Immune Thrombocytopenia Mice Research Articles

Interleukin 9 prevents immune thrombocytopenia in mice via JAK/STAT5 signaling

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by autoimmune-mediated platelet destruction and impaired platelet production, which can lead to an increased risk of bleeding. The clinical management of ITP currently remains a challenge for hematologists. We explored the role of interleukin-9 (IL-9) in the treatment of CD41-induced ITP, and investigated its underlying mechanisms in a CD41-induced ITP mouse model. IL-9 treatment increased the numbers of mature megakaryocytes (CD41+CD42d+) and CD41+Sca-1+ cells in the bone marrow in these model mice, while IL-9 receptor (IL-9R) small interfering RNA (siRNA) inhibited the process. Moreover, phosphorylated signal transducer and activator of transcription 5 (STAT5), as a downstream molecule of IL-9R, was increased after IL-9 treatment. We next investigated the source of IL-9 in bone marrow, osteoblasts produced the highest level of IL-9. These results confirmed that IL-9 could prevent CD41-induced ITP in BALB/c mice by regulating osteoblasts and activating IL-9R/STAT5 signaling in megakaryocytes, thus providing further evidence for IL-9 as a promising therapeutic agent for the treatment of ITP.

ATRA Could Correct the Defective S1P-Mediated Cytoskeletal Reorganization in Proplatelet Formation of ITP

Introduction Sphingosine-1-phosphate (S1P) is now emerging as a vital lipid mediator. Activation of sphingosine kinase (SphK) produces intracellular S1P, which in turn can be secreted out of the cell and act extracellularly by binding to S1P receptors (S1PR). Recent studies suggest that the "inside-out" signaling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). PPF requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases which can be activated by S1P, including Rac1 and Cdc42, have been shown to be master regulators of cytoskeletal rearrangements. The pathogenesis mechanisms of immune thrombocytopenia (ITP) are not entirely understood. 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 S1P-mediated microtubule reorganization is defective in PPF of ITP. All-trans retinoic acid (ATRA), which has demonstrated to be a promising option for ITP patients in our previous study (Lancet Haematology, 2017), could correct the altered microtubule reorganization and promote PPF. Methods Thirty consecutive patients with primary ITP and 20 healthy donors were enrolled in our study. MKs were isolated from bone marrow samples, and they were collected again after ITP patients received ATRA therapy. MK mRNA sequencing by microarray was used to assess the difference of gene expression between ITP and controls. Microtubule regrowth assay was performed to observe microtubule dynamic behavior. In this assay nocodazole was first used to induce complete depolymerization of microtubule network, followed by drug washout to allow microtubule regrowth over time. ATRA was added to the culture medium of MKs to determine the mechanism of ATRA in correcting impaired PPF. Additionally, ITP mice model was established to observe the therapeutic effects of ATRA in PPF. Pf4-Cre/loxP system was used to specifically knock down gene of MKs. Results S1P concentration in bone marrow from ITP patients was lower compared to healthy donors. MKs mRNA sequencing demonstrated that S1P synthetase SphK2 and S1P receptor S1PR1 gene were downregulated while S1P lyase (SPL) gene was upregulated in ITP patients, which caused abnormal S1P signaling. Furthermore, we observed that PPF capacity of MKs in patients with ITP was reduced. Pharmacological disruption of S1PR1 blocked PPF, exogenous S1P corrected impaired PPF. Collectively, deregulation of S1P signaling was associated with impaired PPF in ITP. To verify the downstream role of S1P in regulating PPF, the Rho GTPases detection of MKs revealed a decrease in Cdc42 and Rac1 levels from ITP patients. Immunofluorescence of the differentiated MKs showed that the expression and distribution of β1 tubulin were abnormal from ITP patients. Early PPs from MKs of healthy donors displayed a well-organized tubulin bundles resembling bunches of grapes. In contrast, in MKs from ITP patients, tubulin was disorganized in thick bundles. In addition, TEM analysis of the MKs showed an irregular distribution of granules, tortuous membranes and impaired proplatelet structure. In microtubule regrowth assay, MKs from ITP patients had significantly lower microtubule regrowth at 10 min post-nocodazole washout compared with controls. Together, microtubule alteration resulted in impaired PPF in ITP. We tested whether S1P pathway were required for microtubule reorganization, both SphK2-/- and S1PR1-/- mice displayed significantly reduced S1P, Cdc42 and Rac1, altered microtubule architecture and defective PPF. Taken together, abnormal S1P pathway accounted for impaired microtubule reorganization in ITP. Next, we explored the effect of ATRA on microtubules reorganization in ITP patients, our data showed that in vitro treatment with ATRA restored microtubules structure by upregulating S1P and activating Rho GTPases. In vivo studies showed that ARTA could rescue the impaired PPF in both patients and mice model with ITP. Conclusions The MKs of ITP patients displayed defective cytoskeletal reorganization regulated by S1P pathway. ATRA restored cytoskeletal structure and corrected impaired PPF by upregulating S1P and activating Rho GTPases. It sheds light on a novel mechanism of ITP pathogenesis and provides a basis for the therapeutic potential of ARTA in ITP patients. Disclosures No relevant conflicts of interest to declare.

