RESUMO
Citrulline can be converted into argininosuccinate by argininosuccinate synthetase (ASS1) in the urea cycle and the citrulline-nitric oxide cycle. However, the regulation and biological function of citrulline metabolism remain obscure in the immune system. Unexpectedly, we found that macrophage citrulline declines rapidly after interferon gamma (IFN-γ) and/or lipopolysaccharide (LPS) stimulation, which is required for efficient proinflammatory signaling activation. Mechanistically, IFN-γ and/or LPS stimulation promotes signal transducers and activators of transcription 1 (STAT1)-mediated ASS1 transcription and Janus kinase2 (JAK2)-mediated phosphorylation of ASS1 at tyrosine 87, thereby leading to citrulline depletion. Reciprocally, increased citrulline directly binds to JAK2 and inhibits JAK2-STAT1 signaling. Blockage of ASS1-mediated citrulline depletion suppresses the host defense against bacterial infection in vivo. We therefore define a central role for ASS1 in controlling inflammatory macrophage activation and antibacterial defense through depletion of cellular citrulline and, further, identify citrulline as an innate immune-signaling metabolite that engages a metabolic checkpoint for proinflammatory responses.
Assuntos
Argininossuccinato Sintase/metabolismo , Citrulina/metabolismo , Imunidade Inata , Inflamação/enzimologia , Listeriose/enzimologia , Ativação de Macrófagos , Macrófagos/enzimologia , Animais , Argininossuccinato Sintase/genética , Modelos Animais de Doenças , Células HEK293 , Humanos , Inflamação/genética , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Listeria monocytogenes/imunologia , Listeriose/genética , Listeriose/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Células RAW 264.7 , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de SinaisRESUMO
Macrophages are highly heterogeneous immune cells that fulfill tissue-specific functions. Tissue-derived signals play a critical role in determining macrophage heterogeneity. However, these signals remain largely unknown. The BMP receptor activin receptor-like kinase 1 (ALK1) is well known for its role in blood vessel formation; however, its role within the immune system has never been revealed to our knowledge. Here, we found that BMP9/BMP10/ALK1 signaling controlled the identity and self-renewal of Kupffer cells (KCs) through a Smad4-dependent pathway. In contrast, ALK1 was dispensable for the maintenance of macrophages located in the lung, kidney, spleen, and brain. Following ALK1 deletion, KCs were lost over time and were replaced by monocyte-derived macrophages. These hepatic macrophages showed significantly reduced expression of the complement receptor VSIG4 and alterations in immune zonation and morphology, which is important for the tissue-specialized function of KCs. Furthermore, we found that this signaling pathway was important for KC-mediated Listeria monocytogenes capture, as the loss of ALK1 and Smad4 led to a failure of bacterial capture and overwhelming disseminated infections. Thus, ALK1 signaling instructs a tissue-specific phenotype that allows KCs to protect the host from systemic bacterial dissemination.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Células de Kupffer/enzimologia , Listeria monocytogenes/metabolismo , Listeriose/enzimologia , Listeriose/prevenção & controle , Transdução de Sinais , Receptores de Activinas Tipo II/genética , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Fator 2 de Diferenciação de Crescimento/genética , Fator 2 de Diferenciação de Crescimento/metabolismo , Listeriose/genética , Camundongos , Camundongos KnockoutRESUMO
Infection triggers expansion and effector differentiation of T cells specific for microbial antigens in association with metabolic reprograming. We found that the glycolytic enzyme lactate dehydrogenase A (LDHA) is induced in CD8+ T effector cells through phosphoinositide 3-kinase (PI3K) signaling. In turn, ablation of LDHA inhibits PI3K-dependent phosphorylation of Akt and its transcription factor target Foxo1, causing defective antimicrobial immunity. LDHA deficiency cripples cellular redox control and diminishes adenosine triphosphate (ATP) production in effector T cells, resulting in attenuated PI3K signaling. Thus, nutrient metabolism and growth factor signaling are highly integrated processes, with glycolytic ATP serving as a rheostat to gauge PI3K-Akt-Foxo1 signaling in the control of T cell immunity. Such a bioenergetic mechanism for the regulation of signaling may explain the Warburg effect.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Glicólise , Lactato Desidrogenase 5/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Animais , Linfócitos T CD8-Positivos/enzimologia , Proteína Forkhead Box O1/metabolismo , Humanos , Lactato Desidrogenase 5/genética , Listeria monocytogenes , Listeriose/enzimologia , Listeriose/imunologia , Camundongos , Camundongos Mutantes , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Efeito Warburg em OncologiaRESUMO
