN serum-transfer model of arthritis, recipient C57BL/6 mice are injected with K/BxN serum and a rapidly progressing arthritis occurs due to antibodies specific to the self-protein glucose-6-phosphate isomerase. Using this model, the same dose of TER-119 (50 μg/mouse) which was effective in murine ITP could significantly prevent the induction of inflammatory arthritis. In separate experiments, mice were allowed to develop significant arthritis for 5 days and then injected with TER-119. These arthritic mice underwent a reversal in disease severity with significant and substantial decreases in joint swelling and clinical score evident 24 hours after injection of TER-119. In fact three days after injection with TER-119, clinical scores and ankle width measurements returned to baseline. The function of the Fc region was important in the anti-inflammatory activity of TER-119 as deglycosylation of the Fc region of the antibody (known to inhibit IgG Fc region effector functions) dramatically reduced anti-inflammatory function in the arthritis model. To determine if TER-119 could also ameliorate disease activity in an unrelated (non-autoimmune) inflammatory disease model, TER-119 was also examined in a murine model of transfusion-related acute lung injury (TRALI). TRALI is a syndrome of respiratory distress primarily triggered by leukocyte-reactive antibodies from blood product transfusions and is the leading cause of transfusion-related mortality. TRALI was induced by injecting mice with the anti-MHC class I antibody (34-1-2s) which induced a rapid decrease in body temperature and induced significant lung damage, assessed by the presence of pulmonary edema. Mice pretreated with TER-119 (50 μg) underwent a rescue from hypothermia and exhibited an almost complete inhibition of lung edema. We conclude that TER-119 has significant anti-inflammatory activity and suggest that RBC specific antibodies such as anti-D may have significant therapeutic potential in inflammatory disorders beyond just ITP. DisclosuresSemple:Novartis: Consultancy; Rigel: Consultancy; Amgen: Consultancy; UCB: Consultancy. Lazarus:CSL pharmaceuticals: Research Funding; Momenta pharmaceuticals: Honoraria; Rigel pharmaceuticals: Research Funding; St.Michael's Hosp/The Canadian Blood Services: Other: Patents on monoclonal antibodies as IVIg and anti-D replacements.

TNF-α Blockade Inhibits Pro-Inflammatory Monocytes/Macrophages in ITP

Primary immune thrombocytopenia (ITP) is an acquired autoimmune inflammatory bleeding disorder with elevated expression of tumor necrosis factor-α (TNF-α) (Pehlivan M et. al. Platelets 2011). In our current study, we isolated the CD14+ monocytes from ITP patients and healthy controls, and performed qPCR assay to test the expression of TNF-α. In ITP patients positive for anti-GPIIbIIIa antibody, monocytes expressed significantly higher levels of TNF-α than that in anti-GPIIbIIIa antibody negative patients or healthy controls, indicating that TNF-α may play an important role in anti-GPIIbIIIa antibody mediated Fc-dependent thrombocytopenia.Monocytes and macrophages are the major cell types involved in Fc-dependent platelet clearance in ITP. Monocytes can be divided into three groups based on their CD16 expressions, including the classical (CD14++CD16−), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocytes. Macrophages consist of the classically activated M1 type (TNFα+, iNOS+) and the alternatively activated M2 type (CD206+). The CD16+ monocytes (intermediate and non-classical monocytes) and M1 macrophages are pro-inflammatory with increased TNF-α expression. In our current and previous studies, we discovered a deviation of the monocyte/macrophage system manifesting in an increased percentage of intermediate monocytes in the peripheral blood and elevated M1 macrophages in the spleens of ITP patients (Feng Q et. al. J Thromb Haemost 2017). We carried out in vitro blockade of TNF-α using the monoclonal TNF-α antibody in the peripheral blood mono-nuclear cells (PBMCs) of ITP patients and bone marrow-derived M1 macrophages induced by LPS and IFN-γ. We found that blockade of TNF-α could significantly decrease the percentage of CD16+ monocytes and lower the expression of M1 markers like iNOS, TNF, MCP1. We also isolated the classical monocytes from PBMCs and cultured with M-CSF to generate macrophages. Interestingly, CD16 could be expressed by the classical monocyte-derived macrophagesduring the cultivation, while intervention by TNF-α blockade could reduce the CD16 expression during the macrophage-induction process. This finding further indicates the pro-inflammatory role of TNF-a in the monocyte/macrophage system. Subsequently, we performed western blotting to investigate the mechanisms of TNF-α blockade in regulation of the balance of monocytes/macrophages using cultured bone marrow derived macrophages and found TNF-α blockade could induce autophagy in an Akt/PI3K/mTOR-dependent pathway.To determine the effect of TNF-α blockade on the function of monocytes/macrophages, we performed the phagocytosis assay using FITC-dextran. It was shown that TNF-α blockade could significantly dampen the phagocytic ability of monocyte-derived macrophages from ITP patients and M1 of RAW264.7 cell line. We also isolated the CD4+ T cells from ITP patients and labeled them with CFSE to test the function of monocyte-derived macrophages for stimulating T cell proliferation. The results showed that TNF-α blockade could significantly inhibit the stimulation of T cell proliferation by M1 macrophages.Finally, in the in vivo study, we established the murine passive ITP model by injecting anti-GPIIbIIIa antibody. We then performed flow cytometry to determine the subtypes of monocytes/macrophages after 7 days of injection. The ITP mice were treated with TNF-α antibody at 5mg/kg or isotype IgG control every other day. We observed that TNF-α blockade could ameliorate thrombocytopenia in the murine model by decreasing non-classical monocytes(ly6c-CD11b+CD115+) from peripheral blood and M1 macrophages(F4/80+TNFα+) from abdomen, liver and spleen. Additionally, we injected the DIR-labeled platelets into those ITP mice and found TNF-α blockade could reduce the rentention of platelets in spleen and liver. Our study showed that TNF-α blockade could decrease the antibody-mediated platelet destruction by restoring the monocyte/macrophage subsets in vitro and in vivo. Therefore, TNF-α blockade might be a promising therapeutic strategy for the management of ITP patients especially those positive for anti-GPIIbIIIa auto-antibody. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Anti-inflammatory activity of CD44 antibodies in murine immune thrombocytopenia is mediated by Fcγ receptor inhibition