Thalidomide prevents antibody-mediated immune thrombocytopenia in mice

Immune thrombocytopenia (ITP) is a heterogeneous autoimmune disorder characterized by immune-mediated platelet destruction, leading to lower platelet count. Thalidomide is considered as a novel immunomodulatory drug for treating several autoimmune diseases. Whether thalidomide can ameliorate ITP remains unclear. This study aims to evaluate the effect of thalidomide on ITP mouse model. ITP mouse model was established through intraperitoneal injection of rat anti-mouse integrin GPIIb/CD41 immunoglobulin. Thalidomide (10, 20 or 50 mg/kg body weight) was intraperitoneally injected into mice followed by antibody injection. Then, peripheral blood and plasma was isolated for analysis of platelet count and the level of IFN-γ and IL-17 in plasma. Meanwhile, spleen was extracted to measure the expression of CD68, a macrophage marker. In addition, macrophage cell line RAW264.7 was cultured and treated with thalidomide followed by analysis of cell viability, apoptosis as well as cell cycle. Thalidomide prevented antiplatelet antibody-mediated platelet destruction in ITP mouse model. Compared with vehicle (phosphate-buffered saline), thalidomide significantly inhibited the secretion of IFN-γ and IL-17 in ITP mouse and reduced the expression of CD68 in spleen. After thalidomide treatment, the cell viability of RAW264.7 cell was significantly reduced and the cell number in S phase was also significantly decreased. In addition, the expression of cyclin E2 was significantly reduced. In conclusion, thalidomide prevents antiplatelet antibody-mediated platelet destruction in ITP mouse possibly through reducing the number of macrophages, suggesting that it might be a novel approach for treating ITP.

PS1494 REDUCED ANTIOXIDANT CAPACITY INDUCED PYROPTOSIS IN PLATELETS IN IMMUNE THROMBOCYTOPENIA PATIENTS

Background:The pathophysiology of primary immune thrombocytopenia (ITP) is not fully understood. Antioxidant capacitie (AOC) is reduced in ITP platelets. Mitochondrial reactive oxygen species (ROS) generation can lead to NLRP3 inflammasome activation and pyroptosis. However, the process has not been studied in ITP platelets.Aims:To assess whether the reduced AOC of platelets in ITP can affect the NLRP3 inflammasome signaling pathways and to further explore the pathophysiology of ITP.Methods:We measured mitochondrial membrane potential (MMP) of platelets from patients and healthy donors using JC‐1 by flow cytometry. Next, we investigated AOC of platelets by supplementation of 300 μM H2O2following loading with 50 μM 2’,7’‐dichlorodihydrofluorescein diacetate. ROS level as well as expression of NLRP3, ASC, pro‐caspase‐1 and p20 in platelets after stimulation with H2O2was detected. Besides, co‐localization of NLRP3 and ASC in platelets after stimulation was assessed by immunofluorescence. Blocking experiments were performed using antioxidant N‐Acetyl‐L‐cysteine, NLRP3 inhibitorMCC950 (CP‐456773) and caspase‐1 inhibitorZ‐VAD‐FMK. Furthermore, we studied the pyroptosis of platelets in passive ITP mouse model using NLRP3‐/‐ mice, Caspase‐1‐/‐ mice and wild type mice.Results:The platelets from ITP patients showed significantly reduced antioxidant capacity compared to healthy donors. A significant lower MMP and higher ROS level were detected in ITP platelets. Expression of NLRP3, ASC, pro‐caspase‐1 and p20 was obviously increased and co‐localization of NLRP3 and ASC was more evident in ITP platelets. An increased level of IL‐1β and IL‐18 were observed in culture supernatant. The application of antioxidant N‐Acetyl‐L‐cysteine (NAC), NLRP3 inhibitor MCC950(CP‐456773) and caspase‐1 inhibitor Z‐VAD‐FMK to H2O2‐stimulated ITP platelets significantly inhibit pyroptosis. The indicators of pyroptosis were down‐regulated in NLRP3‐/‐ and Caspase‐1‐/‐ mice.Summary/Conclusion:The reduced AOC of platelets in ITP results in accumulation of intracellular ROS, which contributes to the activation of NLRP3 inflammasome signaling pathways and leads to pyroptosis, which accelerates thrombocytopenia in ITP.image

