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.

The miR-641-STIM1 and SATB1 axes play important roles in the regulation of the Th17/Treg balance in ITP.

Immune thrombocytopenia (ITP) is an autoimmune disease caused by T-cell dysfunction. Recently, several studies have shown that a disturbed Th17/Treg balance contributes to the development of ITP. MicroRNAs (miRNAs) are small noncoding RNA moleculesthat posttranscriptionally regulate gene expression. Emerging evidences have demonstrated that miRNAs play an important role in regulating the Th17/Treg balance. In the present study, we found that miR-641 was upregulated in ITP patients. In primary T cells, overexpression of miR-641 could cause downregulation of its target genes STIM1 and SATB1, thus inducing a Th17 (upregulated)/Treg (downregulated) imbalance. Inhibition of miR-641 by a miR-641 sponge in primary T cells of ITP patients or by antagomiR-641 in an ITP murine model could cause upregulation of STIM1 and SATB1, thus restoring Th17/Treg homeostasis. These results suggested that the miR-641-STIM/SATB1 axis plays an important role in regulating the Th17/Treg balance in ITP.

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.

Genome-wide DNA methylation profiling of CD4+ T lymphocytes identifies differentially methylated loci associated with adult primary refractory immune thrombocytopenia.

BACKGROUND: DNA methylation played a crucial role in the pathogenesis of immune thrombocytopenia (ITP). However, genome-wide DNA methylation analysis has not been applied thus far. The present study aimed to provide the first DNA methylation profiling for ITP. METHODS: Peripheral blood CD4+ T lymphocytes samples were collected from 4 primary refractory ITP cases and 4 age-matched healthy controls, and DNA methylome profiling was performed using Infinium MethylationEPIC BeadChip. Differentially methylated CpG sites were further validated in another independent cohort of 10 ITP patients and 10 healthy controls using qRT-PCR. RESULTS: The DNA methylome profiling identified a total of 260 differentially methylated CpG sites mapping to 72 hypermethylated and 64 hypomethylated genes. These genes were mainly enriched in the actin nucleation of the Arp2/3 complex, vesicle transport, histone H3-K36 demethylation, Th1 and Th2 cell differentiation, and Notch signaling pathway according to the GO and KEGG databases. The mRNA expression of CASP9, C1orf109, and AMD1 were significantly different. CONCLUSIONS: Given the altered DNA methylation profiling of ITP, our study provides new insights into its genetic mechanism and suggests candidate biomarkers for the diagnosis and treatment of ITP.

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.

Tacrolimus prevents complement-mediated Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and pyroptosis of mesenchymal stem cells from immune thrombocytopenia.

The abnormal immunomodulatory functions of mesenchymal stem cells (MSCs) have been implicated in the development of immune thrombocytopenia (ITP). Recent studies have suggested important effects of complement on immune cell function. However, whether complement modulates bone marrow MSCs function in ITP is poorly defined. Tacrolimus has recently been applied to the treatment of autoimmune diseases. Here, we explored whether impaired ITP-MSCs could be targeted by tacrolimus. Our results showed that the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome was activated in ITP MSCs with complement deposition (MSCs-C+ ) and initiated caspase-1-dependent pyroptosis. Transcriptome sequencing results showed abnormal fatty acid metabolism in MSCs-C+ . Enhanced fatty acid ß-oxidation and reactive oxygen species production activated the NLRP3 inflammasome. Adipocytes derived from MSCs-C+ secreted less adiponectin. Adiponectin promoted the differentiation of megakaryocytes and inhibited the destruction of platelets. Tacrolimus inhibited NLRP3 inflammasome activation and MSCs-C+ pyroptosis in vitro and in vivo. Tacrolimus plus danazol elicited a higher sustained response than danazol monotherapy in corticosteroid-resistant patients with ITP. Our findings demonstrate that the activation of the NLRP3 inflammasome in ITP MSCs mediated by complement could be inhibited by tacrolimus, which might be a potential new therapy for ITP.

Insights Into Immunothrombotic Mechanisms in Acute Stroke due to Vaccine-Induced Immune Thrombotic Thrombocytopenia.