Monoclonal immunoglobulin G (IgG) antibodies to CD44 (anti-CD44) are anti-inflammatory in numerous murine autoimmune models, but the mechanisms are poorly understood. Anti-CD44 anti-inflammatory activity shows complete therapeutic concordance with IV immunoglobulin (IVIg) in treating autoimmune disease models, making anti-CD44 a potential IVIg alternative. In murine immune thrombocytopenia (ITP), there is no mechanistic explanation for anti-CD44 activity, although anti-CD44 ameliorates disease similarly to IVIg. Here, we demonstrate a novel anti-inflammatory mechanism of anti-CD44 that explains disease amelioration by anti-CD44 in murine ITP. Macrophages treated with anti-CD44 in vitro had dramatically suppressed phagocytosis through FcγRs in 2 separate systems of IgG-opsonized platelets and erythrocytes. Phagocytosis inhibition by anti-CD44 was mediated by blockade of the FcγR IgG binding site without changing surface FcγR expression. Anti-CD44 of different subclasses revealed that FcγR blockade was specific to receptors that could be engaged by the respective anti-CD44 subclass, and Fc-deactivated anti-CD44 variants lost all FcγR-inhibiting activity. In vivo, anti-CD44 functioned analogously in the murine passive ITP model and protected mice from ITP when thrombocytopenia was induced through an FcγR that could be engaged by the CD44 antibody's subclass. Consistent with FcγR blockade, Fc-deactivated variants of anti-CD44 were completely unable to ameliorate ITP. Together, anti-CD44 inhibits macrophage FcγR function and ameliorates ITP consistent with an FcγR blockade mechanism. Anti-CD44 is a potential IVIg alternative and may be of particular benefit in ITP because of the significant role that FcγRs play in human ITP pathophysiology.

