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1.
Cell ; 180(6): 1115-1129.e13, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32200799

RESUMO

Influenza A virus (IAV) is a lytic RNA virus that triggers receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated pathways of apoptosis and mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necroptosis in infected cells. ZBP1 initiates RIPK3-driven cell death by sensing IAV RNA and activating RIPK3. Here, we show that replicating IAV generates Z-RNAs, which activate ZBP1 in the nucleus of infected cells. ZBP1 then initiates RIPK3-mediated MLKL activation in the nucleus, resulting in nuclear envelope disruption, leakage of DNA into the cytosol, and eventual necroptosis. Cell death induced by nuclear MLKL was a potent activator of neutrophils, a cell type known to drive inflammatory pathology in virulent IAV disease. Consequently, MLKL-deficient mice manifest reduced nuclear disruption of lung epithelia, decreased neutrophil recruitment into infected lungs, and increased survival following a lethal dose of IAV. These results implicate Z-RNA as a new pathogen-associated molecular pattern and describe a ZBP1-initiated nucleus-to-plasma membrane "inside-out" death pathway with potentially pathogenic consequences in severe cases of influenza.


Assuntos
Vírus da Influenza A/genética , Necroptose/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose/genética , Morte Celular/genética , Linhagem Celular Tumoral , Feminino , Vírus da Influenza A/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necrose/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , RNA/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia
2.
J Immunol ; 203(5): 1348-1355, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31358656

RESUMO

Receptor-interacting protein kinase 1 (RIPK1) regulates cell fate and proinflammatory signaling downstream of multiple innate immune pathways, including those initiated by TNF-α, TLR ligands, and IFNs. Genetic ablation of Ripk1 results in perinatal lethality arising from both RIPK3-mediated necroptosis and FADD/caspase-8-driven apoptosis. IFNs are thought to contribute to the lethality of Ripk1-deficient mice by activating inopportune cell death during parturition, but how IFNs activate cell death in the absence of RIPK1 is not understood. In this study, we show that Z-form nucleic acid binding protein 1 (ZBP1; also known as DAI) drives IFN-stimulated cell death in settings of RIPK1 deficiency. IFN-activated Jak/STAT signaling induces robust expression of ZBP1, which complexes with RIPK3 in the absence of RIPK1 to trigger RIPK3-driven pathways of caspase-8-mediated apoptosis and MLKL-driven necroptosis. In vivo, deletion of either Zbp1 or core IFN signaling components prolong viability of Ripk1-/- mice for up to 3 mo beyond parturition. Together, these studies implicate ZBP1 as the dominant activator of IFN-driven RIPK3 activation and perinatal lethality in the absence of RIPK1.


Assuntos
Morte Celular/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/fisiologia , Caspase 8/metabolismo , Linhagem Celular , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia
3.
J Immunol ; 200(10): 3626-3634, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29654208

RESUMO

The cytokine IFN-γ has well-established antibacterial properties against the bacterium Salmonella enterica in phagocytes, but less is known about the effects of IFN-γ on Salmonella-infected nonphagocytic cells, such as intestinal epithelial cells (IECs) and fibroblasts. In this article, we show that exposing human and murine IECs and fibroblasts to IFN-γ following infection with Salmonella triggers a novel form of cell death that is neither pyroptosis nor any of the major known forms of programmed cell death. Cell death required IFN-γ-signaling via STAT1-IRF1-mediated induction of guanylate binding proteins and the presence of live Salmonella in the cytosol. In vivo, ablating IFN-γ signaling selectively in murine IECs led to higher bacterial burden in colon contents and increased inflammation in the intestine of infected mice. Together, these results demonstrate that IFN-γ signaling triggers release of Salmonella from the Salmonella-containing vacuole into the cytosol of infected nonphagocytic cells, resulting in a form of nonpyroptotic cell death that prevents bacterial spread in the gut.


