Role In Immune Thrombocytopenia Research Articles

β2-adrenergic receptor agonist corrects immune thrombocytopenia by reestablishing the homeostasis of T cell differentiation

BackgroundThe sympathetic nerve is known to regulate immune responses in autoimmunity. Aberrant T cell immunity plays a vital role in immune thrombocytopenia (ITP) pathogenesis. The spleen is the primary site of platelet destruction. However, little is known whether and how splenic sympathetic innervation and neuroimmune modulation contribute to ITP pathogenesis. ObjectivesTo determine the sympathetic distribution in the spleen of ITP mice and the association between splenic sympathetic nerves and T cell immunity in ITP development, and to evaluate the treatment potential of β2-adrenergic receptor (β2-AR) in ITP. MethodsChemical sympathectomy was performed in an ITP mouse model with 6-hydroxydopamine and treated with β2-AR agonists to evaluate the effects of sympathetic denervation and activation. ResultsDecreased sympathetic innervation in the spleen of ITP mice was observed. Significantly increased percentages of Th1 and Tc1 cells and reduced percentages of regulatory T cells (Tregs) were also observed in ITP mice with chemical sympathectomy (ITP-syx mice) relative to mice without sympathectomy (controls). Expression of genes associated with Th1, including IFN-γ and IRF8, was significantly upregulated, whereas genes associated with Tregs, including Foxp3 and CTLA4, were significantly downregulated in ITP-syx mice compared with controls. Furthermore, β2-AR restored the percentage of Tregs and increased platelet counts at days 7 and 14 in ITP mice. ConclusionOur findings indicate that decreased sympathetic distribution contributes to ITP pathogenesis by disturbing the homeostasis of T cells and that β2-AR agonists have potential as a novel treatment for ITP.

Accessory spleens recapitulate the immune microenvironment of the main spleen in immune thrombocytopenia.

Accessory spleens (AcS) may play a relevant role in immune thrombocytopenia (ITP) and possibly contribute to ITP relapse following splenectomy. Little is known about the immune microenvironment of AcS in ITP. To address this issue, we compared the histological features of eight matched AcS and main spleen (MS) samples, obtained from adult patients with primary ITP. AcS and MS had overlapping immune architectural features and lymphoid composition, suggesting that similar immunologic events occur in AcS and MS of ITP. These findings may have implications for the potential mechanisms of AcS-mediated ITP relapse. Further studies are needed to confirm these results and to dissect spleen immunity in ITP.

A functional spleen contributes to afucosylated IgG in humans

As a lymphoid organ, the spleen hosts a wide range of immune cell populations, which not only remove blood-borne antigens, but also generate and regulate antigen-specific immune responses. In particular, the splenic microenvironment has been demonstrated to play a prominent role in adaptive immune responses to enveloped viral infections and alloantigens. During both types of immunizations, antigen-specific immunoglobulins G (IgGs) have been characterized by the reduced amount of fucose present on N-linked glycans of the fragment crystallizable (Fc) region. These glycans are essential for mediating the induction of immune effector functions. Therefore, we hypothesized that a spleen may modulate humoral responses and serve as a preferential site for afucosylated IgG responses, which potentially play a role in immune thrombocytopenia (ITP) pathogenesis. To determine the role of the spleen in IgG-Fc glycosylation, we performed IgG subclass-specific liquid chromatography–mass spectrometry (LC–MS) analysis of Fc glycosylation in a large cohort of individuals splenectomized due to trauma, due to ITP, or spherocytosis. IgG-Fc fucosylation was consistently increased after splenectomy, while no effects for IgG-Fc galactosylation and sialylation were observed. An increase in IgG1- and IgG2/3-Fc fucosylation level upon splenectomy has been reported here for the first time, suggesting that immune responses occurring in the spleen may be particularly prone to generate afucosylated IgG responses. Surprisingly, the level of total IgG-Fc fucosylation was decreased in ITP patients compared to healthy controls. Overall, our results suggest a yet unrecognized role of the spleen in either the induction or maintenance of afucosylated IgG responses by B cells.

