Pathogenesis Of Immune Thrombocytopenia Research Articles

The Role of Lymphocyte Subsets in the Pathogenesis of Immune Thrombocytopenia in Children

The Role of Lymphocyte Subsets in the Pathogenesis of Immune Thrombocytopenia in Children

Genetic Variants in Canonical Wnt Signaling Pathway Associated with Pediatric Immune Thrombocytopenia

Through the use of genetic sequencing, molecular variants driving autoimmunity are increasingly identified in patients with chronic and refractory immune cytopenias. With the goal of discovering genetic variants that predispose to pediatric immune thrombocytopenia (ITP) or increase risk for chronic disease, we conducted a genome-wide association study in a large multi-institutional cohort of pediatric patients with ITP. Five-hundred ninety-one patients were genotyped using an Illumina Global Screening Array (GSA) BeadChip. Six variants met genome wide significance in comparison between children with ITP and a cohort of healthy children. One variant in NAV2 was inversely associated with ITP (aOR: 0.52, P=3.2x10-11). Two other variants in close proximity to NKD1 were also inversely associated with ITP (aOR: 0.43, P=8.86x10-15; aOR: 0.48, P=1.84x10-16). These genes have been linked to the canonical Wnt signaling pathway. No variants met genome-wide significance in comparison of those with ITP that self-resolved in less than 1 year versus those who developed chronic ITP. This study identifies genetic variants which may contribute to ITP risk and raises a novel pathway with a potential role in ITP pathogenesis.

Differential alterations of CXCR3, CXCR5 and CX3CR1 in patients with immune thrombocytopenia

As a versatile element for maintaining homeostasis, the chemokine system has been reported to be implicated in the pathogenesis of immune thrombocytopenia (ITP). However, research pertaining to chemokine receptors and related ligands in adult ITP is still limited. The states of several typical chemokine receptors and cognate ligands in the circulation were comparatively assessed through various methodologies. Multiple variable analyses of correlation matrixes were conducted to characterize the correlation signatures of various chemokine receptors or candidate ligands with platelet counts. Our data illustrated a significant decrease in relative CXCR3 expression and elevated plasma levels of CXCL4, 9–11, 13, and CCL3 chemokines in ITP patients with varied platelet counts. Flow cytometry assays revealed eminently diminished CXCR3 levels on T and B lymphocytes and increased CXCR5 on cytotoxic T cell (Tc) subsets in ITP patients with certain platelet counts. Meanwhile, circulating CX3CR1 levels were markedly higher on T cells with a concomitant increase in plasma CX3CL1 level in ITP patients, highlighting the importance of aberrant alterations of the CX3CR1-CX3CL1 axis in ITP pathogenesis. Spearman’s correlation analyses revealed a strong positive association of peripheral CXCL4 mRNA level, and negative correlations of plasma CXCL4 concentration and certain chemokine receptors with platelet counts, which might serve as a potential biomarker of platelet destruction in ITP development. Overall, these results indicate that the differential expression patterns and distinct activation states of peripheral chemokine network, and the subsequent expansion of circulating CXCR5+ Tc cells and CX3CR1+ T cells, may be a hallmark during ITP progression, which ultimately contributes to thrombocytopenia in ITP patients.

The role of platelet desialylation as a biomarker in primary immune thrombocytopenia: mechanisms and therapeutic perspectives.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by the destruction of platelets. Although it was long believed that the critical role of autoantibodies in platelet destruction, primarily through the Fc-dependent platelet clearance pathway, recent findings indicate that the significance of the Fc-independent platelet clearance pathway mediated by hepatocytes, thus shedding light on a previously obscure aspect of ITP pathogenesis. Within this context, the desialylation of platelets has emerged as a pivotal biochemical marker. Consequently, targeting platelet desialylation emerges as a novel therapeutic strategy in the pathogenesis of ITP. Notably, prevailing research has largely focused on antiplatelet antibodies and the glycosylation-associated mechanisms of platelet clearance, while comprehensive analysis of platelet desialylation remains scant. In response, we retrospectively discuss the historical progression, inducing factors, generation process, and molecular regulatory mechanisms underlying platelet desialylation in ITP pathogenesis. By systematically evaluating the most recent research findings, we contribute to a comprehensive understanding of the intricate processes involved. Moreover, our manuscript delves into the potential application of desialylation regulatory strategies in ITP therapy, heralding novel therapeutic avenues. In conclusion, this manuscript not only fills a critical void in existing literature but also paves the way for future research by establishing a systematic theoretical framework. By inspiring new research ideas and offering insights into the development of new therapeutic strategies and targeted drugs, our study is poised to significantly advance the clinical management of ITP.