The cholinergic, purinergic and oxidative stress systems were related to nervous system damage in some pathologies, as well as being involved in pro-inflammatory and anti-inflammatory pathways. The objective was to investigate changes in purinergic, cholinergic systems and oxidative stress related to the neuropathology of listeriosis. Gerbils were used as experimental models. The animals were divided in two groups: control and infected. The animals were orally infected with 5â¯×â¯108 CFU/animal of the pathogenic strain of Listeria monocytogenes. Collected of material was 6 and 12th days post-infection (PI). Infected animals showed moderate mixed inflammatory infiltrates in the liver. The spleen and brain was used for PCR analyses, confirming infection by L. monocytogenes. Increase in number of total leukocytes because of an increase in lymphocytes in infected (Pâ¯<â¯0.001). ATP and ADP hydrolysis by NTPDase was lower at 6 and 12th days PI in infected animals than in the control group. ADA (adenosine deaminase) activity was higher on the 6th day PI (Pâ¯<â¯0.05) and decreased on the 12th day PI (Pâ¯<â¯0.05) in infected animals. AChE (acetylcholinesterase) activity did not differ between groups on the 6th day PI; however, activity decreased in infected group on the 12th day PI (Pâ¯<â¯0.05). On the 12th day PI, an increase of oxygen-reactive species levels and lower catalase and superoxide dismutase activities in the infected group was observed, characterizing a situation of cerebral oxidative stress. The inflammatory and oxidative mechanisms are present in listeriosis in asymptomatic animals, and that ectonucleotidases and cholinesterase's are involved in immunomodulation.
Assuntos
Listeria monocytogenes/patogenicidade , Listeriose/metabolismo , Listeriose/patologia , Acetilcolinesterase/metabolismo , Adenosina Desaminase/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/microbiologia , Encéfalo/patologia , Catalase/metabolismo , DNA Bacteriano/isolamento & purificação , Doenças Transmitidas por Alimentos/microbiologia , Gerbillinae , Hematócrito , Intestino Delgado/patologia , Contagem de Leucócitos , Listeria monocytogenes/genética , Listeriose/enzimologia , Listeriose/transmissão , Fígado/patologia , Nucleotidases/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/análise , Baço/microbiologia , Baço/patologia , Superóxido Dismutase/metabolismoRESUMO
Several evidences have suggested the involvement of enzymes belonging to the phosphotransfer network, formed by creatine kinase (CK), pyruvate kinase (PK) and adenylate kinase (AK), as well the oxidative stress on the pathogenesis of infectious diseases associated with the central nervous system (CNS). Thus, the aim of this study was to evaluate whether listeriosis alters the brain energy metabolism and/or causes oxidative stress in different brain structures of cattle experimentally infected by Listeria monocytogenes. The cytosolic CK activity was inhibited in the cerebral cortex, cerebellum, brainstem and hippocampus of infected animals compared to uninfected animals, while the mitochondrial CK activity was increased. The PK activity was inhibited in all brain structures of infected animals, while the AK activity was unchanged. Na+, K+-ATPase activity decreased in the cerebral cortex, cerebellum and hippocampus of animals infected by L. monocytogenes. Regarding the oxidative strees variables, the cerebellum and brainstem of infected animals showed increased thiobarbituric acid reactive substances, while the catalase activity was inhibited. Glutathione S-transferarase was inhibited in the cerebral cortex and brainstem of infected animals, and it was increased in the cerebellum. L. monocytogenes was quantified in the liver (nâ¯=â¯5/5) and cerebral cortex (nâ¯=â¯4/5) of the infected cattle. Based on these evidences, the nucleocytoplasmic communication between CK isoenzymes was insufficient to avoid an impairment of cerebral bioenergetics. Moreover, the inhibition on brain PK activity caused an impairment in the communication between sites of ATP generation and ATP utilization. The lipid peroxidation and alteration on antioxidant status observed in some brain structures were also involved during the disease. In summary, these alterations contribute to disease pathogenesis linked to CNS during cattle listeriosis.
