Evidence is emerging that platelets are major contributors to innate immune responses in conditions such as acute lung injury (ALI). Platelets form heterotypic aggregates with neutrophils, and we hypothesized that lipoxin mediators regulate formation of neutrophil-platelet aggregates (NPA) and that NPA significantly contribute to ALI. Lipopolysaccharide (LPS)-induced lung injury was accompanied by platelet sequestration, activation, intra-alveolar accumulation, and NPA formation within both blood and alveolar compartments. Using lung intravital microscopy, we observed the dynamic formation of NPA during physiologic conditions, which sharply increased with ALI. Aspirin (ASA) treatment significantly reduced lung platelet sequestration and activation, NPA formation, and lung injury. ASA treatment increased levels of ASA-triggered lipoxin (ATL; 15-epi-lipoxin A4), and blocking the lipoxin A4 receptor (ALX) with a peptide antagonist (Boc2) or using ALX knockouts (Fpr2/3–/–) reversed this protection. LPS increased NPA formation in vitro, which was reduced by ATL, and engagement of ALX by ATL on both neutrophils and platelets was necessary to prevent aggregation. In a model of transfusion-related acute lung injury (TRALI), Boc2 also reversed ASA protection, and treatment with ATL in both LPS and TRALI models protected from ALI. We conclude that ATL regulates neutrophil-platelet aggregation and that platelet-neutrophil interactions are a therapeutic target in lung injury.
Myelofibrosis (MF) is a BCR-ABL1–negative myeloproliferative neoplasm characterized by clonal myeloproliferation, dysregulated kinase signaling, and release of abnormal cytokines. In recent years, important progress has been made in the knowledge of the molecular biology and the prognostic assessment of MF. Conventional treatment has limited impact on the patients’ survival; it includes a wait-and-see approach for asymptomatic patients, erythropoiesis-stimulating agents, androgens, or immunomodulatory agents for anemia, cytoreductive drugs such as hydroxyurea for the splenomegaly and constitutional symptoms, and splenectomy or radiotherapy in selected patients. The discovery of the Janus kinase (JAK)2 mutation triggered the development of molecular targeted therapy of MF. The JAK inhibitors are effective in both JAK2-positive and JAK2-negative MF; one of them, ruxolitinib, is the current best available therapy for MF splenomegaly and constitutional symptoms. However, although ruxolitinib has changed the therapeutic scenario of MF, there is no clear indication of a disease-modifying effect. Allogeneic stem cell transplantation remains the only curative therapy of MF, but due to its associated morbidity and mortality, it is usually restricted to eligible high- and intermediate-2–risk MF patients. To improve current therapeutic results, the combination of JAK inhibitors with other agents is currently being tested, and newer drugs are being investigated.
Increased hepcidin production is key to the development of anemia of inflammation. We investigated whether lexaptepid, an antihepcidin l-oligoribonucleotide, prevents the decrease in serum iron during experimental human endotoxemia. This randomized, double-blind, placebo-controlled trial was carried out in 24 healthy males. At T = 0 hours, 2 ng/kg Escherichia coli lipopolysaccharide was intravenously administered, followed by an intravenous injection of 1.2 mg/kg lexaptepid or placebo at T = 0.5 hours. The lipopolysaccharide-induced inflammatory response was similar in subjects treated with lexaptepid or placebo regarding clinical and biochemical parameters. At T = 9 hours, serum iron had increased by 15.9 ± 9.8 µmol/L from baseline in lexaptepid-treated subjects compared with a decrease of 8.3 ± 9.0 µmol/L in controls (P < .0001). This study delivers proof of concept that lexaptepid achieves clinically relevant hepcidin inhibition enabling investigations in the treatment of anemia of inflammation. This trial was registered at www.clinicaltrial.gov as #NCT01522794.
