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1.
Nat Immunol ; 11(11): 997-1004, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20890285

RESUMO

The detection of intracellular microbial DNA is critical to appropriate innate immune responses; however, knowledge of how such DNA is sensed is limited. Here we identify IFI16, a PYHIN protein, as an intracellular DNA sensor that mediates the induction of interferon-ß (IFN-ß). IFI16 directly associated with IFN-ß-inducing viral DNA motifs. STING, a critical mediator of IFN-ß responses to DNA, was recruited to IFI16 after DNA stimulation. Lowering the expression of IFI16 or its mouse ortholog p204 by RNA-mediated interference inhibited gene induction and activation of the transcription factors IRF3 and NF-κB induced by DNA and herpes simplex virus type 1 (HSV-1). IFI16 (p204) is the first PYHIN protein to our knowledge shown to be involved in IFN-ß induction. Thus, the PYHIN proteins IFI16 and AIM2 form a new family of innate DNA sensors we call 'AIM2-like receptors' (ALRs).


Assuntos
DNA Viral/imunologia , Imunidade Inata , Espaço Intracelular/imunologia , Proteínas Nucleares/imunologia , Fosfoproteínas/imunologia , Animais , Linhagem Celular , Proteínas de Ligação a DNA , Herpesvirus Humano 1/imunologia , Humanos , Interferon beta/imunologia , Interferon beta/metabolismo , Proteínas de Membrana/imunologia , Camundongos , Monócitos/imunologia , Transdução de Sinais
2.
J Biol Chem ; 293(39): 14989-15001, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30111593

RESUMO

Host pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) detect viruses and other pathogens, inducing production of cytokines that cause inflammation and mobilize cells to control infection. Vaccinia virus (VACV) encodes proteins that antagonize these host innate immune responses, and elucidating the mechanisms of action of these viral proteins helped shed light on PRR signaling mechanisms. The VACV virulence factor E3 is one of the most intensely studied VACV proteins and has multiple effects on host cells, many of which cannot be explained by the currently known cellular targets of E3. Here, we report that E3 expression in human monocytes alters TLR2- and TLR8-dependent cytokine induction, and particularly inhibits interleukin (IL)-6. Using MS, we identified DExD/H-box helicase 9 (DHX9) as an E3 target. Although DHX9 has previously been implicated as a PRR for sensing nucleic acid in dendritic cells, we found no role for DHX9 as a nucleic acid-sensing PRR in monocytes. Rather, DHX9 suppression in these cells phenocopied the effects of E3 expression on TLR2- and TLR8-dependent cytokine induction, in that DHX9 was required for all TLR8-dependent cytokines measured, and for TLR2-dependent IL-6. Furthermore, DHX9 also had a cell- and stimulus-independent role in IL-6 promoter induction. DHX9 enhanced NF-κB-dependent IL-6 promoter activation, which was directly antagonized by E3. These results indicate new roles for DHX9 in regulating cytokines in innate immunity and reveal that VACV E3 disrupts innate immune responses by targeting of DHX9.


Assuntos
RNA Helicases DEAD-box/genética , Interações Hospedeiro-Patógeno/genética , Imunidade Inata/genética , Proteínas de Neoplasias/genética , Proteínas de Ligação a RNA/genética , Receptor 2 Toll-Like/genética , Proteínas Virais/genética , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucina-6/genética , Monócitos/virologia , Regiões Promotoras Genéticas , Receptores de Reconhecimento de Padrão/genética , Receptor 8 Toll-Like/genética , Vacínia/genética , Vacínia/virologia , Vaccinia virus/genética , Vaccinia virus/patogenicidade
3.
J Immunol ; 196(6): 2552-60, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26873994

RESUMO

Human NK cells can be classified into phenotypically and functionally distinct subsets based on levels of CD56 receptor. CD56(dim) cells are generally considered more cytotoxic, whereas the CD56(bright) cells are potent producers of IFN-γ. In this study, we define the metabolic changes that occur in peripheral blood NK cells in response to cytokine. Metabolic analysis showed that NK cells upregulate glycolysis and oxidative phosphorylation in response to either IL-2 or IL-12/15 cytokine combinations. Despite the fact that both these cytokine combinations robustly upregulated mammalian Target of Rapamycin Complex 1 in human NK cells, only the IL-2-induced metabolic changes were sensitive to mammalian Target of Rapamycin Complex 1 inhibition by rapamycin. Interestingly, we found that CD56(bright) cells were more metabolically active compared with CD56(dim) cells. They preferentially upregulated nutrient receptors and also differed substantially in terms of their glucose metabolism. CD56(bright) cells expressed high levels of the glucose uptake receptor, Glut1 (in the absence of any cytokine), and had higher rates of glucose uptake compared with CD56(dim) cells. Elevated levels of oxidative phosphorylation were required to support both cytotoxicity and IFN-γ production in all NK cells. Finally, although elevated glycolysis was not required directly for NK cell degranulation, limiting the rate of glycolysis significantly impaired IFN-γ production by the CD56(bright) subset of cells. Overall, we have defined CD56(bright) NK cells to be more metabolically active than CD56(dim) cells, which supports their production of large amounts of IFN-γ during an immune response.


