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.

B cells and antibodies in refractory immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder mediated by pathogenic autoantibodies secreted by plasma cells (PCs) in many patients. In refractory ITP patients, the persistence of splenic and bone marrow autoreactive long-lived PCs (LLPCs) may explain primary failure of rituximab and splenectomy respectively. The reactivation of autoreactive memory B cells generating new autoreactive PCs contributes to relapses after initial response to rituximab. Emerging strategies targeting B cells and PCs aim to prevent the settlement of splenic LLPCs with the combination of anti-BAFF and rituximab, to deplete autoreactive PCs with anti-CD38 antibodies, and to induce deeper B-cell depletion in tissues with novel anti-CD20 monoclonal antibodies and anti-CD19 therapies. Alternative strategies, focused on controlling autoantibody mediated effects, have also been developed, including SYK and BTK inhibitors, complement inhibitors, FcRn blockers and inhibitors of platelet desialylation.

Regulatory factors involved in Th17/Treg cell balance of immune thrombocytopenia.

Immune thrombocytopenia is a common heterogeneous autoimmune disease that is characterized by decreasing peripheral blood platelet counts and increasing risk of bleeding. Studies have shown that an imbalance between T helper 17 (Th17) and Regulatory T (Treg) cells differentiated from CD4+T-cells is a key factor influencing the development and pathogenesis of immune thrombocytopenia. Th17 cells promote the development of chronic inflammatory disorders and induce autoimmune diseases, whereas Treg cells regulate immune homeostasis and prevent autoimmune diseases. Several regulators affecting the production and maintenance of these cells are also essential for proper regulation of Th17/Treg balance; these regulatory factors include cell surface proteins, miRNAs, and cytokine signaling. In this review, we focus on the function and role of balance between Th17 and Treg cells in immune thrombocytopenia, the regulatory factors, and therapeutic goals of this balance in immune thrombocytopenia.

Therapeutic potential of MSCs and MSC-derived extracellular vesicles in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired autoimmune disease involving a variety of immune cells and factors. Despite being a benign disease, it is still considered incurable due to its complex pathogenesis. Mesenchymal stem cells (MSCs), with low immunogenicity, pluripotent differentiation, and immunomodulatory ability, are widely used in a variety of autoimmune diseases. In recent years, impaired bone marrow mesenchymal stem cells (BMMSCs) were found to play an important role in the pathogenesis of ITP; and the therapeutic role of MSCs in ITP has also been supported by increasing evidence with encouraging efficacy. MSCs hold promise as a new approach to treat or even cure refractory ITP. Extracellular vesicles (EVs), as novel carriers in the "paracrine" mechanism of MSCs, are the focus of MSCs. Encouragingly, several studies suggested that EVs may perform similar functions as MSCs to treat ITP. This review summarized the role of MSCs in the pathophysiology and treatment of ITP.

CD44, CD90 and CD96 expression in immune thrombocytopenia purpura (ITP) patients.

Studying the expression of hematopoietic stem cell markers from different sources might be useful in understanding stem cell biology in different niche conditions. The study aimed to assess the difference in cell surface markers (CD44, CD90, CD96) on hematopoietic stem cells in three different niche conditions; umbilical cord blood (UCB), normal bone marrow (NBM) and bone marrow samples from idiopathic (immune) thrombocytopenic purpura (IBM). This study was conducted on 300 cases divided into three study groups; 100 umbilical cord blood units collected from mothers undergoing cesarian section in gynecology and obstetrics department, 100 bone marrow samples from idiopathic (immune) thrombocytopenic purpura patients collected from university children hospital and 100 normal bone marrow samples with no evidence of disease in bone marrow tissue. CD44 was significantly elevated in UCB and NBM groups compared to IBM group (<0.001). There was also a significant elevation of CD90 and CD96 in IBM group compared to NBM group and UCB (<0.001). CD90 and CD96 play a role in the pathogenesis of ITP disorder and could be applied as a targeted therapy to improve the outcome of this disease.

Downregulation of ADAM17 in pediatric immune thrombocytopenia impairs proplatelet formation.

