Hematology
Abstract
Iron overload is the hallmark of hereditary hemochromatosis and a complication of iron-loading anemias such as β-thalassemia. Treatment can be burdensome and have significant side effects, and new therapeutic options are needed. Iron overload in hereditary hemochromatosis and β-thalassemia intermedia is caused by hepcidin deficiency. Although transgenic hepcidin replacement in mouse models of these diseases prevents iron overload or decreases its potential toxicity, natural hepcidin is prohibitively expensive for human application and has unfavorable pharmacologic properties. Here, we report the rational design of hepcidin agonists based on the mutagenesis of hepcidin and the hepcidin-binding region of ferroportin and computer modeling of their docking. We identified specific hydrophobic/aromatic residues required for hepcidin-ferroportin binding and obtained evidence in vitro that a thiol-disulfide interaction between ferroportin C326 and the hepcidin disulfide cage may stabilize binding. Guided by this model, we showed that 7–9 N-terminal amino acids of hepcidin, including a single thiol cysteine, comprised the minimal structure that retained hepcidin activity, as shown by the induction of ferroportin degradation in reporter cells. Further modifications to increase resistance to proteolysis and oral bioavailability yielded minihepcidins that, after parenteral or oral administration to mice, lowered serum iron levels comparably to those after parenteral native hepcidin. Moreover, liver iron concentrations were lower in mice chronically treated with minihepcidins than those in mice treated with solvent alone. Minihepcidins may be useful for the treatment of iron overload disorders.
Authors
Gloria C. Preza, Piotr Ruchala, Rogelio Pinon, Emilio Ramos, Bo Qiao, Michael A. Peralta, Shantanu Sharma, Alan Waring, Tomas Ganz, Elizabeta Nemeth
Abstract
Current therapies for non-Hodgkin lymphoma commonly include CD20 mAb to deplete tumor cells. However, the response is not durable in a substantial proportion of patients. Herein, we report our studies in mice testing the hypothesis that heterogeneity in endogenous tissue CD20+ B cell depletion influences in vivo lymphoma therapy. Using highly effective CD20 mAbs that efficiently deplete endogenous mature B cells and homologous CD20+ primary lymphoma cells through monocyte- and antibody-dependent mechanisms, we found that lymphoma depletion and survival were reduced when endogenous host B cells were not depleted, particularly a rare IL-10–producing B cell subset (B10 cells) known to regulate inflammation and autoimmunity. Even small numbers of adoptively transferred B10 cells dramatically suppressed CD20 mAb–mediated lymphoma depletion by inhibiting mAb-mediated monocyte activation and effector function through IL-10–dependent mechanisms. However, the activation of innate effector cells using a TLR3 agonist that did not activate B10 cells overcame the negative regulatory effects of endogenous B10 cells and enhanced lymphoma depletion during CD20 immunotherapy in vivo. Thus, we conclude that endogenous B10 cells are potent negative regulators of innate immunity, with even small numbers of residual B10 cells able to inhibit lymphoma depletion by CD20 mAbs. Consequently, B10 cell removal could provide a way to optimize CD20 mAb–mediated clearance of malignant B cells in patients with non-Hodgkin lymphoma.
Authors
Mayuka Horikawa, Veronique Minard-Colin, Takashi Matsushita, Thomas F. Tedder
Abstract
Diabetes mellitus is associated with platelet hyperactivity, which leads to increased morbidity and mortality from cardiovascular disease. This is coupled with enhanced levels of thromboxane (TX), an eicosanoid that facilitates platelet aggregation. Although intensely studied, the mechanism underlying the relationship among hyperglycemia, TX generation, and platelet hyperactivity remains unclear. We sought to identify key signaling components that connect high levels of glucose to TX generation and to examine their clinical relevance. In human platelets, aldose reductase synergistically modulated platelet response to both hyperglycemia and collagen exposure through a pathway involving ROS/PLCγ2/PKC/p38α MAPK. In clinical patients with platelet activation (deep vein thrombosis; saphenous vein graft occlusion after coronary bypass surgery), and particularly those with diabetes, urinary levels of a major enzymatic metabolite of TX (11-dehydro-TXB2 [TX-M]) were substantially increased. Elevated TX-M persisted in diabetic patients taking low-dose aspirin (acetylsalicylic acid, ASA), suggesting that such patients may have underlying endothelial damage, collagen exposure, and thrombovascular disease. Thus, our study has identified multiple potential signaling targets for designing combination chemotherapies that could inhibit the synergistic activation of platelets by hyperglycemia and collagen exposure.
