RESUMO
Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4ß7 in conventional T cells while preserving α4ß7 in regulatory T cells, with findings suggesting increased ß1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4ß7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Camundongos , Humanos , Animais , Transplante Homólogo , Receptores Notch/metabolismo , Transdução de Sinais , Doença Enxerto-Hospedeiro/metabolismo , PrimatasRESUMO
ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucemia/tratamento farmacológico , Leucemia/enzimologia , Animais , Antineoplásicos/química , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Desenho de Fármacos , Descoberta de Drogas , Inibidores Enzimáticos/química , Feminino , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Humanos , Leucemia/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/genética , Oncogenes , Domínios Proteicos , Proteínas Recombinantes de Fusão/genéticaRESUMO
The human skin is a significant barrier for protection against pathogen transmission. Rodent models used to investigate human-specific pathogens that target the skin are generated by introducing human skin grafts to immunocompromised rodent strains. Infection-induced immunopathogenesis has been separately studied in humanized rodent models developed with human lymphoid tissue and hematopoietic stem cell transplants. Successful co-engraftment of human skin, autologous lymphoid tissues, and autologous immune cells in a rodent model has not yet been achieved, though it could provide a means of studying the human immune response to infection in the human skin. Here, we introduce the human Skin and Immune System (hSIS)-humanized NOD-scid IL2Rγnull (NSG) mouse and Sprague-Dawley-Rag2tm2hera Il2rγtm1hera (SRG) rat models, co-engrafted with human full-thickness fetal skin, autologous fetal lymphoid tissues, and autologous fetal liver-derived hematopoietic stem cells. hSIS-humanized rodents demonstrate the development of human full-thickness skin, along with autologous lymphoid tissues, and autologous immune cells. These models also support human skin infection following intradermal inoculation with community-associated methicillin-resistant Staphylococcus aureus. The co-engraftment of these human skin and immune system components into a single humanized rodent model could provide a platform for studying human skin infections.
Assuntos
Células Sanguíneas/imunologia , Tecido Linfoide/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , Transplante de Pele , Pele/imunologia , Infecções Estafilocócicas/imunologia , Replicação Viral/imunologia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ratos , Pele/patologia , Infecções Estafilocócicas/terapia , Transplante AutólogoRESUMO
The fungal indole alkaloids are a unique class of complex molecules that have a characteristic bicyclo[2.2.2]diazaoctane ring and frequently contain a spiro-oxindole moiety. While various strains produce these compounds, an intriguing case involves the formation of individual antipodes by two unique species of fungi in the generation of the potent anticancer agents (+)- and (-)-notoamide A. NotI and NotI' have been characterized as flavin-dependent monooxygenases that catalyze epoxidation and semi-pinacol rearrangement to form the spiro-oxindole center within these molecules. This work elucidates a key step in the biosynthesis of the notoamides and provides an evolutionary hypothesis regarding a common ancestor for production of enantiopure notoamides.
Assuntos
Flavinas/metabolismo , Alcaloides Indólicos/metabolismo , Oxigenases de Função Mista/metabolismo , Oxindóis/metabolismo , Compostos de Espiro/metabolismo , Flavinas/química , Alcaloides Indólicos/química , Oxigenases de Função Mista/química , Conformação Molecular , Oxindóis/química , Compostos de Espiro/química , EstereoisomerismoRESUMO
Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced TH17 cell differentiation and increased Treg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of TH17 and Treg cells.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Ácido Litocólico/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Animais , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Ácido Litocólico/química , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/citologia , Células Th17/imunologia , Células Th17/metabolismoRESUMO
A major pathogenic feature associated with HIV infection is lymphoid fibrosis, which persists during antiretroviral therapy (ART). Lymphoid tissues play critical roles in the generation of antigen-specific immune response, and fibrosis disrupts the stromal network of lymphoid tissues, resulting in impaired immune cell trafficking and function, as well as immunodeficiency. Developing an animal model for investigating the impact of HIV infection-induced lymphoid tissue fibrosis on immunodeficiency and immune cell impairment is critical for therapeutics development and clinical translation. Said model will enable in vivo mechanistic studies, thus complementing the well-established surrogate model of SIV infection-induced lymphoid tissue fibrosis in macaques. We developed a potentially novel human immune system-humanized mouse model by coengrafting autologous fetal thymus, spleen, and liver organoids under the kidney capsule, along with i.v. injection of autologous fetal liver-derived hematopoietic stem cells, thus termed the BM-liver-thymus-spleen (BLTS) humanized mouse model. BLTS humanized mouse model supports development of human immune cells and human lymphoid organoids (human thymus and spleen organoids). HIV infection in BLTS humanized mice results in progressive fibrosis in human lymphoid tissues, which was associated with immunodeficiency in the lymphoid tissues, and lymphoid tissue fibrosis persists during ART, thus recapitulating clinical outcomes.
Assuntos
Fibrose/imunologia , Infecções por HIV/imunologia , Fígado/imunologia , Tecido Linfoide/imunologia , Baço/imunologia , Timo/imunologia , Animais , Modelos Animais de Doenças , Feminino , Transplante de Tecido Fetal , Fibrose/patologia , Infecções por HIV/tratamento farmacológico , Células-Tronco Hematopoéticas , Humanos , Fígado/patologia , Transplante de Fígado , Tecido Linfoide/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Organogênese , Baço/patologia , Baço/transplante , Timo/patologia , Timo/transplante , Transplante HeterólogoRESUMO
N-Myc is a global transcription factor that regulates the expression of genes involved in a number of essential cellular processes including: ribosome biogenesis, cell cycle and apoptosis. Upon deregulation, N-Myc can drive pathologic expression of many of these genes, which ultimately defines its oncogenic potential. Overexpression of N-Myc has been demonstrated to contribute to tumorigenesis, most notably for the pediatric tumor, neuroblastoma. Herein, we provide evidence that deregulated N-Myc alters the expression of proteins involved in mitochondrial dynamics. We found that N-Myc overexpression leads to increased fusion of the mitochondrial reticulum secondary to changes in protein expression due to aberrant transcriptional and post-translational regulation. We believe the structural changes in the mitochondrial network in response to N-Myc amplification in neuroblastoma contributes to two important aspects of tumor development and maintenance-bioenergetic alterations and apoptotic resistance. Specifically, we found that N-Myc overexpressing cells are resistant to programmed cell death in response to exposure to low doses of cisplatin, and demonstrated that this was dependent on increased mitochondrial fusion. We speculate that these changes in mitochondrial structure and function may contribute significantly to the aggressive clinical ph9enotype of N-Myc amplified neuroblastoma.