Assuntos
Interferon gama/biossíntese , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Antígeno CD56/biossíntese , Antígeno CD56/imunologia , Citometria de Fluxo , Glicólise/imunologia , Humanos
4.
Cell Mol Life Sci ; 74(9): 1755-1764, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27988795

RESUMO

The cytokine, Interferon (IFN)-α, induces a wide spectrum of anti-viral mediators, via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. STAT1 and STAT2 are well characterised to upregulate IFN-stimulated gene (ISG) expression; but even though STAT3 is also activated by IFN-α, its role in anti-viral ISG induction is unclear. Several viruses, including Hepatitis C and Mumps, reduce cellular STAT3 protein levels, via the promotion of ubiquitin-mediated proteasomal degradation. This viral immune evasion mechanism suggests an undiscovered anti-viral role for STAT3 in IFN-α signalling. To investigate STAT3's functional involvement in this Type I IFN pathway, we first analysed its effect upon the replication of two viruses, Influenza and Vaccinia. Viral plaque assays, using Wild Type (WT) and STAT3-/- Murine Embryonic Fibroblasts (MEFs), revealed that STAT3 is required for the inhibition of Influenza and Vaccinia replication. Furthermore, STAT3 shRNA knockdown also enhanced Influenza replication and hindered induction of several, well characterised, anti-viral ISGs: PKR, OAS2, MxB and ISG15; while STAT3 expression had no effect upon induction of a separate ISG group: Viperin, IFI27, CXCL10 and CCL5. These discoveries reveal, for the first time, an anti-viral role for STAT3 in the IFN-α pathway and characterise a requirement for STAT3 in the expression of specific ISGs. These findings also identify STAT3 as a therapeutic target against viral infection and highlight it as an essential pathway component for endogenous and therapeutic IFN-α responsiveness.


Assuntos
Interferon-alfa/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , 2',5'-Oligoadenilato Sintetase/metabolismo , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Vírus da Influenza A/fisiologia , Camundongos , Proteínas de Resistência a Myxovirus , Vaccinia virus/fisiologia , eIF-2 Quinase/metabolismo
5.
J Immunol ; 193(9): 4477-84, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25261477

RESUMO

The mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cellular metabolism and also has fundamental roles in controlling immune responses. Emerging evidence suggests that these two functions of mTORC1 are integrally linked. However, little is known regarding mTORC1 function in controlling the metabolism and function of NK cells, lymphocytes that play key roles in antiviral and antitumor immunity. This study investigated the hypothesis that mTORC1-controlled metabolism underpins normal NK cell proinflammatory function. We demonstrate that mTORC1 is robustly stimulated in NK cells activated in vivo and in vitro. This mTORC1 activity is required for the production of the key NK cell effector molecules IFN-γ, which is important in delivering antimicrobial and immunoregulatory functions, and granzyme B, a critical component of NK cell cytotoxic granules. The data reveal that NK cells undergo dramatic metabolic reprogramming upon activation, upregulating rates of glucose uptake and glycolysis, and that mTORC1 activity is essential for attaining this elevated glycolytic state. Directly limiting the rate of glycolysis is sufficient to inhibit IFN-γ production and granzyme B expression. This study provides the highly novel insight that mTORC1-mediated metabolic reprogramming of NK cells is a prerequisite for the acquisition of normal effector functions.


Assuntos
Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Ativação Enzimática , Expressão Gênica , Glicólise , Granzimas/genética , Granzimas/metabolismo , Interferon gama/biossíntese , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Poli I-C/farmacologia
6.
Trends Immunol ; 32(12): 574-81, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21940216

RESUMO

Type I interferon (IFN) induction is a crucial anti-pathogen response mediated by innate immune stimulation. Although it has been appreciated for some time that the presence of pathogen DNA within a cell leads to a type I IFN response, it is only in the past few years that some of the key signalling proteins and DNA sensors that regulate this response have been uncovered. Here, we review the nature of these DNA sensors, which include a new family of pattern recognition receptors termed the AIM2-like receptors, and consider the implications of their discovery for understanding emerging principles of innate immune DNA sensing. Furthermore, we discuss how their discovery provides a rationale as to why accumulation of self-DNA mediates IFN-dependent autoimmunity.