Daucosterol regulates JAK2-STAT3 signaling pathway to promote megakaryocyte differentiation

Immune thrombocytopenia (ITP) is an autoimmune disease caused by the loss of immune tolerance to platelet autoantigens, resulting in reduced platelet production and increased platelet destruction. Impaired megakaryocyte differentiation and maturation is a key factor in the pathogenesis and treatment of ITP. Sarcandra glabra, a plant of the Chloranthaceae family, is commonly used in clinical practice to treat ITP, and daucosterol (Dau) is one of its active ingredients. However, whether Dau can treat ITP and the key mechanism of its effect are still unclear. In this study, we found that Dau could effectively promote the differentiation and maturation of megakaryocytes and the formation of polyploidy in the megakaryocyte differentiation disorder model constructed by co-culturing Dami and HS-5 cells. In vivo experiments showed that Dau could not only increase the number of polyploidized megakaryocytes in the ITP rat model, but also promote the recovery of platelet count. In addition, through network pharmacology analysis, we speculated that the JAK2-STAT3 signaling pathway might be involved in the process of Dau promoting megakaryocyte differentiation. Western blot results showed that Dau inhibited the expression of P-JAK2 and P-STAT3. In summary, these results provide a basis for further studying the pharmacological mechanism of Dau in treating ITP.

New findings and new methods on macrophages in primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease with multiple immune cells take part in the pathogenesis. Macrophages play multiple roles in both innate and adaptive immune system. The report by Jiani Mo and colleagues identified new biomarkers and explore the role of mitophagy and ferroptosis in ITP pathogenesis. Commentary on: Mo etal. Comprehensive analysis and prediction model of mitophagy and ferroptosis in primary immune thrombocytopenia. Br J Haematol 2024;204:2429-2442.

Comprehensive analysis and prediction model of mitophagy and ferroptosis in primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is linked to specific pathogenic mechanisms, yet its relationship with mitophagy and ferroptosis is poorly understood. This study aimed to identify new biomarkers and explore the role of mitophagy and ferroptosis in ITP pathogenesis. Techniques such as differential analysis, Mfuzz expression pattern clustering, machine learning, gene set enrichment analysis, single-cell RNA sequencing (scRNA-seq) and immune infiltration analysis were employed to investigate the molecular pathways of pivotal genes. Two-sample Mendelian randomization (TSMR) assessed the causal effects in ITP. Key genes identified in the training set included GABARAPL1, S100A8, LIN28A, and GDF9, which demonstrated diagnostic potential in validation sets. Functional analysis indicated these genes' involvement in ubiquitin phosphorylation, PPAR signalling pathway and T-cell differentiation. Immune infiltration analysis revealed increased macrophage presence in ITP, related to the critical genes. scRNA-seq indicated reduced GABARAPL1 expression in ITP bone marrow macrophages. TSMR linked S100A8 with ITP diagnosis, presenting an OR of 0.856 (95% CI = 0.736-0.997, p = 0.045). The study pinpointed four central genes, GABARAPL1, S100A8, LIN28A, and GDF9, tied to mitophagy and ferroptosis in ITP. It posits that diminished GABARAPL1 expression may disrupts ubiquitin phosphorylation and PPAR signalling, impairing mitophagy and inhibiting ferroptosis, leading to immune imbalance.

Identification of metabolism-related key genes as potential biomarkers for pathogenesis of immune thrombocytopenia

Immune thrombocytopenia (ITP), an acquired autoimmune disease, is characterized by immune-mediated platelet destruction. A biomarker is a biological entity that contributes to disease pathogenesis and reflects disease activity. Metabolic alterations are reported to be associated with the occurrence of various diseases. As metabolic biomarkers for ITP have not been identified. This study aimed to identify metabolism-related differentially expressed genes as potential biomarkers for pathogenesis of ITP using bioinformatic analyses.The microarray expression data of the peripheral blood mononuclear cells were downloaded from the Gene Expression Omnibus database (GSE112278 download link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE112278). Key module genes were intersected with metabolism-related genes to obtain the metabolism-related key candidate genes. The hub genes were screened based on the degree function in the coytoscape sofware. The key ITP-related genes were subjected to functional enrichment analysis. Immune infiltration analysis was performed using a single-sample gene set enrichment analysis algorithm to evaluate the differential infiltration levels of immune cell types between ITP patient and control. Molecular subtypes were identified based on the expression of hub genes. The expression of hub genes in the ITP patients was validated using quantitative real-time polymerase chain reaction analysis. This study identified five hub genes (ADH4, CYP7A1, CYP1A2, CYP8B1, and NR1H4), which were be associated with the pathogenesis of ITP, and two molecular subtypes of ITP. Among these hub genes, CYP7A1 and CYP8B1 involved in cholesterol metabolism,were further verified in clinical samples.