Authors
Wai Ho Tang, Jeremiah Stitham, Scott Gleim, Concetta Di Febbo, Ettore Porreca, Cristiano Fava, Stefania Tacconelli, Marta Capone, Virgilio Evangelista, Giacomo Levantesi, Li Wen, Kathleen Martin, Pietro Minuz, Jeffrey Rade, Paola Patrignani, John Hwa
Abstract
A fundamental goal in cancer research is the identification of the cell types and signaling pathways capable of initiating and sustaining tumor growth, as this has the potential to reveal therapeutic targets. Stem and progenitor cells have been implicated in the genesis of select lymphoid malignancies. However, the identity of the cells in which mature lymphoid neoplasms are initiated remains unclear. Here, we investigate the origin of peripheral T cell lymphomas using mice in which Snf5, a chromatin remodelling–complex subunit with tumor suppressor activity, could be conditionally inactivated in developing T cells. In this model of mature peripheral T cell lymphomas, the cell of origin was a mature CD44hiCD122loCD8+ T cell that resembled a subset of memory cells that has capacity for self-renewal and robust expansion, features shared with stem cells. Further analysis showed that Snf5 loss led to activation of a Myc-driven signaling network and stem cell transcriptional program. Finally, lymphomagenesis and lymphoma proliferation depended upon TCR signaling, establishing what we believe to be a new paradigm for lymphoid malignancy growth. These findings suggest that the self-renewal and robust proliferative capacities of memory T cells are associated with vulnerability to oncogenic transformation. Our findings further suggest that agents that impinge upon TCR signaling may represent an effective therapeutic modality for this class of lethal human cancers.
Authors
Xi Wang, Miriam B.F. Werneck, Boris G. Wilson, Hye-Jung Kim, Michael J. Kluk, Christopher S. Thom, Jonathan W. Wischhusen, Julia A. Evans, Jonathan L. Jesneck, Phuong Nguyen, Courtney G. Sansam, Harvey Cantor, Charles W.M. Roberts
Abstract
STAT1 is the main signal transducer for type I and II IFNs and plays a central role in the regulation of innate and adaptive immune responses. We used Stat1-deficient mice to test the role of donor Stat1 in MHC-matched minor histocompatibility antigen–mismatched (mHA-mismatched) and fully MHC-mismatched models of bone marrow transplantation. Lack of Stat1 in donor splenocytes reduced graft-versus-host disease (GVHD) in both immunogenetic disparities, leading to substantially attenuated morbidity and mortality. Donor Stat1 deficiency resulted in reduced alloantigen-induced activation and expansion of donor T cells and correlated with the expansion of CD4+CD25+Foxp3+ Tregs in vivo. This expansion of Tregs was further confirmed by studies showing that Stat1 deficiency promoted the proliferation, while inhibiting the apoptosis, of natural Tregs, and that absence of Stat1 enhanced the induction of inducible Tregs both in vitro and in vivo. Ex vivo expanded Stat1–/– Tregs were superior to wild-type Tregs in suppressing alloantigen-driven expansion of T cells in vitro and in inhibiting the development of GVHD. These observations demonstrate that Stat1 is a regulator of Tregs and that targeting Stat1 in CD4+ T cells may facilitate in vitro and in vivo expansion of Tregs for therapeutic use.