Vincristine-Loaded Platelets Coated with Anti-CD41 MAbs: A New Proposal for the Treatment of Immune Thrombocytopenia

Objective: Immune thrombocytopenia (ITP) is an autoimmune disorder in which platelet-reactive autoantibodies accelerate the destruction of platelets. Macrophages play a crucial role in ITP through IgG-Fcγ receptor-mediated platelet clearance. Patients responding to initial therapies often troubled with frequent relapses, severe reverse reactions and poor life quantity. Therefore, searching new effective strategies for ITP treatment is in urgent need.Methods: In this study, platelets coated with anti-CD41 antibodies, which can mimic autoantibody-binding platelets in ITP patients, were used as a smart drug delivery system to load vincristine (VCR). Vincristine-loaded platelets coated with anti-CD41 mAbs (CD41-VLP) can selective release VCR in macrophages through IgG-Fcγ receptor-mediated phagocytosis. Scanning electron microscopy (SEM), dynamic light scattering (DLS), and platelet aggregation assays were performed to evaluate the morphological and functional changes of CD41-VLP. The encapsulation efficiency, drug loading and intracellular VCR concentration were determined using high performance liquid chromatography (HPLC). Macrophages were derived from THP-1 induced by phorbol 12-myristate 13-acetate (PMA). The expression of cell differentiation antigen (CD11b and CD14) and was detected by flow cytometry (FCM). The growth inhibition of CD41-VLP on macrophages was detected by Cell Counting Kit-8 (CCK-8) assay and the macrophage apoptosis was quantified by FCM. The possible mechanism of CD41-VLP on macrophages was further investigated by western blotting.Results: platelets coated with anti-CD41 antibodies could load VCR with high drug loading and encapsulation efficiency. There is no significant difference in morphology and function between platelets and CD41-VLP. Meanwhile, VCR was released from CD41-VLP in a pH-triggered behavior and VCR would be rapidly released in an acidic condition. The THP-1 derived macrophages was detected with a strong ability of phagocytosis and a high expression of CD11b and CD14. In the group of macrophages treated with CD41-VLP, intracellular concentration of VCR was significant increased compared with other groups, which demonstrates that Cd41-VLP can selectively deliver VCR to macrophages. In the CD41-VLP group, the growth inhibition rate was also much higher than other groups and the apoptosis rate was significant increased compared with other groups. These results suggest that CD41-VLP can enhance the cytotoxicity of VCR against macrophages. Furthermore, In the CD41-VLP group, the expressions of pro-apoptotic proteins,including Bax, caspase-3 and CyclinB1, were significantly increased and the expression of Bcl-2, anti-apoptotic protein, was decreased. This finding indicates that CD41-VLP might improve the effects of VCR on macrophages through regulating the expression of apoptosis-related proteins.Conclusion: CD41-VLP can improve the cytotoxicity of VCR against macrophages, thus establishing a new targeted therapy for ITP treatment. DisclosuresNo relevant conflicts of interest to declare.

Possible Role of CD11a in Primary Immune Thrombocytopenia Patients on Immunosuppressive Therapy

Background and ObjectivesImmune thrombocytopenia (ITP) is one of the autoimmune diseases that presented by thrombocytopenia and increased risk of bleeding. Etiology of immune thrombocytopenia (ITP) is very complex. Lymphocyte function associated antigen-1 (LFA-1) plays important role in ITP. The aim of this study was evaluation of expression of CD11a on lymphocytes to explore its possible role in primary ITP patients also, regarding severity and response to immunosuppressive treatment.Patients and MethodsThis is a cross-sectional case-control study. Forty adult patients aged (18:58) years, 29 females and 11 males were enrolled as newly diagnosed primary ITP. Forty age and sex matched control subjects were randomly selected. The expression of CD11a on lymphocyte subpopulations (CD3+ T cells, CD3+CD4+ T cells and CD19+ B cells) was analyzed by flowcytometry at the start of the study and after 6 months of follow-up.ResultsThe mean fluorescence intensity (MFI) of CD11a on CD3+ T and CD19+ B lymphocytes was significantly highly increased in ITP patients compared to healthy controls while MFI of CD11a on CD3+ CD4+Tclls was non-significant. MFI of CD11a on CD3+ and CD19+ B lymphocytes showed non-significant elevation with platelet count or bleeding score. MFI of CD11a on CD3+ showed significant highly increased level in refractory ITP compared with responder cases.ConclusionCD11a had possible role in the pathogenesis of ITP. Immunosuppressive therapy in ITP did not affect the level of CD11a expression on T and B lymphocytes. Levels of CD11a do not reflect the severity of ITP neither platelet count nor bleeding score. Increased MFI of CD11a in CD3+T lymphocytes of ITP patients may cause resistance to immunosuppressive therapy.