BACKGROUND: Immune thrombocytopenia (ITP) is the most common etiology of acquired thrombocytopenia diseases in children. ITP is characterized by the immune-mediated decreased formation and excessive destruction of platelets. The pathogenesis and management of pediatric ITP are distinct from adult ITP. A disintegrin and metalloproteinase 17 (ADAM17) mediates the shedding of platelet receptor glycoprotein Ib α (GPIb α) in extracellular domain, functioning in the platelet activation and clearance. Our study aims to probe the roles and mechanisms of ADAM17 in pediatric ITP. METHODS: The differently expressed ADAM17 in megakaryocytes was obtained from children with ITP through the next-generation RNA-Sequence. Hematoxylin-eosin and Giemsa staining were performed for cell morphology identification. Flow cytometry was applied to assess autoantibodies against platelets, subtypes of lymphocytes, the surface expression level of ADAM17 and polyploidization of megakaryocytes, as well as the full-length GP Ib α. RESULTS: ADAM17 was significantly downregulated in megakaryocytes and platelets in children with ITP. Higher values of PDW and positive autoantibodies presence were observed in children with ITP. Loss of ADAM17 in mice led to defects in proplatelet formation and significantly elevated expression of phosphorylated myosin light chain (p-MLC) in megakaryocytes. CONCLUSIONS: Our study indicated that the downregulation of ADAM17 might be an innate cause of inefficient platelet production in pediatric ITP.

Excessive Subchorionic Fibrinoid Deposition as a Component of Massive Perivillous Fibrin Deposition: A Case With Maternal Immune Thrombocytopenia.

Maternal floor infarction (MFI) and massive perivillous fibrin deposition (MPFD) are overlapping placental disorders of unknown etiology, associated with adverse obstetric outcome, and a significant risk of recurrence. We describe a 31-year-old mother with asymptomatic thrombocytopenia throughout pregnancy and a positive lupus anticoagulant. She delivered a normal female neonate at term, whose weight was small for gestational age, with a placenta weighing less than the 10th percentile. Placental examination showed MPFD together with excessive subchorionic fibrinoid deposition. The placenta showed diffuse C4d deposition and an immune-mediated reaction was postulated for the pathogenesis of the placental changes. We suggest that excessive subchorionic fibrinoid deposition may be part of the morphologic spectrum of MFI/MPFD.

Treatment of immune thrombocytopenia (ITP) secondary to malignancy: a systematic review.

Immune thrombocytopenia (ITP) can be associated with lymphoproliferative diseases (LPD) or solid tumors. A systematic review of published literature was conducted to evaluate response to treatment of ITP secondary to malignancy. Primary outcome was overall response (complete response+response) to first-line treatments [steroids alone or in combination with intravenous immunoglobulins (IVIg)]. Among secondary outcomes, overall response to second-line treatments [splenectomy, rituximab or thrombopoietin receptor agonists (TPO-RA)] and death were evaluated. Of the retrieved 238 text articles, 108 were analyzable, for a total of 154 patients: 142 in 105 case reports and 12 in 3 observational studies. Thirty-nine patients had solid tumors, 114 LPD, and 1 both. The median follow up was 19 months (IQR, 9-40). The overall response was 50% (62% in solid tumors, 46% in LPD) after steroids and 47% (67% in solid tumors, 36% in LPD) after steroids+IVIg, which are lower than historical responses observed in primary ITP (≈80%). The overall responses to rituximab (used in LPD only), splenectomy and TPO-RA (70%, 73% and 92%, respectively) were similar to those observed in primary ITP. Seven patients (6%) died due to bleeding events. ITP secondary to malignancy appears to be associated with unsatisfactory response to first-line treatments.

T-follicular helper cell expansion and chronic T-cell activation are characteristic immune anomalies in Evans syndrome.

Pediatric Evans syndrome (pES) is increasingly identified as the presenting manifestation of several inborn errors of immunity. Despite an improved understanding of genetic defects in pES, the underlying immunobiology of pES is poorly defined, and characteristic diagnostic immune parameters are lacking. We describe the immune characteristics of 24 patients with pES and compared them with 22 patients with chronic immune thrombocytopenia (cITP) and 24 healthy controls (HCs). Compared with patients with cITP and HC, patients with pES had increased circulating T-follicular helper cells (cTfh), increased T-cell activation, and decreased naïve CD4+ T cells for age. Despite normal or high immunoglobulin G (IgG) in most pES at presentation, class-switched memory B cells were decreased. Within the cTfh subset, we noted features of postactivation exhaustion with upregulation of several canonical checkpoint inhibitors. T-cell receptor ß chain (TCR-ß) repertoire analysis of cTfh cells revealed increased oligoclonality in patients with pES compared with HCs. Among patients with pES, those without a known gene defect had a similar characteristic immune abnormality as patients with defined genetic defects. Similarly, patients with pES with normal IgG had similar T-cell abnormalities as patients with low IgG. Because genetic defects have been identified in less than half of patients with pES, our findings of similar immune abnormalities across all patients with pES help establish a common characteristic immunopathology in pES, irrespective of the underlying genetic etiology.

