Immune Thrombocytopenia Mice Research Articles

Activation of the HMGB1-TLR4 pathway impacts the functionality of bone marrow mesenchymal stem cells and disrupts macrophage polarization in immune thrombocytopenia.

Recent evidence suggests that immune thrombocytopenia (ITP), a common bleeding disorder, is linked to an imbalance in macrophage polarization and impaired bone marrow mesenchymal stem cells (BMSCs). However, the relationship between macrophage polarization imbalance and functional defects in BMSCs, as well as the involvement of associated molecules in BMSCs' defects, is not well understood. This study aimed to investigate the regulatory effects of high mobility group protein 1 (HMGB1) on the physiological functions of BMSCs, specifically in relation to macrophage polarization imbalance. Patients with ITP showed dysregulation in monocyte/macrophage polarization and impaired BMSCs function. HMGB1 was found to have a negative impact on the ability of BMSCs to regulate the imbalance in macrophage polarization, especially when inflammatory factors are present. The MyD88-dependent pathway downstream of BMSCs was found to be significantly enhanced with HMGB1 treatment. Furthermore, treatment with toll-like receptor 4 (TLR4) inhibitors successfully restored the regulatory capacity of BMSCs in ameliorating macrophage polarization imbalance and effectively inhibited the activation of the MyD88-dependent pathway. Meanwhile, infusion of si-TLR4-BMSCs reversed HMGB1-induced platelet dysfunction and reduced over-polarization to M1-like macrophages in the ITP mouse model. Consequently, targeting the HMGB1-TLR4 pathway could be a potential approach to restore the immunoregulatory function of BMSCs.

NingXueShengBan Decoction Regulates Immune Homeostasis by PI3 K/Akt/mTOR and TLR4/NF-κB Signaling Pathways in Immune Thrombocytopenia Patients

Objective Immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disease. The immune imbalance of T cells is an essential pathological mechanism of ITP, more specific, the balance of Effector T cells (Teff)/Regulatory T cell (Treg) maintains T cell immune homeostasis. Dendritic cells (DC), the largest antigen-presenting cell, interfere with T cell differentiation pathways and exert immune regulatory functions. Our team created and applied the “ NingXueShengBan” (NXSB) decoction. According to our prior studies, we found that NXSB decoction could restore Th17/Treg balance in ITP mice by regulating protein expression in Notch pathway. Therefore, we further research the mechanism of NXSB decoction that regulates ITP immune homeostasis through affecting the interaction between DC and T cells. Methods In this study, we collected peripheral blood from health donors and ITP patients and extracted DC and CD4 + T cells then grouped in control group, model group and NXSB group. Moreover, we established a co-culture model simulating the immune environment in ITP patient to further investigate whether NXSB had therapeutic effects by the interaction between DC and CD4 + T cells. Results In the current study, we found that NXSB decoction potently regulated interaction between DC and T cells by inhibiting DC development and function, specifically, the differentiation of CD4+ T cells was inhibited after NXSB decoction intervened. Our data showed that NXSB decoction inhibited Toll-like receptor 4 (TLR-4), reduced the expression of Nuclear factor kappa-B (NF-κB) and led to the reduction of downstream inflammatory factors, thereby inhibiting the inflammatory response. More importantly, NXSB decoction reduced the DC surface expression of activation and maturation markers. Moreover, we detected that the decreased expression of the phosphorylated Phosphatidylinositol 3-kinase (PI3 K), Protein kinase B (Akt), Mammalian target of rapamycin (mTOR), and their downstream effectors indicated, we found that NXSB decoction could inhibit PI3 K/Akt/mTOR signaling pathways significantly. Conclusions Collectively, our data suggested that NXSB decoction ameliorates ITP-induced immune imbalance via its anti-inflammation and immunosuppressive by regulating TLR-4/ NF-κB and PI3 K/Akt/mTOR signaling pathways.

YAP1 regulates thrombopoiesis by binding to MYH9 in immune thrombocytopenia

AbstractImmune thrombocytopenia (ITP) is a complicated bleeding disease characterized by a sharp platelet reduction. As a dominating element involved in ITP, megakaryocytes (MKs) are responsible for thrombopoiesis. However, the mechanism underlying the dysregulation of thrombopoiesis that occurs in ITP remains unidentified. In this study, we examined the role of Yes-associated protein 1 (YAP1) in thrombopoiesis during ITP. We observed reduced YAP1 expression with cytoskeletal actin misalignment in MKs from patients with ITP. Using an experimental ITP mouse model, we showed that reduced YAP1 expression induced aberrant MK distribution, reduced the percentage of late MKs among the total MKs, and caused submaximal platelet recovery. Mechanistically, YAP1 upregulation by binding of GATA-binding protein 1 to its promoter promoted MK maturation. Phosphorylated YAP1 promoted cytoskeletal activation by binding its WW2 domain to myosin heavy chain 9, thereby facilitating thrombopoiesis. Targeting YAP1 with its activator XMU-MP-1 was sufficient to rescue cytoskeletal defects and thrombopoiesis dysregulation in YAP1+/− mice with ITP and patients. Taken together, these results demonstrate the crucial role of YAP1 in thrombopoiesis, providing potential for the development of diagnostic markers and therapeutic options for ITP.