Assuntos
Citosol/imunologia , DNA/imunologia , Interferon Tipo I/imunologia , Animais , Autoimunidade , Humanos , Imunidade Inata , Interferon Tipo I/biossíntese , Internalização do Vírus
7.
J Biol Chem ; 287(41): 34709-21, 2012 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-22891244

RESUMO

Aberrant Notch signaling has recently emerged as a possible mechanism for the altered neurogenesis, cognitive impairment, and learning and memory deficits associated with Alzheimer disease (AD). Recently, targeting the endocannabinoid system in models of AD has emerged as a potential approach to slow the progression of the disease process. Although studies have identified neuroprotective roles for endocannabinoids, there is a paucity of information on modulation of the pro-survival Notch pathway by endocannabinoids. In this study the influence of the endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol, on the Notch-1 pathway and on its endogenous regulators were investigated in an in vitro model of AD. We report that AEA up-regulates Notch-1 signaling in cultured neurons. We also provide evidence that although Aß(1-42) increases expression of the endogenous inhibitor of Notch-1, numb (Nb), this can be prevented by AEA and 2-arachidonoylglycerol. Interestingly, AEA up-regulated Nct expression, a component of γ-secretase, and this was found to play a crucial role in the enhanced Notch-1 signaling mediated by AEA. The stimulatory effects of AEA on Notch-1 signaling persisted in the presence of Aß(1-42). AEA was found to induce a preferential processing of Notch-1 over amyloid precursor protein to generate Aß(1-40). Aging, a natural process of neurodegeneration, was associated with a reduction in Notch-1 signaling in rat cortex and hippocampus, and this was restored with chronic treatment with URB 597. In summary, AEA has the proclivity to enhance Notch-1 signaling in an in vitro model of AD, which may have relevance for restoring neurogenesis and cognition in AD.


Assuntos
Envelhecimento/metabolismo , Peptídeos beta-Amiloides/farmacologia , Ácidos Araquidônicos/metabolismo , Córtex Cerebral/metabolismo , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/farmacologia , Alcamidas Poli-Insaturadas/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Envelhecimento/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/biossíntese , Peptídeos beta-Amiloides/metabolismo , Animais , Benzamidas/farmacologia , Carbamatos/farmacologia , Células Cultivadas , Córtex Cerebral/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Glicoproteínas de Membrana/biossíntese , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Wistar , Regulação para Cima/efeitos dos fármacos
8.
J Biol Chem ; 286(27): 23688-97, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21606490

RESUMO

Toll-like receptors (TLRs) are pattern-recognition receptors that recognize microbial ligands and subsequently trigger intracellular signaling pathways involving transcription factors such as NFκB and MAPKs such as p38. TLR signaling can regulate both transcriptional and post-transcriptional events leading to altered gene expression and thus appropriate immune responses. The interleukin-1 receptor-associated kinase (IRAK) family comprises four kinases that regulate TLR signaling. However, the role of IRAK-2 has remained unclear, especially in human cells. Recent studies using cells from in-bred Irak2(-/-) mice showed that murine IRAK-2 was not required for early TLR signaling events but had a role in delayed NFκB activation and in cytokine production. IRAK-2 in mice has four splice variants, two of which are inhibitory, whereas human IRAK-2 has no splice variants. Thus IRAK-2 in mice and humans may function differently, and therefore we analyzed the role of IRAK-2 in TLR responses in primary human cells. siRNA knockdown of IRAK-2 expression in human peripheral blood mononuclear cells showed a role for human IRAK-2 in both TLR4- and TLR8-mediated early NFκB and p38 MAPK activation and in induction of TNF mRNA. These data conflict with findings from the in-bred Irak2(-/-) mice but concur with what has been seen in wild-derived mice for TLR2. Moreover, human IRAK-2 was required for regulating MyD88-dependent TNFα mRNA stability via the TNF 3'UTR. Collectively, these data demonstrate for the first time an essential role for IRAK-2 in primary human cells for both transcriptional and post-transcriptional TLR responses.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/metabolismo , Leucócitos Mononucleares/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Transcrição Gênica/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Regiões 3' não Traduzidas/fisiologia , Animais , Células HEK293 , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Leucócitos Mononucleares/citologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Estabilidade de RNA/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Especificidade da Espécie , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 8 Toll-Like/genética , Fator de Necrose Tumoral alfa/genética
9.
MAbs ; 13(1): 1850395, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33459147

RESUMO

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.


