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1.
Mol Cell ; 82(5): 920-932.e7, 2022 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-35245456

RESUMO

IDO1 oxidizes tryptophan (TRP) to generate kynurenine (KYN), the substrate for 1-carbon and NAD metabolism, and is implicated in pro-cancer pathophysiology and infection biology. However, the mechanistic relationships between IDO1 in amino acid depletion versus product generation have remained a longstanding mystery. We found an unrecognized link between IDO1 and cell survival mediated by KYN that serves as the source for molecules that inhibit ferroptotic cell death. We show that this effect requires KYN export from IDO1-expressing cells, which is then available for non-IDO1-expressing cells via SLC7A11, the central transporter involved in ferroptosis suppression. Whether inside the "producer" IDO1+ cell or the "receiver" cell, KYN is converted into downstream metabolites, suppressing ferroptosis by ROS scavenging and activating an NRF2-dependent, AHR-independent cell-protective pathway, including SLC7A11, propagating anti-ferroptotic signaling. IDO1, therefore, controls a multi-pronged protection pathway from ferroptotic cell death, underscoring the need to re-evaluate the use of IDO1 inhibitors in cancer treatment.


Assuntos
Sistema y+ de Transporte de Aminoácidos , Ferroptose , Cinurenina , Neoplasias , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/metabolismo , Cinurenina/farmacologia , Neoplasias/metabolismo , Transdução de Sinais , Triptofano/metabolismo
2.
J Biol Chem ; 298(12): 102629, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36273589

RESUMO

mTORC1 and GCN2 are serine/threonine kinases that control how cells adapt to amino acid availability. mTORC1 responds to amino acids to promote translation and cell growth while GCN2 senses limiting amino acids to hinder translation via eIF2α phosphorylation. GCN2 is an appealing target for cancer therapies because malignant cells can harness the GCN2 pathway to temper the rate of translation during rapid amino acid consumption. To isolate new GCN2 inhibitors, we created cell-based, amino acid limitation reporters via genetic manipulation of Ddit3 (encoding the transcription factor CHOP). CHOP is strongly induced by limiting amino acids and in this context, GCN2-dependent. Using leucine starvation as a model for essential amino acid sensing, we unexpectedly discovered ATP-competitive PI3 kinase-related kinase inhibitors, including ATR and mTOR inhibitors like torins, completely reversed GCN2 activation in a time-dependent way. Mechanistically, via inhibiting mTORC1-dependent translation, torins increased intracellular leucine, which was sufficient to reverse GCN2 activation and the downstream integrated stress response including stress-induced transcriptional factor ATF4 expression. Strikingly, we found that general translation inhibitors mirrored the effects of torins. Therefore, we propose that mTOR kinase inhibitors concurrently inhibit different branches of amino acid sensing by a dual mechanism involving direct inhibition of mTOR and indirect suppression of GCN2 that are connected by effects on the translation machinery. Collectively, our results highlight distinct ways of regulating GCN2 activity.


Assuntos
Aminoácidos , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Aminoácidos/genética , Aminoácidos/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosforilação , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Humanos , Animais , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
3.
J Virol ; 94(3)2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31694942