High dimensional proteomic mapping of bone marrow immune characteristics in immune thrombocytopenia.

To investigate the role of co-stimulatory and co-inhibitory molecules on immune tolerance in immune thrombocytopenia (ITP), this study mapped the immune cell heterogeneity in the bone marrow of ITP at the single-cell level using Cytometry by Time of Flight (CyTOF). Thirty-six patients with ITP and nine healthy volunteers were enrolled in the study. As soluble immunomodulatory molecules, more sCD25 and sGalectin-9 were detected in ITP patients. On the cell surface, co-stimulatory molecules like ICOS and HVEM were observed to be upregulated in mainly central memory and effector T cells. In contrast, co-inhibitory molecules such as CTLA-4 were significantly reduced in Th1 and Th17 cell subsets. Taking a platelet count of 30×109 L-1 as the cutoff value, ITP patients with high and low platelet counts showed different T cell immune profiles. Antigen-presenting cells such as monocytes and B cells may regulate the activation of T cells through CTLA-4/CD86 and HVEM/BTLA interactions, respectively, and participate in the pathogenesis of ITP. In conclusion, the proteomic and soluble molecular profiles brought insight into the interaction and modulation of immune cells in the bone marrow of ITP. They may offer novel targets to develop personalized immunotherapies.

HIF-1α induced by hypoxic condition regulates Treg/Th17 axis polarization in chronic immune thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired immune disorder characterized by increased platelet destruction and reduced platelet (Plt) production. Hypoxia-inducible factor-1α (HIF-1α) have regulatory effects on Treg/Th17 axis balance and may represent relevant factors in the pathogenesis of ITP. Treg/Th17 ratio, serum levels and gene expression were investigated in new diagnosed ITP (NITP) and chronic ITP (CITP). The Treg/Th17 ratio obviously decreased in CITP (P = 0.001). The ratio of Treg/Th17 was correlated with the level of HIF-1α level both in mRNA (r = 0.49, P < 0.0001) and serum level (r = 0.50, P < 0.0001). However, none statistical upregulation of HIF-1α was observed in CITP. In vitro, There was significant polarization difference of Treg/Th17 axis (P = 0.042) and Foxp3-MFI/IL17-MFI (P = 0.0003) in hypoxic condition between NITP and CITP. These findings suggest that HIF-1α induced by hypoxia plays a crucial role in the chronicity of ITP by mediating the imbalance of the Treg/Th17 axis.

Current Understanding of Immune Thrombocytopenia: A Review of Pathogenesis and Treatment Options.

The management of immune thrombocytopenia (ITP) and the prediction of patient response to therapy still represent a significant and constant challenge in hematology. ITP is a heterogeneous disease with an unpredictable evolution. Although the pathogenesis of ITP is currently better known and its etiology has been extensively studied, up to 75% of adult patients with ITP may develop chronicity, which represents a significant burden on patients' quality of life. A major risk of ITP is bleeding, but knowledge on the exact relationship between the degree of thrombocytopenia and bleeding symptoms, especially at a lower platelet count, is lacking. The actual management of ITP is based on immune suppression (corticosteroids and intravenous immunoglobulins), or the use of thrombopoietin receptor agonists (TPO-RAs), rituximab, or spleen tyrosine kinase (Syk) inhibitors. A better understanding of the underlying pathology has facilitated the development of a number of new targeted therapies (Bruton's tyrosine kinase inhibitors, neonatal Fc receptors, strategies targeting B and plasma cells, strategies targeting T cells, complement inhibitors, and newer TPO-RAs for improving megakaryopoiesis), which seem to be highly effective and well tolerated and result in a significant improvement in patients' quality of life. The disadvantage is that there is a lack of knowledge of the predictive factors of response to treatments, which would help in the development of an optimized treatment algorithm for selected patients.

Decreased Expression of IL-35 and Its Receptor Contributes to Impaired Megakaryopoiesis in the Pathogenesis of Immune Thrombocytopenia.