HLA-DRB5 promotes immune thrombocytopenia via activating CD8+ T cells.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by a low platelet (PLT) count and a high risk of bleeding, the clinical treatment for which still needs to be upgraded. Based on the critical role of human leukocyte antigen class II heterodimer β5 (HLA-DRB5) in immune system, we herein investigated its effect on ITP. ITP murine models were established by the injection of guinea pig anti-mouse platelet serum (GP-APS), and the PLT of mouse peripheral blood was counted during the modeling. Quantitative real-time reverse transcription polymerase chain reaction, western blot and immunofluorescence assay was performed to quantify expressions of HLA-DRB5, major histocompatibility complex II (MHC-II) and co-stimulatory molecules (CD80, CD86). Flow cytometry was conducted to analyze the percentage of CD8+ T cells. As a result, the PLT count was decreased in mouse peripheral blood. Expressions of HLA-DRB5, MHC-II and co-stimulatory molecules, as well as the percentage of CD8+ T cells were elevated in peripheral blood of ITP mice. HLA-DRB5 knockdown mitigated ITP by increasing peripheral PLT level, downregulating expressions of MHC-II and co-stimulatory molecules and inactivating CD8+ T cells. Collectively, the downregulation of HLA-DRB5 restores the peripheral PLT count in ITP mice by reducing MHC-II-mediated antigen presentation of macrophages to inhibit the activation of CD8+ T cells.

HLA-DRB5 Overexpression Promotes Platelet Reduction in Immune Thrombocytopenia Mice Model by Facilitating MHC-II-Mediated Antigen Presentation

Introduction: Major histocompatibility complex II (MHC-II)-mediated antigen presentation contributes to the pathogenesis of immune thrombocytopenia (ITP). Human leukocyte antigen (HLA)-DRB5 is an MHC-II molecule and this study aims to investigate its role and mechanisms in ITP development. Methods: Guinea pig anti-mouse platelet (PLT) serum-induced ITP mice received tail vein injection of HLA-DRB5 overexpressing adenoviral vector/immune receptor expressed on myeloid cells-1 (IREM-1) monoclonal antibody (mAb). PLT count changes in mice blood were assessed by a hematology analyzer. MHC-II/CD80/CD86 expression in mice blood was measured by quantitative real-time-PCR and immunofluorescence assay. CD8+ T-cell proportion in mice blood was detected by flow cytometry. Results: HLA-DRB5 overexpression exacerbated PLT reduction since the 5th day of the establishment of ITP mice model and enhanced MHC-II/CD80/CD86 expression upregulation as well as CD8+ T-cell ratio elevation in the blood of ITP mice, while its effects were reversed by IREM-1 mAb. Conclusion: HLA-DRB5 overexpression upregulates MHC-II-mediated antigen presentation to CD8+ T cells, thus lowering PLT count in the ITP mice model.

Glycocalyx-Mimicking Nanoparticles with Differential Organ Selectivity for Drug Delivery and Therapy.

Organ-selective drug delivery is expected to maximize the efficacy of various therapeutic modalities while minimizing their systemic toxicity. Lipid nanoparticles and polymersomes can direct the organ-selective delivery of mRNAs or gene editing machineries, but their delivery is limited to mostly liver, spleen, and lung. A platform that enables delivery to these and other target organs is urgently needed. Here, a library of glycocalyx-mimicking nanoparticles (GlyNPs) comprising five randomly combined sugar moieties is generated, and direct in vivo library screening is used to identify GlyNPs with preferential biodistribution in liver, spleen, lung, kidneys, heart, and brain. Each organ-targeting GlyNP hit show cellular tropism within the organ. Liver, kidney, and spleen-targeting GlyNP hits equipped with therapeutics effectively can alleviate the symptoms of acetaminophen-induced liver injury, cisplatin-induced kidney injury, and immune thrombocytopenia in mice, respectively. Furthermore, the differential organ targeting of GlyNP hits is influenced not by the protein corona but by the sugar moieties displayed on their surface. It is envisioned that the GlyNP-based platform may enable the organ- and cell-targeted delivery of therapeutic cargoes.