The mechanisms controlling the development of dendritic cells (DCs) remain incompletely understood. Using an Mysm1 knockout (Mysm1–/–) mouse model, we identified the histone H2A deubiquitinase Mysm1, as a critical regulator in DC differentiation. Mysm1–/– mice showed a global reduction of DCs in lymphoid organs, whereas development of granulocytes and macrophages were not severely affected. Hematopoietic progenitors and DC precursors were significantly decreased in Mysm1–/– mice and defective in Fms-like tyrosine kinase-3(Flt3) ligand–induced, but not in granulocyte macrophage–colony-stimulating factor (GM-CSF)-induced DC differentiation in vitro. Molecular studies demonstrated that the developmental defect of DCs from common myeloid progenitor (CMP) in Mysm1–/– mice is associated with decreased Flt3 expression and that Mysm1 derepresses transcription of the Flt3 gene by directing histone modifications at the Flt3 promoter region. Two molecular mechanisms were found to be responsible for the selective role of Mysm1 in lineage determination of DCs from CMPs: the selective expression of Mysm1 in a subset of CMPs and the different requirement of Mysm1 for PU.1 recruitment to the Flt3 locus vs GM-CSF-α and macrophage–colony-stimulating factor receptor loci. In conclusion, this study reveals an essential role of Mysm1 in epigenetic regulation of Flt3 transcription and DC development, and it provides a novel mechanism for lineage determination from CMP.
Chronic lymphocytic leukemia (CLL) displays remarkable ethnic predisposition for whites, with relative sparing of African-American and Asian populations. In addition, CLL displays among the highest familial predispositions of all hematologic malignancies, yet the genetic basis for these differences is not clearly defined. The highly polymorphic HLA genes of the major histocompatibility complex play a central role in immune surveillance and confer risk for autoimmune and infectious diseases and several different cancers, the role for which in the development of CLL has not been extensively investigated. The National Marrow Donor Program/Be The Match has collected HLA typing from CLL patients in need of allogeneic hematopoietic stem cell transplant and has recruited millions of volunteers to potentially donate hematopoietic stem cells. HLA genotypes for 3491 US white, 397 African-American, and 90 Hispanic CLL patients were compared with 50 000 controls per population from the donor registry. We identified several HLA alleles associated with CLL susceptibility in each population, reconfirming predisposing roles of HLA-A*02:01 and HLA-DRB4*01:01 in whites. Associations for haplotype DRB4*01:01~DRB1*07:01~DQB1*03:03 were replicated across all 3 populations. These findings provide a comprehensive assessment of the role of HLA in the development of severe CLL.
The monoclonal anti-CD20 antibody rituximab (RTX) depletes B cells in the treatment of lymphoma and autoimmune disease, and contributes to alloantibody reduction in transplantation across immunologic barriers. The effects of RTX on T cells are less well described. T-follicular helper (Tfh) cells provide growth and differentiation signals to germinal center (GC) B cells to support antibody production, and suppressive T-follicular regulatory (Tfr) cells regulate this response. In mice, both Tfh and Tfr are absolutely dependent on B cells for their formation and on the GC for their maintenance. In this study, we demonstrate that RTX treatment results in a lack of GC B cells in human lymph nodes without affecting the Tfh or Tfr cell populations. These data demonstrate that human Tfh and Tfr do not require an ongoing GC response for their maintenance. The persistence of Tfh and Tfr following RTX treatment may permit rapid reconstitution of the pathological GC response once the B-cell pool begins to recover. Strategies for maintaining remission after RTX therapy will need to take this persistence of Tfh into account.
Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family that has been recently linked to tumor development. However, its role in modulating multiple myeloma (MM) biology and disease progression remains unexplored. We first demonstrated that patients with MM present with higher expression of Pyk2 compared with healthy individuals. By using loss-of-function approaches, we found that Pyk2 inhibition led to reduction of MM tumor growth in vivo as well as decreased cell proliferation, cell-cycle progression, and adhesion ability in vitro. In turn, overexpression of Pyk2 promoted the malignant phenotype, substantiated by enhanced tumor growth and reduced survival. Mechanistically, inhibition of Pyk2 reduced activation of Wnt/β-catenin signaling by destabilizing β-catenin, leading to downregulation of c-Myc and Cyclin D1. Furthermore, treatment of MM cells with the FAK/Pyk2 inhibitor VS-4718 effectively inhibited MM cell growth both in vitro and in vivo. Collectively, our findings describe the tumor-promoting role of Pyk2 in MM, thus providing molecular evidence for a novel tyrosine kinase inhibitor as a new therapeutic option in MM.