Recent findings have shown that the level of interleukin-35 (IL-35) is abnormal in several autoimmune diseases. Nonetheless, whether IL-35 participates in the pathogenesis of immune thrombocytopenia (ITP) remains unclear. The current study investigates whether IL-35 modulates megakaryopoiesis. The results show that IL-35 receptors are progressively expressed on bone marrow megakaryocytes during the in vitro differentiation of CD34+ progenitors. IL-35 increases the number of megakaryocyte colony-forming units through the Akt pathway. The level of bone marrow IL-35 is reduced in ITP patients, and the decreased level of IL-35 may inhibit megakaryopoiesis. Then, the potential causes of decreased IL-35 in ITP patients are explored. The primary type of cell that secretes IL-35, known as IL-35-producing regulatory T cells (iTr35), is reduced in ITP patients. Bone marrow mesenchymal stem cells (MSCs) from ITP patients exhibit an impaired capability of inducing iTr35 due to enhanced apoptosis, which may contribute to the reduced level of bone marrow IL-35 in ITP patients. Iguratimod promotes megakaryocyte development and differentiation by elevating the expression of IL-35 receptors on megakaryocytes. Iguratimod improves response rates and reduces bleeding symptoms in corticosteroid-resistant ITP patients.

E3 ubiquitin ligase casitas B-lineage lymphoma-b modulates T-cell anergic resistance via phosphoinositide 3-kinase signaling in patients with immune thrombocytopenia

BackgroundThe E3 ubiquitin ligase casitas B-lineage lymphoma-b (CBLB) is a newly identified component of the ubiquitin-dependent protein degradation system and is considered an important negative regulator of immune cells. CBLB is essential for establishing a threshold of T-cell activation and regulating peripheral T-cell tolerance through various mechanisms. However, the involvement of CBLB in the pathogenesis of immune thrombocytopenia (ITP) is unknown. ObjectivesWe aimed to investigate the expression and role of CBLB in CD4+ T cells obtained from patients with ITP through quantitative proteomics analyses. MethodsCD4+ T cells were transfected with adenoviral vectors overexpressing CBLB to clarify the effect of CBLB on anergic induction of T cells in patients with ITP. DNA methylation levels of the CBLB promoter and 5′ untranslated region (UTR) in patient-derived CD4+ T cells were detected via MassARRAY EpiTYPER assay (Agena Bioscience). ResultsCD4+ T cells from patients with ITP showed resistance to anergic induction, highly activated phosphoinositide 3-kinase–protein kinase B (AKT) signaling, decreased CBLB expression, and 5′ UTR hypermethylation of CBLB. CBLB overexpression in T cells effectively attenuated the elevated phosphorylated protein kinase B level and resistance to anergy. Low-dose decitabine treatment led to significantly elevated levels of CBLB expression in CD4+ T cells from 7 patients showing a partial or complete response. ConclusionThese results indicate that the 5′ UTR hypermethylation of CBLB in CD4+ T cells induces resistance to T-cell anergy in ITP. Thus, the upregulation of CBLB expression by low-dose decitabine treatment may represent a potential therapeutic approach to ITP.

Prevalence of anti-glycoprotein V in patients with primary immune thrombocytopenia

Background Glycoprotein (GP) V is a well-characterized immune target in Varicella-associated and drug-induced thrombocytopenia. Recent studies have focused on the involvement of anti-GP V in the mechanism of platelet clearance as a recent paradigm in the pathophysiology of immune thrombocytopenia (ITP), rather than other anti-GPs like GP IIb/IIIa, GP Ib/IX and less frequently GP Ia/IIa. Objectives This study was conducted to determine the prevalence of anti-GP V in adult ITP Egyptian patients and to detect its impact on the outcome in these patients. Patients and methods This was a cross-sectional study in which, using enzyme-linked immunosorbent assay, the level of serum anti-GP antibodies V was measured in the peripheral blood of 90 patients with primary ITP compared with 20 age-matched and sex-matched normal healthy controls and its relationship with clinical, laboratory parameters, and response to treatment. Results Our study revealed that anti-GP antibodies V level in the cases group was statistically significantly higher as compared with the control group (P=0.005). There was weak statistically significant positive correlation between platelet count and anti-GP V level antibodies in the cases group after treatment with a P value of 0.0548. However, the level of anti-GP V did not show significant correlation with the bleeding score (P=0.123), degree of thrombocytopenia (P=0.967), sex (P=0.067), bone marrow findings (P=0.448), spleen size (P=0.992), or with the response to steroid therapy (P=0.171). Conclusion This study points out that GP V is a frequent immune target in ITP and that the detection of GP-specific autoantibodies being of value in the pathogenesis of ITP. We recommend that further studies including GP V are required before ITP treatment can be tailored according to platelet autoantibody specificity.