During the COVID-19 pandemic, vaccination is the most important countermeasure. Pharmacovigilance concerns however emerged with very rare, but potentially disastrous thrombotic complications following vaccination with ChAdOx1. Platelet factor-4 antibody mediated vaccine-induced immune thrombotic thrombocytopenia (VITT) was described as an underlying mechanism of these thrombotic events. Recent work moreover suggests that mechanisms of immunothrombosis including neutrophil extracellular trap (NET) formation might be critical for thrombogenesis during VITT. In this study, we investigated blood and thrombus specimens of a female patient who suffered severe stroke due to VITT after vaccination with ChAdOx1 in comparison to 13 control stroke patients with similar clinical characteristics. We analyzed cerebral thrombi using histological examination, staining of complement factors, NET-markers, DNase and LL-37. In blood samples at the hyper-acute phase of stroke and 7 days later, we determined cell-free DNA, myeloperoxidase-histone complexes, DNase activity, myeloperoxidase activity, LL-37 and inflammatory cytokines. NET markers were identified in thrombi of all patients. Interestingly, the thrombus of the VITT-patient exclusively revealed complement factors and high amounts of DNase and LL-37. High DNase activity was also measured in blood, implying a disturbed NET-regulation. Furthermore, serum of the VITT-patient inhibited reactive oxygen species-dependent NET-release by phorbol-myristate-acetate to a lesser degree compared to controls, indicating either less efficient NET-inhibition or enhanced NET-induction in the blood of the VITT-patient. Additionally, the changes in specific cytokines over time were emphasized in the VITT-patient as well. In conclusion, insufficient resolution of NETs, e.g. by endogenous DNases or protection of NETs against degradation by embedded factors like the antimicrobial peptide LL-37 might thus be an important factor in the pathology of VITT besides increased NET-formation. On the basis of these findings, we discuss the potential implications of the mechanisms of disturbed NETs-degradation for diagnostic and therapeutic approaches in VITT-related thrombogenesis, other auto-immune disorders and beyond.

[Functional deficiency of bone marrow mesenchymal stem cells in patients with immune thrombocytopenia: An update].

Immune thrombocytopenia (ITP) is an acquired autoimmune hemorrhagic disorder characterized by persistent thrombocytopenia. It may be induced by different pathogenesis due to its heterogeneity, and the therapeutic effects vary on different patients. Bone marrow derived mesenchymal stem cells (BMMSCs) can modulate innate and adaptive immunity, thus resulting in a tolerant microenvironment. Functional defects and immunomodulatory disorders of BMMSCs are significant causes of ITP. Functional effects associated with the activation of the P53 pathway include decreased activity of the phosphatidylinositol 3 kinase/AKT pathway and activation of the TNFAIP3/NF-κB/SMAD7 pathway. Immune dysfunction appears to be correlated with an impaired ability of BMMSCs to induce various types of immune cells in ITP. An in-depth investigation into the pathogenesis of ITP facilitates the treatment of ITP, but larger-scale clinical trials are needed to verify the efficacy of exogenous BMMSCs in the clinical treatment of ITP.

Glucocorticoid receptor modulates myeloid-derived suppressor cell function via mitochondrial metabolism in immune thrombocytopenia.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells and natural inhibitors of adaptive immunity. Intracellular metabolic changes in MDSCs exert a direct immunological influence on their suppressive activity. Our previous study demonstrated that high-dose dexamethasone (HD-DXM) corrected the functional impairment of MDSCs in immune thrombocytopenia (ITP); however, the MDSC population was not restored in nonresponders, and the mechanism remained unclear. In this study, altered mitochondrial physiology and reduced mitochondrial gene transcription were detected in MDSCs from HD-DXM nonresponders, accompanied by decreased levels of carnitine palmitoyltransferase-1 (CPT-1), a rate-limiting enzyme in fatty acid oxidation (FAO). Blockade of FAO with a CPT-1 inhibitor abolished the immunosuppressive function of MDSCs in HD-DXM responders. We also report that MDSCs from ITP patients had lower expression of the glucocorticoid receptor (GR), which can translocate into mitochondria to regulate the transcription of mitochondrial DNA (mtDNA) as well as the level of oxidative phosphorylation. It was confirmed that the expression of CPT-1 and mtDNA-encoded genes was downregulated in GR-siRNA-treated murine MDSCs. Finally, by establishing murine models of active and passive ITP via adoptive transfer of DXM-modulated MDSCs, we confirmed that GR-silenced MDSCs failed to alleviate thrombocytopenia in mice with ITP. In conclusion, our study indicated that impaired aerobic metabolism in MDSCs participates in the pathogenesis of glucocorticoid resistance in ITP and that intact control of MDSC metabolism by GR contributes to the homeostatic regulation of immunosuppressive cell function.