RESUMO

Hemagglutinin (HA) stability, or the pH at which HA is activated to cause membrane fusion, has been associated with the replication, pathogenicity, transmissibility, and interspecies adaptation of influenza A viruses. Here, we investigated the mechanisms by which a destabilizing HA mutation, Y17H (activation pH, 6.0), attenuates virus replication and pathogenicity in DBA/2 mice compared to wild-type (WT) virus (activation pH, 5.5). The extracellular lung pH was measured to be near neutral (pH 6.9 to 7.5). WT and Y17H viruses had similar environmental stability at pH 7.0; thus, extracellular inactivation was unlikely to attenuate the Y17H virus. The Y17H virus had accelerated replication kinetics in MDCK, A549, and RAW 264.7 cells when inoculated at a multiplicity of infection (MOI) of 3 PFU/cell. The destabilizing mutation also increased early infectivity and type I interferon (IFN) responses in mouse bone marrow-derived dendritic cells (DCs). In contrast, the HA-Y17H mutation reduced virus replication in murine airway murine nasal epithelial cell and murine tracheal epithelial cell cultures and attenuated virus replication, virus spread, the severity of infection, and cellular infiltration in the lungs of mice. Normalizing virus infection and weight loss in mice by inoculating them with Y17H virus at a dose 500-fold higher than that of WT virus revealed that the destabilized mutant virus triggered the upregulation of more host genes and increased type I IFN responses and cytokine expression in DBA/2 mouse lungs. Overall, HA destabilization decreased virulence in mice by boosting early infection in DCs, resulting in the greater activation of antiviral responses, including the type I IFN response. These studies reveal that HA stability may regulate pathogenicity by modulating IFN responses.IMPORTANCE Diverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. Rarely, an avian- or swine-origin influenza virus adapts to humans and starts a pandemic. Seasonal and many universal influenza vaccines target the HA surface protein, which is a key component of pandemic influenza viruses. Understanding the HA properties needed for replication and pathogenicity in mammals may guide response efforts to control influenza. Some antiviral drugs and broadly reactive influenza vaccines that target the HA protein have suffered resistance due to destabilizing HA mutations that do not compromise replicative fitness in cell culture. Here, we show that despite not compromising fitness in standard cell cultures, a destabilizing H1N1 HA stalk mutation greatly diminishes viral replication and pathogenicity in vivo by modulating type I IFN responses. This encourages targeting the HA stalk with antiviral drugs and vaccines as well as reevaluating previous candidates that were susceptible to destabilizing resistance mutations.


Assuntos
Células Dendríticas/metabolismo , Hemaglutininas/metabolismo , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Interferon Tipo I/metabolismo , Replicação Viral/fisiologia , Animais , Linhagem Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Hemaglutininas/genética , Hemaglutininas/imunologia , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vacinas contra Influenza , Influenza Humana/virologia , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Infecções por Orthomyxoviridae/virologia , Estabilidade Proteica , Proteínas Virais de Fusão , Virulência
4.
PLoS Pathog ; 13(3): e1006276, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28282440

RESUMO

A pandemic-capable influenza virus requires a hemagglutinin (HA) surface glycoprotein that is immunologically unseen by most people and is capable of supporting replication and transmission in humans. HA stabilization has been linked to 2009 pH1N1 pandemic potential in humans and H5N1 airborne transmissibility in the ferret model. Swine have served as an intermediate host for zoonotic influenza viruses, yet the evolutionary pressure exerted by this host on HA stability was unknown. For over 70 contemporary swine H1 and H3 isolates, we measured HA activation pH to range from pH 5.1 to 5.9 for H1 viruses and pH 5.3 to 5.8 for H3 viruses. Thus, contemporary swine isolates vary widely in HA stability, having values favored by both avian (pH >5.5) and human and ferret (pH ≤5.5) species. Using an early 2009 pandemic H1N1 (pH1N1) virus backbone, we generated three viruses differing by one HA residue that only altered HA stability: WT (pH 5.5), HA1-Y17H (pH 6.0), and HA2-R106K (pH 5.3). All three replicated in pigs and transmitted from pig-to-pig and pig-to-ferret. WT and R106 viruses maintained HA genotype and phenotype after transmission. Y17H (pH 6.0) acquired HA mutations that stabilized the HA protein to pH 5.8 after transmission to pigs and 5.5 after transmission to ferrets. Overall, we found swine support a broad range of HA activation pH for contact transmission and many recent swine H1N1 and H3N2 isolates have stabilized (human-like) HA proteins. This constitutes a heightened pandemic risk and underscores the importance of ongoing surveillance and control efforts for swine viruses.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/metabolismo , Infecções por Orthomyxoviridae/transmissão , Animais , Furões/virologia , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Estabilidade Proteica , Reação em Cadeia da Polimerase em Tempo Real , Suínos/virologia
5.
Proc Natl Acad Sci U S A ; 113(6): 1636-41, 2016 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-26811446

