ALBERT

Beschreibung: 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.

Beschreibung: Introduction: Graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation. Mesenchymal stem cells (MSCs) can modulate immune response and have been used as a treatment for aGVHD. The immune-modulating factors of MSCs are secreted and reside in supernatant fractions that are enriched for extracellular vesicles (EVs). MSC-derived EVs (MSC-EVs) also exhibit immunosuppressive activity, providing many advantages compared to MSCs and have been proven therapeutic in aGVHD. Arsenic trioxide (ATO) exhibits potent antitumor effects and increasing studies indicate its immunosuppressive effects. However, ATO at high concentrations can cause severe adverse effects. If encapsulated in some kind of drug vehicles, ATO can be made less toxic. Therefore, we believed that the combination of MSC-EVs with a low dose of ATO would be an effective therapy for aGVHD. Methods: We used a classical GVHD model (BALB/c→B6) and developed 4 groups: the control group (TCD-BM), the GVHD control group (TCD-BM + spleen T cells), the MSC-EVs treatment group and the MSC-ATO-EVs (MSC-derived ATO-encapsulating EVs) treatment group. OS, GVHD clinical and histological scores were evaluated. A20-luc lymphoma cells were injected to generate the GVL model. Using flow cytometry analysis, we analyzed Th cell subsets, cytokines and transcription factors (Th1*IFN-γ/TNF-α*T-bet, Th2*IL-4*GATA3, Th17*IL-17*RORγt, Treg*IL-10*Foxp3) and sorted CD8+ SLECs, and CD8+ MPECs in BM and spleen of recipients. Dll4 expression was analyzed on DCs. B6 cells were incubated with or without BALB/c spleen cells and complete medium alone, with 10 mM ATO alone. T cell apoptosis was determined with Yopro-1 staining. We used MLR assays to examine Th subsets, cytokines and notch targeted genes with or without ATO or neutralizing Ab specific to Dll4 (anti-Dll4). Results: BALB/c mice receiving B6 TCD-BM alone developed no sign of GVHD, whereas all BALB/c mice receiving B6 donor TCD-BM + spleen T cells died of GVHD. In contrast, injection of MSC-EVs and MSC-ATO-EVs inhibited GVHD in T cell recipients, with 20% and 29% of them surviving without severe GVHD, respectively. These survival rates were accompanied by significantly lower clinical and histological scores. GVL effects mediated by MSC-EVs and MSC-ATO-EVs were comparable to those obtained in the GVHD control group. Compared to the control group, CD4+T and CD8+T cells increased substantially in T cell recipients, resulting in severe GVHD. In contrast, treatment with MSC-ATO-EVs significantly reduced the number of CD4+T and CD8+T cells, while MSC-EVs recipients retained approximately the same number of T cells as the GVHD group. Compared to the GVHD control group, Th2 and Treg cells derived from the spleen increased, while Th1 and Th17 cells were reduced significantly in both the MSC-EVs and MSC-ATO-EVs groups. We also detected lower serum levels of TNF-α and IFNγ as well as lower expression of RORγt and T-bet in blood and BM CD4+ T cells in these two groups, while the expression of GATA3 and Foxp3 increased significantly. Treatment with MSC-ATO-EVs markedly raised the MPEC/SLEC ratio compared to the MSC-EVs and GVHD control groups. We also examined Dll4high DCs in different organs and different groups and found that only MSC-ATO-EVs significantly reduced the Dll4high DCs, especially in the spleen and intestine. Treatment of stimulated B6 CD4+ T and CD8+ T cells with ATO increased production of H2O2. Yopro-1 staining of activated B6 CD4+ T and CD8+ T cells indicated that ATO dramatically triggered apoptosis in those cells. DCs were isolated and cultured with B6 mouse-derived CD4+ T or CD8+ T cells, with or without addition of ATO or anti-Dll4. ATO and anti-Dll4 both led to significant reduction of IFN-γ and TNF-α, while IL-4 and IL-10 increased slightly. We next assessed the notch pathway targeted genes in T cells and found there were significantly increased GATA3 and reduced Dtx expression levels. Conclusion: Altogether, our findings demonstrate that MSC-ATO-EVs might be a highly promising therapy for aGVHD through reducing T cell amounts and modulating Th subsets and CD8+ T cell differentiation. These effects can be explained with the inhibition of the Dll4-notch pathway by ATO. Therefore, further exploitation of the potential application of ATO in aGVHD and the mechanisms of action of ATO may improve outcomes after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Introductions Immune thrombocytopenia (ITP) is an acquired thrombocytopenia caused by immune-mediated platelet destruction and impaired platelet production. As the immunosuppressive therapy, high-dose dexamethasone (HD-DXM) has been recommended as the standard first-line treatment. Nevertheless, approximately half of ITP patients relapse within 6 months and require further therapies. All-trans retinoic acid (ATRA) is a derivative of vitamin A that has an immunomodulatory effect on hemopoiesis. Our previous study demonstrated that ATRA could induce a more rapid and long-lasting response in patients with corticosteroid-resistant or relapsed ITP (Lancet haematology, 2017). In addition, we found that ATRA corrected impaired platelet production by regulating the complement-IL-1β loop and TNFAIP3/NF-κB/SMAD7 