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.

A functional spleen contributes to afucosylated IgG in humans.

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

Primary Immune Thrombocytopenia and Essential Thrombocythemia: So Different and yet Somehow Similar-Cases Series and a Review of the Literature.

This article collects several published cases in which immune thrombocytopenic purpura (ITP) is followed by essential thrombocythemia (ET) and vice versa. This surprising clinical condition is possible, but very rare and difficult to diagnose and manage. We have made an attempt to analyse the possible causes of the sequential appearance of ITP and ET taking into consideration the following: alteration of the thrombopoietin (TPO) receptor, the role of autoimmunity and inflammation, and cytokine modulation. A better understanding of these interactions may provide opportunities to determine predisposing factors and aid in finding new treatment modalities both for ITP and ET patients.

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.

IL-23/Th17 pathway and IL-17A gene polymorphism in Egyptian children with immune thrombocbytopenic purpura.

BACKGROUND: Immune thrombocytopenic purpura (ITP) is an acquired complex autoimmune thrombocytopenia. Uncontrolled cellular immune response is one of the key triggers for the loss of immune tolerance in ITP patients. The purpose of this study was to investigate the association of IL-23/Th17, IL-17A and IL-17A rs2275913 gene polymorphism with ITP in Egyptian children. METHODS: 60 patients with ITP and 50 healthy control children from Minia city- Egypt were involved. Serum levels of IL-23 and IL-17A were determined by enzyme-linked immunosorbent assay. The frequency of Th17 cells was measured using flow cytometer. Genotyping for IL-17A was performed via polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Comparing children with ITP to controls, serum levels of IL-23 and IL-17A as well as Th17 cells percentage were significantly increased (p <  0.001). Also, higher levels of these ILs and Th17 cells percentage were associated with decreased platelet count within ITP patients (p <  0.001). Analysis of genotype frequencies for IL-17A rs2275913 polymorphism and its alleles (A, G) showed no significant difference between cases and controls. Likewise, no significant differences were demonstrated between acute and chronic ITP regarding both IL-17A rs2275913 polymorphism prevalence and levels of IL-23, IL-17A plus Th17 cells percentage. The frequency of A alleles was 85 and 86% within patients and controls, respectively. CONCLUSIONS: Elevated levels of IL-23, IL-17A and Th17 cells may be involved in ITP pathogenesis while IL-17A polymorphism rs2275913 is not prevalent in Egyptian children with ITP.

Elucidating the Mechanism of Action of the Attributed Immunomodulatory Role of Eltrombopag in Primary Immune Thrombocytopenia: An In Silico Approach.

Eltrombopag is a thrombopoietin receptor (MPL) agonist approved for the treatment of primary immune thrombocytopenia (ITP). Recent evidence shows that some patients may sustain platelet counts following eltrombopag discontinuation. The systemic immunomodulatory response that resolves ITP in some patients could result from an increase in platelet mass, caused either by the direct action of eltrombopag on megakaryocytes through MPL stimulation, or potential MPL-independent actions on other cell types. To uncover the possible mechanisms of action of eltrombopag, in silico analyses were performed, including a systems biology-based approach, a therapeutic performance mapping system, and structural analyses. Through manual curation of the available bibliography, 56 key proteins were identified and integrated into the ITP interactome analysis. Mathematical models (94.92% mean accuracy) were obtained to elucidate potential MPL-dependent pathways in non-megakaryocytic cell subtypes. In addition to the effects on megakaryocytes and platelet numbers, the results were consistent with MPL-mediated effects on other cells, which could involve interferon-gamma, transforming growth factor-beta, peroxisome proliferator-activated receptor-gamma, and forkhead box protein P3 pathways. Structural analyses indicated that effects on three apoptosis-related proteins (BCL2L1, BCL2, BAX) from the Bcl-2 family may be off-target effects of eltrombopag. In conclusion, this study proposes new hypotheses regarding the immunomodulatory functions of eltrombopag in patients with ITP.

The Role of Leptin in Childhood Immune Thrombocytopenia (ITP): An Anti-Inflammatory Agent?