Effect and mechanism of low-dose chidamide on the treatment of primary immune thrombocytopenia

目的探讨低剂量（0.1 mg/kg）西达本胺治疗原发免疫性血小板减少症（ITP）的作用及机制。方法①应用C57BL/6J小鼠建立ITP被动模型，灌胃给予0、0.01、0.1、0.5、5.0 mg/kg西达本胺，观察治疗前后ITP小鼠模型外周血血小板计数。②应用C57BL/6J小鼠建立ITP主动模型，灌胃给予0.1 mg/kg西达本胺，观察治疗前后ITP小鼠模型外周血血小板计数；4周后处死小鼠，流式细胞术检测脾细胞中CD4+CD25+Foxp3+自然调节性T细胞（nTreg）比例并应用ELISA方法检测小鼠外周血IL-6水平。③分离ITP患者外周血单个核细胞，与低剂量西达本胺共培养72 h后检测nTreg细胞比例；免疫磁珠法分离CD4+CD25+调节性T细胞（Treg细胞）以及CD4+CD25−效应T细胞，将二者以1∶4比例混合共培养，加入低剂量西达本胺干预，检测Treg细胞对效应T细胞增殖的抑制作用。结果①低剂量西达本胺可明显提高ITP被动模型鼠外周血血小板水平。②低剂量西达本胺可显著提高ITP动物模型外周血血小板水平，降低出血相关死亡率。③低剂量西达本胺可显著提高ITP动物模型脾细胞中nTreg比例、降低血清IL-6水平。④低剂量西达本胺可显著提高ITP患者外周血单个核细胞培养体系中nTreg细胞比例、增强Treg细胞对效应T细胞增殖的抑制作用。结论低剂量西达本胺可促进nTreg生成、增强Treg细胞的免疫抑制功能、降低IL-6水平，促进免疫耐受，对ITP有较好的治疗作用。

Transplanting of Adipose Derived Stem Cells Up-Regulate CD4+CD25+Foxp3+ T Cells in a Murine Model of Passive Chronic Immune Thrombocytopenia

Background: Immune thrombocytopenia (ITP) is an autoimmune disorder in which antibodies and/or T cells lead to enhanced peripheral platelet destruction and reduced bone marrow platelet production. The dysregulated CD4+CD25+Foxp3+ T regulatory cells (Tregs) play a critical role in the pathophysiology of ITP. Human adipose-derived mesenchymal stem cells (hADSCs) were recently found to suppress effector T cell responses and to have beneficial effects in various immune disorders. However, it is not clear how ADSCs could affect ITP. This study is aimed to investigate the therapeutic effect of ADSCs on the treatment of ITP mice and explore the underlying mechanisms. Methods: A passive immune thrombocytopenia animal model was induced by injection of the monoclonal antiplatelet antibody MWReg30, and ADSC injection was performed on the 2nd day after ITP induction. PLT counts have been measured by an automatic blood cell counter daily after ADSC transplant for 6 days. The frequency of Tregs in the blood was analyzed by flow cytometry. mRNAs and proteins of Foxp3, TGF-β1 and IL-10 in the spleen, thymus and lymph nodes were detected by RT-PCR and western blotting respectively. Findings: Transplant of ADSCs on ITP mice led to increased PLT counts, an up-regulation of Tregs in the blood, and an up-regulation of Foxp3, TGF-β1 and IL-10 mRNA and protein in the spleen, thymus and lymph nodes as compared with the untreated ITP mice. Conclusion: ADSCs transplant may be an available method in therapy of ITP. ADSCs transplant may affect proliferation and function of Tregs in ITP, and Foxp3, TGF-β1 and IL-10 may be involved in this process. Funding: This work was supported by the Youth Program of National Natural Science Founds (8150091), The Doctoral Start Project of Natural Science Foundation of Guangdong Province(2015A030310022) and the Youth Program of Natural Science Founds of Hunan Province (2018JJ3472). Declaration of Interest: The authors declare that they have no conflict of interest. Ethical Approval: All experimental procedures conformed to the Guidelines of Animal Experiments from the Chinese Committee of Medical Ethics and were approved by the Commission of Animal Research, Sun Yat-sen University Medical Center. Raw human aspirates from patients undergoing lipectomy were collected after obtaining informed consent according to procedures approved by the Ethics Committee of the Chinese Academy of Medical Sciences and the First Affiliated Hospital of South China University.