Effects of Eltrombopag on In Vitro Macrophage Polarization in Pediatric Immune Thrombocytopenia.

Immune Thrombocytopenia (ITP) is an autoimmune disease characterized by autoantibodies-mediated platelet destruction, a prevalence of M1 pro-inflammatory macrophage phenotype and an elevated T helper 1 and T helper 2 lymphocytes (Th1/Th2) ratio, resulting in impairment of inflammatory profile and immune response. Macrophages are immune cells, present as pro-inflammatory classically activated macrophages (M1) or as anti-inflammatory alternatively activated macrophages (M2). They have a key role in ITP, acting both as effector cells, phagocytizing platelets, and, as antigen presenting cells, stimulating auto-antibodies against platelets production. Eltrombopag (ELT) is a thrombopoietin receptor agonist licensed for chronic ITP to stimulate platelet production. Moreover, it improves T and B regulatory cells functions, suppresses T-cells activity, and inhibits monocytes activation. We analyzed the effect of ELT on macrophage phenotype polarization, proposing a new possible mechanism of action. We suggest it as a mediator of macrophage phenotype switch from the M1 pro-inflammatory type to the M2 anti-inflammatory one in paediatric patients with ITP, in order to reduce inflammatory state and restore the immune system function. Our results provide new insights into the therapy and the management of ITP, suggesting ELT also as immune-modulating drug.

Reduced intracellular antioxidant capacity in platelets contributes to primary immune thrombocytopenia via ROS-NLRP3-caspase-1 pathway

Primary immune thrombocytopenia (ITP) is a common acquired autoimmune hemorrhagic disease characterized by a low platelet count and increased risk of bleeding. However, some patients do not respond well to current therapeutic approaches. Further studies on pathogenesis and pathophysiology of ITP are needed to discover new therapeutic targets. We explored the role of enhanced intracellular oxidative stress and NLRP3 inflammasome activation of platelets in ITP. The expression of NLRP3 inflammasome was assessed in platelets from active ITP patients and healthy donors. Both the mRNA and protein expression level of platelet NLRP3 inflammasome was upregulated in ITP patients compared with healthy donors. Besides, the elevated caspase-1 activity and increased co-localization of NLRP3 and its adaptor molecule ASC indicated activation of NLRP3 inflammasome in ITP platelets. Significantly decreased intracellular antioxidant capacity was observed in ITP platelets. H2O2 supplementation elevated the expression of NLRP3 inflammasome and increased IL-1β secretion in ITP platelets. Preincubating ITP platelets with NAC down-regulated the expression of NLRP3 inflammasome. Pretreating ITP platelets with NLRP3 inhibitor MCC950 or caspase-1 inhibitor Z-YVAD-FMK significantly reduced the proportion of pyroptotic cells in H2O2-treated ITP platelets and suppressed IL-1β secretion in supernatants. Hence, platelet NLRP3 inflammasome activation resulted from reduced intracellular antioxidant capacity plays a critical role in ITP and might have potential diagnostic or therapeutic implications.

Histology of the spleen in immune thrombocytopenia: clinical-pathological characterization and prognostic implications.

