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1.
Mol Cell ; 76(3): 485-499.e8, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31495563

RESUMO

Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.


Assuntos
Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Fibroblastos/efeitos dos fármacos , Interferon gama/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Quinase 8 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Fibroblastos/enzimologia , Fibroblastos/virologia , Células HCT116 , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , RNA Polimerase II/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Vesiculovirus/patogenicidade
2.
Immunity ; 38(2): 250-62, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23352233

RESUMO

Gene regulation by cytokine-activated transcription factors of the signal transducer and activator of transcription (STAT) family requires serine phosphorylation within the transactivation domain (TAD). STAT1 and STAT3 TAD phosphorylation occurs upon promoter binding by an unknown kinase. Here, we show that the cyclin-dependent kinase 8 (CDK8) module of the Mediator complex phosphorylated regulatory sites within the TADs of STAT1, STAT3, and STAT5, including S727 within the STAT1 TAD in the interferon (IFN) signaling pathway. We also observed a CDK8 requirement for IFN-γ-inducible antiviral responses. Microarray analyses revealed that CDK8-mediated STAT1 phosphorylation positively or negatively regulated over 40% of IFN-γ-responsive genes, and RNA polymerase II occupancy correlated with gene expression changes. This divergent regulation occurred despite similar CDK8 occupancy at both S727 phosphorylation-dependent and -independent genes. These data identify CDK8 as a key regulator of STAT1 and antiviral responses and suggest a general role for CDK8 in STAT-mediated transcription. As such, CDK8 represents a promising target for therapeutic manipulation of cytokine responses.


Assuntos
Quinase 8 Dependente de Ciclina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Fator de Transcrição STAT1/genética , Animais , Quinase 8 Dependente de Ciclina/imunologia , Quinase 8 Dependente de Ciclina/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/virologia , Células Hep G2 , Humanos , Interferon gama/imunologia , Interleucina-6/imunologia , Interleucina-6/farmacologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Polimerase II/imunologia , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Vesiculovirus/fisiologia
3.
J Immunol ; 204(6): 1607-1620, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32024700

RESUMO

Autoinflammatory diseases are characterized by dysregulation of the innate immune system, leading to spontaneous inflammation. Pstpip2cmo mouse strain is a well-characterized model of this class of disorders. Because of the mutation leading to the lack of adaptor protein PSTPIP2, these animals suffer from autoinflammatory chronic multifocal osteomyelitis similar to several human syndromes. Current evidence suggests that it is driven by hyperproduction of IL-1ß by neutrophil granulocytes. In this study, we show that in addition to IL-1ß, PSTPIP2 also negatively regulates pathways governing reactive oxygen species generation by neutrophil NOX2 NADPH oxidase. Pstpip2cmo neutrophils display highly elevated superoxide production in response to a range of stimuli. Inactivation of NOX2 NADPH oxidase in Pstpip2cmo mice did not affect IL-1ß levels, and the autoinflammatory process was initiated with similar kinetics. However, the bone destruction was almost completely alleviated, suggesting that dysregulated NADPH oxidase activity is a key factor promoting autoinflammatory bone damage in Pstpip2cmo mice.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Osso e Ossos/patologia , Proteínas do Citoesqueleto/metabolismo , NADPH Oxidase 2/metabolismo , Osteomielite/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Osso e Ossos/imunologia , Linhagem Celular , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Humanos , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , NADPH Oxidase 2/genética , Neutrófilos/imunologia , Neutrófilos/metabolismo , Osteomielite/genética , Osteomielite/patologia , Cultura Primária de Células , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Superóxidos/imunologia , Superóxidos/metabolismo
5.
PLoS Pathog ; 13(11): e1006696, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29112952