Regulatory role of ceRNA network in B lymphocytes of patients with immune thrombocytopenia

ObjectiveHigh-throughput sequencing was used to screen expressing differences of miRNA, lncRNA, and mRNA in CD19+ B peripheral blood samples of newly diagnosed immune thrombocytopenia (ITP) patients and healthy controls. The study aimed to explore the regulatory role of ceRNA network in the pathogenesis of dysfunctional CD19 + B lymphocytes of ITP patients.MethodsCD19+ B lymphocytes were isolated from peripheral blood samples of ITP patients and their healthy counterparts. High-throughput sequencing was used to screen for the expression of miRNA, lncRNA, and mRNA of ITP patients and healthy controls, which were analysed by the ceRNA network. Moreover, qPCR was used to verify the differential expression of miRNA, lncRNA, and mRNA in ITP patients and healthy controls. The correlation between differentially expressed miRNA, lncRNA, mRNA, and B lymphocyte subsets was also analysed.ResultsThe CD19+ B lymphocytes of 4 newly diagnosed ITP patients and 4 healthy controls were sequenced and analysed. There were 65 differentially expressed lncRNA and 149 mRNA forming a ceRNA network showed that 12 lncRNA and 136 differentially expressed mRNA were closely associated. Similarly, miR-144-3p, miR-374c-3p, and miR-451a were highly expressed in ITP patients, as confirmed by qPCR, which was consistent with the high-throughput sequence results. LOC102724852 and CCL20 were highly expressed in ITP patients, while LOC105378901, LOC112268311, ALAS2, and TBC1D3F were not as compared to healthy controls, which was consistent with the high-throughput sequence results. In addition, the expression of miR-374c-3p, LOC112268311, LOC105378901, and CXCL3 were correlated with the percentage of B lymphocyte subsets.ConclusionsThe ceRNA network of miRNA, lncRNA, and mRNA in peripheral CD19 + B lymphocytes plays an essential role in the pathogenesis of ITP.

Quercetin, a key active ingredient of Jianpi Zishen Xiehuo Formula, suppresses M1 macrophage polarization and platelet phagocytosis by inhibiting STAT3 activation based on network pharmacology.

Primary immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disease, and abnormal M1 macrophage polarization participates in the pathogenesis of ITP. Jianpi Zishen Xiehuo (JZX) Formula has a good therapeutic effect on ITP. However, its key active ingredients and molecular mechanisms remain unclear. In this study, we explored the key active ingredients and potential targets of JZX in treating ITP using network pharmacology combined with in vitro experimental verification. A total of 157 active ingredients of JZX were identified from public databases, and quercetin was the most important one. One hundred sixty-five intersection targets of active ingredients in JZX, ITP, and macrophage polarization were obtained by Venn diagram. The top three potential targets were signal transducer and activator of transcription 3 (STAT3), protein kinase B (PKB/AKT) 1, and c-JUN through protein-protein interaction analysis. Molecular docking showed that quercetin had strong binding affinities with them all. In vitro experiment, CD16+ monocytes increased in ITP patients compared with healthy controls, which indicated a M1/M2 polarization imbalance in ITP. The expression levels of M1 polarization markers, CD86, CD80, and inducible nitric oxide synthase (iNOS), M1 polarization-associated cytokines, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), and antibody-opsonized platelet phagocytosis significantly increased in THP-1 macrophages stimulated with lipopolysaccharide (LPS). Quercetin markedly inhibited the expressions of M1 markers, decreased the levels of TNF-α and IL-6, and down-regulated the phosphorylated STAT3 (p-STAT3) protein, which confirmed the prediction by network pharmacology and molecular docking. Importantly, quercetin significantly reduced the phagocytosis of antibody opsonised platelet. In conclusion, quercetin suppressed platelet phagocytosis in M1 macrophages via its anti-inflammatory effects and may serve as a potential drug forthe treatment of ITP. Quercetin could be a key ingredient for JZX against ITP.

Baricitinib in Combination with High-Dose Dexamethasone As First-Line Treatment for Newly Diagnosed Immune Thrombocytopenia: A Prospective, Multicenter, Randomized Trial

