Yulu Wu

Imbalanced expression of Bcl-xL and Bax in platelets treated with plasma from immune thrombocytopenia.

Immune thrombocytopenia is a heterogeneous autoimmune disease, characterized by accelerated platelet destruction and impaired platelet production. Bcl-xL and Bax play an opposite role in the regulation of apoptotic process with Bcl-xL for cell survival and Bax for cell apoptosis. Given the critical roles in the regulation of platelet apoptosis, whether Bcl-xL or Bax was involved in the pathogenesis of ITP remains unknown. The aim of this study is to evaluate the expression profile of Bcl-xL and Bax in platelets treated with ITP plasma. Normal washed platelets were treated with plasma from 20 active ITP patients or 10 age and gender-matched control to mimic the ITP in vivo environment. Mitochondrial depolarization, platelet apoptosis and activation were measured by flow cytometry. Expression of Bcl-xL, Bax and caspase-3 were also measured by quantitative real-time PCR and western blot. Our results demonstrated increased mitochondrial depolarization, platelet apoptosis and activation in platelets after treated with ITP plasma in comparison to control. In addition, decreased expression of Bcl-xL, increased expression of Bax and activity of caspase-3 were also observed. Furthermore, a negative correlation of Bcl-xL with Bax was found in platelets treated with ITP plasma. In conclusion, imbalanced expression of Bcl-xL and Bax might be associated with platelet apoptosis in ITP and therapeutically targeting them might be a novel approach in the treatment of ITP.

Aberrant expression of RUNX3 in patients with immune thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disease, characterized by dysregulation of cellular immunity. Previous studies demonstrated that immune imbalance between Th1 and Th2 was associated with the pathogenesis of ITP. Runt-related transcription factor 3 (RUNX3) is a member of the runt domain-containing family of transcription factors and plays an important role in the regulation of T cell differentiation into Th1 cells. Whether RUNX3 was involved in the pathogenesis of ITP remains unclear. In this study, 47 active ITP patients, 18 ITP with remission and 26 age and gender matched healthy control were included. Peripheral blood mononuclear cells (PBMCs) were isolated from ITP and control for isolation of RNA and plasma which were used to measure mRNA level of RUNX3 and T-box transcription factor (T-bet) by quantitative real-time PCR and interferon γ (IFN-γ) plasma level by ELISA. Meanwhile, protein was also extracted from PBMCs for Western blot analysis of RUNX3 expression. Our results showed a significantly higher expression of RUNX3, T-bet and plasma level of IFN-γ in active ITP patients compared to control. No differences were observed between ITP with remission and control. Furthermore, a positive correlation of RUNX3 with T-bet was found in active ITP patients. In conclusion, aberrant expression of RUNX3 was associated with the pathogenesis of ITP and therapeutically targeting it might be a novel approach in ITP treatment.

Long non‐coding RNAs expression profiles in hepatocytes of mice after hematopoietic stem cell transplantation

Hepatic veno‐occlusive disease (HVOD), one serious complication following hematopoietic stem cell transplantation (HSCT), is mainly initiated by the damage to sinusoidal endothelial cells and hepatocytes. Long non‐coding RNAs (lncRNAs) play an important role in the proliferation of hepatocytes and liver regeneration. lncRNAs profile in hepatocytes post‐HSCT remains unclear. The aim of this study is to evaluate the profile of lncRNAs in hepatocytes of mice after HSCT. Mice HSCT model was established through infusion of 5 × 106 bone marrow mononuclear cells. On day 7, 14 and 33 after HSCT, mice were sacrificed for analysis of liver pathology, function and index. Total RNA was extracted from hepatocytes of mice on day 14 for microarray analysis of the expression profiles of lncRNAs by Arraystar Mouse lncRNA Microarray v2.0. Obvious edema and spotty necrosis of hepatocytes with inflammatory cells infiltration were observed post‐HSCT. Meanwhile, increased levels of alkaline phosphatase, aspartate transaminase, and total bilirubin, as well as elevated liver index were also found. 2,918 up‐regulated and 1,911 down‐regulated lncRNAs in hepatocytes were identified. Some of differentially expressed mRNAs had adjacent lncRNAs that were also significantly dysregulated, with the same dysregulation direction. T‐cell receptor (up‐regulation) and VEGF signaling pathway (down‐regulation) were identified as one of the most enriched pathways. Dysregulated lncRNAs might be involved in hepatocytes damage after HSCT, suggesting targeting them might be a novel approach in amelioration of hepatocytes damage.