Assuntos
Adenilato Quinase/metabolismo , Encéfalo/enzimologia , Doenças dos Bovinos/enzimologia , Creatina Quinase/metabolismo , Listeria monocytogenes/fisiologia , Listeriose/veterinária , Piruvato Quinase/metabolismo , Adenilato Quinase/genética , Animais , Antioxidantes/metabolismo , Encéfalo/metabolismo , Encéfalo/microbiologia , Bovinos , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/microbiologia , Creatina Quinase/genética , Creatina Quinase Mitocondrial/genética , Creatina Quinase Mitocondrial/metabolismo , Metabolismo Energético , Listeriose/enzimologia , Listeriose/metabolismo , Listeriose/microbiologia , Oxidantes/metabolismo , Estresse Oxidativo , Fosforilação , Piruvato Quinase/genéticaRESUMO
The oxidoreductase RECON is a high-affinity cytosolic sensor of bacterium-derived cyclic dinucleotides (CDNs). CDN binding inhibits RECON's enzymatic activity and subsequently promotes inflammation. In this study, we sought to characterize the effects of RECON on the infection cycle of the intracellular bacterium Listeria monocytogenes, which secretes cyclic di-AMP (c-di-AMP) into the cytosol of infected host cells. Here, we report that during infection of RECON-deficient hepatocytes, which exhibit hyperinflammatory responses, L. monocytogenes exhibits significantly enhanced cell-to-cell spread. Enhanced bacterial spread could not be attributed to alterations in PrfA or ActA, two virulence factors critical for intracellular motility and intercellular spread. Detailed microscopic analyses revealed that in the absence of RECON, L. monocytogenes actin tail lengths were significantly longer and there was a larger number of faster-moving bacteria. Complementation experiments demonstrated that the effects of RECON on L. monocytogenes spread and actin tail lengths were linked to its enzymatic activity. RECON enzyme activity suppresses NF-κB activation and is inhibited by c-di-AMP. Consistent with these previous findings, we found that augmented NF-κB activation in the absence of RECON caused enhanced L. monocytogenes cell-to-cell spread and that L. monocytogenes spread correlated with c-di-AMP secretion. Finally, we discovered that, remarkably, increased NF-κB-dependent inducible nitric oxide synthase expression and nitric oxide production were responsible for promoting L. monocytogenes cell-to-cell spread. The work presented here supports a model whereby L. monocytogenes secretion of c-di-AMP inhibits RECON's enzymatic activity, drives augmented NF-κB activation and nitric oxide production, and ultimately enhances intercellular spread.IMPORTANCE To date, bacterial CDNs in eukaryotes are solely appreciated for their capacity to activate cytosolic sensing pathways in innate immunity. However, it remains unclear whether pathogens that actively secrete CDNs benefit from this process. Here, we provide evidence that secretion of CDNs leads to enhancement of L. monocytogenes cell-to-cell spread. This is a heretofore-unknown role of these molecules and suggests L. monocytogenes may benefit from their secretion in certain contexts. Molecular characterization revealed that, surprisingly, nitric oxide was responsible for the enhanced spread. Pathogens act to prevent nitric oxide production or, like L. monocytogenes, they have evolved to resist its direct antimicrobial effects. This study provides evidence that intracellular bacterial pathogens not only tolerate nitric oxide, which is inevitably encountered during infection, but can also capitalize on the changes this pleiotropic molecule enacts on the host cell.
Assuntos
Estradiol Desidrogenases/imunologia , Hepatócitos/enzimologia , Listeria monocytogenes/fisiologia , Listeriose/enzimologia , Oxirredutases/metabolismo , Animais , AMP Cíclico/metabolismo , Estradiol Desidrogenases/genética , Hepatócitos/imunologia , Hepatócitos/microbiologia , Humanos , Listeria monocytogenes/genética , Listeriose/imunologia , Listeriose/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/genética , NF-kappa B/imunologia , Oxirredutases/genéticaRESUMO
IL-1 receptor-associated kinase (IRAK) 4 is a central enzyme of the TLR pathways. This study tested the hypothesis that IRAK4 kinase activity is prerequisite for regulating innate immunity during infections with intracellular bacteria. To this end, we analyzed responses of macrophages obtained from mice expressing wild-type (WT) IRAK4 or its kinase-inactive K213M mutant (IRAK4KI ) upon infection with intracellular bacteria Listeria monocytogenes or Mycobacterium smegmatis. In contrast to robust induction of cytokines by macrophages expressing kinase-sufficient IRAK4, IRAK4KI macrophages expressed decreased TNF-α, IL-6, IL-1ß, and C-C motif chemokine ligand 5 upon infection with L. monocytogenes or M. smegmatis. Bacterial infection of IRAK4KI macrophages led to attenuated activation of IRAK1, MAPKs and NF-κB, impaired induction of inducible NO synthase mRNA and secretion of NO, but resulted in elevated microbial burdens. Compared with WT animals, systemic infection of IRAK4KI mice with M. smegmatis or L. monocytogenes resulted in decreased levels of serum IL-6 and CXCL-1 but increased bacterial burdens in the spleen and liver. Thus, a loss of IRAK4 kinase activity underlies deficient cytokine and microbicidal responses during infection with intracellular bacteria L. monocytogenes or M. smegmatis via impaired activation of IRAK1, MAPKs, and NF-κB but increases bacterial burdens, correlating with decreased induction of NO.