RESUMO

Influenza pandemics require that a virus containing a hemagglutinin (HA) surface antigen previously unseen by a majority of the population becomes airborne-transmissible between humans. Although the HA protein is central to the emergence of a pandemic influenza virus, its required molecular properties for sustained transmission between humans are poorly defined. During virus entry, the HA protein binds receptors and is triggered by low pH in the endosome to cause membrane fusion; during egress, HA contributes to virus assembly and morphology. In 2009, a swine influenza virus (pH1N1) jumped to humans and spread globally. Here we link the pandemic potential of pH1N1 to its HA acid stability, or the pH at which this one-time-use nanomachine is either triggered to cause fusion or becomes inactivated in the absence of a target membrane. In surveillance isolates, our data show HA activation pH values decreased during the evolution of H1N1 from precursors in swine (pH 5.5-6.0), to early 2009 human cases (pH 5.5), and then to later human isolates (pH 5.2-5.4). A loss-of-function pH1N1 virus with a destabilizing HA1-Y17H mutation (pH 6.0) was less pathogenic in mice and ferrets, less transmissible by contact, and no longer airborne-transmissible. A ferret-adapted revertant (HA1-H17Y/HA2-R106K) regained airborne transmissibility by stabilizing HA to an activation pH of 5.3, similar to that of human-adapted isolates from late 2009-2014. Overall, these studies reveal that a stable HA (activation pH ≤ 5.5) is necessary for pH1N1 influenza virus pathogenicity and airborne transmissibility in ferrets and is associated with pandemic potential in humans.


Assuntos
Ácidos/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Pandemias , Animais , Evolução Biológica , Furões/virologia , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H1N1/patogenicidade , Masculino , Camundongos Endogâmicos DBA , Mutação/genética , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Estabilidade Proteica , Suínos , Ativação Viral , Replicação Viral
6.
J Virol ; 91(2)2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27807237

RESUMO

Whether influenza virus replication in macrophages is productive or abortive has been a topic of debate. Utilizing a panel of 28 distinct human, avian, and swine influenza viruses, we found that only a small subset can overcome cellular blocks to productively replicate in murine and primary human macrophages. Murine macrophages have two cellular blocks. The first block is during viral entry, where virions with relatively acid-stable hemagglutinin (HA) proteins are rendered incapable of pH-induced triggering for membrane fusion, resulting in lysosomal degradation. The second block is downstream of viral replication but upstream of late protein synthesis. In contrast, primary human macrophages only have one cellular block that occurs after late protein synthesis. To determine the impact of abortive replication at different stages of the viral life cycle or productive replication on macrophage function, we assessed cytotoxicity, nitric oxide or reactive oxygen species production, and phagocytosis. Intriguingly, productive viral replication decreased phagocytosis of IgG-opsonized bioparticles and Fc receptor CD16 and CD32 surface levels, a function, to our knowledge, never before reported for an RNA virus. These data suggest that replication in macrophages affects cellular function and plays an important role in pathogenesis during infection in vivo IMPORTANCE: Macrophages are a critical first line of defense against respiratory pathogens. Thus, understanding how viruses evade or exploit macrophage function will provide greater insight into viral pathogenicity and antiviral responses. We previously showed that only a subset of highly pathogenic avian (HPAI) H5N1 influenza virus strains could productively replicate in murine macrophages through a hemagglutinin (HA)-mediated mechanism. These studies expand upon this work and demonstrate that productive replication is not specific to unique HPAI H5N1 viruses; an H1N1 strain (A/WSN/33) can also replicate in macrophages. Importantly, we identify two cellular blocks limiting replication that can be overcome by an avian-like pH of activation for nuclear entry and a yet-to-be-identified mechanism(s) to overcome a postnuclear entry block. Overcoming these blocks reduces the cell's ability to phagocytose IgG-opsonized bioparticles by decreasing Fc receptor surface levels, a mechanism previously thought to occur during bacterial and DNA viral infections.