Assuntos
Anticorpos Biespecíficos/imunologia , Complexo CD3/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Enterotoxina/imunologia , Animais , Anticorpos Biespecíficos/farmacocinética , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Hibridomas , Macaca fascicularis/imunologia , Macaca fascicularis/metabolismo , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Neoplasias/metabolismo , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacocinética , Anticorpos de Cadeia Única/uso terapêutico , Linfócitos T/imunologia , Linfócitos T/metabolismo
10.
J Biol Chem ; 282(46): 33435-33443, 2007 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-17878161

RESUMO

Toll-like receptor (TLR) signaling is known to involve interleukin-1 receptor-associated kinases (IRAKs), however the particular role of IRAK-2 has remained unclear. Further, although IRAK-1 was originally thought to be central for the TLR-NFkappaB signaling axis, recent data have shown that it is dispensable for NFkappaB activation for some TLRs and demonstrated an alternative role for it in interferon regulatory factor activation. Here we show that IRAK-2 is critical for the TLR-mediated NFkappaB activation pathway. The poxviral TLR antagonist A52 inhibited NFkappaB activation by TLR2, -3, -4, -5, -7, and -9 ligands, via its interaction with IRAK-2, while not affecting interferon regulatory factor activation. Knockdown of IRAK-2 expression by small interfering RNA suppressed TLR3, TLR4, and TLR8 signaling to NFkappaB in human cell lines, and importantly, TLR4-mediated chemokine production in primary human cells. IRAK-2 usage by different TLRs was distinct, because it acted downstream of the TLR adaptors MyD88 and Mal but upstream of TRIF. Expression of IRAK-2, but not IRAK-1, led to TRAF6 ubiquitination, an event critical for NFkappaB activation. Further, IRAK-2 loss-of-function mutants, which could not activate NFkappaB, were incapable of promoting TRAF6 ubiquitination. Thus we propose that IRAK-2 plays a more central role than IRAK-1 in TLR signaling to NFkappaB.


Assuntos
Regulação da Expressão Gênica , Quinases Associadas a Receptores de Interleucina-1/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Receptores Toll-Like/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Inativação Gênica , Genes Reporter , Humanos , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Homologia de Sequência de Aminoácidos
11.
J Immunol ; 174(11): 6829-38, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15905524

RESUMO

Human herpesviruses, including EBV, persist for life in infected individuals. During the lytic replicative cycle that is required for the production of infectious virus and transmission to another host, many viral Ags are expressed. Especially at this stage, immune evasion strategies are likely to be advantageous to avoid elimination of virus-producing cells. However, little is known about immune escape during productive EBV infection because no fully permissive infection model is available. In this study, we have developed a novel strategy to isolate populations of cells in an EBV lytic cycle based on the expression of a reporter gene under the control of an EBV early lytic cycle promoter. Thus, induction of the viral lytic cycle in transfected EBV(+) B lymphoma cells resulted in concomitant reporter expression, allowing us, for the first time, to isolate highly purified cell populations in lytic cycle for biochemical and functional studies. Compared with latently infected B cells, cells supporting EBV lytic cycle displayed down-regulation of surface HLA class I, class II, and CD20, whereas expression levels of other surface markers remained unaffected. Moreover, during lytic cycle peptide transport into the endoplasmic reticulum, was reduced to <30% of levels found in latent infection. Because steady-state levels of TAP proteins were unaffected, these results point toward EBV-induced interference with TAP function as a specific mechanism contributing to the reduced levels of cell surface HLA class I. Our data implicate that EBV lytic cycle genes encode functions to evade T cell recognition, thereby creating a window for the generation of viral progeny.


Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Transportadores de Cassetes de Ligação de ATP , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/virologia , Linhagem Celular Tumoral , Separação Celular , Regulação para Baixo/imunologia , Infecções por Vírus Epstein-Barr/virologia , Genes Reporter , Antígenos HLA-D/biossíntese , Antígenos HLA-D/metabolismo , Herpesvirus Humano 4/genética , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Transporte Proteico/genética , Transporte Proteico/imunologia , Ratos , Proteínas Virais/biossíntese , Proteínas Virais/genética , Ativação Viral/genética , Ativação Viral/imunologia , Replicação Viral/genética , Replicação Viral/imunologia
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