Elevated expression of NLRP3 in patients with immune thrombocytopenia

Immune thrombocytopenia (ITP) is a heterogeneous autoimmune disease, which is characterized by dysregulation of T cell-mediated autoimmunity. NLRP3, a largest and mostly well-studied inflammasome, has been shown to be important in the regulation of adaptive immune response, especially in T cell response. Given the closely association of imbalance of T cell response with ITP, whether NLRP3 is involved in the pathogenesis of ITP remains poorly understood. In this study, 69 active ITP patients, 21 ITP in remission and 24 age- and gender-matched healthy controls were included. Peripheral blood mononuclear cells (PBMCs) were isolated from ITP and control for isolation of RNA and plasma, which were used to measure mRNA level of NLRP3 and adaptor protein ASC by quantitative real-time PCR and IL-18 plasma level by ELISA. Meanwhile, protein was also extracted from PBMCs for Western blot analysis of NLRP3 expression. Our results showed a significantly higher expression of NLRP3, ASC and plasma IL-18 level in patients with active ITP when compared to control. The expression of NLRP3, ASC and plasma IL-18 level was significantly lower in patients in remission than that in active ITP, and no difference was observed when compared to control. Furthermore, a significantly positive correlation of NLRP3 with ASC was observed in patients with active ITP. In conclusion, increased expression of NLRP3 was associated with the pathogenesis of ITP and therapeutically targeting it might be a new strategy in the treatment of ITP.

Busulfan Triggers Intrinsic Mitochondrial-Dependent Platelet Apoptosis Independent of Platelet Activation

As a nonspecific alkylating antineoplastic agent, busulfan has been widely used in the treatment of patients with chronic myeloid leukemia. In vitro and in vivo studies demonstrated busulfan-induced cell apoptosis. Whether busulfan triggers platelet apoptosis remains unclear. This study aimed to evaluate the role of busulfan in platelet apoptosis. Isolated human platelets were incubated with busulfan followed by analysis of platelet apoptosis by flow cytometry or western blot, including mitochondrial depolarization, expression of Bcl-2, and Bax and caspase 3 activation. Meanwhile, platelet activation, expression of glycoprotein Ibα (GPIbα), glycoprotein VI (GPVI), and IIb3 and platelet aggregation in response to collagen and adenosine diphosphate (ADP) were measured. Additionally, busulfan was injected into mice with or without administration of caspase inhibitor QVD-Oph to investigate its effect on platelet lifespan. Our results showed that busulfan-treated platelets displayed increased mitochondrial membrane depolarization, decreased expression of Bcl-2, increased expression of Bax and caspase 3 activation in dose-dependent manner, which were inhibited by QVD-Oph. Platelet activation was not observed in busulfan-treated platelets as showed by no increased P-selectin expression and PAC-1 binding. However, busulfan reduced collagen- or ADP-induced platelet aggregation without affecting expression of GPIbα, GPVI, and IIb3. Furthermore, busulfan reduced circulating platelet lifespan which was ameliorated by QVD-Oph in mice. In conclusion, busulfan triggers mitochondrial-dependent platelet apoptosis and reduces platelet lifespan in mice. These data suggest targeting caspase activation might be beneficial in the prophylaxis of platelet apoptosis-associated thrombocytopenia after administration of busulfan.