Bim contributes to resistance to various standard and novel agents. Here we demonstrate that Bim plays a functional role in bortezomib resistance in multiple myeloma (MM) cells and that targeting Bim by combining histone deacetylase inhibitors (HDACIs) with BH3 mimetics (eg, ABT-737) overcomes bortezomib resistance. BH3-only protein profiling revealed high Bim levels (Bimhi) in most MM cell lines and primary CD138+ MM samples. Whereas short hairpin RNA Bim knockdown conferred bortezomib resistance in Bimhi cells, adaptive bortezomib-resistant cells displayed marked Bim downregulation. HDACI upregulated Bim and, when combined with ABT-737, which released Bim from Bcl-2/Bcl-xL, potently killed bortezomib-resistant cells. These events were correlated with Bim-associated autophagy attenuation, whereas Bim knockdown sharply increased autophagy in Bimhi cells. In Bimlow cells, autophagy disruption by chloroquine (CQ) was required for HDACI/ABT-737 to induce Bim expression and lethality. CQ also further enhanced HDACI/ABT-737 lethality in bortezomib-resistant cells. Finally, HDACI failed to diminish autophagy or potentiate ABT-737–induced apoptosis in bim–/– mouse embryonic fibroblasts. Thus, Bim deficiency represents a novel mechanism of adaptive bortezomib resistance in MM cells, and Bim-targeting strategies combining HDACIs (which upregulate Bim) and BH3 mimetics (which unleash Bim from antiapoptotic proteins) overcomes such resistance, in part by disabling cytoprotective autophagy.
The distinction between acquired aplastic anemia (AA) and hypocellular myelodysplastic syndrome (hMDS) is often difficult, especially nonsevere AA. We postulated that somatic mutations are present in a subset of AA, and predict malignant transformation. From our database, we identified 150 AA patients with no morphological evidence of MDS, who had stored bone marrow (BM) and constitutional DNA. We excluded Fanconi anemia, mutations of telomere maintenance, and a family history of BM failure (BMF) or cancer. The initial cohort of 57 patients was screened for 835 known genes associated with BMF and myeloid cancer; a second cohort of 93 patients was screened for mutations in ASXL1, DNMT3A, BCOR, TET2, and MPL. Somatic mutations were detected in 19% of AA, and included ASXL1 (n = 12), DNMT3A (n = 8) and BCOR (n = 6). Patients with somatic mutations had a longer disease duration (37 vs 8 months, P < .04), and shorter telomere lengths (median length, 0.9 vs 1.1, P < .001), compared with patients without mutations. Somatic mutations in AA patients with a disease duration of >6 months were associated with a 40% risk of transformation to MDS (P < .0002). Nearly one-fifth of AA patients harbor mutations in genes typically seen in myeloid malignancies that predicted for later transformation to MDS.
Only a minority of myelodysplastic syndrome (MDS) patients respond to hypomethylating agents (HMAs), but strong predictors of response are unknown. We sequenced 40 recurrently mutated myeloid malignancy genes in tumor DNA from 213 MDS patients collected before treatment with azacitidine (AZA) or decitabine (DEC). Mutations were examined for association with response and overall survival. The overall response rate of 47% was not different between agents. Clonal TET2 mutations predicted response (odds ratio [OR] 1.99, P = .036) when subclones unlikely to be detected by Sanger sequencing (allele fraction <10%) were treated as wild-type (WT). Response rates were highest in the subset of TET2 mutant patients without clonal ASXL1 mutations (OR 3.65, P = .009). Mutations of TP53 (hazard ratio [HR] 2.01, P = .002) and PTPN11 (HR 3.26, P = .006) were associated with shorter overall survival but not drug response. Murine-competitive bone marrow transplantation followed by treatment with AZA demonstrated that Tet2-null cells have an engraftment advantage over Tet2-WT cells. AZA significantly decreased this advantage for Tet2-null cells (P = .002) but not Tet2-WT cells (P = .212). Overall, Tet2 loss appears to sensitize cells to treatment with AZA in vivo, and TET2 mutations can identify patients more likely to respond to HMAs.
During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly "transdifferentiate" into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal–induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor–dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF–pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo–isolated G-CSF–mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils.