Immunoglobulin Levels As Predictors of the Development of Chronic Disease in Pediatric Immune Thrombocytopenia

Background: Immune thrombocytopenia (ITP) is the most common acquired bleeding disorder in children. Although ~75% of these patients will go on to have spontaneous resolution of disease, up to 25% will develop chronic ITP, with significant and prolonged bleeding symptomatology and impaired quality of life. At this time, there is no definitive method to predict the development of chronic disease at the time of ITP diagnosis. Aims: We aim to identify clinical biomarkers predictive of the development of chronic ITP at the time of diagnosis among pediatric ITP patients. Methods: The clinical records of 280 pediatric ITP patients enrolled in a biological banking study at a large pediatric tertiary care referral center from July 1, 2015 to July 1, 2019 were reviewed in accordance with IRB-approved protocols. ITP diagnosis and chronicity of disease (vs. spontaneous resolution within 1 year of diagnosis) was confirmed by a pediatric hematologist, as defined by current international expert committee standards (Neunert et al, Blood, 2011;117(16):4190-207; Provan et al, Blood, 2010;115(2):168-186). Patients with non-classical ITP were excluded (1 child with drug-induced ITP in the setting of acyclovir therapy, and 1 neonate with passive ITP in the setting of maternal lupus); and patients who were lost to follow-up or had ongoing disease of &amp;lt;1 year duration were subsequently excluded. Patient characteristics including age, gender, ethnicity, presence of concurrent autoimmune disease, and time to resolution were collected. Pertinent biomarkers including immunoglobulin levels (IgG, IgA, and IgM), anti-nuclear antibody (ANA), C-reactive protein (CRP), and immature platelet fraction (IPF) which were obtained at the time of diagnosis were documented, if if obtained prior to the administration of any ITP-directed therapy, and within 2 weeks of diagnosis. Chi-squared test or Fisher's exact test was utilized to compare nonparametric categorical data. Mann-Whitney U test was used to compare nonparametric continuous data. A multivariate backwards logistic regression model was conducted to determine independent associations with the development of chronic ITP. Statistical analyses were performed using SPSS Statistics 26 (IBM, Armonk, New York). A Bonferroni correction was applied to correct for multiple comparisons. A p value &amp;lt; 0.05 was defined as statistically significant. Results: We identified 251 pediatric ITP patients with sufficient length of follow-up to define acute (&amp;lt;1 year) vs. chronic (&amp;gt;1 year) disease within our 4-year study period. This included 132 patients with acute ITP (53%) and 119 patients who went on to develop chronic ITP (47%). Mean age of diagnosis among those with acute ITP was 5.5 years, while mean age of diagnosis among those with chronic ITP was 7.7 years. Within the entire cohort, 126 (50%) were male, and 124 (49%) were of Hispanic ethnicity. Fifty-eight had ITP attributable to systemic autoimmune disease - 17% within the Acute ITP group, and 30% within the Chronic ITP group. Among all patients (n=251), 188 with evaluable biomarkers (immunoglobulin levels, ANA, CRP, or IPF) at the time of diagnosis were identified. These included 174 patients with Ig levels at diagnosis, 43 patients with ANA titers at diagnosis, 25 patients with CRP levels at diagnosis, and 78 patients with IPF at diagnosis. By univariate analysis, we found that abnormalities in immunoglobulin levels at diagnosis were significantly associated with the development of chronic ITP. Low IgG levels (p = 0.015) were more prevalent in chronic (10.3%) vs. acute ITP (1.9%). High IgG levels (p = 0.015) were more prevalent in chronic (6.7%) vs. acute ITP (1.9%). High IgM levels (p = 0.03) were more prevalent in chronic (26.5%) vs. acute ITP (13.3%). Of note, the presence of Evans syndrome (p = 0.0005) and other systemic autoimmune disease (p = 0.011) were also significantly associated with the development of chronic ITP. Subsequent multivariate analysis identified low IgG level at diagnosis to be independently predictive of chronic ITP (p = 0.043, with an odds ratio of 5.7). Conclusion: Although further study is needed within a larger international pediatric ITP cohort, the findings from this institutional study indicate that biomarkers obtained in routine clinical care at the time of ITP diagnosis, specifically immunoglobulin levels, can be utilized to help predict development of chronic disease among pediatric ITP patients. Disclosures Despotovic: Amgen: Research Funding; Dova: Honoraria; Novartis: Research Funding.