RESUMO

Klebsiella pneumoniae is a significant cause of nosocomial pneumonia and an alarming pathogen owing to the recent isolation of multidrug resistant strains. Understanding of immune responses orchestrating K. pneumoniae clearance by the host is of utmost importance. Here we show that type I interferon (IFN) signaling protects against lung infection with K. pneumoniae by launching bacterial growth-controlling interactions between alveolar macrophages and natural killer (NK) cells. Type I IFNs are important but disparate and incompletely understood regulators of defense against bacterial infections. Type I IFN receptor 1 (Ifnar1)-deficient mice infected with K. pneumoniae failed to activate NK cell-derived IFN-γ production. IFN-γ was required for bactericidal action and the production of the NK cell response-amplifying IL-12 and CXCL10 by alveolar macrophages. Bacterial clearance and NK cell IFN-γ were rescued in Ifnar1-deficient hosts by Ifnar1-proficient NK cells. Consistently, type I IFN signaling in myeloid cells including alveolar macrophages, monocytes and neutrophils was dispensable for host defense and IFN-γ activation. The failure of Ifnar1-deficient hosts to initiate a defense-promoting crosstalk between alveolar macrophages and NK cell was circumvented by administration of exogenous IFN-γ which restored endogenous IFN-γ production and restricted bacterial growth. These data identify NK cell-intrinsic type I IFN signaling as essential driver of K. pneumoniae clearance, and reveal specific targets for future therapeutic exploitations.


Assuntos
Interferon Tipo I/imunologia , Células Matadoras Naturais/imunologia , Infecções por Klebsiella/imunologia , Macrófagos Alveolares/imunologia , Transdução de Sinais/imunologia , Animais , Resistência a Múltiplos Medicamentos/imunologia , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Cross-Talk/imunologia , Infecções Respiratórias/imunologia
6.
Cell Microbiol ; 20(12): e12943, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30112857

RESUMO

Candida glabrata is a common human fungal commensal and opportunistic pathogen. This fungus shows remarkable resilience as it can form recalcitrant biofilms on indwelling catheters, has intrinsic resistance against azole antifungals, and is causing vulvovaginal candidiasis. As a nosocomial pathogen, it can cause life-threatening bloodstream infections in immune-compromised patients. Here, we investigate the potential role of the high osmolarity glycerol response (HOG) MAP kinase pathway for C. glabrata virulence. The C. glabrata MAP kinase CgHog1 becomes activated by a variety of environmental stress conditions such as osmotic stress, low pH, and carboxylic acids and subsequently accumulates in the nucleus. We found that CgHog1 allows C. glabrata to persist within murine macrophages, but it is not required for systemic infection in a mouse model. C. glabrata and Lactobacilli co-colonise mucosal surfaces. Lactic acid at a concentration produced by vaginal Lactobacillus spp. causes CgHog1 phosphorylation and accumulation in the nucleus. In addition, CgHog1 enables C. glabrata to tolerate different Lactobacillus spp. and their metabolites when grown in co-culture. Using a phenotypic diverse set of clinical C. glabrata isolates, we find that the HOG pathway is likely the main quantitative determinant of lactic acid stress resistance. Taken together, our data indicate that CgHog1 has an important role in the confrontation of C. glabrata with the common vaginal flora.


Assuntos
Antibiose/fisiologia , Candida glabrata/fisiologia , Proteínas Fúngicas/metabolismo , Lactobacillus/fisiologia , Animais , Candida glabrata/efeitos dos fármacos , Candida glabrata/patogenicidade , Candidíase/microbiologia , Núcleo Celular/metabolismo , Feminino , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/farmacologia , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Vagina/microbiologia
7.
PLoS Pathog ; 12(12): e1006032, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27973535