Mesenchymal stem cells improve platelet counts in mice with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder. The breakdown of immune tolerance (regulatory T [Treg] cells and suppressor cytokines) plays an important role in ITP pathophysiology, especially in refractory ITP. Bone marrow-derived mesenchymal stem cells (BM-MSCs) show immunomodulatory properties and have been extensively utilized for autoimmune diseases. However, it has not been fully elucidated how BM-MSCs affect ITP. In this study, we explore the therapeutic mechanism of BM-MSCs on ITP in mice. Dose-escalation passive ITP mice were inducted by injection of MWReg30. BALB/c mice were randomly divided into two groups: ITP with BM-MSC transplantation and ITP controls. The serum levels of cytokines (interleukin 10 [IL-10] and transforming growth factor-β1 [TGF-β1]) were examined by enzyme-linked immunosorbent assays. The frequency of Treg cells in both peripheral blood and spleen mononuclear cells was analyzed by flow cytometry, and the forkhead box P3 (Foxp3) messenger RNA (mRNA) level was measured by real-time polymerase chain reaction. After BM-MSC treatment, the platelet (PLT) counts were significantly elevated. Meanwhile, cytokines (TGF-β1 and IL-10), the ratios of Treg cells, and the Foxp3 mRNA expression level were significantly higher in the BM-MSC group. Our results show that BM-MSCs can improve PLT counts mainly by secreting suppressive cytokines and upregulating Tregs, which may provide new therapeutic potential for human ITP.

Low-dose chidamide restores immune tolerance in ITP in mice and humans

AbstractIncreased macrophage phagocytosis of antibody-coated platelets, as well as decreased numbers and/or impaired function of CD4+CD25+Foxp3+ regulatory T (Treg) cells, has been shown to participate in the pathogenesis of immune thrombocytopenia (ITP). Low-dose histone deacetylase inhibitors (HDACi's) are anti-inflammatory and immunomodulatory agents that can enhance immunosuppression in graft-versus-host disease by increasing the number and function of Foxp3+ Treg cells, but it is unclear whether they have the potential to promote immune tolerance and platelet release in ITP. In this study, we performed in vitro and in vivo experiments and found that a low-dose HDACi (chidamide) alleviated thrombocytopenia in passive and active murine models of ITP. Further, low-dose HDACi's attenuated macrophage phagocytosis of antibody-coated platelets, stimulated the production of natural Foxp3+ Treg cells, promoted the peripheral conversion of T cells into Treg cells, and restored Treg cell suppression in vivo and in vitro. Finally, we confirmed that low-dose HDACi's could regulate CTLA4 expression in peripheral blood mononuclear cells through modulation of histone H3K27 acetylation. Low-dose HDACi treatment in ITP could be offset by blocking the effect of CTLA4. Therefore, we propose that low-dose chidamide administration has potential as a novel treatment for ITP in the clinic.

Vincristine-loaded platelets coated with anti-CD41 mAbs: a new macrophage targeting proposal for the treatment of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder in which platelet-reactive autoantibodies accelerate the destruction of platelets. Macrophages play an important role in ITP through Fc receptor (FcR)-mediated antigen presenting and platelet clearance. In this study, a novel drug delivery system of vincristine-loaded platelets coated with anti-CD41 mAbs (CD41-VCR-PLT, CD41-VLP) was successfully established. The therapeutic effects and safety of CD41-VLP in vitro and in vivo were evaluated, and the possible mechanism was also explored. The results showed that PLT-CD41 could load VCR with high drug loading (DL) and encapsulation efficiency (EE), which were up to 41.16 ± 1.92% and 60.73 ± 2.79%, respectively, where platelets had no obvious morphological or functional changes. CD41-VLP could facilitate vincristine accumulation in macrophages, where the intracellular VCR concentration was 30.72 ± 3.11% at 72 h, which was significantly increased compared with the other groups (P < 0.01), thus inhibiting macrophage cell viability and inducing apoptosis. The cell viability inhibition rate and total apoptosis rate were 73.06 ± 5.26% and 69.70 ± 4.26%, respectively, both much higher than those of the other groups (P < 0.05). In the ITP mouse model, CD41-VLP increased the platelet count in peripheral blood, which was 720 ± 197.98 × 109 L-1, and significantly improved the platelet count compared with that in the VCR group (P < 0.05); moreover, it reduced the systemic toxicity and peripheral neurotoxicity of vincristine. The possible mechanism was that CD41-VLP could precisely target M1 macrophages in spleen and liver tissues through FcγR, thus reducing the platelet destruction caused by M1 macrophages. Therefore, CD41-VLP provides a new targeted therapy for ITP treatment.

Low-Dose Decitabine Could Improve the Imbalance of M1/M2 Ratio in Immune Thrombocytopenia