To investigate the effect of leptin in childhood ITP, we measured plasma leptin in 39 children with acute ITP, after treatment and in remission, and in 33 healthy age/BMI-matched controls. We also cultured ITP and control peripheral blood mononuclear cells (PBMCs) with recombinant leptin to assess its direct effect on pro/anti-inflammatory cytokine gene expression. A significant increase in leptin was observed in children with active disease compared to controls. A significant inverse correlation of leptin with platelet count was also observed in children with acute ITP. Leptin remained high after treatment with IVIg, whereas steroid treatment lowered leptin below control levels. In remission, leptin was in the control range. Cytokine gene expression was significantly increased in children with acute ITP compared with controls, with highest expression for IFN-γ and IL-10. IVIg/steroid treatment significantly decreased IFN-γ and IL-10 expression. In remission, IFN-γ and IL-10 expression remained low. Addition of leptin to PBMCs isolated from patients in remission resulted in a significant increase in IL-10 gene expression compared to controls. Further experiments with purified T-cells and monocytes identified monocytes as the source of leptin-induced IL-10. We suggest that leptin acts as an active anti-inflammatory agent in childhood ITP by promoting IL-10 secretion by monocytes.

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.

The role and mechanism of miR-557 in inhibiting the differentiation and maturation of megakaryocytes in immune thrombocytopenia.

Specific miRNA in immune thrombocytopenia (ITP) was screened to explore its intervention effects and mechanisms in ITP. MTT assay and CFSE staining were used to detect the effects of gradient concentrations of thrombopoietin (TPO) on cell proliferation. Expressions of differentially expressed miRNAs were analysed via qRT-PCR in TPO-induced megakaryocytes and ITP plasma. Effects of miR-557 on cell physiological functions were examined by MTT and flow cytometry. Expressions of miR-557, apoptosis-associated genes and Akt/ERK pathways were detected by qRT-PCR and Western blot as needed. Multinucleation of TPO-induced megakaryocytes was determined by megakaryocyte colonies. The toe skin and intestinal bleeding of the ITP rat model were observed and evaluated. Effects of miR-557 on the numbers of platelets, megakaryocytes, and peripheral blood platelets and the expressions of CD4+ T cells, Treg cells, TGF-ß, IL-6 and miR-557 in the ITP rats were detected by Giemsa staining, flow cytometry, ELISA and qRT-PCR. MiR-557 was identified as an specific miRNA associated with both ITP and TPO treatment. MiR-557 inhibitor enhanced the physiological functions of TPO-induced megakaryocytes, while miR-557 mimic had the opposite effect. At the molecular level, the expressions of miR-557, cleaved Caspase-3 and Bax were further silenced by inhibitor, on the contrary, the expressions of bcl-2, p-Akt and p-ERK were upregulated. Animal experiments showed that, miR-557 inhibitor increased the numbers of platelets and megakaryocytes, and improved the symptoms of ITP model rats. Our results indicated that miR-557 inhibitor improved ITP by regulating apoptosis-related genes and cellular immunity and activating the Akt/ERK pathway.

Extracellular vesicles derived from miR-199a-5p-modified adipose-derived mesenchymal stem cells alleviate immune thrombocytopenia by inhibiting T helper 17 differentiation.

The abnormal differentiation of T helper 17 (Th17) cells is considered a vital promoter of immune thrombocytopenia (ITP) progression. Therefore, this study investigated the role of miR-199a-5p in Th17 differentiation and determined whether extracellular vesicles (EVs) derived from miR-199a-5p-modified adipose-derived mesenchymal stem cells (ADSCs) could relieve ITP by inhibiting Th17 differentiation. The miR-199a-5p level was lessened in the spleen tissues of mice with ITP, while the signal transducer and activator of transcription 3 (STAT3) expression and the population of Th17 in CD4+T cells were boosted. Functionally, miR-199a-5p overexpression lowered IL-17 secretion and the proportion of Th17/CD4+T cells. Further investigation showed that miR-199a-5p directly targeted STAT3 mRNA, and negatively modulated its expression. STAT3 overexpression was found to facilitate Th17 differentiation, which was subsequently abolished by miR-199a-5p overexpression. EVs isolated from miR-199a-5p-modified ADSCs (miR-199a-5p-EVs) highly expressed miR-199a-5p and could restrain CD4+T cells polarized toward a Th17 phenotype in vitro. Administering of miR-199a-5p-EVs elevated platelet counts and decreased the proportion of Th17/CD4+T cells in mice with ITP. Taken together, EVs derived from miR-199a-5p-modified ADSCs vividly repressed Th17 differentiation by transferring miR-199a-5p to CD4+T cells, thus ameliorating experimental ITP.