NLRP3 regulates platelet integrin αIIbβ3 outside-in signaling, hemostasis and arterial thrombosis

In addition to their hemostatic function, platelets play an important role in regulating the inflammatory response. The platelet NLRP3 inflammasome not only promotes interleukin-1β secretion, but was also found to be upregulated during platelet activation and thrombus formation in vitro. However, the role of NLRP3 in platelet function and thrombus formation in vivo remains unclear. In this study, we aimed to investigate the role of NLRP3 in platelet integrin αIIbβ3 signaling transduction. Using NLRP3−/− mice, we showed that NLRP3-deficient platelets do not have significant differences in expression of the platelet-specific adhesive receptors αIIbβ3 integrin, GPIba or GPVI; however, NLRP3−/− platelets transfused into wild-type mice resulted in prolonged tail-bleeding time and delayed arterial thrombus formation, as well as exhibiting impaired spreading on immobilized fibrinogen and defective clot retraction, concomitant with decreased phosphorylation of c-Src, Syk and PLCγ2 in response to thrombin stimulation. Interestingly, addition of exogenous recombinant interleukin-1β reversed the defect in NLRP3−/− platelet spreading and clot retraction, and restored thrombin-induced phosphorylation of c-Src/Syk/PLCγ2, whereas an anti-interleukin-1β antibody blocked spreading and clot retraction mediated by wild-type platelets. Using the direct NLRP3 inhibitor, CY-09, we demonstrated significantly reduced human platelet aggregation in response to threshold concentrations of collagen and ADP, as well as impaired clot retraction in CY-09-treated human platelets, supporting a role for NLRP3 also in regulating human platelet αIIbβ3 outside-in signaling. This study identifies a novel role for NLRP3 and interleukin-1β in platelet function, and provides a new potential link between thrombosis and inflammation, suggesting that therapies targeting NLRP3 or interleukin-1β might be beneficial for treating inflammation-associated thrombosis.

An increased expression profile of Th9/IL-9 correlated with Th17/IL-17 in patients with immune thrombocytopenia

Abstract Immune thrombocytopenia (ITP) is a heterogeneous autoimmune disease, characterized by dysregulation of cellular immunity. Th9 cells were recently identified as a new subtype of Th cells, characterized by preferential production of IL-9. Given the pleiotropic function of IL-9, Th9 cells are demonstrated to be involved in various autoimmune diseases. However, whether Th9 cells are involved in the pathogenesis of ITP remains unclear. In this study, 49 active ITP patients, 39 ITP with remission and 20 healthy controls were included. Peripheral blood mononuclear cells (PBMCs) were isolated from ITP and controls for measuring Th9 and Th17 cells by flow cytometry. Meanwhile, RNA was isolated from PBMCs for the measurement of the mRNA level of PU.1, IRF4, BATF, and RORγt by quantitative real-time PCR. Plasma levels of IL-9 and IL-17 were detected by ELISA. Our results showed that higher expressions of Th9, IL-9, and associated transcription factors (PU.1, IRF4, and BATF) were found in active ITP patients and restored to the normal level (except IL-9) in patients in remission. Meanwhile, Th9 cells and the IL-9 plasma level were positively correlated with Th17 cells and the IL-17 level in ITP patients, respectively. Moreover, a positive correlation of IRF4 or BATF with RORγt was found. In conclusion, an aberrant expression profile of Th9/IL-9 was associated with pathogenesis of ITP possibly through cooperatively working with Th17/IL-17 and therapeutically targeting Th9/IL-9 might be a novel approach in the treatment of ITP.

An absence of platelet activation following thalidomide treatment in vitro or in vivo

Increased risk of thromboembolism and platelet hyperreactivity has been reported in patients receiving thalidomide therapy. Whether thalidomide induces platelet activation directly or through other factors remains unclear. The aim of this study was to evaluate the effect of thalidomide on platelet activation under resting conditions in vitro and in vivo. Isolated human or mouse platelets were treated with different concentrations of thalidomide (10, 50 and 100 μg/ml) for 60 min at 37°C followed by analysis of platelet surface expression of platelet receptors GPIbα, GPVI, αIIbβ3 and P-selectin, and PAC-1 or fibrinogen binding, by flow cytometry and collagen- or ADP-induced platelet aggregation. In addition, thalidomide (200 mg/kg) was intraperitoneally injected into mice for analysis of the effect of thalidomide on platelet activation in vivo. No increased expression of P-selectin, PAC-1 or fibrinogen binding was observed in either human and mouse platelets after thalidomide treatment in vitro for 60 min at 37°C. Thalidomide treatment also did not affect expression of GPIbα, GPVI or αIIbβ3, nor did it affect collagen- or ADP-induced platelet aggregation at threshold concentrations. However, while mice injected with thalidomide displayed no increased surface expression of platelet P-selectin or αIIbβ3, there was a significantly shortened tail bleeding time, thrombin time, prothrombin time together with higher levels of Factor IX and fibrinogen. In conclusion, thalidomide at therapeutic doses does not directly induce platelet activation under resting conditions in vitro or in vivo, but results in increased procoagulant activity, which could explain the thalidomide-dependent prothrombotic tendency in patients.