Immune thrombocytopenia (ITP) is an acquired disorder, characterized by immune-mediated platelet destruction. The spleen plays a key pathogenic role in ITP and splenectomy is a valuable second-line therapy for this disease. Little is known on ITP spleen histology and response to splenectomy is unpredictable. This study aims to characterize ITP spleen histology and assess possible predictors of splenectomy outcome. A series of 23 ITP spleens were retrospectively assessed for the following histological parameters: density of lymphoid follicles (LFs), marginal zones (MZs), T helper and cytotoxic T cells; presence of reactive germinal centers (GCs); width of perivascular T cell sheaths; and red pulp features. Clinical and histological data were matched with postsplenectomy platelet counts to assess their prognostic relevance. Three histological patterns were documented: a hyperplastic white pulp pattern, a non-activated white pulp pattern (lacking GCs), and a white pulp-depleted pattern. Poor surgical responses were associated with presplenectomy high-dose steroid administration, autoimmune comorbidities and low T follicular helper cell density. The combination of such parameters stratified patients into different splenectomy response groups. The removal of accessory spleens was also associated with better outcome. ITP spleens are histologically heterogeneous and clinical-pathological parameters may help predict the splenectomy outcome.

Tacrolimus ameliorates thrombocytopenia in an ITP mouse model.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by lower platelet count resulting from immune cells-mediated platelet clearance. Tacrolimus is an immunosuppressive agent which selectively inhibits T cell activation. Whether tacrolimus plays a role in ITP remains unclear. This study aimed to investigate the effect of tacrolimus on ITP in mice. An ITP mouse model was established by injection of rat anti-mouse integrin GPIIb/CD41 immunoglobulin and treated with tacrolimus followed by isolation of peripheral blood mononuclear cells and plasma. The mRNA expression of T-bet, GATA3, and Foxp3 was measured by RT-PCR, and level of IFN-γ, IL-12p70, IL-4, IL-13, and TGF-β in plasma was measured by ELISA. Tacrolimus inhibited antiplatelet antibody-mediated platelet clearance in ITP mouse model. Meanwhile, tacrolimus-treated ITP mice displayed a significant decrease in the mRNA expression of T-bet and plasma level of IFN-γ and IL-12p70 compared with ITP mice but without differences when compared with normal mice. Furthermore, the expression of GATA3, Foxp3, and plasma level of IL-4 and TGF-β were upregulated in tacrolimus-treated ITP mice without significant differences to normal mice (except TGF-β). Tacrolimus prevents antiplatelet antibody-mediated thrombocytopenia in ITP mice possibly through regulating T cell differentiations, suggesting it might be a novel approach for preventing ITP.

Cellular immune dysregulation in the pathogenesis of immune thrombocytopenia.

: Immune thrombocytopenia (ITP) is an acquired autoimmune hemorrhagic disease characterized by immune-mediated increased platelet destruction and decreased platelet production, resulting from immune intolerance to autoantigen. The pathogenesis of ITP remains unclear, although dysfunction of T and B lymphocytes has been shown to be involved in the pathogenesis of ITP. More recently, it is found that dendritic cells, natural killer, and myeloid-derived suppressor cells also play an important role in ITP. Elucidating its pathogenesis is expected to provide novel channels for the targeted therapy of ITP. This article will review the role of different immune cells in ITP.

A CARD9 single-nucleotide polymorphism rs4077515 is associated with reduced susceptibility to and severity of primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by a low platelet count and consequent increased risk of bleeding. The etiology underlying this condition remains poorly understood. The aim of this study is to evaluate the association of a single nucleotide polymorphism (SNP) rs4077515 in the caspase recruitment domain-containing protein 9 (CARD9) gene with the pathogenesis and therapy of ITP. Two hundred ninety-four patients with ITP and 324 age-matched healthy participants were recruited in this case-control study. Genotyping of CARD9 rs4077515 polymorphism was performed by Sanger sequencing. Our results revealed that a polymorphism rs4077515 in CARD9 gene is associated with decreased risk of susceptibility to and severity of ITP (susceptibility: codominant, AA vs. GG, OR = 0.175, 95% CI = 0.054-0.776, p = 0.001; recessive, GG + AG vs. AA, OR = 6.183, 95% CI = 2.287-16.715, p < 0.001; severity: allele, A vs. G, OR = 0.685, 95% CI = 0.476-0.985, p = 0.041; codominant, AG vs. GG, OR = 0.571, 95% CI = 0.350-0.931, p = 0.025; dominant, AA + AG vs. GG, OR = 0.558, 95% CI = 0.343-0.907, p = 0.019). The existence of the allele A, the mutant AA genotype and the heterozygous AG genotype of CARD9 rs4077515, plays a protective role in ITP. However, CARD9 rs4077515 polymorphism had no effect on corticosteroid sensitivity or refractoriness of ITP.