BackgroundPrimary immune thrombocytopenia (ITP) is an acquired autoimmune hemorrhagic disease characterized by increased immune-mediated platelet destruction and insufficient platelet production. As the impaired balance of M1 macrophage and M2 macrophage subsets increases platelet destruction, the M1/M2 ratio has become the focus of attention. The demethylating agent, decitabine (DAC) has been successfully used in the treatment of myelodysplastic syndrome with a moderate response in thrombocytopenia. Interestingly, at low doses, DAC induces megakaryocyte differentiation and maturation in vitro via inducing hypomethylation, thus having the potential to promote platelet release in ITP patients. In addition, several reports suggest that DAC can promote the polarization of macrophages to M2 macrophages. Therefore, we hypothesized that low dose of DAC treatment could lead to shrinkage of platelet destruction in murine model of ITP via promoting the M2 macrophage polarization.MethodsPlatelets isolated from Wistar rats were transferred into CBA/Ht mice via intraperitoneal injection weekly to induce a marine model of primary ITP. Starting on day 8, ITP mice were treated with low-dose DAC (0.25 mg/kg/d, n = 8) or PBS (n = 8) for two consecutive weeks intraperitoneally. Platelet counts were recorded every three days. The spleens were removed at day 10, from which M1 macrophages (F4/80+ CD11c+) and M2 macrophages (F4/80+ CD206+) were identified by flow cytometry analysis and immunofluorescence.ResultsThe platelet levels in ITP group began to descend sharply at the second week after transferring platelets when compared with the control group (735.0 ± 25.2 × 109vs. 390.3 ± 70.0 × 109, p < 0.05). Mice in DAC-treated group recovered from thrombocytopenia by day 10 (713.8 ± 29.1 × 109vs. 311.0 ± 4.5 × 109, p < 0.001). In the following days, it still showed significantly higher level of platelet in DAC-treated group than ITP group despite the trend to decline. Comparing with the ITP group, immunofluorescence revealed that spleen samples showed stronger M2 expression pattern in DAC group, and the ratio of M1/M2 declined regardless of the fact that both the percentage of M1 macrophage and M2 macrophage was increased when DAC was applied.ConclusionsLow dose DAC can promote the polarization of both M1 and M2 macrophage, especially M2 macrophage, thus reversing the unbalanced M1/M2 ratio in ITP group. Further studies are required to investigate the mechanism underlies the specialized polarization of macrophages. DisclosuresNo relevant conflicts of interest to declare.

18β-Glycyrrhetic Acid Modulates Th1/Th17/Th22/Regulatory T Cells Homeostasis Via HMGB1/NF-κB Signaling Pathway in Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disorder. Loss of peripheral immune tolerance in ITP patients leads to autoreactive T cells proliferation and production of auto-antibodies against platelet cell surface antigen. It was reported that the homeostasis of CD4+ T-cell subsets, including the balance of T helper1(Th1)/T helper2(Th2) and T helper17(Th17)/CD4+CD25+FOXP3+ regulatory T cells (Tregs), was disturbed in ITP patients.High-mobility group box 1 (HMGB1), a non-histone nuclear protein, plays a critical role not only inside of the cell as a DNA chaperone, but also outside of the cell as the damage associated molecular pattern molecule (DAMP). In our previous studies, we found a significant increase of HMGB1 expression in peripheral blood mononuclear cells (PBMCs) and blood plasma in ITP patients compared with healthy controls by q-PCR and ELISA analysis. (Fig. 1)18β-glycyrrhetinic acid (18β-GA) is a novel pharmacological inhibitor of HMGB1 cytokine activities, which attributed effects like antioxidant, anti-viral, and anti-cancer activities, clinically used for the treatment of allergic purpura, chronic urticaria, ulcerative colitis possibly by upregulating Tregs. However, whether 18β-GA could regulate the immune homeostasis in ITP is unknown.To examine whether 18β-GA could facilitate the development and function of Tregs in ITP, PBMCs were separated from ITP patients and healthy controls, and cultured with DMSO or different doses of 18β-GA (12.5μM/25μM/50μM/100μM). We observed a dose-dependent increase in the number of Tregs both in the culturing of PBMCs from ITP patients and healthy controls by flow cytometric analysis. (Fig. 2) And 18β-GA could significantly induce the apoptosis of PBMCs at high doses (50μM and 100μM), so we selected 25μM as the optimal dose in our in vitro study. In addition, we isolated the CD4+ naïve T cells to investigate whether 18β-GA could directly induce the development of Tregs. It was shown that 18β-GA could directly activate and induce the differentiation of Tregs from CD4+ naïve T cells. (Fig. 3)Furthermore, we isolated the CD4+CD25+FOXP3+ Tregs from ITP patients, and then cocultured with CD4+CD25- effector T cells from the same patient at a ratio of 1:4, with the administration of 25μM 18β-GA or DMSO, and stimulated with anti-CD3 antibodies, anti-CD28 antibodies, and rhIL-2. After incubation for 5 days, the proliferation of effector T cells was analyzed by flow cytometry. The results showed that Tregs significantly inhibited the proliferation of effector T cells, and the addition of 18β-GA could enhance the suppressive effect of Tregs.To survey whether 18β-GA could modulate the homeostasis of the CD4+ T-cell subsets and to explore the mechanism, we isolated the CD4+ T cells from ITP patients and cultured with 25μM 18β-GA or recombinant HMGB1 (rHMGB1) for 5 days. The levels of Th1, Th17 and Th22 showed significant decreases and the level of Tregs increased in the 18β-GA group, compared with the DMSO group by flow cytometry. (Fig. 4) However, this change was apparently attenuated in the rHMGB1 group. After incubation for 5 days, the culture supernatants were analyzed by ELISA, and the cells were used for analyses by q-PCR. We also observed a decrease in the level of HMGB1 expression from results of ELISA and q-PCR in the 18β-GA group, and the de-phosphorylation of NF-κB on the downstream pathway of HMGB1, as well as the decrease of mRNA levels of cytokines such as IL-1β, IL-6, and TNFα. We confirmed that 18β-GA could directly affect CD4+ T cells and modulate Th1/Th17/Th22/Tregs homeostasis by reducing the expression of HMGB1.Finally, we established a passive murine model of ITP by injecting anti-CD41 antibody every 36 hours and divided ITP mice randomly into two groups with intraperitoneal injection of 18β-GA or the same dose of phosphate buffered saline as a control group. After 7 days, we examined the expression of Tregs by flow cytometry. It was observed that 18β-GA increased the numbers of Tregs in the peripheral blood, lymph nodes and spleen, compared with the control group.Our findings demonstrated the regulatory role of 18β-GA in maintaining CD4+ T cells homeostasis in immune thrombocytopenia. 18β-GA could reduce the pro-inflammatory Th1, Th17 and Th22 cells whereas enhance the number and functions of Tregs by reducing the expression of HMGB1. Therefore, 18β-GA might be a promising therapeutic strategy for the management of ITP patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