Assuntos
Vírus da Influenza A/fisiologia , Macrófagos/fisiologia , Macrófagos/virologia , Replicação Viral , Animais , Linhagem Celular , Chlorocebus aethiops , Cães , Endossomos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Influenza Humana/imunologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Lisossomos/metabolismo , Camundongos , Neuraminidase/genética , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismo
7.
Antimicrob Agents Chemother ; 60(4): 2118-31, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26787699

RESUMO

Most cases of severe influenza are associated with pulmonary complications, such as acute respiratory distress syndrome (ARDS), and no antiviral drugs of proven value for treating such complications are currently available. The use of monoclonal antibodies targeting the stem of the influenza virus surface hemagglutinin (HA) is a rapidly developing strategy for the control of viruses of multiple HA subtypes. However, the mechanisms of action of these antibodies are not fully understood, and their ability to mitigate severe complications of influenza has been poorly studied. We evaluated the effect of treatment with VIS410, a human monoclonal antibody targeting the HA stem region, on the development of ARDS in BALB/c mice after infection with influenza A(H7N9) viruses. Prophylactic administration of VIS410 resulted in the complete protection of mice against lethal A(H7N9) virus challenge. A single therapeutic dose of VIS410 given 24 h after virus inoculation resulted in dose-dependent protection of up to 100% of mice inoculated with neuraminidase inhibitor-susceptible or -resistant A(H7N9) viruses. Compared to the outcomes in mock-treated controls, a single administration of VIS410 improved viral clearance from the lungs, reduced virus spread in lungs in a dose-dependent manner, resulting in a lower lung injury score, reduced the extent of the alteration in lung vascular permeability and protein accumulation in bronchoalveolar lavage fluid, and improved lung physiologic function. Thus, antibodies targeting the HA stem can reduce the severity of ARDS and show promise as agents for controlling pulmonary complications in influenza.


Assuntos
Anticorpos Monoclonais/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Subtipo H7N9 do Vírus da Influenza A/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Síndrome do Desconforto Respiratório/prevenção & controle , Animais , Líquido da Lavagem Broncoalveolar/virologia , Permeabilidade Capilar/efeitos dos fármacos , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Relação Dose-Resposta Imunológica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Subtipo H7N9 do Vírus da Influenza A/crescimento & desenvolvimento , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Infecções por Orthomyxoviridae/complicações , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/mortalidade , Síndrome do Desconforto Respiratório/virologia , Análise de Sobrevida , Carga Viral/efeitos dos fármacos
8.
J Virol ; 88(23): 13811-20, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25253342