PS1497 NEUTROPHIL EXTRACELLULAR TRAPS (NETS), BLOOD CELL COUNTS AND THROMBOTIC RISK IN ITP PATIENTS

Background:Patients with immune thrombocytopenia (ITP) are at increased risk of vascular events (VE), but the precise mechanisms of this prothrombotic state remain largely unknown. Neutrophil extracellular traps (NETs) are DNA‐containing structures released by neutrophils that are increasingly being reported in patients with infection and thrombosis associated with various autoimmune and non‐immune disorders. However, the contribution of NETs in the prothrombotic state in ITP patients is largely unknown.Aims:To determine if ITP patients have enhanced intrinsic potential for NET formation and to evaluate their prothrombotic role in ITP, their correlation with platelet activation, and also with specific therapeutic approaches.Methods:Sixty‐three ITP patients at different stages of disease and 30 healthy controls were included. NETs were evaluated by quantifying cell free DNA (cfDNA) using SYTOX Green, and by assessing the levels of Citrullinated Histone H3 complexed to DNA (H3Cit‐DNA) via a sandwich ELISA. Platelet and white blood cell (WBC) counts were assessed by a cell counter (Sysmex), and platelet glycoproteins, size, and degranulation were evaluated by flow cytometry.Results:The levels of cfDNA and H3Cit‐DNA in the peripheral blood of ITP patients were higher than in controls (p = 0.006, and p = 0.001, respectively). The percentage of patients with detectable H3Cit‐DNA complexes (optical density &gt;0.199) was significantly different to that of controls (26.2% vs. 6.7%; p = 0.029). In patients, positivity for H3Cit‐DNA correlated with higher cfDNA (Pearson correlation: R = 0.407; p = 0.01). No significant differences in the levels of cfDNA or H3Cit‐DNA were detected between the patients in remission (n = 24) and those with active ITP (n = 39). Seven ITP patients (11.1%) had experienced previous VE and presented with increased cfDNA (0.207 vs. 0.152 ng/ml; p = 0.034), WBC count (9.6 vs. 8.0 x109/L; p = 0.037), and neutrophil count (5.4 vs. 4.3x109/L; p = 0.027) compared to patients with no history of thrombotic events. Only one of the patients with VE was under low dose prednisone. Previous thrombosis was neither associated to H3Cit‐cfDNA positivity, platelet counts, mean fluorescence intensity of platelet glycoprotein expression (GPIba, aIIb), size (FSC), nor percentage of degranulated platelets (CD62, CD63). When we evaluated whether other acquired risk factors correlated with cfDNA, neither previous splenectomy, hypertension, hypercholesterolemia, smoking, age, nor diabetes correlated with cfDNA. Notably, treatment with thrombopoietin receptor agonists, corticosteroids, or immunosuppresants at sampling were neither related to circulating NETs cfDNA or H3Cit‐DNA) nor to platelet activation.Summary/Conclusion:Our results show that plasma NET levels are elevated in ITP patients, and that this increase is especially marked in patients who have suffered from a vascular event. The fact that patients with previous thrombosis had increased WBC and neutrophil counts suggests that there may be more NETosis in these individuals, probably indicating a dysregulation between production and clearance. Other factors (platelet activation, acquired thrombotic risk factors or current therapy) did not seem to influence NET formation or platelet degradation. Similar to patients with cancer and myeloproliferative disorders, where leukocytosis has been associated with increased thromboembolic risk, we propose an association between NET generation and neutrophil counts leading to increased risk of thrombosis in ITP, which may provide new therapeutic targets to combat VE in ITP.Funding: ISCIII and FEDER [PI17/00051].

