ROCK2 inhibition attenuates CTL-mediated platelet destruction by suppressing IL-6/STAT3-driven glycolysis in primary immune thrombocytopenia

Rituximab, a monoclonal anti-CD20 antibody, has proven to be an excellent therapeutic agent for haematological diseases. Low-dose rituximab (LDR; 100 mg weekly for 4 weeks) has emerged as a promising alternative to the standard dosing (375 mg/m²) for autoimmune haematological disorders, offering comparable efficacy with reduced adverse events and costs. In this review, the authors examine LDR’s role in immune thrombocytopenia, thrombotic thrombocytopenic purpura, and autoimmune haemolytic anaemia (AIHA) in the adult population. For immune thrombocytopenia, studies demonstrate that LDR achieves high overall response rates in both patients with relapsed/refractory disease and those who are newly diagnosed, confirming its non-inferiority to standard dosing. Combination therapies including thrombopoietin agonists further enhance efficacy. Single-dose rituximab shows comparable efficacy to LDR, but reduces indirect costs derived from hospital visits. In thrombotic thrombocytopenic purpura, LDR combined with plasma exchange plus steroids reduces relapse rates, in-hospital stays, and procedures, though prospective data remains limited. For AIHA, LDR combined with steroids or other agents (such as the immunomodulatory drug, bortezomib) induces high response rates, particularly in warm AIHA. Overall, LDR exhibits favourable safety (with minimal infectious complications) and cost-effectiveness across these disorders. While current evidence supports LDR as a front-line or salvage therapy, further research is needed to optimise dosing, identify ideal candidates, and validate combination strategies in randomised trials.

Primary immune thrombocytopenia (ITP) is a condition characterized by immune-mediated platelet loss due to excessive destruction and/or insufficient production. IL-12p35 is involved in autoimmune uveitis; however, its role in ITP remains unknown. In the study, CD19+ B and CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of patients with ITP and healthy individuals. CD4+CD25- T cells were cocultured with the CD19+ B cells treated with anti-IL-12p35 antibody or IL-12p35 recombinant protein. The impact of IL-12p35 on CD19+ B cells was assessed by enzyme-linked immunosorbent assay, Western blotting, quantitative real-time PCR and flow cytometry. PBMCs from patients with ITP showed lower proportions of IL-10+CD19+ B cells and decreased mRNA levels of IL-12p35-coding gene IL12A than those from healthy individuals. Anti-IL-12p35 antibody-treated CD19+ B cells could reduce the population of IL-10+ in CD19+ B cells and regulate the differentiation of CD4+CD25- T cells, while recombinant IL-12p35 demonstrated the opposite effects. Moreover, the increased IL-10+ B-cell population and HIF-1α expression in CD19+ B cells induced by recombinant IL-12p35 were reversed by HIF-1α and JAK2/STAT3 inhibitors. In conclusion, CD19+IL-10+ B cells induced by IL-12p35 regulate CD4+ T-cell subsets in patients with ITP via the JAK2/STAT3/HIF-1α signalling pathway.

B-Cell Distribution in Immune Thrombocytopenia and Its Relation to Ageing

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

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.

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

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.

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

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.

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.

: 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.

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.

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].

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.

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 (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.