Introduction: High-dose dexamethasone is among the standard initial therapeutic options for patients with newly diagnosed immune thrombocytopenia (ITP). Although most patients achieve initial remission, optimal first-line therapy remains a challenge due to the low rates of sustained response and the high incidence of corticosteroid-dependency. Antiplatelet autoantibodies and splenic macrophages play central roles in the pathogenesis of ITP. Baricitinib, a Janus-associated kinase 1/2 (JAK1/2) inhibitor, can inhibit antigen presentation by innate immune cells and subsequent T-cell activation while also alleviating B-cell abnormalities and antibody production. The results of our single-arm pilot study suggested that baricitinib might increase the rate of durable response in patients with steroid-resistant or relapsed ITP (NCT05446831, presented at EHA2023, P1594). Accordingly, we further conducted a randomized, controlled trial to evaluate the efficacy and safety of baricitinib plus high-dose dexamethasone as first-line therapy for adulthood ITP. Methods: This multicenter, open-label, randomized, controlled, phase 2 trial was conducted in 10 different tertiary medical centers in China. Adult patients with newly diagnosed, treatment-naïve, confirmed primary ITP who had a platelet count &amp;lt;30×10⁹/L, or a platelet count &amp;lt;50×10⁹/L with clinically significant bleeding, were eligible for this study. Patients were randomly assigned at a 1:1 ratio to receive baricitinib (2 mg daily for 6 consecutive months) plus high-dose dexamethasone (40 mg daily for 4 consecutive days, repeated in the case of lack of response by Day 10) or high-dose dexamethasone alone. The primary endpoint was durable response at the 6-month follow-up. Efficacy endpoints were analyzed in the intention-to-treat population. Safety profiles were analyzed in all patients receiving at least one dose of study medications. The study protocol was approved by the ethics committee or institutional review board of each participating center and was registered with ClinicalTrials.gov (NCT 05932524). Results: A total of 172 patients were screened, of which 132 patients were eligible for this study. Patients were randomly assigned to receive baricitinib plus high-dose dexamethasone (n=66) or high-dose dexamethasone monotherapy (n=66). Four patients did not receive allocated treatment, leaving 128 patients in the safety analysis set. At the 6-month follow-up, a significantly higher proportion of participants in the baricitinib plus high-dose dexamethasone group (42/66, 63.6%) than in the high-dose dexamethasone monotherapy group (25/66, 37.9%) achieved durable response (OR 2.870, 95% CI 1.416-5.816; p=0.003). Initial response was achieved by 52 (78.8%) patients in the combination group and 47 (71.2%) patients in the monotherapy group. No significant difference was observed in the median time from initial treatment to response between the two groups. Complete response (at any time during the treatment) was achieved by 40 (60.6%) patients in the combination group and 35 (53.0%) patients in the monotherapy group. Patients responding to treatment in the combination group showed significantly longer durations of response than responders in the monotherapy group. Patients in both groups reported significantly improved health-related quality of life. The most common adverse events were infections (15/64, 23.4%) and insomnia (12/64, 18.8%) in the combination group, and insomnia (10/64, 15.6%) and anxiety or mood disorders (9/64, 14.1%) in the monotherapy group. No grade 4 or worse adverse events or treatment-related deaths were reported in either group. No thrombotic events, major adverse cardiovascular events (MACE), treatment-related bleeding or anemia were observed. Treatment discontinuation due to an adverse event was reported in 2 (3.1%) patients in the combination group and 3 (4.7%) patients in the monotherapy group. Conclusions: This is the first study reporting JAK inhibition as a first-line therapy for ITP. Baricitinib at 2 mg daily plus high-dose dexamethasone was well tolerated in patients with newly diagnosed ITP and might contribute to achieving a higher rate of durable response.

Adiponectin Promote TPO Response for Treating ITP By Relating Rab6A-myosin9 Trafficking Vehicle Carrying c-Mpl to Cell Surface

Impaired megakaryocyte maturation is one of the pathogenic mechanisms of primary immune thrombocytopenia (ITP). Exploring the regulatory mechanisms of megakaryocyte maturation is important for the clinical treatment of ITP. The thrombopoietin (TPO) receptor c-Mpl on the membrane surface of megakaryocytes plays a critical role in the cellular reception of TPO signals and megakaryocyte maturation. But how the distribution of c-Mpl on the membrane surface of megakaryocytes influences the pathogenesis of ITP and the specific regulatory mechanisms are still unclear. In this project, we found that the distribution of c-Mpl on the membrane surface of megakaryocytes of ITP patients was significantly reduced, confirming that the abnormal distribution of c-Mpl is associated with the pathogenesis of ITP. Further, we applied immunoprecipitation and protein profiling to find the intracellular c-Mpl transport-related protein molecules. Motor protein myosin 9 and G protein-associated protein Rab6A were screened out. Next, we down-regulated the expression of myosin 9 and Rab6A, proved that aberrant expression of both leads to reduced transportation of the c-Mpl to membranes and impaired megakaryocyte maturation, identifying the regulatory complex for c-Mpl transport to membranes was composed of myosin 9 and Rab6A. Moreover, we confirmed that the formation of the above complex is dependent on GTP activation of Rab6A by transient overexpression of wild-type Rab6A (wt), dominant-negative (T27N) and GTPase-deficient (Q72L) Rab6A, clarified the working mechanism of how the Rab6A-myosin9 trafficking vehicle regulate c-Mpl transport to membranes. At the same time, we demonstrated that the abnormally low expression of Adiponectin in the plasma of ITP patients affects the transportation of the c-Mpl to membranes by inhibiting the interaction between Rab6A and myosin 9, thereby reducing the responsiveness of megakaryocytes to TPO and inhibiting the activation of its corresponding intracellular signaling pathways. Finally, using a mouse model of ITP disease, we demonstrated that Adiponectin could mediate the transportation of the c-Mpl to membranes by regulating the Rab6A-myosin9 complex and can improve the efficacy of TPO in the treatment of ITP. In conclusion, the present study identified a novel trafficking complex mediating the transportation of the c-Mpl to membranes, clarified the abnormal mechanism of this complex in ITP, and confirms that Adiponectin can regulate this complex to improve the efficacy of TPO, which provides a new idea for the clinical treatment of ITP.