Assuntos
Imunidade Inata , Síndromes de Imunodeficiência/imunologia , Quinases Associadas a Receptores de Interleucina-1/imunologia , Listeriose/imunologia , Macrófagos/microbiologia , Infecções por Mycobacterium não Tuberculosas/imunologia , Animais , Citocinas/metabolismo , Feminino , Síndromes de Imunodeficiência/genética , Quinases Associadas a Receptores de Interleucina-1/deficiência , Quinases Associadas a Receptores de Interleucina-1/genética , Listeria monocytogenes , Listeriose/enzimologia , Fígado/microbiologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Infecções por Mycobacterium não Tuberculosas/enzimologia , Mycobacterium smegmatis , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Doenças da Imunodeficiência Primária , Baço/microbiologiaRESUMO
The host employs both cell-autonomous and system-level responses to limit pathogen replication in the initial stages of infection. Previously, we reported that the eukaryotic initiation factor 2α (eIF2α) kinases heme-regulated inhibitor (HRI) and protein kinase R (PKR) control distinct cellular and immune-related activities in response to diverse bacterial pathogens. Specifically for Listeria monocytogenes, there was reduced translocation of the pathogen to the cytosolic compartment in HRI-deficient cells and consequently reduced loading of pathogen-derived antigens on major histocompatibility complex class I (MHC-I) complexes. Here we show that Hri-/- mice, as well as wild-type mice treated with an HRI inhibitor, are more susceptible to listeriosis. In the first few hours of L. monocytogenes infection, there was much greater pathogen proliferation in the liver of Hri-/- mice than in the liver of Hri+/+ mice. Further, there was a rapid increase of serum interleukin-6 (IL-6) levels in Hri+/+ mice in the first few hours of infection whereas the increase in IL-6 levels in Hri-/- mice was notably delayed. Consistent with these in vivo findings, the rate of listeriolysin O (LLO)-dependent pathogen efflux from infected Hri-/- macrophages and fibroblasts was significantly higher than the rate seen with infected Hri+/+ cells. Treatment of cells with an eIF2α kinase activator enhanced both the HRI-dependent and PKR-dependent infection phenotypes, further indicating the pharmacologically malleability of this signaling pathway. Collectively, these results suggest that HRI mediates the cellular confinement and killing of virulent L. monocytogenes in addition to promoting a system-level cytokine response and that both are required to limit pathogen replication during the first few hours of infection.
Assuntos
Listeria monocytogenes/fisiologia , Listeriose/enzimologia , Proteínas Serina-Treonina Quinases/imunologia , Animais , Feminino , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Listeria monocytogenes/genética , Listeriose/genética , Listeriose/imunologia , Listeriose/microbiologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Recent evidence on HDAC6 function underlines its role as a key protein in the innate immune response to viral infection. However, whether HDAC6 regulates innate immunity during bacterial infection remains unexplored. To assess the role of HDAC6 in the regulation of defence mechanisms against intracellular bacteria, we used the Listeria monocytogenes (Lm) infection model. Our data show that Hdac6-/- bone marrow-derived dendritic cells (BMDCs) have a higher bacterial load than Hdac6+/+ cells, correlating with weaker induction of IFN-related genes, pro-inflammatory cytokines and nitrite production after bacterial infection. Hdac6-/- BMDCs have a weakened phosphorylation of MAPK signalling in response to Lm infection, suggesting altered Toll-like receptor signalling (TLR). Compared with Hdac6+/+ counterparts, Hdac6-/- GM-CSF-derived and FLT3L-derived dendritic cells show weaker pro-inflammatory cytokine secretion in response to various TLR agonists. Moreover, HDAC6 associates with the TLR-adaptor molecule Myeloid differentiation primary response gene 88 (MyD88), and the absence of HDAC6 seems to diminish the NF-κB induction after TLR stimuli. Hdac6-/- mice display low serum levels of inflammatory cytokine IL-6 and correspondingly an increased survival to a systemic infection with Lm. The impaired bacterial clearance in the absence of HDAC6 appears to be caused by a defect in autophagy. Hence, Hdac6-/- BMDCs accumulate higher levels of the autophagy marker p62 and show defective phagosome-lysosome fusion. These data underline the important function of HDAC6 in dendritic cells not only in bacterial autophagy, but also in the proper activation of TLR signalling. These results thus demonstrate an important regulatory role for HDAC6 in the innate immune response to intracellular bacterial infection.