Low-affinity immunoglobulin gamma Fc region receptor III-B (FcγRIIIB, CD16B) deficiency in patients with blood and immune system disorders.

Low-affinity immunoglobulin gamma Fc region receptor III-B (FcγRIIIB) deficiency is present in ˜0·05% of the general population. Among our patients, FcγRIIIB deficiency was less frequent in those with immune-system disorders (one of 1815 patients, 0·05%) than in those with blood disorders (nine of 2147 patients, 0·42%, P = 0·023): mainly primary immune thrombocytopenia (4·34%), therapy related myeloid neoplasms (1·16%) and myelodysplastic syndrome with excess blasts (1·28%). Four of the nine (44·4%) patients with blood disorders were diagnosed with or quickly evolved to acute myeloid leukaemia (AML), suggesting that FcγRIIIB deficiency could be an adverse prognostic factor for progression to AML that should be confirmed in large multicentre studies.

Decreasing lncRNA PVT1 causes Treg/Th17 imbalance via NOTCH signaling in immune thrombocytopenia.

Objectives: Immune thrombocytopenia (ITP) is an autoimmune disease. T helper cell 17 (Th17) cells are increased in peripheral blood of ITP patients. NOTCH signaling is involved in Th17 cell differentiation and function. Besides, lncRNA Plasmacytoma variant translocation 1 (PVT1) was decreased in experimental autoimmune encephalomyelitis, and overexpressing PVT1 inhibited Th17 cell differentiation. Here, we aimed to investigate the effect of lncRNA PVT1 on ITP and its related mechanism.Methods: The number of Th17 cells and Treg cells was carried out using flow cytometry. PVT1 levels were detected by quantitative real-time PCR. Interleukin-17 (IL-17) levels and transforming growth factor-ß (TGF-ß) levels were detected by enzyme-linked immunosorbent assay. Protein levels of retinoid acid-related orphan receptor γ t (RORγt), forkhead box P3 (Foxp3), and NOTCH1 were carried out by western blot. NOTCH1 ubiquitylation was detected by ubiquitination assay.Results: PVT1 was down-regulated and Th17 cells were up-regulated in ITP patients. Overexpression of PVT1 decreased the number of Th17 cells, and also decreased the levels of IL-17, RORγt, and NOTCH1. Besides, PVT1 could bind to NOTCH1 and mediated NOTCH1 degradation by increasing its ubiquitination. Additionally, excessive expression of PVT1 could increase the levels of PVT1, reduce the amount of Th17 cells, as well as the levels of IL-17, RORγt, and NOTCH1, while co-overexpressing NOTCH1 reversed the results.Conclusion: PVT1 was down-regulated in ITP patients. Overexpressing PVT1 might reduce Th17 cell differentiation by down-regulating NOTCH1, and further alleviated the development of ITP.

Immunological characteristics and effect of cyclosporin in patients with immune thrombocytopenia.

OBJECTIVE: Immune thrombocytopenia (ITP) is well-known as an antibody-mediated autoimmune disease, and it is easy to get response but often turns to relapse or refractory. Cyclosporin is a traditional immunosuppressant and had a good effect on ITP patients. In this paper, we retrospectively analyze the immunological characteristics and therapeutic effect of cyclosporin in 220 patients with ITP. METHODS: All newly diagnosed ITP patients in the Department of Hematology, Tianjin Medical University General Hospital from June 2018 to December 2020 were enrolled and divided into four groups according to the expression of autoantibodies and the occurrence of prodromal infection. The basic data and immune indexes of ITP patients in each group were collected. The clinical immunological characteristics of patients in each group and the therapeutic effect of cyclosporin in each group were analyzed. RESULTS: Multi-autoantibody ITP patients were more likely to have low serum albumin and high gamma globulin, and the ratio of albumin to globulin decreased. In addition, the level of IgA and IgG increased and the level of complement C3 and C4 decreased more frequently than those in other groups. The number of CD3+T lymphocytes, especially CD3+CD4+T lymphocytes, decreased in ANA+ITP patients. The number of CD16+CD56+NK cells, pDC/DC ratio, and pDC/mDC ratio were higher than those in other groups. The expression of IL-6 and the proportion of CD19+B lymphocytes increased in two groups of ITP patients with abnormal autoantibodies. The patients of pro-infected group were more likely to suffer from coagulation disorder. After treatment with cyclosporin, the response rate increased and the 3-month relapse rate decreased in all ITP patients, and the therapeutic effect of patients with high megakaryocyte number was significantly higher than that of patients with low megakaryocyte number. The impact factors that influence the effect of glucocorticoid and(or) IVIG were the number of CD3+CD8+T lymphocytes, CD4/CD8 cell ratio, and the number of CD19+B lymphocytes. The independent impact factor of cyclosporin therapeutic response rate was the number of CD3+T lymphocytes. CONCLUSIONS: ITP is a heterogeneous disease, recurrence may occur during or rapidly after treatment. Cyclosporine included treatment can improve the effective rate of ITP and reduce the relapse rate within 3 months. The number of CD3+T lymphocytes was the only impact factor that influence the therapeutic effect of cyclosporin in ITP patients.