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

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

Terbutaline Modulates Macrophage Homeostasis and Macrophage-T-Cell Interactions in Immune Thrombocytopenia

Background: Increased platelet destruction by macrophage phagocytosis contributes to immune thrombocytopenia (ITP). Neural immune modulation in autoimmune diseases has been given considerable interest. Our preliminary studies showed a decreased sympathetic innervation in the bone marrow and spleen of ITP mice and that abnormal T cell immunity caused by sympathetic modulation promoted the progression of ITP ( Blood Advances 2019; J Thromb Haemost, 2023). Moreover, previous studies indicated a potential interaction between T cells and macrophages ( Immunity, 2017). However, the regulation of spleen sympathetic denervation on macrophage function and the interaction between splenic macrophages and T cells under the sympathetic modulation in ITP remain unknown. Methods: Whole-tissue immunolabeling and 3D imaging of the human spleen were performed to compare the sympathetic distribution between ITP with splenectomy and non-ITP with traumatic splenectomy. RNA sequencing was used to detect the transcriptional changes and metabolomics and seahorse extracellular flux analyses were carried out to determine the metabolic reprogramming in splenic macrophages of ITP mice. The expression of cell surface markers of macrophages and T cells and cytokines was assessed by flow cytometry, western blot, and enzyme-linked immunosorbent assay. Chemical sympathectomy with 6-hydroxydopamine (6-OHDA), a β2-AR agonist terbutaline, and a β2-AR antagonist ICI 118,551 were used to detect the sympathetic modulation. Genetic lineage tracing was used to compare serum norepinephrine (NE) and its origin in the spleen of WT mice with or without chemical sympathectomy. Results: A panicle-shaped sympathetic neural architecture was revealed in the spleen of both ITP and non-ITP patients. The distribution of sympathetic nerves in the spleen and the level of serum NE were both significantly decreased in ITP patients. Immunofluorescence staining revealed an anatomical colocalization between sympathetic nerves, macrophages, and T cells in the spleen, suggesting potential sympathetic modulation of macrophages and a potential interaction between splenic macrophages and T cells. RNA-sequencing data of splenic macrophages in ITP mice compared with wild type (WT) mice revealed higher expression of M1-specific genes, including IL1b, Tnf and IL6, and lower expression of M2-specific genes, including IL10 and Arg1, indicating an M1 polarization tendency. In splenic macrophages isolated from ITP mice, we observed an enrichment of glycolysis pathway genes compared to WT mice, including Hk2, Pkm2and Ldha. An increase in the extracellular acidification rate and a decrease in the oxygen consumption rate were also found in splenic macrophages of ITP mice, indicating a switch to glycolysis. To investigate the interaction between splenic macrophages and T cells under sympathetic modulation in ITP, we measured the expression of cytokines that might have an influence on macrophage polarization and T cell differentiation and observed upregulation of interleukin (IL)-12, IL-18 and interferon (IFN)-γ. We also used macrophages from ITP mice and WT mice as antigen-presenting cells for naïve CD4 + T cells, which were interrogated for IFN-γ production following stimulation with OVA 323-339. We observed increased IFN-γ production and upregulation of IL-12 and IL-18 in response to macrophages from ITP mice, and we also observed diminished production of IL-12, IL-18 and IFN-γ when pretreated with aIL-12 mAb and aIL-18 mAb. Moreover, chemical sympathectomy with 6-OHDA was carried out in WT mice and significantly decreased serum NE and platelet counts at D7 and D14 were observed. Increased expression of M1-specific markers and upregulation of IL-12, IL-18 and IFN-γ were also determined. The β2-AR agonist terbutaline was shown to decrease the expression of proinflammatory factors and inhibit M1 polarization, and its effects were reversed by the β2-AR antagonist ICI 118,551. Conclusions: Splenic sympathetic denervation-mediated proinflammatory M1 polarization, metabolic alterations, and proinflammatory interactions between macrophages and T cells were involved in ITP pathogenesis. The β2-AR agonist terbutaline was demonstrated to restore the homeostasis of splenic macrophages, indicating a novel potential ITP treatment strategy.

Platelet-Derived TGF-β1 Induces Functional Reprogramming of Myeloid-Derived Suppressor Cells in Immune Thrombocytopenia