RESUMO

UNLABELLED: Lassa virus is an Old World Arenavirus which causes Lassa hemorrhagic fever in humans, mostly in West Africa. Lassa fever is an important public health problem, and a safe and effective vaccine is urgently needed. The infection causes immunosuppression, probably due to the absence of activation of antigen-presenting cells (dendritic cells and macrophages), low type I interferon (IFN) production, and deficient NK cell function. However, a recombinant Lassa virus carrying D389A and G392A substitutions in the nucleoprotein that abolish the exonuclease activity and IFN activation loses its inhibitory activity and induces strong type I IFN production by dendritic cells and macrophages. We show here that during infection by this mutant Lassa virus, antigen-presenting cells trigger efficient human NK cell responses in vitro, including production of IFN-γ and cytotoxicity. NK cell activation involves close contact with both antigen-presenting cells and soluble factors. We report that infected dendritic cells and macrophages express the NKG2D ligands major histocompatibility complex (MHC) class I-related chains A and B and that they may produce interleukin-12 (IL-12), IL-15, and IL-18, all involved in NK cell functions. NK cell degranulation is significantly increased in cocultures, suggesting that NK cells seem to kill infected dendritic cells and macrophages. This work confirms the inhibitory function of Lassa virus nucleoprotein. Importantly, we demonstrate for the first time that Lassa virus nucleoprotein is involved in the inhibition of antigen-presenting cell-mediated NK cell responses. IMPORTANCE: The pathogenesis and immune responses induced by Lassa virus are poorly known. Recently, an exonuclease domain contained in the viral nucleoprotein has been shown to be able to inhibit the type I IFN response by avoiding the recognition of viral RNA by cell sensors. Here, we studied the responses of NK cells to dendritic cells and macrophages infected with a recombinant Lassa virus in which the exonuclease functions have been abolished and demonstrated that NK cells are strongly activated and presented effective functions. These results show that the strategy developed by Lassa virus to evade innate immunity is also effective on NK cells, explaining the weak NK cell activation observed with the wild-type virus. By providing a better understanding of the interactions between Lassa virus and the host immune system, these results are important for the field of arenavirus biology and may be useful for a vaccine approach against Lassa fever.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Proteínas do Capsídeo/imunologia , Exonucleases/imunologia , Células Matadoras Naturais/imunologia , Vírus Lassa/imunologia , Animais , Proteínas do Capsídeo/genética , Degranulação Celular , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Exonucleases/genética , Humanos , Tolerância Imunológica , Interferon gama/metabolismo , Interleucinas/metabolismo , Macrófagos/imunologia , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Mutação de Sentido Incorreto
9.
J Virol ; 88(23): 13923-7, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25253344

RESUMO

Lassa virus (LASV), which causes a viral hemorrhagic fever, inhibits the innate immune response. The exonuclease (ExoN) domain of its nucleoprotein (NP) is implicated in the suppression of retinoic acid-inducible gene I (RIG-I) signaling. We show here that a LASV in which ExoN function has been abolished strongly activates innate immunity and that this effect is dependent on RIG-I signaling. These results highlight the key role of NP ExoN function in the immune evasion that occurs during LASV infection.


Assuntos
Exonucleases/imunologia , Tolerância Imunológica , Imunidade Inata , Vírus Lassa/imunologia , Vírus Lassa/fisiologia , Nucleoproteínas/imunologia , Transdução de Sinais , Células Cultivadas , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , Exonucleases/metabolismo , Humanos , Nucleoproteínas/metabolismo , Receptores Imunológicos
10.
Eur J Immunol ; 42(7): 1822-32, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22585682

RESUMO

Lassa virus (LASV) and Mopeia virus (MOPV) are closely related Arenaviruses. LASV causes hemorrhagic fever, whereas MOPV is not pathogenic. Both viruses display tropism for APCs such as DCs and macrophages. During viral infections, NK cells are involved in the clearance of infected cells and promote optimal immune responses by interacting with APCs. We used an in vitro model of human NK and APC coculture to study the role of NK cells and to characterize their interactions with APCs during LASV and MOPV infections. As expected, NK cells alone were neither infected nor activated by LASV and MOPV, and infected DCs did not activate NK cells. By contrast, LASV- and MOPV-infected macrophages activated NK cells, as shown by the upregulation of CD69, NKp30, and NKp44, the downregulation of CXCR3, and an increase in NK-cell proliferation. NK cells acquired enhanced cytotoxicity, as illustrated by the increase in granzyme B (GrzB) expression and killing of K562 targets, but did not produce IFN-γ. Contact between NK cells and infected macrophages and type I IFNs were essential for activation; however, NK cells could not kill infected cells and control infection. Overall, these findings show that MOPV- as well as pathogenic LASV-infected macrophages mediate NK-cell activation.