GM-CSF and IL-4 are not involved in IVIG-mediated amelioration of ITP in mice: a role for IL-11 cannot be ruled out.

Previously, we have reported that interleukin (IL)-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-11, but not IL-33, are up-regulated in two strains of mice with immune thrombocytopenia (ITP) that are responsive to intravenous immunoglobulin (IVIg) treatment. Previously, IL-4 was ruled out in the mechanism of IVIg; however, other publications have suggested this cytokine as a major player in the mechanism of IVIg action. Thus, we sought to further investigate a role for IL-4 and, in addition, GM-CSF and IL-11 in the mechanism of action of IVIg using a murine model of ITP. A passive platelet antibody model was used to generate ITP in IL-4 receptor knock-out (IL-4R-/- ), IL-11 receptor knock-out (IL-11Rα-/- ) and GM-CSF knock-out (Csf2-/- ) mice. We also used a neutralizing antibody to IL-11 and recombinant human IL-11 (rhIL-11) in addition to depleting basophils in vivo to study the effect of IVIg to ameliorate ITP. Our results showed that basophils, IL-4 and GM-CSF were unimportant in both ITP induction and its amelioration by IVIg. The role of IL-11 in these processes was less clear. Even though IL-11Rα-/- mice with ITP responded to IVIg similarly to wild-type (WT) mice, treatment of ITP WT mice with rhIL-11 instead of IVIg showed an increase in platelet numbers and WT mice administered anti-IL-11 showed a significant reduction in the ability of IVIg to ameliorate the ITP. Our findings indicate that neither IL-4, basophils or GM-CSF have roles in IVIg amelioration of ITP; however, a role for IL-11 requires further study.

Autoantibody against integrin αvβ3 contributes to thrombocytopenia by blocking the migration and adhesion of megakaryocytes

Background The pathogenesis of immune thrombocytopenia (ITP) has not been fully clarified. Anti-αvβ3 integrin autoantibody is detected in chronic ITP patients, but its contribution to ITP is still unclear. Objectives To clarify the potential role of anti-αvβ3 integrin autoantibody in chronic ITP and the related mechanism. Methods Relationship between levels of anti-αvβ3 autoantibody and platelets in chronic ITP patients was evaluated. The influence of anti-αvβ3 antibody on megakaryocyte (MK) survival, differentiation, migration and adhesion was assessed, and the associated signal pathways were investigated. Platelet recovery and MKs' distribution were observed in an ITP mouse model pretreated with different antibodies. Result In this study, we showed that the anti-αvβ3 autoantibody usually coexists with anti-αIIbβ3 autoantibody in chronic ITP patients, and patients with both autoantibodies have lower platelets. In invitro studies, we showed that the anti-αvβ3 antibody had no significant effect on the survival and proliferation of MKs, whereas it decreased formations of proplatelet significantly. Anti-αvβ3 antibody impeded stromal cell derived facor-1 alpha (SDF-1α)- mediated migration and inhibited the phosphorylation of protein kinase B. Anti-αvβ3 antibody significantly inhibited MKs' adhesion to endothelial cells and Fibrogen. The phosphorylation of focal adhesion kinase and proto-oncogene tyrosine-protein kinase Src induced by adhesion was inhibited when MKs were pretreated with anti-αvβ3 antibody. In invivo studies, we showed that injection with anti-αv antibody delayed platelet recovery in a mouse model of ITP. Conclusions These findings demonstrate that the autoantibody against integrin αv β3 may aggravate thrombocytopenia in ITP patients by impeding MK migration and adhesion to the vascular niche, which provides new insights into the pathogenesis of ITP.

Transplantation of bone-marrow-derived mesenchymal stem cells into a murine model of immune thrombocytopenia.