Assuntos
Autofagia/imunologia , Desacetilase 6 de Histona/imunologia , Imunidade Inata , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Receptores Toll-Like/imunologia , Animais , Células Dendríticas/imunologia , Feminino , Desacetilase 6 de Histona/deficiência , Desacetilase 6 de Histona/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucina-6/sangue , Listeriose/enzimologia , Listeriose/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/imunologia , Transdução de Sinais/imunologiaRESUMO
PURPOSE: Listeria monocytogenes (Lm) can cross the intestinal barrier in humans and then disseminates into different organs. Invasion of the liver occurs even in sublethal infections, however, knowledge of affected physiological processes is scarce. This study employed a sublethal murine infection model to investigate liver responses systematically by proteomics. EXPERIMENTAL DESIGN: Liver samples from three stages of the sublethal infection covering the initial invasion, the peak of infection, and the clearance phase (1, 3, 9 days postinoculation) were analyzed in comparison to samples from noninfected mice. Apart from flow cytometry and RT-PCRs for immune status control, liver responses were analyzed by quantitative peptide sequencing (HPLC-Orbitrap Fusion) using 4-plex iTRAQ-labeling. RESULTS: Accurate MS characterized about 3600 proteins and statistics revealed 15% of the hepatic proteome as regulated. Immunological data as well as protein regulation dynamics strongly indicate stage-specific hepatic responses in sublethal infections. Most notably, this study detected a comprehensive deregulation of drug metabolizing enzymes at all stages, including 25 components of the cytochrome P450 system. CONCLUSIONS AND CLINICAL RELEVANCE: Sublethal Lm infection deregulates hepatic drug metabolizing pathways. This finding indicates the need to monitor drug administration along Lm infections, especially in all patients needing constant medication.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Regulação para Baixo , Listeriose/enzimologia , Fígado/enzimologia , Sequência de Aminoácidos , Animais , Biotransformação , Feminino , Listeriose/imunologia , Listeriose/metabolismo , Fígado/imunologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , ProteômicaRESUMO
Listeria monocytogenes is a bacterial pathogen which establishes intracellular parasitism in various cells, including macrophages and nonhematopoietic cells, such as hepatocytes. It has been reported that several proinflammatory cytokines have pivotal roles in innate protection against L. monocytogenes infection. We found that a proinflammatory cytokine, interleukin 22 (IL-22), was expressed by CD3(+) CD4(+) T cells at an early stage of L. monocytogenes infection in mice. To assess the influence of IL-22 on L. monocytogenes infection in hepatocytes, cells of a human hepatocellular carcinoma line, HepG2, were treated with IL-22 before L. monocytogenes infection in vitro. Gene expression analysis of the IL-22-treated HepG2 cells identified phospholipase A2 group IIA (PLA2G2A) as an upregulated antimicrobial molecule. Addition of recombinant PLA2G2A to the HepG2 culture significantly suppressed L. monocytogenes infection. Culture supernatant of the IL-22-treated HepG2 cells contained bactericidal activity against L. monocytogenes, and the activity was abrogated by a specific PLA2G2A inhibitor, demonstrating that HepG2 cells secreted PLA2G2A, which killed extracellular L. monocytogenes. Furthermore, colocalization of PLA2G2A and L. monocytogenes was detected in the IL-22-treated infected HepG2 cells, which suggests involvement of PLA2G2A in the mechanism of intracellular killing of L. monocytogenes by HepG2 cells. These results suggest that IL-22 induced at an early stage of L. monocytogenes infection enhances innate immunity against L. monocytogenes in the liver by stimulating hepatocytes to produce an antimicrobial molecule, PLA2G2A.
Assuntos
Fosfolipases A2 do Grupo II/metabolismo , Hepatócitos/enzimologia , Imunidade Inata , Interleucinas/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Fígado/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Fosfolipases A2 do Grupo II/genética , Células Hep G2 , Hepatócitos/imunologia , Hepatócitos/microbiologia , Humanos , Interleucinas/genética , Listeria monocytogenes/efeitos dos fármacos , Listeriose/enzimologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Interleucina 22RESUMO
Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2) regulates fatty acid oxidation (FAO) by inhibiting carnitine palmitoyltransferase 1 (CPT1), a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses.