RESUMO

Pathogen clearance and host resilience/tolerance to infection are both important factors in surviving an infection. Cells of the myeloid lineage play important roles in both of these processes. Neutrophils, monocytes, macrophages, and dendritic cells all have important roles in initiation of the immune response and clearance of bacterial pathogens. If these cells are not properly regulated they can result in excessive inflammation and immunopathology leading to decreased host resilience. Programmed cell death (PCD) is one possible mechanism that myeloid cells may use to prevent excessive inflammation. Myeloid cell subsets play roles in tissue repair, immune response resolution, and maintenance of homeostasis, so excessive PCD may also influence host resilience in this way. In addition, myeloid cell death is one mechanism used to control pathogen replication and dissemination. Many of these functions for PCD have been well defined in vitro, but the role in vivo is less well understood. We created a mouse that constitutively expresses the pro-survival B-cell lymphoma (bcl)-2 protein in myeloid cells (CD68(bcl2tg), thus decreasing PCD specifically in myeloid cells. Using this mouse model we explored the impact that decreased cell death of these cells has on infection with two different bacterial pathogens, Legionella pneumophila and Streptococcus pyogenes. Both of these pathogens target multiple cell death pathways in myeloid cells, and the expression of bcl2 resulted in decreased PCD after infection. We examined both pathogen clearance and host resilience and found that myeloid cell death was crucial for host resilience. Surprisingly, the decreased myeloid PCD had minimal impact on pathogen clearance. These data indicate that the most important role of PCD during infection with these bacteria is to minimize inflammation and increase host resilience, not to aid in the clearance or prevent the spread of the pathogen.


Assuntos
Apoptose/imunologia , Doença dos Legionários/imunologia , Células Mieloides/imunologia , Infecções Estreptocócicas/imunologia , Animais , Citometria de Fluxo , Imunidade Inata , Legionella pneumophila/imunologia , Camundongos , Camundongos Transgênicos , Streptococcus pyogenes/imunologia
8.
Nucleic Acids Res ; 44(D1): D90-5, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26602692

RESUMO

AREsite2 represents an update for AREsite, an on-line resource for the investigation of AU-rich elements (ARE) in human and mouse mRNA 3'UTR sequences. The new updated and enhanced version allows detailed investigation of AU, GU and U-rich elements (ARE, GRE, URE) in the transcriptome of Homo sapiens, Mus musculus, Danio rerio, Caenorhabditis elegans and Drosophila melanogaster. It contains information on genomic location, genic context, RNA secondary structure context and conservation of annotated motifs. Improvements include annotation of motifs not only in 3'UTRs but in the whole gene body including introns, additional genomes, and locally stable secondary structures from genome wide scans. Furthermore, we include data from CLIP-Seq experiments in order to highlight motifs with validated protein interaction. Additionally, we provide a REST interface for experienced users to interact with the database in a semi-automated manner. The database is publicly available at: http://rna.tbi.univie.ac.at/AREsite.


Assuntos
Regiões 3' não Traduzidas , Bases de Dados de Ácidos Nucleicos , RNA/química , Animais , Genômica , Humanos , Camundongos , Anotação de Sequência Molecular , Conformação de Ácido Nucleico , Motivos de Nucleotídeos
9.
Mol Syst Biol ; 12(5): 868, 2016 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-27178967

RESUMO

Precise regulation of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. However, a global model integrating regulation and functional consequences of inflammation-associated mRNA decay remains to be established. Using time-resolved high-resolution RNA binding analysis of the mRNA-destabilizing protein tristetraprolin (TTP), an inflammation-limiting factor, we qualitatively and quantitatively characterize TTP binding positions in the transcriptome of immunostimulated macrophages. We identify pervasive destabilizing and non-destabilizing TTP binding, including a robust intronic binding, showing that TTP binding is not sufficient for mRNA destabilization. A low degree of flanking RNA structuredness distinguishes occupied from silent binding motifs. By functionally relating TTP binding sites to mRNA stability and levels, we identify a TTP-controlled switch for the transition from inflammatory into the resolution phase of the macrophage immune response. Mapping of binding positions of the mRNA-stabilizing protein HuR reveals little target and functional overlap with TTP, implying a limited co-regulation of inflammatory mRNA decay by these proteins. Our study establishes a functionally annotated and navigable transcriptome-wide atlas (http://ttp-atlas.univie.ac.at) of cis-acting elements controlling mRNA decay in inflammation.


Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , RNA Mensageiro/química , Tristetraprolina/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Células HEK293 , Humanos , Macrófagos/efeitos dos fármacos , Camundongos , Estabilidade de RNA , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
10.
Cytokine ; 89: 21-26, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-26586165

RESUMO

Expression of cytokines and chemokines is regulated at multiple steps during the transfer of the genetic information from DNA sequence to the functional protein. The multilayered control of cytokine expression reflects the need of the immune system to precisely and rapidly adjust the magnitude and duration of immune responses to external cues. Common features of the regulation of cytokine expression are temporal and highly dynamic changes in cytokine mRNA stability. Failures in the timing and extent of mRNA decay can result in disease. Recent advances in transcriptome-wide approaches began to shed light into the complex network of cis-acting sequence elements and trans-acting factors controlling mRNA stability. These approaches led to the discovery of novel unexpected paradigms but they also revealed new questions. This review will discuss the control of cytokine mRNA stability both in the context of high content approaches as well as focused mechanistic studies and animal models. The article highlights the need for systems biology approaches as important means to understand how cytokine mRNA decay helps maintain the immune and tissue homeostasis, and to explore options for therapeutical exploitation of mRNA stability regulation.


Assuntos
Citocinas/imunologia , Regulação da Expressão Gênica/imunologia , Estabilidade de RNA/imunologia , RNA Mensageiro/imunologia , Transcriptoma/imunologia , Animais , Humanos
11.
PLoS Pathog ; 7(5): e1001345, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21625574

RESUMO

Streptococcus pyogenes is a Gram-positive human pathogen that is recognized by yet unknown pattern recognition receptors (PRRs). Engagement of these receptor molecules during infection with S. pyogenes, a largely extracellular bacterium with limited capacity for intracellular survival, causes innate immune cells to produce inflammatory mediators such as TNF, but also type I interferon (IFN). Here we show that signaling elicited by type I IFNs is required for successful defense of mice against lethal subcutaneous cellulitis caused by S. pyogenes. Type I IFN signaling was accompanied with reduced neutrophil recruitment to the site of infection. Mechanistic analysis revealed that macrophages and conventional dendritic cells (cDCs) employ different signaling pathways leading to IFN-beta production. Macrophages required IRF3, STING, TBK1 and partially MyD88, whereas in cDCs the IFN-beta production was fully dependent on IRF5 and MyD88. Furthermore, IFN-beta production by macrophages was dependent on the endosomal delivery of streptococcal DNA, while in cDCs streptococcal RNA was identified as the IFN-beta inducer. Despite a role of MyD88 in both cell types, the known IFN-inducing TLRs were individually not required for generation of the IFN-beta response. These results demonstrate that the innate immune system employs several strategies to efficiently recognize S. pyogenes, a pathogenic bacterium that succeeded in avoiding recognition by the standard arsenal of TLRs.


Assuntos
DNA Bacteriano/metabolismo , Células Dendríticas , Macrófagos , RNA Bacteriano/metabolismo , Streptococcus pyogenes/imunologia , Animais , Células Cultivadas , Celulite (Flegmão)/microbiologia , Celulite (Flegmão)/mortalidade , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Inativação Gênica , Imunidade Inata , Fator Regulador 3 de Interferon , Fatores Reguladores de Interferon , Interferon beta/biossíntese , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fator 88 de Diferenciação Mieloide , Infiltração de Neutrófilos/imunologia , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases , RNA Interferente Pequeno , Receptores de Reconhecimento de Padrão , Transdução de Sinais/imunologia , Streptococcus pyogenes/genética
12.
Nucleic Acids Res ; 39(Database issue): D66-9, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21071424

RESUMO

AREsite is an online resource for the detailed investigation of AU-rich elements (ARE) in vertebrate mRNA 3'-untranslated regions (UTRs). AREs are one of the most prominent cis-acting regulatory elements found in 3'-UTRs of mRNAs. Various ARE-binding proteins that possess RNA stabilizing or destabilizing functions are recruited by sequence-specific motifs. Recent findings suggest an essential role of the structural mRNA context in which these sequence motifs are embedded. AREsite is the first database that allows to quantify the structuredness of ARE motif sites in terms of opening energies and accessibility probabilities. Moreover, we also provide a detailed phylogenetic analysis of ARE motifs and incorporate information about experimentally validated targets of the ARE-binding proteins TTP, HuR and Auf1. The database is publicly available at: http://rna.tbi.univie.ac.at/AREsite.