Introduction: Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by isolated thrombocytopenia and varying degrees of bleeding symptoms. Platelets are anucleate cytoplasmic cell fragments that play dual physiological roles as both cellular mediators of thrombosis and immune modulatory cells. Platelets are the largest storage pool of TGF-β1, a crucial enforcer of immune homeostasis and tolerance. Studies have shown that TGF-β1 released by platelets contributes to the pathogenesis of pulmonary arterial hypertension and cervical carcinoma. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells with potent immunosuppressive functions. Our previous studies have shown that MDSCs are impaired in number and function in patients with ITP. The objective of this study was to elucidate the underlying molecular mechanisms of MDSCs reprogramming by platelet-derived TGF-β1. Methods: A transwell assay was used to determine the impact of platelets on MDSCs induced from peripheral blood mononuclear cells (PBMCs) of ITP patients and healthy controls. The percentage of circulating MDSCs in ITP patients was detected before and after administration of thrombopoietin receptor agonists (TPO-RAs). Besides, anti-CD61 immune-sensitized splenocytes were transferred into SCID mice to establish an active murine model of ITP. Subsequently, MDSCs from TPO-RA-treated ITP mice were transplanted as cell therapy to rescue treatment-free ITP mice. In specific, adeno-associated viral (AAV) vectors efficiently infected MDSCs to enable cell-specific Smad2/3 knock-down. Finally, platelet specific TGF-β1-deficient mice were used to validate the effect of platelet-derived TGF-β1 on MDSCs. Results: TGF-β1 levels in the supernatant of MDSCs co-cultured with platelets of high concentration was significantly higher than that in MDSCs co-cultured with platelets of low concentration or alone. FACS analysis showed that platelet mass enhanced the inhibitory effects of MDSCs on the proliferation of CD4 + T cells, which could be offset by TGF-β1 inhibitor (TGF-βi). Besides, TPO-RAs treatment induced significantly increased percentage of circulating MDSCs in responders with chronic ITP. Likewise, TPO-RA significantly expanded MDSC population in active ITP murine models. Furthermore, after receiving donor MDSCs from TPO-RA-treated ITP mice, arrested differentiation of plasma cells, reduced Fas ligand expression on cytotoxic T cells, and re-balanced T cell subsets were found in the recipient mice, indicating augmented inhibitory functions of MDSCs. Mechanistically, transcriptome analysis confirmed the participation of TGF-β/Smad pathway in donor MDSCs from TPO-RA-treated ITP mice, which was offset by Smad2/3 knockdown. Finally, by constructing ITP models using Pf4 cre-Tgfb1 fl/fl and Pf4 cre+Tgfb1fl/fl mice, we found that TGF-β1 inhibition and platelet TGF-β1-deficence, both, could counteract the elevated platelet count and MDSCs percentage, as well as improved immunosuppressive capacity of MDSCs trigged by TPO-RA modulation. Conclusion: To sum up, it was demonstrated that platelet TGF-β1 induced the expansion and functional reprogramming of MDSCs via TGF-β/Smad pathway. These data indicate that platelet recovery not only serves as an endpoint of remission, but also paves the way for tolerance induction in immune-mediated thrombocytopenia. Our exploratory study deciphered immunological changes induced by TPO-RAs and helped to identify molecular mechanisms responsible for long-term remission after TPO-RA discontinuation.

The Combination of Iguratimod and Danazol Versus Danazol As a Treatment of Corticosteroid-Resistant/Relapse Immune Thrombocytopenia: A Randomized, Controlled, Multicenter, Open-Label Trial

Introduction: The treatment of choice in corticosteroid-resistant or relapsed immune thrombocytopenia (ITP) is still controversial due to the unclear mechanism and unpredictable efficacy of available therapies. The small molecule compound iguratimod is widely used as a novel antirheumatic drug to treat several autoimmune diseases. According to studies involving ITP mice, iguratimod may represent a new approach for the prevention and treatment of anti-platelet antibody-mediated ITP by modulating T-cell differentiation. Since iguratimod and danazol share disparate mechanisms in treating ITP, a combination of iguratimod and danazol may work synergistically based on a “double-hit” mechanism targeting both platelet production and destruction. The aim of this study was to evaluate the efficacy and safety of iguratimod and danazol versus danazol alone in patients with corticosteroid-resistant or relapsed primary immune thrombocytopenia. Methods: Across seven tertiary medical centers in China, adult ITP patients from this randomized, controlled, multicenter, open-label trial participated. Eligible corticosteroid-resistant or relapsed ITP patients with a platelet count <30×10 9/L or bleeding symptoms at enrollment were randomlyallocated by masked statisticians in a 1:1 ratio to receive oral iguratimod at 25 mg twice daily plus oral danazol at 200 mg twice daily or oral danazol monotherapy at 200 mg twice daily for 12 weeks. The group assignment was not masked for clinicians or patients. Following the first eight weeks of treatment, patients were assessed every two weeks thereafter. The primary endpoint was a 6-month sustained response (SR), defined as a platelet count of 30×10 9/L or more and at least a doubling of the baseline platelet count (partial response) or a platelet count of 100×10 9/L or more (complete response) and the absence of bleeding without rescue medication. This trial was registered with ClinicalTrials.gov (NCT05281068). Results: From September 1, 2021, to December 31, 2022, a total of 106 patients were screened for eligibility; 35 were excluded, and 71 were randomly assigned: 37 to the iguratimod plus danazol group and 34 to the danazol group. The median age of the patients was 41 years, and 69.0% (49/71) of them were females. All patients enrolled failed to respond to the first-line treatment with corticosteroids, and 21 (29.6%) of all patients had received three or more therapies. The data cutoff for the analyses was July 1, 2023. The median follow-up was 14 months (IQR 11-15) in the iguratimod plus danazol group and 12.5 months (11-15) in the danazol group. Six-month follow-up results showed that iguratimod plus danazol was more likely to produce SR than danazol alone (20 [54.1%] of 37 vs. 9 [26.5%] of 34; odds ratio 1.82, 95% CI 1.22-2.70, p = 0.003). The combination group had 25 (67.6%) patients with at least one response (overall response), and 13 (35.1%) patients had a complete response by the end of the follow-up; whereas 15 (44.1%) of 34 patients in the monotherapy group had an overall response, and three (8.8%) had a complete response. A target platelet count of 30×10 9/L was achieved in 18 (48.6%) of 37 patients given iguratimod plus danazol after 4 weeks and five (14.7%) of 34 patients given danazol monotherapy, indicating that the combination therapy significantly increased the initial response rate ( p = 0.002). Both groups experienced similar serious adverse events (AEs), rescue treatment, and treatment side effects, and all patients tolerated the treatment well, without any grade 4 AEs or treatment-related deaths reported. Conclusions: Patients with corticosteroid-resistant or relapsed ITP responds well to iguratimod plus danazol. This regimen achieved a rapid and long-lasting response. It is necessary to conduct further studies to determine the optimal dosage and a variety of combination therapies using several immune-suppressing agents, including iguratimod, to achieve a sustained response in patients with corticosteroid-resistant or relapsed ITP.