: Several reports have demonstrated T regulatory cells may play an important role in the pathophysiology of immune thrombocytopenia (ITP). As the immunomodulator, bone-marrow-derived mesenchymal stem cells (MSCs) (BM-MSCs) regulate T regulatory cells and show therapeutic effects on autoimmune diseases. However, it is not clear how BM-MSCs affect ITP. In this study, we explored the specific effects of BM-MSCs on ITP in mice. Using a murine model of ITP, mice were randomly divided into three groups: normal control group, ITP control group and ITP and BM-MSCs group. Platelet (PLT) levels were monitored by an automatic blood cell counter, and T regulatory cells were analyzed by flow cytometry. Compared with the untreated ITP mice, the PLT level of the ITP mice was significantly increased after BM-MSCs treatment. In the BM-MSCs group, T regulatory cells were significantly decreased. These findings demonstrate that bone-marrow-derived MSCs are effective in improving PLT levels and reducing the T regulatory cells mediating proinflammatory response in ITP mice.

Icaritin Improves Antibody-Induced Thrombocytopenia in a Mouse Model by Regulating T-cell Polarization.

Previous studies have shown that icaritin (ICT) has significant protective effects on immune thrombocytopenia (ITP), and the present study aimed to discuss the mechanism of this protective effect from the aspect of regulating T-cell polarization by an antibody-induced ITP mice model. Mice were given rat anti-mouse CD41 antibody (MWReg30) by intraperitoneal injection for 7 d to produce ITP model. At the same time, ICT was administrated at 10 mg/kg/d orally for 9 d. Peripheral blood platelets were counted by hematology analyzer. Spleen index was also tested. Spleen T-helper cell (Th), cytotoxic T-cell (CTL), Th1, Th2, Th17, regulatory T-cell (Treg), and follicular helper T-cell (Tfh) were quantified by flow cytometry. Serum Th1/Th2/Th17 cytokines were tested by mouse Th1/Th2/Th17 cytometric bead array (CBA) kit and transforming growth factor beta (TGF-β) were analyzed by enzyme-linked immunosorbent assay (ELISA) kit. The results indicated that ICT (10 mg/kg) protected against MWReg30-induced ITP, as evidenced by increased blood platelets and decreased spleen index. In addition, the imbalance of Th/CTL in ITP mice spleen was regulated by ICT. Meanwhile, ICT inhibited Th1, Th17, and Tfh and improved Th2 and Treg in ITP mice spleen. Furthermore, the results of CBA and ELISA suggested that ICT decreased serum Th1- and Th17-related cytokines and increased Th2 cytokines, as well as promoted the release of TGF-β. These results demonstrated that the protective effect of ICT on ITP was mediated by regulating T-cell polarization.

Assessment of Th17/Treg cells and Th cytokines in an improved immune thrombocytopenia mouse model

ABSTRACTObjectives: The improved passive immune thrombocytopenia (ITP) mouse model has been extensively utilized for the study of ITP. However, how closely this model matches the human inflammation state and immune background is unclear. Our study aimed to explore the profile of Th cytokines and Th17/Treg cells in the model.Methods: We induced the ITP mouse model by dose-escalation injection of MWReg30. The serum levels of cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-17A, and TGF-β1) were measured by enzyme-linked immunosorbent assay and the frequency of Th17 and Treg cells was measured by flow cytometry. The mRNA expression of Foxp3 and RORrt was measured by real-time PCR.Results: The serum levels of cytokines IFN-γ, TGF-β1, IL-4, and IL-10 were significantly lower in ITP mice. The secretion of serum proinflammatory cytokines IL-2 and IL-17A and the percentage of Th17 cells showed no statistically significant increase. In ITP mice the frequency of Treg cells and mRNA expression of Foxp3 was significantly lower in splenocytes.Conclusion: Our data suggest that the improved passive ITP mouse model does not mimic the autoimmune inflammatory process of human ITP. Compared with human ITP, this model has a similar change in frequency of Treg cells, which may directly or indirectly result from antibody-mediated platelet destruction due to attenuated release of TGF-β.

Mouse immune thrombocytopenia is associated with Th1 bias and expression of activating Fcγ receptors.

Immune thrombocytopenia (ITP) is an autoimmune disease mediated by anti-platelet autoantibodies. We recently established a mouse ITP model exhibiting regulatory T-cell (Treg) deficiency, although only one-third of the Treg-deficient mice developed ITP. To clarify mechanisms involved in the emergence of platelet-specific autoimmunity in this model, we examined the T helper (Th)-cell balance and macrophage Fcγ receptor (FcγR) expression profiles in Treg-deficient mice with and without ITP. Splenocytes from both populations of Treg-deficient mice and control BALB/c mice were subjected to flow cytometry-based analyses to evaluate Th cell subset proportions and the expression of activating and inhibitory FcγRs on macrophages. In addition, IgG subclass distribution of anti-platelet autoantibodies in splenocyte culture supernatants was determined by flow cytometry using IgG subclass-specific antibodies. Treg-deficient ITP mice exhibited a significantly higher proportion of Th1 cells than either Treg-deficient non-ITP or control mice. The predominant anti-platelet autoantibody subclasses in the ITP mice were Th1-associated IgG2a and IgG2b. Furthermore, the FcγRI/FcγRIIB expression ratio in splenic macrophages was higher in the Treg-deficient ITP than in the Treg-deficient non-ITP and control mice. In summary, Th1 polarization and macrophages' activating FcγR expression profile are associated with the development of ITP in Treg-deficient mice.