Assuntos
Morte Celular/imunologia , Interferon gama/imunologia , Fagócitos/imunologia , Piroptose/imunologia , Infecções por Salmonella/imunologia , Salmonella enterica/imunologia , Células 3T3 , Animais , Linhagem Celular , Citosol/imunologia , Citosol/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Fibroblastos/imunologia , Fibroblastos/microbiologia , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Fator Regulador 1 de Interferon/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Camundongos , Fagócitos/microbiologia , Fator de Transcrição STAT1/imunologia , Infecções por Salmonella/microbiologia
4.
Cytokine ; 98: 27-32, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-27773552

RESUMO

Salmonella enterica is a facultative intracellular bacterium that is the leading cause of food borne illnesses in humans. The cytokine IFN-γ has well-established antibacterial properties against Salmonella and other intracellular microbes, for example its capacity to activate macrophages, promote phagocytosis, and destroy phagocytosed microbes by free radical-driven toxification of phagosomes. But IFN-γ induces the expression of hundreds of uncharacterized genes, suggesting that this cytokine deploys additional antimicrobial strategies that await discovery. Recently, one such mechanism, mediated by a family of IFN-inducible small GTPases called Guanylate Binding Proteins (GBPs) has been uncovered. GBPs were shown to facilitate the pyroptotic clearance of Salmonella from infected macrophages by rupturing the protective intracellular vacuole this microbe forms around itself. Once this protective vacuole is lost, exposed Salmonella activates pyroptosis, which destroys the infected cell. In this review, we summarize such emerging roles for IFN-γ in restricting Salmonella pathogenesis.


Assuntos
Interferon gama/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Animais , Autofagia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Inflamassomos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Fagocitose , Fagossomos/imunologia , Fagossomos/microbiologia , Piroptose , Infecções por Salmonella/microbiologia , Infecções por Salmonella/fisiopatologia
5.
Nat Med ; 28(3): 557-567, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35241842

RESUMO

Myelodysplastic syndromes (MDS) are heterogeneous neoplastic disorders of hematopoietic stem cells (HSCs). The current standard of care for patients with MDS is hypomethylating agent (HMA)-based therapy; however, almost 50% of MDS patients fail HMA therapy and progress to acute myeloid leukemia, facing a dismal prognosis due to lack of approved second-line treatment options. As cancer stem cells are the seeds of disease progression, we investigated the biological properties of the MDS HSCs that drive disease evolution, seeking to uncover vulnerabilities that could be therapeutically exploited. Through integrative molecular profiling of HSCs and progenitor cells in large patient cohorts, we found that MDS HSCs in two distinct differentiation states are maintained throughout the clinical course of the disease, and expand at progression, depending on recurrent activation of the anti-apoptotic regulator BCL-2 or nuclear factor-kappa B-mediated survival pathways. Pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden in experimental systems. Further, patients with MDS who progressed after failure to frontline HMA therapy and whose HSCs upregulated BCL-2 achieved improved clinical responses to venetoclax-based therapy in the clinical setting. Overall, our study uncovers that HSC architectures in MDS are potential predictive biomarkers to guide second-line treatments after HMA failure. These findings warrant further investigation of HSC-specific survival pathways to identify new therapeutic targets of clinical potential in MDS.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Síndromes Mielodisplásicas , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Células-Tronco Hematopoéticas/patologia , Humanos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas
6.
ACS Med Chem Lett ; 12(6): 1011-1016, 2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34141086

RESUMO

BCL-XL, an antiapoptotic member of the BCL-2 family of proteins, drives tumor survival and maintenance and thus represents a key target for cancer treatment. Herein we report the rational design of a novel series of selective BCL-XL inhibitors exemplified by A-1293102. This molecule contains structural elements of selective BCL-XL inhibitor A-1155463 and the dual BCL-XL/BCL-2 inhibitors ABT-737 and navitoclax, while representing a distinct pharmacophore as assessed by an objective cheminformatic evaluation. A-1293102 exhibited picomolar binding affinity to BCL-XL and both efficiently and selectively killed BCL-XL-dependent tumor cells. X-ray crystallographic analysis demonstrated a key hydrogen bonding network in the P2 binding pocket of BCL-XL, while the bent-back moiety achieved efficient occupancy of the P4 pocket in a manner similar to that of navitoclax. A-1293102 represents one of the few distinct structural series of selective BCL-XL inhibitors, and thus serves as a useful tool for biological studies as well as a lead compound for further optimization.