Assuntos
Células Matadoras Naturais/imunologia , Febre Lassa/imunologia , Vírus Lassa/imunologia , Macrófagos/imunologia , Animais , Processos de Crescimento Celular/imunologia , Chlorocebus aethiops , Técnicas de Cocultura , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Regulação Viral da Expressão Gênica , Granzimas/genética , Granzimas/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Células K562 , Células Matadoras Naturais/virologia , Febre Lassa/virologia , Ativação Linfocitária/imunologia , Macrófagos/virologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Células Vero
11.
J Virol ; 85(16): 8293-306, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21632749

RESUMO

The events leading to death in severe cases of Lassa fever (LF) are unknown. Fatality seems to be linked to high viremia and immunosuppression, and cellular immunity, rather than neutralizing antibodies, appears to be essential for survival. We previously compared Lassa virus (LV) with its genetically close but nonpathogenic homolog Mopeia virus (MV), which was used to model nonfatal LF. We showed that strong and early activation of antigen-presenting cells (APC) may play a crucial role in controlling infection. Here we developed an in vitro model of dendritic-cell (DC)-T-cell coculture in order to characterize human T-cell responses induced by MV- or LV-infected DCs. Our results show very different responses to infection with LV and MV. MV strongly and durably stimulated CD8(+) and CD4(+) T cells, showing early and high activation, a strong proliferative response, and acquisition of effector and memory phenotypes. Furthermore, robust and functional CD4(+) and CD8(+) cytotoxic T lymphocytes (CTL) were generated. LV, however, induced only weak memory responses. Thus, this study allows an improved understanding of the pathogenesis and immune mechanisms involved in the control of human LV.


Assuntos
Arenavirus do Velho Mundo/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/virologia , Linfócitos T Citotóxicos/imunologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , ELISPOT , Citometria de Fluxo , Humanos , Memória Imunológica , Vírus Lassa/imunologia , Ativação Linfocitária , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Virologie (Montrouge) ; 16(6): 390-401, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31910557

RESUMO

Lassa fever is a hemorrhagic fever endemic to West Africa and caused by Lassa virus, an Old-World arenavirus. It may be fatal, but most patients recover from acute disease and some experience asymptomatic infection. The immune mechanisms associated with these different outcomes have not yet been fully elucidated, but considerable progress has recently been made, through the use of in vitro human models and non-human primates, the only relevant animal model that mimics the pathophysiology and immune responses induced in patients. We discuss here the roles of the various components of the innate and adaptive immune systems in Lassa virus infection and in the control of viral replication and pathogenesis.

13.
Elife ; 102021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33646117

RESUMO

Interleukin-4-induced-1 (IL4i1) is an amino acid oxidase secreted from immune cells. Recent observations have suggested that IL4i1 is pro-tumorigenic via unknown mechanisms. As IL4i1 has homologs in snake venoms (L-amino acid oxidases [LAAO]), we used comparative approaches to gain insight into the mechanistic basis of how conserved amino acid oxidases regulate cell fate and function. Using mammalian expressed recombinant proteins, we found that venom LAAO kills cells via hydrogen peroxide generation. By contrast, mammalian IL4i1 is non-cytotoxic and instead elicits a cell protective gene expression program inhibiting ferroptotic redox death by generating indole-3-pyruvate (I3P) from tryptophan. I3P suppresses ferroptosis by direct free radical scavenging and through the activation of an anti-oxidative gene expression program. Thus, the pro-tumor effects of IL4i1 are likely mediated by local anti-ferroptotic pathways via aromatic amino acid metabolism, arguing that an IL4i1 inhibitor may modulate tumor cell death pathways.


Assuntos
Aminoácidos/metabolismo , Ferroptose/efeitos dos fármacos , L-Aminoácido Oxidase/metabolismo , L-Aminoácido Oxidase/toxicidade , Animais , Morte Celular , Linhagem Celular , Linhagem Celular Tumoral , Venenos Elapídicos/enzimologia , Regulação da Expressão Gênica , Humanos , Peróxido de Hidrogênio/metabolismo , Camundongos , Oxirredução
14.
Cancer Res ; 81(19): 5047-5059, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34301764