Assuntos
Acetil-CoA Carboxilase/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunidade Celular , Mitocôndrias/imunologia , Acetil-CoA Carboxilase/genética , Animais , Linfócitos T CD8-Positivos/enzimologia , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/imunologia , Carnitina O-Palmitoiltransferase/metabolismo , Ácidos Graxos/genética , Ácidos Graxos/imunologia , Ácidos Graxos/metabolismo , Listeria monocytogenes/imunologia , Listeriose/enzimologia , Listeriose/genética , Listeriose/imunologia , Coriomeningite Linfocítica/enzimologia , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Transgênicos , Mitocôndrias/enzimologia , Mitocôndrias/genéticaRESUMO
The deubiquitinating enzyme CYLD is an important tumor suppressor and inhibitor of immune responses. In contrast to full-length CYLD, the immunological function of the naturally occurring short splice variant of CYLD (sCYLD) is insufficiently described. Previously, we showed that DCs, which lack full-length CYLD but express sCYLD, exhibit augmented NF-κB and DC activation. To explore the function of sCYLD in infection, we investigated whether DC-specific sCYLD regulates the pathogenesis of listeriosis. Upon Listeria monocytogenes infection of CD11c-Cre Cyld(ex7/8 fl/fl) mice, infection of CD8α(+) DCs, which are crucial for the establishment of listeriosis in the spleen, was not affected. However, NF-κB activity of CD11c-Cre Cyld(ex7/8 fl/fl) DCs was increased, while activation of ERK and p38 was normal. In addition, CD11c-Cre Cyld(ex7/8 fl/fl) DCs produced more TNF, IL-10, and IL-12 upon infection, which led to enhanced stimulation of IFN-γ-producing NK cells. In addition CD11c-Cre Cyld(ex7/8 fl/fl) DCs presented Listeria Ag more efficiently to CD8(+) T cells resulting in a stronger pathogen-specific CD8(+) T-cell proliferation and more IFN-γ production. Collectively, the improved innate and adaptive immunity and survival during listeriosis identify the DC-specific FL-CYLD/sCYLD balance as a potential target to modulate NK-cell and Ag-specific CD8(+) T-cell responses.
Assuntos
Cisteína Endopeptidases/imunologia , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Listeriose/enzimologia , Listeriose/imunologia , Animais , Apresentação de Antígeno , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/imunologia , Cisteína Endopeptidases/genética , Citocinas/biossíntese , Células Dendríticas/metabolismo , Enzima Desubiquitinante CYLD , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Isoenzimas/genética , Isoenzimas/imunologia , Células Matadoras Naturais/imunologia , Leucócitos/imunologia , Leucócitos/patologia , Listeria monocytogenes/imunologia , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/metabolismo , Baço/imunologia , Baço/patologia , Regulação para CimaRESUMO
During an immune response against a microbial pathogen, activated naive T lymphocytes give rise to effector cells that provide acute host defense and memory cells that provide long-lived immunity. It has been shown that T lymphocytes can undergo asymmetric division, enabling the daughter cells to inherit unequal amounts of fate-determining proteins and thereby acquire distinct fates from their inception. In this study, we show that the absence of the atypical protein kinase C (PKC) isoforms, PKCζ and PKCλ/ι, disrupts asymmetric CD8(+) T lymphocyte division. These alterations were associated with aberrant acquisition of a pre-effector transcriptional program, detected by single-cell gene expression analyses, in lymphocytes that had undergone their first division in vivo and enhanced differentiation toward effector fates at the expense of memory fates. Together, these results demonstrate a role for atypical PKC in regulating asymmetric division and the specification of divergent CD8(+) T lymphocyte fates early during an immune response.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Divisão Celular/imunologia , Imunidade Inata , Isoenzimas/imunologia , Listeriose/imunologia , Proteína Quinase C/imunologia , Animais , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/microbiologia , Linfócitos T CD8-Positivos/patologia , Diferenciação Celular , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Células Dendríticas/patologia , Regulação da Expressão Gênica , Memória Imunológica , Isoenzimas/genética , Isoenzimas/metabolismo , Listeria monocytogenes/imunologia , Listeriose/enzimologia , Listeriose/microbiologia , Listeriose/patologia , Camundongos , Camundongos Knockout , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Transdução de Sinais , Análise de Célula Única , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/microbiologia , Subpopulações de Linfócitos T/patologiaRESUMO
Bacterial pathogens often interfere with host tyrosine phosphorylation cascades to control host responses and cause infection. Given the role of tyrosine phosphorylation events in different human infections and our previous results showing the activation of the tyrosine kinase Src upon incubation of cells with Listeria monocytogenes, we searched for novel host proteins undergoing tyrosine phosphorylation upon L. monocytogenes infection. We identify the heavy chain of the non-muscle myosin IIA (NMHC-IIA) as being phosphorylated in a specific tyrosine residue in response to L. monocytogenes infection. We characterize this novel post-translational modification event and show that, upon L. monocytogenes infection, Src phosphorylates NMHC-IIA in a previously uncharacterized tyrosine residue (Tyr-158) located in its motor domain near the ATP-binding site. In addition, we found that other intracellular and extracellular bacterial pathogens trigger NMHC-IIA tyrosine phosphorylation. We demonstrate that NMHC-IIA limits intracellular levels of L. monocytogenes, and this is dependent on the phosphorylation of Tyr-158. Our data suggest a novel mechanism of regulation of NMHC-IIA activity relying on the phosphorylation of Tyr-158 by Src.