Immune cells surveil aberrantly sialylated O-glycans on megakaryocytes to regulate platelet count.

Immune thrombocytopenia (ITP) is a platelet disorder. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti-Thomsen-Friedenreich antigen (TF antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF antigen in these patients. The O-glycan sialyltransferase St3gal1 adds sialic acid specifically on the TF antigen. To understand if TF antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1 in megakaryocytes (MK) (St3gal1MK-/-). TF antigen exposure was restricted to MKs and resulted in thrombocytopenia. Deletion of Jak3 in St3gal1MK-/- mice normalized platelet counts implicating involvement of immune cells. Interferon-producing Siglec H-positive bone marrow (BM) immune cells engaged with O-glycan sialic acid moieties to regulate type I interferon secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following inhibition of interferon and Siglec H receptors. Single-cell RNA-sequencing determined that TF antigen exposure by MKs primed St3gal1MK-/- BM immune cells to release type I interferon. Single-cell RNA-sequencing further revealed a new population of immune cells with a plasmacytoid dendritic cell-like signature and concomitant upregulation of the immunoglobulin rearrangement gene transcripts Igkc and Ighm, suggesting additional immune regulatory mechanisms. Thus, aberrant TF antigen moieties, often found in pathological conditions, regulate immune cells and thrombopoiesis in the BM, leading to reduced platelet count.

Exacerbation of immune thrombocytopenia following COVID-19 vaccination.

There is concern that COVID-19 vaccination may adversely affect immune thrombocytopenia (ITP) patients. Fifty-two consecutive chronic ITP patients were prospectively followed after COVID-19 vaccination. Fifteen percent had no worsening of clinical symptoms but no post-vaccination platelet count; 73% had no new symptoms and no significant platelet count decline. However, 12% had a median platelet count drop of 96% within 2-5 days post vaccination with new bleeding symptoms; after rescue therapy with corticosteroids +/- intravenous immunoglobulin (IVIG), platelets recovered to >30 × 109 /l a median three days later. ITP exacerbation occurred independently of remission status, concurrent ITP treatment, or vaccine type. Safety of a second vaccine dose needs careful assessment.

Apoptosis in platelets from adult patients with chronic idiopathic thrombocytopenic purpura.

Adult chronic idiopathic thrombocytopenic purpura (cITP) is a chronic and usually life-long haemorrhagic disorder in which enhanced platelet destruction and weakened platelet production lead to thrombocytopenia. Platelets were isolated from blood samples collected from 40 adult patients with cITP and 40 healthy volunteers. Mitochondrial membrane potential (ΔΨm) and plasma membrane phosphatidylserine externalization were determined by flow cytometry, and activation of caspase-3 and expressions of Bax, Bak and Bcl-xL were analysed by western blotting. Flow cytometry showed increased mitochondrial depolarization and lower ΔΨm in platelets from adult patients with cITP. In addition, plasma membrane phosphatidylserine externalization was observed on platelets from adult patients with cITP, but rarely from healthy volunteers. Western blot analysis of platelet proteins revealed that, in adult cITP patients, caspase-3 was activated, which cleaved gelsolin and to release a 47-kDa fragment. Moreover, the expressions of Bax and Bak were elevated, and Bcl-xL was decreased markedly in platelets from adult patients with cITP. Our findings reveal, based on loss of mitochondrial membrane potential (Δψm), phosphatidylserine exposure, caspase-3 activation, enhanced expression of Bax and Bak, and attenuated expression of Bcl-xL, that platelet death in the pathogenesis of thrombocytopenia in chronic ITP in adults is apoptotic.

COVID-19 vaccine-induced immune thrombotic thrombocytopenia: A review of the potential mechanisms and proposed management.