Iguratimod Could Correct the Defective IL-35-Mediated Megakaryopoiesis in Immune Thrombocytopenia

Introduction: IL-35 is an inhibitory cytokine of the IL-12 family, and comprises an EBI3 subunit and a p35 subunit. Previous data indicated that serum levels of IL-35 were significantly lower in immune thrombocytopenia (ITP) patients than in healthy controls. Moreover, the concentration of plasma IL-35 in ITP patients achieving complete response returned to normal, but was not corrected in those who failed to achieve response. However, the underlying pathological function of IL-35 in impaired megakaryopoiesis in ITP remains unexplored. Our study aimed to evaluate the immunoregulatory role of IL-35 in megakaryocytes (MKs) differentiation. Iguratimod, a traditional therapy for inflammatory disorders, has been demonstrated to promote megakaryopoiesis. The effect of iguratimod on the treatment of ITP was retrospectively reviewed. Methods: Bone marrow samples were obtained from newly-diagnosed primary ITP patients and healthy donors. CD34+ cells from ITP patients and healthy donors were differentiated into MKs with and without IL-35 to determine the mechanism of IL-35 in megakaryopoiesis. Iguratimod was added to the culture medium of MKs to determine its function in MKs differentiation. Additionally, ITP mouse model was established to further validate the function of IL-35. Results: In comparison with healthy controls, ITP patients showed lower bone marrow levels of IL-35. IL-35 receptor expression was progressively induced on bone marrow MKs from healthy controls during the differentiation of CD34+ progenitors. In contrast, MKs from ITP patients exhibited defective expression of the IL-35 receptor. We then determined the effect of IL-35 on the megakaryocytic potential of CD34+ progenitor cells in culture medium containing TPO. IL-35 increased the number of medium-sized and large-sized colonies, and increased the total number of CD41+ cells. In IL-35-treated MKs, higher levels of Akt phosphorylation were observed. Pretreatment with LY294002, an inhibitor of Akt phosphorylation, blocked the differentiation of MKs induced by IL-35. LY294002 inhibited proplatelet formation and aggregation of β1-tubulin in MKs stimulated by IL-35. Flow cytometry analysis revealed that platelet production decreased in the LY294002 pretreatment group. These results demonstrated that IL-35 can enhance the proliferation and differentiation of MKs through Akt pathways. The decreased level of bone marrow IL-35 may partially explain the defective megakaryopoiesis in ITP. We then explored the potential causes of the decreased IL-35 levels in ITP. Flow cytometry analysis showed that the percentage of bone marrow iTr35 cells, the primary sources of IL-35, was significantly lower in ITP patients. Furthermore, we cocultured CD4+ T cells with MSCs from healthy controls and ITP patients, respectively. The percentage of iTr35 cells was lower when cultured with ITP-MSCs. These results showed that ITP-MSCs exhibited an impaired capability of inducing iTr35 cells, which might contribute to the reduced level of bone marrow IL-35 in ITP patients. We further investigated the effects of iguratimod on the differentiation of CD34+ progenitors into MKs. When CD34+ cells were cultured with TPO alone, the addition of iguratimod upregulated the expression of IL-35 receptors on MKs from ITP patients and a murine model of this disease, and consequently promoted the formation of MK colonies. In the retrospective study of iguratimod, a total of 68 adult patients with corticosteroid-resistant primary ITP were included. Initial response was achieved in 42/68 (62%) patients, and complete response was achieved in 25/68 (37%) patients. In patients who achieved initial response, the median time to response was 28 days. Sustained response at 6 months was seen in 35/68 (51%). Bleeding symptoms were reduced from 37/68 (54%) patients at baseline to 14/68 (21%) patients at 6 months. Most adverse events were mild (grade 1-2), and there were no grade 4 or worse adverse events in our patients. The most common adverse events were elevated ALT, gastrointestinal disorders, and nausea. Conclusions: ITP-MSCs exhibited an impaired capability of inducing iTr35 cells, which contributed to the reduced level of bone marrow IL-35 in ITP. IL-35 promoted MKs differentiation through the Akt pathway. Iguratimod promoted megakaryopoiesis by inducing the expression of IL-35 receptors on MKs. Iguratimod had good efficacy and safety in the treatment of ITP.