PD-1/PD-L Pathway Potentially Involved in ITP Immunopathogenesis.

The binding of programmed death 1 (PD-1) to its ligands PD-L1 and PD-L2 on antigen-presenting cells turns off autoreactive T cells and induces peripheral tolerance. Aberrant PD-1/PD-L signalling could result in a breakdown of peripheral tolerance and lead to autoimmune diseases. In this study, we detected PD-1 and PD-L expression on T cells and dendritic cells (DCs) in immune thrombocytopenia (ITP) patients with active disease by flow cytometry. The effects of PD-L1-Fc fusion protein (PD-L1-Fc) on T cells and on secretion of interferon-γ (IFN-γ) and interleukin-2 (IL-2) were detected by flow cytometry and enzyme-linked immunosorbent assay, respectively. Compared with healthy controls, PD-1 expression was significantly increased in CD4+ T cells and CD8+ T cells from patients with active ITP. However, PD-L1 expression on monocyte-derived DCs was lower in patients with active ITP than in healthy controls. In vitro assays revealed that PD-L1-Fc increased T cell apoptosis, inhibited activation and proliferation of CD4+ T cells and CD8+ T cells and decreased IFN-γ and IL-2 secretion in patients with active ITP. These results suggest that the aberrant PD-1/PD-L negative co-stimulatory pathway may play a role in ITP. Enhancing PD-1/PD-L signalling might be a promising therapeutic approach for ITP patients by enhancing T cell apoptosis, inhibiting T cell activation and proliferation and reducing secretion of inflammatory factors.

Vincristine-loaded platelets coated with anti-CD41 mAbs: a new macrophage targeting proposal for the treatment of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder in which platelet-reactive autoantibodies accelerate the destruction of platelets. Macrophages play an important role in ITP through Fc receptor (FcR)-mediated antigen presenting and platelet clearance. In this study, a novel drug delivery system of vincristine-loaded platelets coated with anti-CD41 mAbs (CD41-VCR-PLT, CD41-VLP) was successfully established. The therapeutic effects and safety of CD41-VLP in vitro and in vivo were evaluated, and the possible mechanism was also explored. The results showed that PLT-CD41 could load VCR with high drug loading (DL) and encapsulation efficiency (EE), which were up to 41.16 ± 1.92% and 60.73 ± 2.79%, respectively, where platelets had no obvious morphological or functional changes. CD41-VLP could facilitate vincristine accumulation in macrophages, where the intracellular VCR concentration was 30.72 ± 3.11% at 72 h, which was significantly increased compared with the other groups (P < 0.01), thus inhibiting macrophage cell viability and inducing apoptosis. The cell viability inhibition rate and total apoptosis rate were 73.06 ± 5.26% and 69.70 ± 4.26%, respectively, both much higher than those of the other groups (P < 0.05). In the ITP mouse model, CD41-VLP increased the platelet count in peripheral blood, which was 720 ± 197.98 × 109 L-1, and significantly improved the platelet count compared with that in the VCR group (P < 0.05); moreover, it reduced the systemic toxicity and peripheral neurotoxicity of vincristine. The possible mechanism was that CD41-VLP could precisely target M1 macrophages in spleen and liver tissues through FcγR, thus reducing the platelet destruction caused by M1 macrophages. Therefore, CD41-VLP provides a new targeted therapy for ITP treatment.