signaling pathway in ITP (Br J Haematol, 2018; Haematologica, 2019). These findings suggested that the addition of ATRA to HD-DXM in treatment-naive patients could improve sustained response rate based on the synergetic function. Here, we present the results of the first prospective, multicenter, randomized, controlled trial with the largest cohort to date comparing the efficacy and safety of HD-DXM plus ATRA vs HD-DXM as first-line therapy in newly diagnosed adult primary ITP patients. Methods In this open-label, randomized, phase 2 trial, we enrolled adult ITP patients from 6 different tertiary medical centers in China. Eligible subjects had confirmed newly-diagnosed, treatment-naive ITP; platelet counts 50×109/L without any additional ITP-modifying therapy at the 6-month follow up. Key secondary endpoints were initial response by day 14 (OR: platelet count ≥30×109/L and at least 2-fold increase of the baseline platelet count and absence of bleeding; CR: platelet count ≥100×109/L), duration of response (DOR), bleeding scores, and adverse events (AEs). Results Between June 2015, and July 2018, 300 patients were randomly allocated into either ATRA plus HD-DXM (n=150) or HD-DXM monotherapy (n=150). At the 6-month follow-up, the proportion of patients with SR was significantly higher in the ATRA plus HD-DXM group than in the HD-DXM monotherapy group (61% vs 37%, p= 0.009). So far, the increase of almost one quarter is very encouraging. The combination of ATRA and HD-DXM resulted in a higher incidence of early OR at day 14 compared with HD-DXM monotherapy (83% vs 67%, P=0.031), and the CR rate was 60.0% (ATRA+HD-DXM) vs 40.6% (HD-DXM) (P=0.015). Throughout the follow-up period, overall DOR was greater in the combination group, estimated by the Kaplan-Meier analysis. Fewer patients in the combination group than in the monotherapy group relapsed during follow-up (p〈 0.001), and the median time to relapse was 75 days (ATRA+HD-DXM) vs 48 days (HD-DXM). Bleeding was more effectively controlled in the ATRA plus HD-DXM arm, with fewer bleeding events and lower bleeding scores. There was no difference between the 2 groups in terms of rescue treatments. All subjects tolerated the treatment well, and no grade 4 adverse events or treatment-related death were reported. No statistically significant differences were observed in the incidence of treatment-related AEs between the two groups. Conclusions ATRA plus HD-DXM is an effective and safe treatment for ITP as a first-line therapy that can provide a sustained prolonged response in adults. This therapy could be a new treatment option for ITP subjects. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Introduction Virus-induced thrombocytopenia is a severe complication in immunocompromised hosts. Among patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT), human cytomegalovirus (HCMV) infection contributes to a variety of end-organ diseases and hematological complications, leading to increased mortality. Even with antiviral treatment, HCMV remains a potentially lethal infection due to the lack of understanding of the underlying mechanisms of host-virus interactions. The key to solving this problem is to identify the factors that predispose patients to HCMV infection and carry out targeted therapy. Here, we investigated the megakaryo/thrombopoiesis process, including the thrombopoietin (TPO)/c-Mpl pathway, after HCMV infection in vivo and in vitro, screened for susceptible subsets of megakaryocytes (MKs) and explored novel therapeutic targets for HCMV infection. Methods To test whether thrombocytopenia induced by HCMV results from an impaired megakaryo/thrombopoiesis process, we studied the impact of HCMV in an in vivo model of HCMV DNAemia patients following allo-HSCT and an in vitro model of bone marrow CD34+-derived MKs infected with serum from HCMV DNAemia patients. Forty patients who had received allo-HSCT were enrolled in this study, among whom 18 recipients had HCMV DNAemia and 22 were HCMV negative, and bone marrow-derived mononuclear cells (MNCs) from patients were tested for CD41, vWF, pp65, c-Mpl, PDGFR, αvβ3 and TLR2 using flow cytometry (FCM). Transmission electron microscopy (TEM) was used to detect HCMV capsids inside MKs. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. Finally, inhibitors of PDGFR (IMC-3G3 and Gleevec), αvβ3 and TLR2 were cocultured with MKs. Results Our data showed that pp65+ cells accounted for 40.59±6.12% of total CD41+vWF+ MKs from HCMV DNAemia patients, and there was a significant increase in the expression of αvβ3, PDGFR and TLR2 in pp65+ MKs compared with that in control patients. Furthermore, the percentage of PDGFR+αvβ3+ MKs emerged as an independent factor associated with HCMV infection in multivariate analysis (p = 0.008). MKs in HCMV-infected patients showed increased apoptosis and necrosis and different patterns of MK ploidy distribution compared with those in HCMV-negative patients, with a decreased proportion from 16N to 64N and a peak at 8N. Meanwhile, the expression of TPO receptor c-Mpl was lower in pp65+ MKs from HCMV DNAemia patients (0.77±0.38% in pp65+ MKs from HCMV DNAemia patients, 1.75±0.40% in pp65- MKs from HCMV DNAemia patients, 1.97±0.67% in MKs from HCMV-negative patients, and 2.06±0.29% in MKs from healthy controls, p