Authors
Huihui Ma, Caisheng Lu, Judith Ziegler, Ailing Liu, Antonia Sepulveda, Hideho Okada, Suzanne Lentzsch, Markus Y. Mapara
Abstract
Thrombosis is initiated by tissue factor (TF), a coagulation cofactor/receptor expressed in the vessel wall, on myeloid cells, and on microparticles (MPs) with variable procoagulant activity. However, the molecular pathways that generate prothrombotic TF in vivo are poorly defined. The oxidoreductase protein disulfide isomerase (PDI) is thought to be involved in the activation of TF. Here, we found that in mouse myeloid cells, ATP-triggered signaling through purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7 receptor; encoded by P2rx7) induced activation (decryption) of TF procoagulant activity and promoted release of TF+ MPs from macrophages and SMCs. The generation of prothrombotic MPs required P2X7 receptor–dependent production of ROS leading to increased availability of solvent-accessible extracellular thiols. An antibody to PDI with antithrombotic activity in vivo attenuated the release of procoagulant MPs. In addition, P2rx7–/– mice were protected from TF-dependent FeCl3-induced carotid artery thrombosis. BM chimeras revealed that P2X7 receptor prothrombotic function was present in both hematopoietic and vessel wall compartments. In contrast, an alternative anti-PDI antibody showed activities consistent with cellular activation typically induced by P2X7 receptor signaling. This anti-PDI antibody restored TF-dependent thrombosis in P2rx7–/– mice. These data suggest that PDI regulates a critical P2X7 receptor–dependent signaling pathway that generates prothrombotic TF, defining a link between inflammation and thrombosis with potential implications for antithrombotic therapy.
Authors
Christian Furlan-Freguia, Patrizia Marchese, András Gruber, Zaverio M. Ruggeri, Wolfram Ruf
Abstract
Deep vein thrombosis (DVT) is a major cause of pulmonary thromboembolism, a leading cause of death in individuals with DVT. Several lines of evidence indicate proinflammatory cytokines such as TNF-α are involved in thrombus formation and resolution, but the roles of IFN-γ remain unclear. To address this issue, we performed ligation of the inferior vena cava to induce DVT in WT and IFN-γ–deficient (Ifng–/–) mice. In WT mice, intrathrombotic IFN-γ levels were elevated progressively as the postligation interval was extended. Thrombus size was substantially smaller at 10 and 14 days in Ifng–/– mice than in WT mice. Intrathrombotic collagen content was remarkably reduced at more than 10 days after the ligation in Ifng–/– mice compared with WT mice. The expression and activity of MMP-9, but not MMP-2, was higher at the late phase in Ifng–/– mice than in WT mice. Moreover, intrathrombotic recanalization was increased in Ifng–/– mice, with enhanced Vegf gene expression, compared with that in WT mice. Activation of the IFN-γ/Stat1 signal pathway suppressed PMA-induced Mmp9 and Vegf gene expression in peritoneal macrophages. Furthermore, administration of anti–IFN-γ mAbs accelerated thrombus resolution in WT mice. Collectively, these findings indicate that IFN-γ can have detrimental roles in thrombus resolution and may be a good molecular target for the acceleration of thrombus resolution in individuals with DVT.
Authors
Mizuho Nosaka, Yuko Ishida, Akihiko Kimura, Yumi Kuninaka, Masanori Inui, Naofumi Mukaida, Toshikazu Kondo
Abstract
Transcription intermediary factor 1γ (TIF1γ) was suggested to play a role in erythropoiesis. However, how TIF1γ regulates the development of different blood cell lineages and whether TIF1γ is involved in human hematological malignancies remain to be determined. Here we have shown that TIF1γ was a tumor suppressor in mouse and human chronic myelomonocytic leukemia (CMML). Loss of Tif1g in mouse HSCs favored the expansion of the granulo-monocytic progenitor compartment. Furthermore, Tif1g deletion induced the age-dependent appearance of a cell-autonomous myeloproliferative disorder in mice that recapitulated essential characteristics of human CMML. TIF1γ was almost undetectable in leukemic cells of 35% of CMML patients. This downregulation was related to the hypermethylation of CpG sequences and specific histone modifications in the gene promoter. A demethylating agent restored the normal epigenetic status of the TIF1G promoter in human cells, which correlated with a reestablishment of TIF1γ expression. Together, these results demonstrate that TIF1G is an epigenetically regulated tumor suppressor gene in hematopoietic cells and suggest that changes in TIF1γ expression may be a biomarker of response to demethylating agents in CMML.