RESUMO

Immune cells regulate tumor growth by mirroring their function as tissue repair organizers in normal tissues. To understand the different facets of immune-tumor collaboration through genetics, spatial transcriptomics, and immunologic manipulation with noninvasive, longitudinal imaging, we generated a penetrant double oncogene-driven autochthonous model of neuroblastoma. Spatial transcriptomic analysis showed that CD4+ and myeloid populations colocalized within the tumor parenchyma, while CD8+ T cells and B cells were peripherally dispersed. Depletion of CD4+ T cells or CCR2+ macrophages, but not B cells, CD8+ T cells, or natural killer (NK) cells, prevented tumor formation. Tumor CD4+ T cells displayed unconventional phenotypes and were clonotypically diverse and antigen independent. Within the myeloid fraction, tumor growth required myeloid cells expressing arginase-1. Overall, these results demonstrate how arginine-metabolizing myeloid cells conspire with pathogenic CD4+ T cells to create permissive conditions for tumor formation, suggesting that these protumorigenic pathways could be disabled by targeting myeloid arginine metabolism. SIGNIFICANCE: A new model of human neuroblastoma provides ways to track tumor formation and expansion in living animals, allowing identification of CD4+ T-cell and macrophage functions required for oncogenesis.


Assuntos
Arginase/genética , Linfócitos T CD4-Positivos/metabolismo , Suscetibilidade a Doenças , Células Mieloides/metabolismo , Neuroblastoma/etiologia , Neuroblastoma/metabolismo , Animais , Arginase/metabolismo , Biomarcadores , Células da Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Biologia Computacional/métodos , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Neuroblastoma/patologia , Oncogenes , Análise de Célula Única , Transcriptoma
15.
Nat Commun ; 11(1): 431, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969567

RESUMO

Multinucleated giant cells (MGCs) are implicated in many diseases including schistosomiasis, sarcoidosis and arthritis. MGC generation is energy intensive to enforce membrane fusion and cytoplasmic expansion. Using receptor activator of nuclear factor kappa-Β ligand (RANKL) induced osteoclastogenesis to model MGC formation, here we report RANKL cellular programming requires extracellular arginine. Systemic arginine restriction improves outcome in multiple murine arthritis models and its removal induces preosteoclast metabolic quiescence, associated with impaired tricarboxylic acid (TCA) cycle function and metabolite induction. Effects of arginine deprivation on osteoclastogenesis are independent of mTORC1 activity or global transcriptional and translational inhibition. Arginine scarcity also dampens generation of IL-4 induced MGCs. Strikingly, in extracellular arginine absence, both cell types display flexibility as their formation can be restored with select arginine precursors. These data establish how environmental amino acids control the metabolic fate of polykaryons and suggest metabolic ways to manipulate MGC-associated pathologies and bone remodelling.


Assuntos
Arginina/metabolismo , Células Gigantes/imunologia , Animais , Artrite/genética , Artrite/metabolismo , Artrite/fisiopatologia , Remodelação Óssea , Ciclo do Ácido Cítrico , Feminino , Células Gigantes/citologia , Humanos , Interleucina-4/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese , Ligante RANK/genética , Ligante RANK/metabolismo
16.
mSphere ; 1(2)2016.
Artigo em Inglês | MEDLINE | ID: mdl-27303732