Changes of IL-21 and Its Mediated JAK/STAT Signaling Pathway in Patients with Immune Thrombocytopenia

To explore the correlation between JAK/STAT signaling pathways and pathogenesis of immune thrombocytopenia(ITP). Twenty-six newly-diagnosed ITP patients was included in this study. They all meet the clinical and hematological criteria for the diagnosis of ITP, and patients with coronary heart disease, severe refractory hypertension, diabetes or with severe liver or kidney function incompetence were ruled out. 24 healthy control without autoimmune diseases, viral infectious diseases and with normal liver and kidney functions were also included. The expressions of Jak3, p-Jak3 mRNA, Stat3, and p-Stat3 were tested and the changes in levels of IL-21 mRNA, IL-21 cell secretion after DEX intervention and AG490 blockade were measured. Compared with the healthy control, patients with ITP had significantly high expressions of Jak3, p-Jak3 mRNA, Stat3 and p-Stat3 protein, which significantly reduced after AG490 blocking (P<0.01). The expression of IL-21 mRNA and the secretion of IL-21 obviously decreased after DEX intervention, but increased after AG490 blocking(P<0.01). The pathogenesis of ITP associates with the activation of JAK/STAT signaling pathways, and IL-21-mediated JAK/STAT signaling pathways play regulatory role in ITP.

Autoantibodies to thrombopoietin and the thrombopoietin receptor in patients with immune thrombocytopenia.

Autoantibodies to thrombopoietin (TPO, also termed THPO) or the TPO receptor (cMpl, also termed MPL) could play a pathological role in immune thrombocytopenia (ITP). In this study, we tested for autoantibodies against TPO, cMpl, or the TPO/cMpl complex in ITP and other thrombocytopenic disorders. Using an inhibition step with excess TPO in fluid-phase to improve binding specificity, the prevalence of anti-TPO autoantibodies was: active ITP: 9/32 (28%); remission ITP: 0/14 (0%); non-immune thrombocytopenias: 1/10 (10%); and healthy controls: 1/11 (9%). Similarly, using an inhibition step with excess cMpl, the prevalence of specific anti-cMpl autoantibodies was: active ITP: 7/32 (22%); remission ITP: 1/14 (7%); non-immune thrombocytopenias: 3/10 (30%); and healthy controls: 0/11 (0%). Two active ITP patients had autoantibodies against the TPO/cMpl complex, but not against TPO or cMpl alone. Anti-TPO or anti-cMpl autoantibodies were found in 44% of ITP patients, and in 40% of patients with other thrombocytopenic disorders. These autoantibodies did not correlate with ITP disease severity or number of ITP treatments received; however, in this cohort, 3 patients failed to respond to TPO receptor agonist medications, and of those, 2 had anti-TPO autoantibodies. This suggests that anti-TPO and anti-cMpl autoantibodies are associated with thrombocytopenia, and may be clinically relevant in a subset of ITP patients.

Progranulin facilitates the increase of platelet count in immune thrombocytopenia

IntroductionProgranulin (PGRN) is emerging as a critical immune mediator involved in a variety of autoimmune disorders. However, its role in immune thrombocytopenia (ITP) remains unclear. Materials and methodsIn this study, the enzyme-linked immunosorbent assay was used for determining the plasma levels of PGRN in ITP patients vs. healthy controls. In addition, the role of PGRN in ITP was investigated in two kinds of ITP murine models. Further, we explored whether PGRN functioned by affecting the number of T regulatory cells (Tregs) using flow cytometry. ResultsWe first observed that plasma levels of PGRN were significantly elevated in ITP patients (n = 52) compared to healthy controls (n = 40), and the levels of PGRN declined in patients after receiving treatment. Additionally, we found a negative correlation between plasma PGRN levels and platelet count of ITP patients, suggesting that PGRN is involved in the pathogenesis of ITP. PGRN deficiency further decreased platelet count in a passive-transfer ITP murine model. By contrast, administration of recombinant PGRN increased platelet count in SCID mice with chronic ITP. Meanwhile, PGRN deficiency impaired proliferation of Tregs in the passive transfer ITP murine model. These data suggest that PGRN may exert a protective role in ITP by promoting Treg proliferation. ConclusionOur study revealed a new regulator involved in the pathogenesis of ITP and provided a potential strategy for management of ITP.

Fcγ receptor expression on splenic macrophages in adult immune thrombocytopenia.