PS1494 REDUCED ANTIOXIDANT CAPACITY INDUCED PYROPTOSIS IN PLATELETS IN IMMUNE THROMBOCYTOPENIA PATIENTS

Background:The pathophysiology of primary immune thrombocytopenia (ITP) is not fully understood. Antioxidant capacitie (AOC) is reduced in ITP platelets. Mitochondrial reactive oxygen species (ROS) generation can lead to NLRP3 inflammasome activation and pyroptosis. However, the process has not been studied in ITP platelets.Aims:To assess whether the reduced AOC of platelets in ITP can affect the NLRP3 inflammasome signaling pathways and to further explore the pathophysiology of ITP.Methods:We measured mitochondrial membrane potential (MMP) of platelets from patients and healthy donors using JC‐1 by flow cytometry. Next, we investigated AOC of platelets by supplementation of 300 μM H2O2following loading with 50 μM 2’,7’‐dichlorodihydrofluorescein diacetate. ROS level as well as expression of NLRP3, ASC, pro‐caspase‐1 and p20 in platelets after stimulation with H2O2was detected. Besides, co‐localization of NLRP3 and ASC in platelets after stimulation was assessed by immunofluorescence. Blocking experiments were performed using antioxidant N‐Acetyl‐L‐cysteine, NLRP3 inhibitorMCC950 (CP‐456773) and caspase‐1 inhibitorZ‐VAD‐FMK. Furthermore, we studied the pyroptosis of platelets in passive ITP mouse model using NLRP3‐/‐ mice, Caspase‐1‐/‐ mice and wild type mice.Results:The platelets from ITP patients showed significantly reduced antioxidant capacity compared to healthy donors. A significant lower MMP and higher ROS level were detected in ITP platelets. Expression of NLRP3, ASC, pro‐caspase‐1 and p20 was obviously increased and co‐localization of NLRP3 and ASC was more evident in ITP platelets. An increased level of IL‐1β and IL‐18 were observed in culture supernatant. The application of antioxidant N‐Acetyl‐L‐cysteine (NAC), NLRP3 inhibitor MCC950(CP‐456773) and caspase‐1 inhibitor Z‐VAD‐FMK to H2O2‐stimulated ITP platelets significantly inhibit pyroptosis. The indicators of pyroptosis were down‐regulated in NLRP3‐/‐ and Caspase‐1‐/‐ mice.Summary/Conclusion:The reduced AOC of platelets in ITP results in accumulation of intracellular ROS, which contributes to the activation of NLRP3 inflammasome signaling pathways and leads to pyroptosis, which accelerates thrombocytopenia in ITP.image

Mesenchymal stem cells improve platelet counts in mice with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder. The breakdown of immune tolerance (regulatory T [Treg] cells and suppressor cytokines) plays an important role in ITP pathophysiology, especially in refractory ITP. Bone marrow-derived mesenchymal stem cells (BM-MSCs) show immunomodulatory properties and have been extensively utilized for autoimmune diseases. However, it has not been fully elucidated how BM-MSCs affect ITP. In this study, we explore the therapeutic mechanism of BM-MSCs on ITP in mice. Dose-escalation passive ITP mice were inducted by injection of MWReg30. BALB/c mice were randomly divided into two groups: ITP with BM-MSC transplantation and ITP controls. The serum levels of cytokines (interleukin 10 [IL-10] and transforming growth factor-β1 [TGF-β1]) were examined by enzyme-linked immunosorbent assays. The frequency of Treg cells in both peripheral blood and spleen mononuclear cells was analyzed by flow cytometry, and the forkhead box P3 (Foxp3) messenger RNA (mRNA) level was measured by real-time polymerase chain reaction. After BM-MSC treatment, the platelet (PLT) counts were significantly elevated. Meanwhile, cytokines (TGF-β1 and IL-10), the ratios of Treg cells, and the Foxp3 mRNA expression level were significantly higher in the BM-MSC group. Our results show that BM-MSCs can improve PLT counts mainly by secreting suppressive cytokines and upregulating Tregs, which may provide new therapeutic potential for human ITP.