RESUMO

Highly pathogenic influenza A(H5N8) viruses from clade 2.3.4.4 were introduced to North America by migratory birds in the fall of 2014. Reassortment of A(H5N8) viruses with avian viruses of North American lineage resulted in the generation of novel A(H5N2) viruses with novel genotypes. Through sequencing of recent avian influenza viruses, we identified PB1 and NP gene segments very similar to those in the viruses isolated from North American waterfowl prior to the introduction of A(H5N8) to North America, highlighting these bird species in the origin of reassortant A(H5N2) viruses. While they were highly virulent and transmissible in poultry, we found A(H5N2) viruses to be low pathogenic in mice and ferrets, and replication was limited in both hosts compared with those of recent highly pathogenic avian influenza (HPAI) H5N1 viruses. Molecular characterization of the hemagglutinin protein from A(H5N2) viruses showed that the receptor binding preference, cleavage, and pH of activation were highly adapted for replication in avian species and similar to those of other 2.3.4.4 viruses. In addition, North American and Eurasian clade 2.3.4.4 H5NX viruses replicated to significantly lower titers in differentiated normal human bronchial epithelial cells than did seasonal human A(H1N1) and highly pathogenic A(H5N1) viruses isolated from a human case. Thus, despite their having a high impact on poultry, our findings suggest that the recently emerging North American A(H5N2) viruses are not expected to pose a substantial threat to humans and other mammals without further reassortment and/or adaptation and that reassortment with North American viruses has not had a major impact on viral phenotype. IMPORTANCE Highly pathogenic H5 influenza viruses have been introduced into North America from Asia, causing extensive morbidity and mortality in domestic poultry. The introduced viruses have reassorted with North American avian influenza viruses, generating viral genotypes not seen on other continents. The experiments and analyses presented here were designed to assess the impact of this genetic diversification on viral phenotypes, particularly as regards mammalian hosts, by comparing the North American viruses with their Eurasian precursor viruses.

17.
PLoS Negl Trop Dis ; 8(1): e2637, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24421914

RESUMO

The pathogenesis of Lassa fever (LF), a hemorrhagic fever endemic to West Africa, remains unclear. We previously compared Lassa virus (LASV) with its genetically close, but nonpathogenic homolog Mopeia virus (MOPV) and demonstrated that the strong activation of antigen-presenting cells (APC), including type I IFN production, observed in response to MOPV probably plays a crucial role in controlling infection. We show here that human macrophages (MP) produce large amounts of CC and CXC chemokines in response to MOPV infection, whereas dendritic cells (DC) release only moderate amounts of CXC chemokines. However, in the presence of autologous T cells, DCs produced CC and CXC chemokines. Chemokines were produced in response to type I IFN synthesis, as the levels of both mediators were strongly correlated and the neutralization of type I IFN resulted in an inhibition of chemokine production. By contrast, LASV induced only low levels of CXCL-10 and CXCL-11 production. These differences in chemokine production may profoundly affect the generation of virus-specific T-cell responses and may therefore contribute to the difference of pathogenicity between these two viruses. In addition, a recombinant LASV (rLASV) harboring the NP-D389A/G392A mutations, which abolish the inhibition of type I IFN response by nucleoprotein (NP), induced the massive synthesis of CC and CXC chemokines in both DC and MP, confirming the crucial role of arenavirus NP in immunosuppression and pathogenicity. Finally, we confirmed, using PBMC samples and lymph nodes obtained from LASV-infected cynomolgus monkeys, that LF was associated with high levels of CXC chemokine mRNA synthesis, suggesting that the very early synthesis of these mediators may be correlated with a favourable outcome.


Assuntos
Células Apresentadoras de Antígenos/metabolismo , Quimiocinas CC/metabolismo , Quimiocinas CXC/metabolismo , Febre Lassa/imunologia , Febre Lassa/veterinária , Vírus Lassa/imunologia , Doenças dos Primatas/imunologia , Animais , Humanos , Evasão da Resposta Imune , Masculino , Nucleoproteínas/imunologia , Proteínas Virais/imunologia
18.
Viruses ; 4(11): 2766-85, 2012 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-23202504

RESUMO

Lassa fever is a hemorrhagic fever endemic to West Africa and caused by Lassa virus, an Old World arenavirus. It may be fatal, but most patients recover from acute disease and some experience asymptomatic infection. The immune mechanisms associated with these different outcomes have not yet been fully elucidated, but considerable progress has recently been made, through the use of in vitro human models and nonhuman primates, the only relevant animal model that mimics the pathophysiology and immune responses induced in patients. We discuss here the roles of the various components of the innate and adaptive immune systems in Lassa virus infection and in the control of viral replication and pathogenesis.


Assuntos
Febre Lassa/imunologia , Vírus Lassa/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Humanos , Imunidade Humoral , Células Matadoras Naturais/imunologia , Linfócitos T/imunologia
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