Assuntos
Listeria monocytogenes/fisiologia , Listeriose/enzimologia , Miosina não Muscular Tipo IIA/metabolismo , Processamento de Proteína Pós-Traducional , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Carga Bacteriana , Células CACO-2 , Ativação Enzimática , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Listeriose/microbiologia , FosforilaçãoRESUMO
The bacterial pathogen Listeria monocytogenes induces internalization into mammalian cells and uses actin-based motility to spread within tissues. Listeria accomplishes this intracellular life cycle by exploiting or antagonizing several host GTPases. Internalization into human cells is mediated by the bacterial surface proteins InlA or InlB. These two modes of uptake each require a host actin polymerization pathway comprised of the GTPase Rac1, nucleation promotion factors, and the Arp2/3 complex. In addition to Rac1, InlB-mediated internalization involves inhibition of the GTPase Arf6 and participation of Dynamin and septin family GTPases. After uptake, Listeria is encased in host phagosomes. The bacterial protein GAPDH inactivates the human GTPase Rab5, thereby delaying phagosomal acquisition of antimicrobial properties. After bacterial-induced destruction of the phagosome, cytosolic Listeria uses the surface protein ActA to stimulate actin-based motility. The GTPase Dynamin 2 reduces the density of microtubules that would otherwise limit bacterial movement. Cell-to-cell spread results when motile Listeria remodel the host plasma membrane into protrusions that are engulfed by neighbouring cells. The human GTPase Cdc42, its activator Tuba, and its effector N-WASP form a complex with the potential to restrict Listeria protrusions. Bacteria overcome this restriction through two microbial factors that inhibit Cdc42-GTP or Tuba/N-WASP interaction.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Listeria monocytogenes/fisiologia , Listeriose/enzimologia , Animais , Humanos , Listeriose/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Granzymes are generally recognized for their capacity to induce various pathways of perforin-dependent target cell death. Within this serine protease family, Granzyme M (GrzM) is unique owing to its preferential expression in innate effectors such as natural killer (NK) cells. During Listeria monocytogenes infection, we observed markedly reduced secretion of macrophage inflammatory protein-1 alpha (MIP-1α) in livers of GrzM-deficient mice, which resulted in significantly impaired NK cell recruitment. Direct stimulation with IL-12 and IL-15 demonstrated that GrzM was required for maximal secretion of active MIP-1α. This effect was not due to reduced protein induction but resulted from heightened intracellular accumulation of MIP-1α, with reduced release. These results demonstrate that GrzM is a critical mediator of innate immunity that can regulate chemotactic networks and has an important role in the initiation of immune responses and pathogen control.