Assuntos
Regiões 3' não Traduzidas , Adenina/análise , Bases de Dados de Ácidos Nucleicos , Uracila/análise , Animais , Sequência de Bases , Sequência Conservada , Genômica , Humanos , Camundongos , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Análise de Sequência de RNA
13.
Elife ; 122023 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-36961408

RESUMO

Tristetraprolin (TTP) is a critical negative immune regulator. It binds AU-rich elements in the untranslated-regions of many mRNAs encoding pro-inflammatory mediators, thereby accelerating their decay. A key but poorly understood mechanism of TTP regulation is its timely proteolytic removal: TTP is degraded by the proteasome through yet unidentified phosphorylation-controlled drivers. In this study, we set out to identify factors controlling TTP stability. Cellular assays showed that TTP is strongly lysine-ubiquitinated, which is required for its turnover. A genetic screen identified the ubiquitin E3 ligase HUWE1 as a strong regulator of TTP proteasomal degradation, which we found to control TTP stability indirectly by regulating its phosphorylation. Pharmacological assessment of multiple kinases revealed that HUWE1-regulated TTP phosphorylation and stability was independent of the previously characterized effects of MAPK-mediated S52/S178 phosphorylation. HUWE1 function was dependent on phosphatase and E3 ligase binding sites identified in the TTP C-terminus. Our findings indicate that while phosphorylation of S52/S178 is critical for TTP stabilization at earlier times after pro-inflammatory stimulation, phosphorylation of the TTP C-terminus controls its stability at later stages.


Assuntos
Tristetraprolina , Ubiquitina-Proteína Ligases , Fosforilação , Tristetraprolina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteólise , Ubiquitina/metabolismo , Estabilidade de RNA/genética
14.
Mol Syst Biol ; 7: 560, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22186734

RESUMO

For a successful yet controlled immune response, cells need to specifically destabilize inflammatory mRNAs but prevent premature removal of those still used. The regulatory circuits controlling quality and timing in the global inflammatory mRNA decay are not understood. Here, we show that the mRNA-destabilizing function of the AU-rich element-binding protein tristetraprolin (TTP) is inversely regulated by the p38 MAPK activity profile such that after inflammatory stimulus the TTP-dependent decay is initially limited to few mRNAs. With time, the TTP-dependent decay gradually spreads resulting in cumulative elimination of one third of inflammation-induced unstable mRNAs in macrophages in vitro. We confirmed this sequential decay model in vivo since LPS-treated mice with myeloid TTP ablation exhibited similar cytokine dysregulation profile as macrophages. The mice were hypersensitive to LPS but otherwise healthy with no signs of hyperinflammation seen in conventional TTP knockout mice demonstrating the requirement for myeloid TTP in re-installment but not maintenance of immune homeostasis. These findings reveal a TTP- and p38 MAPK-dominated regulatory mechanism that is vital for balancing acute inflammation by a temporally and qualitatively controlled mRNA decay.


Assuntos
Inflamação/genética , Estabilidade de RNA/imunologia , Tristetraprolina/imunologia , Animais , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Inflamação/imunologia , Lipopolissacarídeos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estabilidade de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma , Tristetraprolina/genética , Tristetraprolina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
J Immunol ; 185(6): 3544-53, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20713887

RESUMO

IL-1beta is an important proinflammatory cytokine with a major role in several inflammatory diseases. Expression of IL-1beta is tightly regulated at the level of transcription, mRNA stability, and proteolytic processing. In this study, we report that IL-1beta expression in response to LPS is also regulated at the translational level. LPS-induced IL-1beta protein levels in macrophages derived from murine bone marrow are markedly increased in the absence of tyrosine kinase 2 (Tyk2). Increased IL-1beta is found intra- and extracellularly, irrespective of the efficiency of IL-1beta processing. We show that the absence of Tyk2 results both in higher translational rates and in enhanced association of IL-1beta mRNA with polysomes. Induction and stability of IL-1beta mRNA are not affected by the lack of Tyk2. We show further that the Tyk2-dependent translational inhibition is mediated by autocrine/paracrine type I IFN signaling and requires signal transducer and activator of transcription 1. Enhanced IL-1beta production in Tyk2- and IFN receptor 1-deficient macrophages is also observed following Listeria monocytogenes infection. Taken together, the data describe a novel mechanism for the control of IL-1beta synthesis.