Crucial Role of Complement Activation and IL-1β in Bone Marrow Niche of Immune Thrombocytopenia

It is well known that complement activation and cytokine dysregulation are critical in the pathogenesis of immune thrombocytopenia (ITP). We previously showed impaired proliferative capacity and reduced ability to inhibit T-cell proliferation of mesenchymal stem cells (MSCs) from ITP patients (Zhang JM, et al. Stem Cell Transl Med 2016), indicating that the bone marrow (BM) niche might be involved in ITP etiology. Recent studies have defined sympathetic nerve fibers and ensheathing Schwann cells as regulators in the BM niche (Arranz L, et al. Nature 2014). Given that the initial pathogenic mechanisms underlying ITP have not yet been identified, we hypothesized that complement activation played a role in the ITP BM niche including nestin+MSCs and Schwann cells.Thirty-one consecutive patients with primary ITP and 19 healthy donors were enrolled in this prospective study. Samples from patients with ITP were divided into the two groups according to whether complement activation product C1q, C3b, C4d or C5b-9 was detectable on the surface of nestin+MSCs: the complement+nestin+MSC-ITP group and the complement-nestin+MSC-ITP group. Here we found that the complement+nestin+MSC-ITP group had fewer nestin+MSCs, lower proliferative capacity, less Cxcl12 production and a reduced capacity for inhibiting T cell proliferation, as well as higher levels of the pro-inflammatory cytokines interleukin-1β (IL-1β) and TNF-α in bone marrow supernatant compared with the complement-nestin+MSC-ITP group and the nestin+MSC-control group, indicating that activation of the complement cascade and enhancement of the inflammatory response are involved in the ITP BM niche.To further define the possible mechanism for the vulnerability of nestin+MSC-ITP to the complement system, messenger RNA (mRNA) from 5 samples from each group respectively were analyzed by PrimeView™ Human Gene Expression Array (Affymetrix). Diminished levels of complement factor H mRNA consistent with factor H secretion was revealed in the complement+nestin+MSC-ITP group. Furthermore, the mRNA levels of C3aR, C5aR, IL-1R and caspase1 were up-regulated, whereas mRNA levels of CD46, CD55 and CD59 showed no statistically significant differences in this group. Upon further exposure to TNF-α, the complement+nestin+MSC-ITP group insignificantly up-regulated factor H compared with the other two groups.Interestingly, in the complement+nestin+MSC-ITP group, complement activation products were detected on the outer surface of Schwann cells. Immunostaining of glial fibrillary acidic protein (Gfap) to visualize Schwann cells showed undesirable innervation in bone marrow of the complement+nestin+MSC-ITP group. GFAP mRNA expression was concurrently reduced in this group. To characterize the supporting role of Schwann cells, Schwann cells and nestin+MSCs derived from neonatal BMCD45-CD31-Ter119-Nes-GFP+Pdgfrα-/+ cells were co-cultured in vitro with different concentrations of IL-1β for 24 h. The results indicated that sympathetic neuropathy could sensitize nestin+MSCs to cell death triggered by IL-1β. Schwann cells were additionally incubated with an antagonist of interleukin-1 receptor (IL-1Ra), which consequently reversed IL-1β mediated cell damage. Together, these data suggested that IL-1β contributes to neuroglial damage, which compromised nestin+MSC survival.To further define potential regulators that can reverse the alternations of the ITP BM niche, we developed an ITP murine model. ITP mice were randomly assigned to two groups and treated with either prednisone or IL-1Ra. Both of these two groups exhibited improved proliferative capacity and inhibition of T cell proliferation of nestin+MSCs and recovered Schwann cells innervations. No statistically differences were observed between the two groups.In conclusion, the impaired nestin+MSCs and Schwann cells by complement activation and IL-1β in the BM niche play a role in the pathogenesis of ITP. The effect of IL-1Ra in correcting the dysfunction of nestin+MSCs and Schwann cells suggests a potential novel therapeutic approach of IL-1Ra for patients with ITP. DisclosuresNo relevant conflicts of interest to declare.

Prednison provokes serum and vasoactive substances in a mice model of immune thrombocytopenia.