Assuntos
Quimiocina CCL3/metabolismo , Granzimas/metabolismo , Imunidade Inata , Células Matadoras Naturais/enzimologia , Listeriose/enzimologia , Animais , Células Cultivadas , Quimiotaxia de Leucócito , Técnicas de Cocultura , Modelos Animais de Doenças , Granzimas/deficiência , Granzimas/genética , Humanos , Interleucinas/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/microbiologia , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Listeriose/genética , Listeriose/imunologia , Listeriose/microbiologia , Camundongos , Camundongos Knockout , Fatores de TempoRESUMO
The bacterial pathogen Listeria monocytogenes uses actin-based motility to spread from infected human cells to surrounding healthy cells. Cell-cell spread involves the formation of thin extensions of the host plasma membrane ('protrusions') containing motile bacteria. In cultured enterocytes, the Listeria protein InlC promotes protrusion formation by binding and antagonizing the human scaffolding protein Tuba. Tuba is a known activator of the GTPase Cdc42. In this work, we demonstrate an important role for Cdc42 in controlling Listeria spread. Infection of the enterocyte cell line Caco-2 BBE1 induced a decrease in the level of Cdc42-GTP, indicating that Listeria downregulates this GTPase. Genetic data involving RNA interference indicated that bacterial impairment of Cdc42 may involve inhibition of Tuba. Experiments with dominant negative and constitutively activated alleles of Cdc42 demonstrated that the ability to inactivate Cdc42 is required for efficient protrusion formation by Listeria. Taken together, these findings indicate a novel mechanism of bacterial spread involving pathogen-induced downregulation of host Cdc42.
Assuntos
Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Células CACO-2 , Extensões da Superfície Celular/metabolismo , Regulação para Baixo , Repressão Enzimática , Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Junções Intercelulares/enzimologia , Junções Intercelulares/ultraestrutura , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/ultraestrutura , Listeriose/enzimologia , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
In this study we investigated the role of Bruton's tyrosine kinase (Btk) in the immune response to the Gram-positive intracellular bacterium Listeria monocytogenes (Lm). In response to Lm infection, Btk was activated in bone marrow-derived macrophages (BMMs) and Btk (-/-) BMMs showed enhanced TNF-α, IL-6 and IL-12p40 secretion, while type I interferons were produced at levels similar to wild-type (wt) BMMs. Although Btk-deficient BMMs displayed reduced phagocytosis of E. coli fragments, there was no difference between wt and Btk (-/-) BMMs in the uptake of Lm upon infection. Moreover, there was no difference in the response to heat-killed Lm between wt and Btk (-/-) BMMs, suggesting a role for Btk in signaling pathways that are induced by intracellular Lm. Finally, Btk (-/-) mice displayed enhanced resistance and an increased mean survival time upon Lm infection in comparison to wt mice. This correlated with elevated IFN-γ and IL-12p70 serum levels in Btk (-/-) mice at day 1 after infection. Taken together, our data suggest an important regulatory role for Btk in macrophages during Lm infection.
Assuntos
Listeria monocytogenes/fisiologia , Listeriose/enzimologia , Listeriose/microbiologia , Macrófagos/enzimologia , Macrófagos/microbiologia , Proteínas Tirosina Quinases/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Células da Medula Óssea/patologia , Citocinas/biossíntese , Suscetibilidade a Doenças , Ativação Enzimática/efeitos dos fármacos , Immunoblotting , Lipopeptídeos/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Listeriose/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Fagocitose/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Fagossomos/microbiologia , Proteínas Tirosina Quinases/deficiênciaRESUMO
Activation of the transcription factor NF-κB is critical for cytokine production and T cell survival after TCR engagement. The effects of persistent NF-κB activity on T cell function and survival are poorly understood. In this study, using a murine model that expresses a constitutively active form of inhibitor of NF-κB kinase ß (caIKKß) in a T cell-specific manner, we demonstrate that chronic inhibitor of NF-κB kinase ß signaling promotes T cell apoptosis, attenuates responsiveness to TCR-mediated stimulation in vitro, and impairs T cell responses to bacterial infection in vivo. caIKKß T cells showed increased Fas ligand expression and caspase-8 activation, and blocking Fas/Fas ligand interactions enhanced cell survival. T cell unresponsiveness was associated with defects in TCR proximal signaling and elevated levels of B lymphocyte-induced maturation protein 1, a transcriptional repressor that promotes T cell exhaustion. caIKKß T cells also showed a defect in IL-2 production, and addition of exogenous IL-2 enhanced their survival and proliferation. Conditional deletion of B lymphocyte-induced maturation protein 1 partially rescued the sensitivity of caIKKß T cells to TCR triggering. Furthermore, adoptively transferred caIKKß T cells showed diminished expansion and increased contraction in response to infection with Listeria monocytogenes expressing a cognate Ag. Despite their functional defects, caIKKß T cells readily produced proinflammatory cytokines, and mice developed autoimmunity. In contrast to NF-κB's critical role in T cell activation and survival, our study demonstrates that persistent IKK-NF-κB signaling is sufficient to impair both T cell function and survival.