Assuntos
Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/imunologia , TYK2 Quinase/fisiologia , Animais , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Interferon Tipo I/fisiologia , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , TYK2 Quinase/deficiência , TYK2 Quinase/genética , Regulação para Cima/genética , Regulação para Cima/imunologia
16.
Sci Adv ; 8(9): eabj7293, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35235356

RESUMO

Interleukin-1α (IL-1α) and IL-1ß are inflammatory cytokines with important roles in health and disease. They trigger the same receptor and elicit comparable cellular responses but, for poorly understood reasons, are not redundant in vivo. Here, we decoupled IL-1α and IL-1ß functions that drive protective responses against invasive infection with group A Streptococcus. IL-1ß was essential for pathogen clearance, hence resistance to infection, by inducing granulocyte colony-stimulating factor at the infection site and establishing emergency granulopoiesis. In contrast, IL-1α governed reprogramming of liver metabolic pathways associated with tolerance to infection. The IL-1α-dominated hepatic regulation corresponded to high IL-1α levels in the liver during infection. Conversely, IL-1ß was critical for the regulation of the spleen transcriptome, which correlated with ample IL-1ß expression in this tissue. The results identify distinct and organ-specific roles of IL-1α versus IL-1ß and implicate spatial restriction of their expression and bioavailability during infection as the underlying mechanism.


Assuntos
Interleucina-1alfa , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo
17.
Sci Signal ; 15(764): eabq5389, 2022 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-36512641

RESUMO

Promoters of antimicrobial genes function as logic boards, integrating signals of innate immune responses. One such set of genes is stimulated by interferon (IFN) signaling, and the expression of these genes [IFN-stimulated genes (ISGs)] can be further modulated by cell stress-induced pathways. Here, we investigated the global effect of stress-induced p38 mitogen-activated protein kinase (MAPK) signaling on the response of macrophages to IFN. In response to cell stress that coincided with IFN exposure, the p38 MAPK-activated transcription factors CREB and c-Jun, in addition to the IFN-activated STAT family of transcription factors, bound to ISGs. In addition, p38 MAPK signaling induced activating histone modifications at the loci of ISGs and stimulated nuclear translocation of the CREB coactivator CRTC3. These actions synergistically enhanced ISG expression. Disrupting this synergy with p38 MAPK inhibitors improved the viability of macrophages infected with Listeria monocytogenes. Our findings uncover a mechanism of transcriptional synergism and highlight the biological consequences of coincident stress-induced p38 MAPK and IFN-stimulated signal transduction.


Assuntos
Interferon gama , Interferons , Interferons/genética , Interferons/farmacologia , Interferons/metabolismo , Interferon gama/metabolismo , Macrófagos/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transcrição Gênica , Fatores de Transcrição/metabolismo , Fosforilação
18.
Cell Microbiol ; 12(2): 199-216, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19811500

RESUMO

The opportunistic human fungal pathogen Candida glabrata is confronted with phagocytic cells of the host defence system. Survival of internalized cells is thought to contribute to successful dissemination. We investigated the reaction of engulfed C. glabrata cells using fluorescent protein fusions of the transcription factors CgYap1 and CgMig1 and catalase CgCta1. The expression level and peroxisomal localization of catalase was used to monitor the metabolic and stress status of internalized C. glabrata cells. These reporters revealed that the phagocytosed C. glabrata cells were exposed to transient oxidative stress and starved for carbon source. Cells trapped within macrophages increased their peroxisome numbers indicating a metabolic switch. Prolonged phagocytosis caused a pexophagy-mediated decline in peroxisome numbers. Autophagy, and in particular pexophagy, contributed to survival of C. glabrata during engulfment. Mutants lacking CgATG11 or CgATG17, genes required for pexophagy and non-selective autophagy, respectively, displayed reduced survival rates. Furthermore, both CgAtg11 and CgAtg17 contribute to survival, since the double mutant was highly sensitive to engulfment. Inhibition of peroxisome formation by deletion of CgPEX3 partially restored viability of CgATG11 deletion mutants during engulfment. This suggests that peroxisome formation and maintenance might sequester resources required for optimal survival. Mobilization of intracellular resources via autophagy is an important virulence factor that supports the viability of C. glabrata in the phagosomal compartment of infected innate immune cells.