Splenic macrophages play a key role in immune thrombocytopenia (ITP) pathogenesis by clearing opsonized platelets. Fcγ receptors (FcγR) participate in this phenomenon, but their expression on splenic macrophages and their modulation by treatment have scarcely been studied in human ITP. We aimed to compare the phenotype and function of splenic macrophages between six controls and 24 ITP patients and between ITP patients according to the treatments they received prior to splenectomy. CD86, human leucocyte antigen D-related (HLA-DR) and FcγR expression were measured by flow cytometry on splenic macrophages. The major FcγR polymorphisms were determined and splenic macrophage function was assessed by a phagocytosis assay. The expression of the activation markers CD86 and HLA-DR was higher on splenic macrophages during ITP compared to controls. While the expression of FcγR was not different between ITP and controls, the phagocytic function of splenic macrophages was reduced in ITP patients treated with intravenous immunoglobulin (IVIg) within the 2weeks prior to splenectomy. The FCGR3A (158V/F) polymorphism, known to increase the affinity of FcγRIII to IgG, was over-represented in ITP patients. Thus, these are the first results arguing for the fact that the therapeutic use of IVIg during human chronic ITP does not modulate FcγR expression on splenic macrophages but decreases their phagocytic capabilities.

Circulating myeloid-derived suppressor cells predict disease activity and treatment response in patients with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a disease characterized by isolated thrombocytopenia. Abnormal effector T cell activation is an important mechanism in the pathogenesis of ITP. Regulatory T cells (Treg) have a strong immunosuppressive function for T cell activation and their importance in the pathophysiology and clinical treatment of ITP has been confirmed. Myeloid-derived suppressor cells (MDSCs) are other immunosuppressive cells, which can also suppress T cell activation by secreting arginase, iNOS and ROS, and are essential for Treg cells’ differentiation and maturation. Therefore, we speculate that MDSCs might also be involved in the immune-dysregulation mechanism of ITP. In this study, we tested MDSCs and Treg cells in peripheral blood samples of twenty-five ITP patients and ten healthy donors. We found that MDSCs and Treg cells decreased simultaneously in active ITP patients. Relapsed ITP patients showed lower MDSCs levels compared with new patients. All patients received immunosuppressive treatment including dexamethasone alone or in combination with intravenous immune globulin. We found that MDSCs’ level after treatment correlated with platelet recovery. Our study is the first that focused on MDSCs’ role in ITP. Based on our results, we concluded that circulating MDSCs could predict disease activity and treatment response in ITP patients. This preliminary conclusion indicates a substantial significance of MDSCs in the pathophysiology and clinical treatment of ITP, which deserves further investigation.

Aberrant Phosphorylation of STAT3 Protein of the CD4+ T Cells in Patients with Primary Immune Thrombocytopenia

Background: Primary immune thrombocytopenia (ITP) is an autoimmune mediated bleeding disorder characterized by decreased platelet count. Dysfunctional cellular immunity is considered important in the pathogenesis of ITP. Signal transducer and activators of transcription 3 (STAT3) is critical for the differentiation of T cells, abnormal activation of STAT3 pathway has been confirmed in a variety of autoimmune diseases. However little is known in its role in ITP. The present studiesaimed to analyze the STAT3 protein phosphorylation of CD4+ T cells before / after therapy, response group versus no response group, in newly diagnosed ITP patients.Methods: Twenty-nine adult patients with newly diagnosed ITP and sixteen healthy controls enrolled in this study. The IL-6 level was measured by ELISA; the phosphorylation STAT3 protein and the percentage of Th17 cells of the CD4+ T cells were analyzed by flow cytometry; the STAT3 mRNA expression was analyzed by Real-time PCR.Results: The levels of IL-6 in the response group were higher than that in the controls. The basal level of pSTAT3 in the response group was higher than that in the no response group. The basal level of pSTAT3 correlated with the response. There was no statistical difference between the three groups both in the STAT3 mRNA expression and the percentage of Th17 cells of the CD4+ T cells.Conclusion: The higher basal level of pSTAT3 might have good response with the corticosteroids therapy. This suggested the level of pSTAT3 might has prognostic role in ITP patients. DisclosuresNo relevant conflicts of interest to declare.