Increased ADAM10 expression in patients with immune thrombocytopenia

ABSTRACT Immune thrombocytopenia (ITP) is an autoimmune disease, which is characterized by abnormal of T immunity. A disintegrin and metalloproteinase (ADAM) 10, a member of proteinase family, has been demonstrated to regulate T cell proliferation and effector function. Considering the closely association of dysregulation of T cell function with ITP, whether ADAM10 involves in the pathogenesis of ITP remains unclear. In this study, 54 active ITP patients, 18 ITP in remission and 24 age and gender matched healthy control were enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated from patients and control for isolation of RNA and plasma which were used to measure mRNA level of ADAM10 and tissue inhibitor of metalloproteinase 3 (TIMP3) by quantitative real‐time PCR and soluble level of FasL and lymphocyte activation gene‐3 (LAG‐3) in plasma by ELISA. Meanwhile, T cell activation was measured by flow cytometry. Our results showed significantly higher expression of ADAM10 and lower expression of TIMP3 in active ITP patients compared with control, which were all restored into normal level in remission patients. Consistent with the expression profile of ADAM10, increased soluble plasma level of FasL and LAG‐3 were observed in active ITP patients and reduced to normal level in patients in remission. Furthermore, increased T cell activation as demonstrated by higher expression of HLA‐DR and CD69 were found in active ITP patients. In conclusion, elevated expression of ADAM10 was associated with the pathogenesis and development of ITP and therapeutically targeting it might be a novel approach for the treatment of ITP. HighlightsHigher ADAM10 and lower TIMP3 expression in active ITP patientsADAM10 and TIMP3 expression is restored into normal level in ITP patients with remission.Increased FasL and LAG‐3 level in the plasma of active patients and recovered to normal level in remission patientsIncreased T cell activation in active ITP patients

Macrophages ameliorate bone marrow inflammatory injury and promote hematopoiesis in mice following hematopoietic stem cell transplantation

Bone marrow macrophages have been demonstrated to serve a critical role in promoting maintenance and retention of hematopoietic stem cells (HSCs). Our previous study indicated increased macrophages infiltration in bone marrow after HSC transplantation (HSCT). However, it is not well understood whether macrophages affect hematopoietic reconstitution after HSCT. The present study aimed to investigate the role of macrophages in hematopoietic reconstitution after HSCT. BALB/c mice were divided into HSCT, HSCT+Clodronate Liposomes, HSCT+PBS Liposomes, HSCT+RS102895 and HSCT+Vehicle groups and sacrificed on day 7, 14, 21, 28 and 35 after HSCT. Analysis was performed to detect the changes of bone marrow pathology by H&E staining and the number of macrophages was assessed by immunohistochemical staining and western blot analysis. The number of c-kit+sca-1+ and c-kit+ was measured by flow cytometry. Mice with a depletion of bone marrow macrophages displayed significantly reduced overall survival, delayed hematopoietic recovery, a reduced number of hematopoietic stem/progenitor cells and bone marrow cells as well as exaggerated bone marrow injury. However, compared with the HSCT+Vehicle group, mice with an increased number of bone marrow macrophages exhibited no difference of overall survival, had accelerated hematopoietic reconstitution, a higher number of hematopoietic stem/progenitor cells and bone marrow cells and ameliorated bone marrow injury. In conclusion, the present study indicated that bone marrow macrophages serve a protective role in bone marrow injury and may promote hematopoiesis in mice after HSCT, suggesting manipulation of macrophages may be a novel strategy for improving the efficacy of HSCT.