Authors
Romain Aucagne, Nathalie Droin, Jérôme Paggetti, Brice Lagrange, Anne Largeot, Arlette Hammann, Amandine Bataille, Laurent Martin, Kai-Ping Yan, Pierre Fenaux, Régine Losson, Eric Solary, Jean-Noël Bastie, Laurent Delva
Abstract
Hemolytic transfusion reactions (HTRs) can produce serious and potentially life-threatening complications in sickle cell disease (SCD) patients; however, the mechanisms underlying these complications remain undetermined. We established a model of alloimmune, IgG-mediated HTRs in a well-characterized humanized murine model of SCD. HTRs induced acute vaso-occlusive crisis (VOC), resulting in shortened survival of SCD mice. Acute VOC was associated with elevated circulating inflammatory chemokine levels, including striking elevation of the levels of the neutrophil chemoattractant CXCL1. Recombinant CXCL1 administration was sufficient to induce acute VOC in SCD mice, characterized by leukocyte recruitment in venules, capture of circulating red blood cells, reduction of venular flow, and shortened survival. In contrast, blockade of the CXCL1 receptor, CXCR2, prevented HTR-elicited acute VOC and prolonged survival in SCD mice. These results indicate that CXCL1 is a key inflammatory mediator of acute VOC in SCD mice. Targeted inhibition of CXCL1 and/or CXCR2 may therefore represent a new therapeutic approach for acute VOC in SCD patients.
Authors
Jung-Eun Jang, Eldad A. Hod, Steven L. Spitalnik, Paul S. Frenette
Abstract
IL-15 may have a role in the development of T cell large granular lymphocyte (T-LGL) or NKT leukemias. However, the mechanisms of action and the identity of the cell subset that undergoes leukemic transformation remain elusive. Here we show that in both mice and humans, NKp46 expression marks a minute population of WT NKT cells with higher activity and potency to become leukemic. Virtually 100% of T-LGL leukemias in IL-15 transgenic mice expressed NKp46, as did a majority of human T-LGL leukemias. The minute NKp46+ NKT population, but not the NKp46– NKT population, was selectively expanded by overexpression of endogenous IL-15. Importantly, IL-15 transgenic NKp46– NKT cells did not become NKp46+ in vivo, suggesting that NKp46+ T-LGL leukemia cells were the malignant counterpart of the minute WT NKp46+ NKT population. Mechanistically, NKp46+ NKT cells possessed higher responsiveness to IL-15 in vitro and in vivo compared with that of their NKp46– NKT counterparts. Furthermore, interruption of IL-15 signaling using a neutralizing antibody could prevent LGL leukemia in IL-15 transgenic mice. Collectively, our data demonstrate that NKp46 identifies a functionally distinct NKT subset in mice and humans that appears to be directly susceptible to leukemic transformation when IL-15 is overexpressed. Thus, IL-15 signaling and NKp46 may be useful targets in the treatment of patients with T-LGL or NKT leukemia.
Authors
Jianhua Yu, Takeki Mitsui, Min Wei, Hsiaoyin Mao, Jonathan P. Butchar, Mithun Vinod Shah, Jianying Zhang, Anjali Mishra, Christopher Alvarez-Breckenridge, Xingluo Liu, Shujun Liu, Akihiko Yokohama, Rossana Trotta, Guido Marcucci, Don M. Benson Jr., Thomas P. Loughran Jr., Susheela Tridandapani, Michael A. Caligiuri
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)