Bone Marrow CD8 + Tissue-Resident Memory T Cell Promote B Cell Differentiation and Inhibit Platelet Production in Patients with Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease in which enhanced anti-platelet T cell activity and dysfunction of CD8 + T cells are involved(Audia et al., 2013; Semple &amp; Freedman, 1991). Persistent secretion of antibodies by long-lived plasma cells and memory B cells in the bone marrow contributes to ITP pathogenesis (Roeser, Lazarus, &amp; Mahevas, 2023). However, the role of memory T cells, especially tissue-resident memory T cells (TRM), in ITP remains underexplored. TRM develop in specific tissues and play crucial role in local immunity. TRM exert their effects through mechanisms including the induction of local humoral responses, contributing to autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (Chang et al., 2021; Gu, Song, Roh, Jung, &amp; Kim, 2021). Bone marrow contains one of the largest pools of memory CD8 + T cells. However, the involvement of bone marrow TRM in ITP pathogenesis remains poorly understood. In this study, we observed a significantly higher proportion of CD8 + TRM (CD8 +CD45RA -CD45RO +CD69 +) in the bone marrow of ITP patients compared to healthy controls. However, no significant differences were observed in CD4 + TRM, CD8 + TCM and CD8 + TEM between the two groups. CD8 + TRM from ITP patients exhibited increased levels of IFN-γ, NLRP3, IL-1β, TNF-α, and decreased levels of IL-10 compared to those from healthy controls. Furthermore, CD8 + TRM proportions were significantly higher in glucocorticoid responders than non-responders, suggesting a potential role of CD8 + TRMs in mediating glucocorticoid therapeutic effects. Transcriptome sequencing revealed upregulated genes, including IGLC2, ASAP2, HIST2H2AA4, IGLV2-11, C1QA, ATP5MC1, IL5RA, and CYC1, and downregulated genes, including CCR9, HSPE1P18, RGCC, and ITGAE, in TRM cells from ITP patients compared to controls. GO/KEGG analysis of the top 500 upregulated genes highlighted pathways associated with B cell-associated immunity. To explore the effect of TRM on B cells, bone marrow CD19 + B cells were co-cultured with autologous TRM or memory T cells without TRM (TM) in the presence of IgM, and the proportion of memory B cells (CD19 +CD27 +CD38 -) and plasma cells (CD19 -CD24 -CD38 +) were measured by flow cytometry. The supernatants were collected, and cytokines and IgG level were tested by ELISA. Results showed that TRMs from ITP patients promoted the differentiation of memory B cells and IgG antibody production compared with those from healthy controls. However, no significant difference was observed in the proportion of naive B cells and plasma cells between the two groups. TRM, but not TM augmented B cell differentiation into memory B cells or plasma cells both in ITP patients and healthy controls. Additionally, TRM-primed B cells showed a stronger effect on antibody production and resulted in a higher level of IgG in supernatant compared with TM primed B cells. In line with these findings, the levels of B cell activating factor (BAFF), a proliferation inducing ligand (APRIL), and sCD40L, which were critical cytokines for B cell survival and differentiation, were significantly higher in supernatants from ITP patients than those from healthy controls. Moreover, the levels of BAFF, APRIL and CD40L in co-culture system with TRM were significantly increased compared to the system with TM, providing insights into the impact of TRM on B cell differentiation and function. To further investigate the role of TRM in regulating platelet production in ITP patients, we cocultured TRM with CD34 +cells in the presence of thrombopoietin (TPO), stem cell factor (SCF) and interleukin (IL)-3. Results showed that TRM did not suppress the production of CD41 + megakaryocytes from CD34 + cells, but reduced megakaryocyte apoptosis and increased the percentage of polyploidy (≥4N) megakaryocytes compared with TM. Additionally, TRM inhibited reticulated platelet formation and platelet production. Notably, dexamethasone (DXM) modulation alleviated certain suppressive effects on platelet production. In summary, our findings reveal the involvement of impaired bone marrow CD8 + TRM in the pathogenesis of ITP through the promotion of B cell differentiation and inhibition of platelet production. These results shed new light on the potential of immune therapy targeting TRM in the management of ITP.