Study on the Treatment of ITP Mice with IVIG Sourced from Distinct Sex-Special Plasma (DSP-IVIG).

Intravenous immunoglobulin (IVIG) is a first-line drug prepared from human plasma for the treatment of autoimmune diseases (AIDs), especially immune thrombocytopenia (ITP). Significant differences exist in protein types and expression levels between male and female plasma, and the prevalence of autoimmune diseases varies between sexes. The present study seeks to explore potential variations in IVIG sourced from distinct sex-specific plasma (DSP-IVIG), including IVIG sourced from female plasma (F-IVIG), IVIG sourced from male plasma (M-IVIG), and IVIG sourced from a blend of male and female plasma (Mix-IVIG). To address this question, we used an ITP mouse model and a monocyte-macrophage inflammation model treated with DSP IVIG. The analysis of proteomics in mice suggested that the pathogenesis and treatment of ITP may involve FcγRs mediated phagocytosis, apoptosis, Th17, cytokines, chemokines, and more. Key indicators, including the mouse spleen index, CD16+ macrophages, M1, M2, IL-6, IL-27, and IL-13, all indicated that the efficacy in improving ITP was highest for M-IVIG. Subsequent cell experiments revealed that M-IVIG exhibited a more potent ability to inhibit monocyte phagocytosis. It induced more necrotic M2 cells and fewer viable M2, resulting in weaker M2 phagocytosis. M-IVIG also demonstrated superiority in the downregulation of surface makers CD36, CD68, and CD16 on M1 macrophages, a weaker capacity to activate complement, and a stronger binding ability to FcγRs on the THP-1 surface. In summary, DSP-IVIG effectively mitigated inflammation in ITP mice and monocytes and macrophages. However, M-IVIG exhibited advantages in improving the spleen index, regulating the number and typing of M1 and M2 macrophages, and inhibiting macrophage-mediated inflammation compared to F-IVIG and Mix-IVIG.

Integrin αvβ8-Mediated Activation of TGF-β1 Is Involved in Regulating the Sustained Response after TPO-Ras Withdrawal in ITP Patients

Background: Thrombopoietin receptor agonists（TPO-RAs）are effective and safe in immune thrombocytopenia (ITP), and are currently the most important second-line drugs. The effect of TPO-RAs is transient, platelet counts usually return to baseline within 2 weeks unless treatment is continued. Only one-third of patients can maintain after drug discontinuation, but the mechanism is unknown and worthy of further exploration. Method and Results：In this study we observed 66 patients treated with TPO-RAs and followed-up to three months after drug withdrawal. Patients were divided into three groups: non-response group (NR), sustained group (Group A), and non- sustained group (Group B) (Fig1-A). The blood samples were collected at the drug discontinuation, and we found that compared with total TGF-β1 levels，plasma activated TGF-β1 levels were extremely lower in NR patients and were also reduced in group B (Fig1-B,C). At the same time, regulatory T cells have the same trend. By using the qPCR and Western blot we preliminary found that integrin αvβ8 regulate the activation of TGF-β1 to promote the formation of Tregs, which affects the sustained response to TPO-RAs withdrawal. Furthermore, lower levels of activated TGF-β1 in the bone marrow were associated with higher numbers of megakaryocytes, fewer plate-producing megakaryocytes, and decreased proportion of hyperploid megakaryocytes (≥16N). Through in vitro cell culture, we found that recombinant human integrin avb8 protein can promote Treg differentiation and inhibit T cell activation in ITP patients' PBMCs, but this phenomenon can be reversed by anti-β8 antibody. Then, we constructed PF4-TGF-β1 conditional knockout mice (Cre+ mice) and constructed of an ITP passive model by injecting CD41 antibody and found that the Cre+ mice have higher platelets at baseline, while the platelets dropped rapidly than Cre- mice after CD41 antibody injection (Fig2-A). Using rhTPO to treat the ITP mice, we found that Cre+ mice were less effective than Cre- mice, and the sustained response time was shorter after drug withdrawal. When sacrificed the mice, decreased Treg cells and elevated M1 macrophages were detected in Cre+ mice. Meanwhile, HE staining of bone marrow found a greater number of megakaryocytes, but the cell diameter decreased significantly (Fig2-B, D). Next, we treated the mice with rhTPO in combined with recombinant mice integrin avb8 (50ng/kg or 250ng/kg), the therapeutic efficacy and sustained response time in the high-dose avb8 group were significantly improved (Fig2-C) We also constructed a more representative ITP active model of autoimmune diseases, by immunizing CD61KO mice to produce autoantibodies and infusing spleen cells back into SCID mice. Simultaneously through transfection siRNA or shRNA to activate or silence mouse T cell integrin β8 and reinfuse it into SCID mice. We found that activating integrin β8 in mice had a more significant therapeutic effect and maintained a longer response time. Finally, through single-cell sequencing analysis, the smads signaling pathways，integrin binding pathways and AP-1 family signaling pathways were significantly enriched in group A patients. Further study confirmed that TPO-RAs can regulate the AP-1 family, which upregulated the β8 subunit, resulting in enhanced activation of TGF-β1 (Fig3-A, B). Conclusion: Integrin αvβ8 activates TGF-β1 and upregulates Tregs to restore immune intolerance in ITP patients, meanwhile, activated TGF-β1 also promotes hyperploidy megakaryocyte differentiation in bone marrow, which co-regulates the sustained response after TPO-RAs discontinuation.