With over 600 million coronavirus (COVID-19) vaccine doses administered globally, adverse events are constantly monitored. Recently however, reports of thrombosis and thrombocytopenia following vaccination with the ChAdOx1 nCoV-19 vaccine have emerged. This paper aims to review the available literature and guidelines pertaining to vaccine-induced immune thrombotic thrombocytopenia (VITT) and the proposed guidelines, while offering a potential approach that unifies the available evidence. While the risk of VITT remains extremely low and the benefits outweigh the risks, experimental studies are needed to clarify the pathophysiology behind VITT and possibly decrease the risk of thrombosis and other adverse events occurring. However, treatment should not be delayed in suspected cases, and IV immunoglobulin and non-heparin anticoagulation should be initiated.

Mechanisms of Immunothrombosis in Vaccine-Induced Thrombotic Thrombocytopenia (VITT) Compared to Natural SARS-CoV-2 Infection.

Herein, we consider venous immunothrombotic mechanisms in SARS-CoV-2 infection and anti-SARS-CoV-2 DNA vaccination. Primary SARS-CoV-2 infection with systemic viral RNA release (RNAaemia) contributes to innate immune coagulation cascade activation, with both pulmonary and systemic immunothrombosis - including venous territory strokes. However, anti-SARS-CoV-2 adenoviral-vectored-DNA vaccines -initially shown for the ChAdOx1 vaccine-may rarely exhibit autoimmunity with autoantibodies to Platelet Factor-4 (PF4) that is termed Vaccine-Induced Thrombotic Thrombocytopenia (VITT), an entity pathophysiologically similar to Heparin-Induced Thrombocytopenia (HIT). The PF4 autoantigen is a polyanion molecule capable of independent interactions with negatively charged bacterial cellular wall, heparin and DNA molecules, thus linking intravascular innate immunity to both bacterial cell walls and pathogen-derived DNA. Crucially, negatively charged extracellular DNA is a powerful adjuvant that can break tolerance to positively charged nuclear histone proteins in many experimental autoimmunity settings, including SLE and scleroderma. Analogous to DNA-histone interactons, positively charged PF4-DNA complexes stimulate strong interferon responses via Toll-Like Receptor (TLR) 9 engagement. A chain of events following intramuscular adenoviral-vectored-DNA vaccine inoculation including microvascular damage; microbleeding and platelet activation with PF4 release, adenovirus cargo dispersement with DNA-PF4 engagement may rarely break immune tolerance, leading to rare PF4-directed autoimmunity. The VITT cavernous sinus cerebral and intestinal venous territory immunothrombosis proclivity may pertain to venous drainage of shared microbiotal-rich areas of the nose and in intestines that initiates local endovascular venous immunity by PF4/microbiotal engagement with PF4 autoantibody driven immunothrombosis reminiscent of HIT. According to the proposed model, any adenovirus-vectored-DNA vaccine could drive autoimmune VITT in susceptible individuals and alternative mechanism based on molecular mimicry, vaccine protein contaminants, adenovirus vector proteins, EDTA buffers or immunity against the viral spike protein are secondary factors. Hence, electrochemical DNA-PF4 interactions and PF4-heparin interactions, but at different locations, represent the common denominator in HIT and VITT related autoimmune-mediated thrombosis.

Comparative study of IgG binding to megakaryocytes in immune and myelodysplastic thrombocytopenic patients.

Immune thrombocytopenia (ITP) is a disorder in which autoantibodies are responsible for destruction and decreased production of platelets. In the meantime, thrombocytopenia is frequent in patients with myelodysplastic syndromes (MDS) and immune clearance of megakaryocytes could be a reason. The aim of the present study is to evaluate and compare IgG binding to megakaryocytes in bone marrow of ITP and MDS patients to determine megakaryocytes targeting by autoantibodies in vivo as a mechanism of platelet underproduction in these disorders. The study was carried out on 20 ITP (group I) patients, 20 thrombocytopenic patients with (MDS) (group II), and 20 non-ITP patients as a control (group III) who were admitted to Minia University Hospital. Serial histological sections from bone marrow biopsies were stained for IgG. All patients in group I and 50% of group II patients showed bleeding tendency and the difference was significant (p < 0.001). No patient experienced fatigue in group I while 35% of patients in group II complained of easy fatigability, and the difference was significant (p < 0.008). High IgG antibody binding was found in ITP and MDS compared to the control group but no significant difference between ITP and MDS patients (14/20 (70%) vs. 13/20 (65%)) (p value = 0.736). Antibody binding to megakaryocytes in a proportion of MDS patients suggests that immune-mediated mechanism underlies platelet underproduction in those patients.