7.
Nat Commun ; 8(1): 1931, 2017 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-29203926

RESUMO

The risk of emerging pandemic influenza A viruses (IAVs) that approach the devastating 1918 strain motivates finding strain-specific host-pathogen mechanisms. During infection, dendritic cells (DC) mature into antigen-presenting cells that activate T cells, linking innate to adaptive immunity. DC infection with seasonal IAVs, but not with the 1918 and 2009 pandemic strains, induces global RNA degradation. Here, we show that DC infection with seasonal IAV causes immunogenic RIPK3-mediated cell death. Pandemic IAV suppresses this immunogenic DC cell death. Only DC infected with seasonal IAV, but not with pandemic IAV, enhance maturation of uninfected DC and T cell proliferation. In vivo, circulating T cell levels are reduced after pandemic, but not seasonal, IAV infection. Using recombinant viruses, we identify the HA genomic segment as the mediator of cell death inhibition. These results show how pandemic influenza viruses subvert the immune response.


Assuntos
Morte Celular/imunologia , Células Dendríticas/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Imunidade Adaptativa/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Imunidade Inata/imunologia , Técnicas In Vitro , Influenza Humana/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia
9.
PLoS One ; 11(7): e0158774, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27391363

RESUMO

The kinase RIPK3 is a key regulator of cell death responses to a growing number of viral and microbial agents. We have found that influenza A virus (IAV)-mediated cell death is largely reliant on RIPK3 and that RIPK3-deficient mice are notably more susceptible to lethal infection by IAV than their wild-type counterparts. Recent studies demonstrate that RIPK3 also participates in regulating gene transcription programs during host pro-inflammatory and innate-immune responses, indicating that this kinase is not solely an inducer of cell death and that RIPK3-driven transcriptional responses may collaborate with cell death in promoting clearance of IAV. Here, we carried out DNA microarray analyses to determine the contribution of RIPK3 to the IAV-elicited host transcriptional response. We report that RIPK3 does not contribute significantly to the RLR-activated transcriptome or to the induction of type I IFN genes, although, interestingly, IFN-ß production at a post-transcriptional step was modestly attenuated in IAV-infected ripk3-/- fibroblasts. Overall, RIPK3 regulated the expression of <5% of the IAV-induced transcriptome, and no genes were found to be obligate RIPK3 targets. IFN-ß signaling was also found to be largely normal in the absence of RIPK3. Together, these results indicate that RIPK3 is not essential for the host antiviral transcriptional response to IAV in murine fibroblasts.


Assuntos
Proteína DEAD-box 58/metabolismo , Fibroblastos/virologia , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Interferon Tipo I/metabolismo , Interferon beta/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Proteína DEAD-box 58/genética , Feminino , Fibroblastos/metabolismo , Interferon Tipo I/genética , Interferon beta/genética , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia
10.
Cell Host Microbe ; 20(5): 674-681, 2016 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-27746097

RESUMO

Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation and implicate DAI as a sensor of RNA viruses.


Assuntos
Morte Celular , Glicoproteínas/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A/imunologia , RNA Viral/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Genômica , Glicoproteínas/deficiência , Camundongos , Camundongos Knockout , Mutação , Proteínas Quinases/metabolismo , Proteínas de Ligação a RNA
11.
Cell Host Microbe ; 20(1): 13-24, 2016 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-27321907

RESUMO

Influenza A virus (IAV) is a lytic virus in primary cultures of many cell types and in vivo. We report that the kinase RIPK3 is essential for IAV-induced lysis of mammalian fibroblasts and lung epithelial cells. Replicating IAV drives assembly of a RIPK3-containing complex that includes the kinase RIPK1, the pseudokinase MLKL, and the adaptor protein FADD, and forms independently of signaling by RNA-sensing innate immune receptors (RLRs, TLRs, PKR), or the cytokines type I interferons and TNF-α. Downstream of RIPK3, IAV activates parallel pathways of MLKL-driven necroptosis and FADD-mediated apoptosis, with the former reliant on RIPK3 kinase activity and neither on RIPK1 activity. Mice deficient in RIPK3 or doubly deficient in MLKL and FADD, but not MLKL alone, are more susceptible to IAV than their wild-type counterparts, revealing an important role for RIPK3-mediated apoptosis in antiviral immunity. Collectively, these results outline RIPK3-activated cytolytic mechanisms essential for controlling respiratory IAV infection.


Assuntos
Apoptose , Proteína de Domínio de Morte Associada a Fas/metabolismo , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/imunologia , Necrose , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/fisiologia , Células Epiteliais/virologia , Proteína de Domínio de Morte Associada a Fas/genética , Fibroblastos/fisiologia , Fibroblastos/virologia , Humanos , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/patologia , Proteínas Quinases/genética , Multimerização Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/genética
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