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

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

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

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

When immune thrombocytopenia gets on your nerves: β2-adrenergic receptor agonists as a potential treatment?

When immune thrombocytopenia gets on your nerves: β2-adrenergic receptor agonists as a potential treatment?

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.

Low-Dose Rhein Sensitizes Platelet Production to Thrombopoietin Receptor Agonists (TPO-RA) in Immune Thrombocytopenia

Introduction: Primary immune thrombocytopenia (ITP) is an immune-mediated disorder characterized by isolated thrombocytopenia and risk of hemorrhage. Impaired megakaryocyte maturation and insufficient platelet formation, induced by platelet-specific autoantibodies or ITP plasma, play vital roles in its pathogenesis. Although thrombopoietin receptor agonists (TPO-RA) is recommended the first choice among second-line treatment of ITP, clinical data have indicated that nearly half of ITP patients failed or relapsed during eltrombopag maintenance, and increasing drug dose cannot reverse therapeutic response. Diacerein, hydrolyzed into its active metabolite rhein after oral administration, is a non-steroidal anti-inflammatory drug used in osteoarthritis. Our team first observed that 3 ITP patients with low platelet levels (30-50 ×109/L) during eltrombopag treatment, achieved significant platelet response (≥100×109/L) after taking diacerein concomitantly due to the complaint of arthralgia. These data suggested that diacerein may have sensitization effect on TPO-RA, and this novel combination is expected to boost thrombopoiesis in ITP. Thus, in vitro and in vivo experiments were performed to explore the role of TPO-RA plus rhein in megakaryocyte growth and platelet formation in ITP. Methods: Twenty-eight eltrombopag-inefficient or relapsed ITP patients and 15 healthy volunteers were included in this study. We sorted CD34+ cells from healthy umbilical cord blood mononuclear cells, then cultured them in SFEM medium containing thrombopoietin, interleukin-3, stem cell factor and 10% serum from either ITP patients or healthy donors. Different doses of eltrombopag combined with rhein (0-100 μM) were added on the fourth day, and cell suspension was collected on the 14th day. Flow cytometry, RNA sequencing, proteomic sequencing and cytokine assay were performed to evaluate the overlaying effect and underlying mechanisms of rhein on eltrombopag. In addition, active ITP murine models were established to evaluate the therapeutic effects of romiplostim plus rhein in vivo. Results: Compared with eltrombopag, eltrombopag combined with low-dose rhein significantly increased the proportion of CD41+, CD41+CD42+, CD41+CD61+ megakaryocytes in vitro. We further found that eltrombopag plus low-dose rhein significantly promoted polyploid maturation and apoptosis of CD41+ megakaryocytes, as well as platelet production in vitro (Fig 1). The ITP mice exhibited profound thrombocytopenia, and their platelet counts decreased significantly by the second week after irradiation. Our data showed that romiplostim plus low-dose rhein significantly alleviated thrombocytopenia than romiplostim monotherapy on day 21 and day 28 (Fig 2). Mice receiving different doses of rhein monotherapy were comparable in platelet counts with control group. Flow cytometry analysis suggested that the combination therapy significantly increased megakaryocyte percentage and polyploidization than romiplostim in ITP mice. Subsequently, RNA sequencing and proteomic analysis revealed that the combination of eltrombopag and low-dose rhein promoted megakaryocyte growth and platelet formation, decreased proinflammatory cytokines, and upregulated phosphorylated PI3K in vitro. Cytokine analysis indicated that eltrombopag plus low-dose rhein significantly increased supernatant level of TGF-β, and reduced IFN-α, IFN-γ, IL-12p70, IL-17A and IL-18 than eltrombopag. Furthermore, it was confirmed that the addition of rhein to TPO-RA significantly promoted megakaryocyte PI3K phosphorylation in vitro and in vivo. PI3K inhibition offset the enhanced effect of eltrombopag plus low-dose rhein over eltrombopag in megakaryocyte maturation and platelet formation. Conclusions: In sum, our data reveal a sensitizing role of rhein to TPO-RA in megakaryocyte polyploidization and platelet formation in ITP, and this novel combination therapy warrants further investigation as an optimized strategy for ITP patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Enhancing regulatory T-cell function via inhibition of high mobility group box 1 protein signaling in immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is the most common acquired autoimmune bleeding disorder. Abnormally increased levels of High Mobility Group Box 1 (HMGB1) protein associate with thrombocytopenia and therapeutic outcome in ITP. Previous studies proposed that a natural inhibitor of HMGB1, 18β-glycyrrhetinic acid (18β-GA), could be used for its anti-inflammatory and immune-modulatory effects, although its ability to correct immune balance in ITP is unclear. In this study, we showed that plasma HMGB1 correlated negatively with platelet counts in ITP patients, and confirmed that 18β-GA stimulated the production of regulatory T cells (Treg), restored the balance of CD4+ T-cell subsets and enhanced the suppressive function of Treg through blocking the effect on HMGB1 in patients with ITP. HMGB1 short hairpin RNA interference masked the effect of 18β-GA in Treg of ITP patients. Furthermore, we found that 18β-GA alleviated thrombocytopenia in mice with ITP. Briefly, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to induce a murine model of severe ITP. The proportion of circulating Treg increased significantly, while the level of plasma HMGB1 and serum antiplatelet antibodies decreased significantly in ITP mice along 18β-GA treatment. In addition, 18β-GA reduced phagocytic activity of macrophages towards platelets both in ITP patients and ITP mice. These results indicate that 18β-GA has the potential to restore immune balance in ITP via inhibition of HMGB1 signaling. In short, this study reveals the role of HMGB1 in ITP, which may serve as a potential target for thrombocytopenia therapy.