Assuntos
Autofagia/fisiologia , Candida glabrata/metabolismo , Candida glabrata/fisiologia , Fagocitose/fisiologia , Animais , Autofagia/genética , Northern Blotting , Southern Blotting , Candida glabrata/genética , Células Cultivadas , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fagocitose/genética
19.
J Immunol ; 183(2): 1197-206, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19542371

RESUMO

IL-10 is essential for inhibiting chronic and acute inflammation by decreasing the amounts of proinflammatory cytokines made by activated macrophages. IL-10 controls proinflammatory cytokine and chemokine production indirectly via the transcription factor Stat3. One of the most physiologically significant IL-10 targets is TNF-alpha, a potent proinflammatory mediator that is the target for multiple anti-TNF-alpha clinical strategies in Crohn's disease and rheumatoid arthritis. The anti-inflammatory effects of IL-10 seem to be mediated by several incompletely understood transcriptional and posttranscriptional mechanisms. In this study, we show that in LPS-activated bone marrow-derived murine macrophages, IL-10 reduces the mRNA and protein levels of TNF-alpha and IL-1alpha in part through the RNA destabilizing factor tristetraprolin (TTP). TTP is known for its central role in destabilizing mRNA molecules containing class II AU-rich elements in 3' untranslated regions. We found that IL-10 initiates a Stat3-dependent increase of TTP expression accompanied by a delayed decrease of p38 MAPK activity. The reduction of p38 MAPK activity releases TTP from the p38 MAPK-mediated inhibition, thereby resulting in diminished mRNA and protein levels of proinflammatory cytokines. These findings establish that TTP is required for full responses of bone marrow-derived murine macrophages to IL-10.


Assuntos
Inflamação/imunologia , Interleucina-10/imunologia , Macrófagos/imunologia , Tristetraprolina/fisiologia , Animais , Células da Medula Óssea , Células Cultivadas , Citocinas/antagonistas & inibidores , Macrófagos/citologia , Camundongos , Estabilidade de RNA , Tristetraprolina/genética , Regulação para Cima/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
Proc Natl Acad Sci U S A ; 105(26): 8944-9, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18574148

RESUMO

The transcription factor Stat1 plays an essential role in responses to interferons (IFNs). Activation of Stat1 is achieved by phosphorylation on Y701 that is followed by nuclear accumulation. For full transcriptional activity and biological function Stat1 must also be phosphorylated on S727. The molecular mechanisms underlying the IFN-induced S727 phosphorylation are incompletely understood. Here, we show that both Stat1 Y701 phosphorylation and nuclear translocation are required for IFN-induced S727 phosphorylation. We further show that Stat1 mutants lacking the ability to stably associate with chromatin are poorly serine-phosphorylated in response to IFN-gamma. The S727 phosphorylation of these mutants is restored on IFN-beta treatment that induces the formation of the ISGF3 complex (Stat1/Stat2/Irf9) where Irf9 represents the main DNA binding subunit. These findings indicate that Stat1 needs to be assembled into chromatin-associated transcriptional complexes to become S727-phosphorylated and fully biologically active in response to IFNs. This control mechanism, which may be used by other Stat proteins as well, restricts the final activation step to the chromatin-tethered transcription factor.


Assuntos
Cromatina/metabolismo , Interferon beta/farmacologia , Interferon gama/farmacologia , Fosfosserina/metabolismo , Fator de Transcrição STAT1/química , Fator de Transcrição STAT1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromatina/efeitos dos fármacos , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína
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