Atorvastatin Restored the CD4+ T Cell Homeostasis By Regulating the Axis of Effector T Cells and Regulatory T Cells in Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder, accounting for about 1/3 of clinical hemorrhagic diseases. 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 underpin the pathophysiology of ITP.Atorvastatin (AT), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, could competitively combine with HMG-CoA reductase and inhibit the production of cholesterol, accompanying with the decrease of some intermediate metabolites, such as small GTPase. Recent studies have found that statins could regulate the homeostasis of effector T cells and Tregs in some autoimmune diseases and enhance bone marrow endothelial cell function in corticosteroid-resistant ITP. However, whether AT could target the Tregs/effector T cell-axis to restore the peripheral immune tolerance in ITP is unknown.To assess the effect of AT in ITP, CD4+ T cells were isolated magnetically from peripheral blood mononuclear cells of ITP patients and cultured with different doses of AT (0μM, 5μM, 10μM, 20μM) for 3 days. The activation of CD4+ T cells were analyzed by flow cytometry. It was shown that AT could significantly inhibit the expression of CD25 on CD4+ T cells, CD4+CD45RA+ naïve T cells and CD4+CD45RO+ memory T cells and impede the switching from CD45RA to RO dose-dependently. Moreover, AT was also effective in reducing the early activation of CD4+ T cells by decreasing the expression of CD69. The dampened activation of CD4+ T cells could be reversed after blocking AT by L-mevalonate (L-MA). These results suggested that AT can inhibit the activation of CD4+ T cells and naïve T cells in vitro.We further analyzed the influence of AT on the proliferation, apoptosis and cell cycle progression of CD4+ T cells. The isolated CD4+ T cells were labeled with CFSE and cultured with AT for 7 days. AT was observed to significantly inhibit the proliferation of CD4+ T cells in a dose-dependent manner and found to induce the apoptosis of CD4+ T cells with the cell arrest in G1 phase.In line with the previous studies about the promotion of Tregs after AT treatment, our in vitro study showed that the ratio of CD4+CD25+Foxp3+ Tregs among CD4+CD25+ T cells were elevated after AT treatment, suggesting that AT could increase the proportion of Treg in activated CD4+ T cells.Furthermore, as it was reported that AT could target some small GTPase to exert its regulation on T cells, we tested the regulation role of AT on the activation of Rho, Rac and Ras by western blot. It was shown that the expression of Ras and Rho of CD4+ T cells was decreased after AT administration in the culture system, and further influence on activation of small GTPase will be confirmed by pull-down assays.Finally, in the in vivo study, we established the murine passive ITP models by injecting anti-CD41 antibody and divided them randomly into AT group (AT 40mg/kg/d) and control group (same dose of PBS). The platelet count were detected every other day and the expression of CD25 and Foxp3 on CD4+T in thymus, lymph nodes, spleen and peripheral blood of mice were determined after 7 days. There was no difference on the expression of CD25+ on CD4+ T cells in peripheral blood, lymph nodes, thymus and spleen between the two groups. But increased number of Tregs in the lymph nodes, peripheral blood and spleen of the AT group and decreased number of Tregs in thymus were observed compared to the control group, suggesting that AT could induce the development of peripheral Tregs and facilitate the migration of Tregs from thymus to peripheral organs in the ITP murine models. Due to the short period of the mice model, we didn't observe a significant increase in the platelet number after AT treatment.Our current results showed that AT played an important role in regulating peripheral immune tolerance by inhibiting the activation, proliferation, expansion and survival of CD4+ T cells, whereas increasing the number of Tregs with dampened GTPase activation. The regulatory role of AT was recapitulated in the ITP murine models. This novel mechanism of AT underlies the potential therapeutic strategy for ITP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Antiplatelet antibody-induced thrombocytopenia does not correlate with megakaryocyte abnormalities in murine immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by increased peripheral immune platelet destruction and megakaryocyte defects in the bone marrow. Although ITP was originally thought to be primarily due to antibody-mediated autoimmunity, it is now clear that T cells also play a significant role in the disease. However, the exact interplay between platelet destruction, megakaryocyte dysfunction and the elements of both humoral and cell-mediated immunity in ITP remains incompletely defined. While most studies have focused on immune platelet destruction in the spleen, an additional possibility is that the antiplatelet antibodies can also destroy bone marrow megakaryocytes. To address this, we negated the effects of T cells by utilizing an invivo passive ITP model where BALB/c mice were administered various anti-αIIb, anti-β3 or anti-GPIb antibodies or antisera and platelet counts and bone marrow megakaryocytes were enumerated. Our results show that after 24hours, all the different antiplatelet antibodies/sera induced variable degrees of thrombocytopenia in recipient mice. Compared with naïve control mice, however, histological examination of the bone marrow revealed that only 2 antibody preparations (mouse-anti-mouse β3 sera and an anti- αIIb monoclonal antibody (MWReg30) could affect bone marrow megakaryocyte counts. Our study shows that while most antiplatelet antibodies induce acute thrombocytopenia, the majority of them do not affect the number of megakaryocytes in the bone marrow. This suggests that other mechanisms may be responsible for megakaryocyte abnormalities seen during immune thrombocytopenia.