In this study, we test the assumption that the hematopoietic progenitor/colony-forming cells of the embryonic yolk sac (YS), which are endowed with megakaryocytic potential, differentiate into the first platelet-forming cells in vivo. We demonstrate that from embryonic day (E) 8.5 all megakaryocyte (MK) colony-forming cells belong to the conventional hematopoietic progenitor cell (HPC) compartment. Although these cells are indeed capable of generating polyploid MKs, they are not the source of the first platelet-forming cells. We show that proplatelet formation first occurs in a unique and previously unrecognized lineage of diploid platelet-forming cells, which develop within the YS in parallel to HPCs but can be specified in the E8.5 Runx1-null embryo despite the absence of the progenitor cell lineage.
Fetal hemoglobin (HbF) induction can ameliorate the clinical severity of sickle cell disease and β-thalassemia. We previously reported that activation of the eukaryotic initiation factor 2α (eIF2α) stress pathway increased HbF through a posttranscriptional mechanism. In this study, we explored the underlying means by which salubrinal, an activator of eIF2α signaling, enhances HbF production in primary human erythroid cells. Initial experiments eliminated changes in globin messenger RNA (mRNA) stability or cellular location and reduction of adult hemoglobin as possible salubrinal mechanisms. We then determined that salubrinal selectively increased the number of actively translating ribosomes on -globin mRNA. This enhanced translation efficiency occurred in the recovery phase of the stress response as phosphorylation of eIF2α and global protein synthesis returned toward baseline. These findings highlight -globin mRNA translation as a novel mechanism for regulating HbF production and as a pharmacologic target for induction of HbF.
We developed an approach of T-cell-replete haploidentical hematopoietic stem cell transplantation (HSCT) with low-dose anti-T-lymphocyte globulin and prospectively compared outcomes of all contemporaneous T-cell-replete HSCT performed at our center using matched sibling donors (MSDs), unrelated donors (URDs), and haploidentical related donors (HRDs). From 2008 to 2013, 90 patients underwent MSD-HSCT, 116 underwent URD-HSCT, and 99 underwent HRD-HSCT. HRDs were associated with higher incidences of grades 2 to 4 (42.4%) and severe acute graft-versus-host disease (17.2%) and nonrelapse mortality (30.5%), compared with MSDs (15.6%, 5.6%, and 4.7%, respectively; P < .05), but were similar to URDs, even fully 10/10 HLA-matched URDs. For high-risk patients, a superior graft-versus-leukemia effect was observed in HRD-HSCT, with 5-year relapse rates of 15.4% in HRD-HSCT, 28.2% in URD-HSCT (P = .07), and 49.9% in MSD-HSCT (P = .002). Furthermore, 5-year disease-free survival rates were not significantly different for patients undergoing transplantation using 3 types of donors, with 63.6%, 58.4%, and 58.3% for MSD, URD, and HRD transplantation, respectively (P = .574). Our data indicate that outcomes after HSCT from suitably matched URDs and HRDs with low-dose anti-T-lymphocyte globulin are similar and that HRD improves outcomes of patients with high-risk leukemia. This trial was registered at www.chictr.org (Chinese Clinical Trial Registry) as #ChiCTR-OCH-12002490.
We analyzed the influence of donor killer-cell immunoglobulin-like receptor (KIR) gene haplotypes on the risk for relapse and the probability of event-free survival (EFS) in children with acute lymphoblastic leukemia who received human leukocyte antigen–haploidentical transplantation of ex vivo T-cell-depleted peripheral blood stem cells. The KIR gene haplotype was evaluated in 85 donors, and the KIR B content score was determined in the 63 KIR haplotype B donors. Patients transplanted from a KIR haplotype B donor had a significantly better EFS than those transplanted from a KIR haplotype A donor (50.6% vs 29.5%, respectively; P = .033). Moreover, a high donor KIR B-content score was associated with a significantly reduced risk for relapse (Log-rank test for trend, P = .026). These data indicate that KIR genotyping should be included in the donor selection algorithm for haploidentical transplantation in children with acute lymphoblastic leukemia with the aim of choosing, whenever possible, a KIR haplotype B donor with a high KIR B-content score.