S290: ATRA CAN CORRECT DEFECTIVE HIF-1Α/S1P AXIS-MEDIATED CYTOSKELETAL REORGANIZATION IN PROPLATELET FORMATION OF ITP

Background: Bone marrow physiological hypoxia plays a crucial role in haematopoietic stem cell homeostasis. Hypoxia inducible factor (HIF) is central to mediating the cellular response to hypoxia. HIF-1α expression was decreased in the bone marrow of patients with immune thrombocytopenia (ITP), and HIF-1α activation was shown to enhance megakaryopoiesis in mice. Recent studies suggest that “inside-out” signalling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that HIF-1α/S1P axis-mediated cytoskeletal reorganization was defective in the PPF of ITP. All-trans retinoic acid (ATRA), which has been shown to be a promising treatment option for ITP patients in our clinical studies (Blood, 2021; Lancet haematology, 2017; Lancet haematology, 2021), could restore cytoskeletal reorganization and correct impaired PPF. Aims: This study aimed to explore the role of hypoxia inducible factor-1α (HIF-1α) in proplatelet formation (PPF) and the underlying mechanisms of ATRA treatment in ITP patients. Methods: Thirty consecutive patients with newly diagnosed ITP and 30 healthy donors were included in our study. MKs were isolated from bone marrow samples. Targeted and untargeted metabolomic profiling through metabolomic analysis was performed to explore the relationship between the metabolome and ITP. Confocal microscopy and transmission electron microscopy were used to observe the PPF and cytoskeleton structure of ITP MKs. An ITP mouse model was established to observe the therapeutic effects of ATRA in the PPF. Results: In the present study, we observed that MKs displayed altered cytoskeletal reorganization and impaired proplatelet formation (PPF) in ITP patients. Targeted and untargeted metabolite profiling revealed a decreased sphingosine-1-phosphate (S1P) level in ITP. Downregulated sphingosine kinase 2 (SPHK2) expression in MKs accounted for the low level of S1P in ITP. S1P is essential for S1P receptor 1 (S1PR1) and Rac1 activation, Src family kinases (SFKs) activity, and subsequent cytoskeletal reorganization and PPF regulation. Moreover, we demonstrated that HIF-1α mediated SPHK2 activation and S1P production. Decreased HIF-1α levels were found in the MKs of patients with ITP, contributing to impaired PPF. We then investigated the effect of ATRA on PPF in ITP patients. ATRA upregulated HIF-1α and SPHK2 expression, increased S1P production and corrected impaired PPF in vitro. In an ITP mouse model, ATRA alleviated thrombocytopenia and restored cytoskeletal reorganization. ATRA corrected impaired PPF by upregulating HIF-1α expression. The exposure of ITP MKs to selective RARα (AM580) or RARγ (BMS961) agonists did not change PPF. However, the treatment of ITP MKs with a selective RARβ agonist (CD2314) significantly increased PPF. Furthermore, we found that the effect of ATRA on enhancing PPF in ITP MKs was reversed in the presence of an RARβ antagonist (CD2665). These data suggest that impaired PPF in ITP MKs is corrected by ATRA in a RARβ-dependent manner. Summary/Conclusion: Together, our data show that the HIF-1α/S1P axis mediates altered cytoskeletal reorganization and impaired PPF in ITP and suggest that ATRA correction of impaired PPF is a potential mechanistic explanation for the clinical efficacy of ATRA in ITP.