Validation of Differentially Expressed Micrornas in Primary Immune Thrombocytopenia Suggests Mir-199a-5p Is a Potential Diagnostic Biomarker

Introduction Currently, there is no specific diagnostic test for immune thrombocytopenia (ITP). MicroRNAs (miRNA) are small, noncoding RNAs involved in the post-transcriptional regulation of gene expression. Aberrant expression of miRNAs has been suggested to have potential diagnostic value. Previous explorative studies have identified several differentially expressed miRNAs in ITP with potential diagnostic value; however, such findings are usually difficult to reproduce. Therefore, we aimed to validate some of these miRNAs in a larger ITP cohort. Methods Based on previous studies in ITP, we selected 12 miRNAs for validation in patients with primary ITP. The following miRNA were selected: miR-199a-5p, miR-33a-5p, miR-195-5p, miR-130a-3p, miR-144-3p, miR-146a-5p, miR-222-3p, miR-374-5p, miR-486-5p, miR-1341-5p, miR-766-3p, miR-409-3p. Blood samples (n=61) were obtained from 2 cohorts of primary ITP and healthy controls. The first cohort included samples from 24 patients with newly diagnosed or persistent ITP from the PROLONG study (NCT00344149); all patients had been treated with corticosteroids +/- IVIG but relapsed or failed to respond. The second cohort included 37 samples obtained from the ITP biobank at Østfold Hospital, Norway; patients had different disease phases and had received different therapies. In addition, 28 samples were obtained from healthy controls. The miRNAs were quantified in citrated plasma using droplet digital PCR (Bio-Rad). Results The mean age of the ITP patients was 51 years (62% female) and of the controls 47 years (64% female). The mean platelet count for all patients was 48.8 ·10 9/L. PROLONG patients had a lower mean platelet count (33 ·10 9/L) than patients from ITP biobank (59 ·10 9/L). Mann-Whitney test showed that miR-199a-5p (p=0.001), miR-33a-5p (p=0.010), miR-374-5p (p=0.009) and miR-146a-5p (p=0.026) were significantly differentially expressed (downregulated) in ITP compared with controls. Subgroup analysis of PROLONG patients showed that 3 additional miRNAs (7 of the 12 miRNAs) were significantly differentially expressed (downregulated) in these ITP patients: miR-130a-3p (p=0.005), miR-766-3p (p=0.010) and miR-409-3p (p=0.001). ROC curve analysis showed good discrimination for miR-199a-5p in all patients with an AUC of 0.718 (95% CI 0.599-0.836), and satisfactory for miR-33a-5p with an AUC of 0.671 (CI 0.549-0.793), 0.673 (CI 0.554-0.792) for miR-374-5p and 0.632 (CI 0.505-0.759) for miR-409-3p (Figure A). Subgroup analysis for each of the two cohorts separately showed higher AUC in PROLONG patients for miR-199a-5p (AUC=0.824; 95% CI 0.708-0.940), miR-374-5p (AUC=0.806; 95% CI 0.688-0.924), and for miR-33a-5p (AUC=0.732; 95% CI 0.597-0.868). The combination of all three miRNAs: miR-199a-5p, miR-33a-5p and miR-374-5p in PROLONG patients resulted in a slight improvement of the AUC to 0.865 (CI 0.767-0.962) (Figure B). Conclusions This study confirms that miR-199a-5p, miR-33a-5p, miR-374-5p and miR-146a-5p are differentially expressed in primary ITP compared with healthy controls. miR-199a-5p shows a good ability to discriminate primary ITP patients from healthy controls. In the PROLONG cohort, both miR-199a-5p and miR-374-5p showed a very good discrimination, probably due to the fact that these patients had early ITP. These findings should be further validated in a prospective cohort of newly diagnosed ITP patients and in patients with other types of thrombocytopenia to explore their potential diagnostic value. Some of these miRNAs have important regulatory functions, particularly in inflammation and apoptosis, so it is essential to evaluate their function and role in the pathogenesis of ITP and their potential use as therapeutic targets.