The main objective of this study was to investigate the variations of β-endorphin (β-EP), vasoactive intestinal peptide (VIP), serotonin (5-HT) and norepinephrine (NE) of immune thrombocytopenia (ITP) mice as well as the regulatory mechanism of prednison. Sixty BALB/c mice were randomly divided into control group, model group and prednison intervention group. ITP mice model was duplicated by injecting with glycoprotein-antiplatelet serum (GP-APS) except in control group. After ITP disease model was successful established, prednison was used in prednison intervention group. The β-EP, VIP, 5-HT and NE contents of ITP mice were detected by enzyme linked immunosorbent assay (ELISA). Compared with the values in control group, the detection values of VIP and 5-HT in model group declined, while the detection values of β-EP and NE increased. Compared with prednison intervention group, the detection values of VIP and 5-HT in model group increased, while the detection values of β-EP and NE showed no significant change. In this study, the β-EP, VIP, 5-HT and NE contents in ITP mice injected with GP-APS were changed by prednison. It shows that prednison as the first-line therapy for ITP with effective hemostasis function is likely to increasing the contents of VIP and 5-HT. These results suggest the therapeutic value of prednison for the treatment of ITP.

Thrombopoietin receptor agonists shift the balance of Fcγ receptors toward inhibitory receptor IIb on monocytes in ITP

AbstractElevated expression of the activating Fcγ receptor (FcγR) I and FcγRIIa together with decreased expression of the inhibitory FcγRIIb are involved in the pathogenesis of primary immune thrombocytopenia (ITP). Thrombopoietin receptor agonists (TPO-RAs) have been used clinically for the management of ITP; however, little is known about the effect of TPO-RAs on FcγR modulation in ITP. In this prospective study, we measured the alteration in monocyte FcγR expression from 21 corticosteroid-resistant/relapsed patients with chronic ITP receiving eltrombopag therapy. Results showed that the mRNA and protein levels of FcγRIIb were significantly elevated after 6-week eltrombopag treatment. Concurrently, FcγRI and IIa levels decreased remarkably, whereas FcγRIII expression did not change. In vitro phagocytosis assays indicated that a shift in the balance of FcγR toward inhibitory FcγRIIb on monocytes was accompanied with a considerable decrease in monocyte/macrophage phagocytic capacity. The response to eltrombopag therapy in patients with ITP was associated with FcγR phenotype and functional changes of monocytes/macrophages. Moreover, the plasma transforming growth factor-β1 (TGF-β1) concentrations increased significantly in eltrombopag responders. Modulation of monocyte FcγR balance by TPO-RAs was also found in a murine model of ITP established by transferring splenocytes from immunized CD61 knockout mice into CD61+ severe combined immunodeficient mice. Romiplostim administration in ITP mice significantly upregulated inhibitory FcγRII expression and downregulated activating FcγRI expression. These findings showed that recovery of platelet counts after TPO-RA treatment in ITP is associated with the restoration of FcγR balance toward the inhibitory FcγRIIb on monocytes, and suggested that thrombopoietic agents have a profound effect on immune modulation in ITP. This study is registered at ClinicalTrials.gov as #NCT01864512.

The spleen dictates platelet destruction, anti-platelet antibody production, and lymphocyte distribution patterns in a murine model of immune thrombocytopenia.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.

Successful Treatment of Thrombocytopenia with Staphylococcal Protein A (PRTX-100) in a Murine Model of Immune Thrombocytopenia (ITP)

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder in which autoantibodies and autoreactive T cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. Several therapeutic options e.g. steroids, IVIg, Rituximab and thrombopoietin mimetics, are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. PRTX-100 is a highly purified formulation of Staphylococcal Protein A that has been evaluated in clinical trials with rheumatoid arthritis patients. Here, we analyzed the efficacy of PRTX-100 in raising platelet counts in a well-established murine model of ITP that demonstrates both antibody- and T cell-mediated thrombocytopenia. Platelet glycoprotein (GP) IIIa (CD61) knockout (KO) mice were immunized with CD61+ platelets and ITP was initiated by transfer of their splenocytes into severe combined immunodeficient (SCID) mice. On day 10, post transfer, the SCID mice were treated with either placebo, 1g/kg IVIg (ip, biweekly) or doses of PRTX-100 (iv, biweekly, 2.5-250 ug/kg) and platelet counts were measured weekly. Results show that control SCID mice transferred with 1x104 splenocytes from immune CD61 KO mice became thrombocytopenic at day 7 post-transfer and remained extremely thrombocytopenic throughout the 28 day protocol (3 mice died from bleeding diatheses). Similar observations were found with SCID mice given placebo. In contrast, however, transferred SCID mice treated with either IVIg or the PRTX-100 doses had platelet counts that increased to within normal levels within 1-2 weeks after treatment and none of the mice died (Table 1). Table 1Platelet counts (mean+SD) in SCID ITP mice (N=3-8/group) treated with the indicated compounds.TreatmentPre-bleedDay 14Day 21Day 28No treatment848+20289+4651+4278+51Placebo (250 ug/kg)848+202113+5946+2063+40IVIg (1 g/kg)848+202290+211490+289698+350PRTX-100 (250 ug.kg)848+202101+37303+112505+226PRTX-100 (25 ug.kg)848+202203+88290+67254+70PRTX-100 (2.5 ug.kg)848+202275+202405+289631+401These results demonstrate that PRTX-100 was effective in elevating platelet counts in a murine model of human ITP and support the proof of principle that PRTX-100 may be beneficial in patients with ITP. DisclosuresNo relevant conflicts of interest to declare.