RESUMO
Staphylococcus aureus (S. aureus) is a common pathogen involved in community- and hospital-acquired infections. Its biofilm formation ability predisposes it to device-related infections. Methicillin-resistant S. aureus (MRSA) strains are associated with more serious infections and higher mortality rates and are more complex in terms of antibiotic resistance. It is still controversial whether MRSA are indeed more virulent than methicillin-susceptible S. aureus (MSSA) strains. A difference in biofilm formation by both types of bacteria has been suggested, but how only the presence of the SCCmec cassette or mecA influences this phenotype remains unclear. In this review, we have searched for literature studying the difference in biofilm formation by MRSA and MSSA. We highlighted the relevance of the icaADBC operon in the PIA-dependent biofilms generated by MSSA under osmotic stress conditions, and the role of extracellular DNA and surface proteins in the PIA-independent biofilms generated by MRSA. We described the prominent role of surface proteins with the LPXTG motif and hydrolases for the release of extracellular DNA in the MRSA biofilm formation. Finally, we explained the main regulatory systems in S. aureus involved in virulence and biofilm formation, such as the SarA and Agr systems. As most of the studies were in vitro using inert surfaces, it will be necessary in the future to focus on biofilm formation on extracellular matrix components and its relevance in the pathogenesis of infection by both types of strains using in vivo animal models.
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BACKGROUND: Aspiration pneumonia (AP), which is a major cause of death in the elderly, does present with typical symptoms in the early stages of onset, thus it is difficult to detect and treat at an early stage. In this study, we identified biomarkers that are useful for the detection of AP and focused on salivary proteins, which may be collected non-invasively. Because expectorating saliva is often difficult for elderly people, we collected salivary proteins from the buccal mucosa. METHODS: We collected samples from the buccal mucosa of six patients with AP and six control patients (no AP) in an acute-care hospital. Following protein precipitation using trichloroacetic acid and washing with acetone, the samples were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS). We also determined the levels of cytokines and chemokines in non-precipitated samples from buccal mucosa. RESULTS: Comparative quantitative analysis of LC-MS/MS spectra revealed 55 highly (P values < 0.10) abundant proteins with high FDR confidence (q values < 0.01) and high coverage (> 50%) in the AP group compared with the control group. Among the 55 proteins, the protein abundances of four proteins (protein S100-A7A, eukaryotic translation initiation factor 1, Serpin B4, and peptidoglycan recognition protein 1) in the AP group showed a negative correlation with the time post-onset; these proteins are promising AP biomarker candidates. In addition, the abundance of C-reactive protein (CRP) in oral samples was highly correlated with serum CRP levels, suggesting that oral CRP levels may be used as a surrogate to predict serum CRP in AP patients. A multiplex cytokine/chemokine assay revealed that MCP-1 tended to be low, indicating unresponsiveness of MCP-1 and its downstream immune pathways in AP. CONCLUSION: Our findings suggest that oral salivary proteins, which are obtained non-invasively, can be utilized for the detection of AP.
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Hepatocellular death increases with hepatic steatosis aggravation, although its regulation remains unclear. Here we show that hepatic steatosis aggravation shifts the hepatocellular death mode from apoptosis to necroptosis, causing increased hepatocellular death. Our results reveal that the transcription factor ATF3 acts as a master regulator in this shift by inducing expression of RIPK3, a regulator of necroptosis. In severe hepatic steatosis, after partial hepatectomy, hepatic ATF3-deficient or -overexpressing mice display decreased or increased RIPK3 expression and necroptosis, respectively. In cultured hepatocytes, ATF3 changes TNFα-dependent cell death mode from apoptosis to necroptosis, as revealed by live-cell imaging. In non-alcoholic steatohepatitis (NASH) mice, hepatic ATF3 deficiency suppresses RIPK3 expression and hepatocellular death. In human NASH, hepatocellular damage is correlated with the frequency of hepatocytes expressing ATF3 or RIPK3, which overlap frequently. ATF3-dependent RIPK3 induction, causing a modal shift of hepatocellular death, can be a therapeutic target for steatosis-induced liver damage, including NASH.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Camundongos , Masculino , Humanos , Animais , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fatores de Transcrição/metabolismo , Necroptose , Apoptose , Hepatócitos/metabolismo , Morte Celular , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator 3 Ativador da Transcrição/metabolismoRESUMO
Inflammasome activation exacerbates infectious disease caused by pathogens such as Listeria monocytogenes, Staphylococcus aureus, and severe acute respiratory syndrome coronavirus 2. Although these pathogens activate host inflammasomes to regulate pathogen expansion, the mechanisms by which pathogen toxins contribute to inflammasome activation remain poorly understood. Here we show that activation of inflammasomes by Listeria infection is promoted by amino acid residue T223 of listeriolysin O (LLO) independently of its pore-forming activity. LLO T223 is critical for phosphorylation of the inflammasome adaptor ASC at amino acid residue Y144 through Lyn-Syk signaling, which is essential for ASC oligomerization. Notably, a Listeria mutant expressing LLO T223A is impaired in inducing ASC phosphorylation and inflammasome activation. Furthermore, the virulence of LLO T223A mutant is markedly attenuated in vivo due to impaired ability to activate the inflammasome. Our results reveal a function of a pathogen toxin that exacerbates infection by promoting phosphorylation of ASC.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Inflamassomos/metabolismo , Listeria monocytogenes/patogenicidade , Transdução de Sinais , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas Adaptadoras de Sinalização CARD/química , Proteínas Adaptadoras de Sinalização CARD/deficiência , Proteínas Adaptadoras de Sinalização CARD/genética , Edição de Genes , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Interleucina-18/metabolismo , Listeria monocytogenes/metabolismo , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Fosforilação , Quinase Syk/genética , Quinase Syk/metabolismo , Virulência , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Listeria monocytogenes (LM) infection induces pyroptosis, a form of regulated necrosis, in host macrophages via inflammasome activation. Here, we examined the role of Mint3 in macrophages, which promotes glycolysis via hypoxia-inducible factor-1 activation, during the initiation of pyroptosis following LM infection. Our results showed that Mint3-deficient mice were more resistant to lethal listeriosis than wild-type (WT) mice. Additionally, the mutant mice showed higher levels of IL-1ß/IL-18 in the peritoneal fluid during LM infection than WT mice. Moreover, ablation of Mint3 markedly increased the activation of caspase-1, maturation of gasdermin D, and pyroptosis in macrophages infected with LM in vitro, suggesting that Mint3 depletion promotes pyroptosis. Further analyses revealed that Mint3 depletion upregulates inflammasome assembly preceding pyroptosis via glycolysis reduction and reactive oxygen species production. Pharmacological inhibition of glycolysis conferred resistance to listeriosis in a Mint3-dependent manner. Moreover, Mint3-deficient mice treated with the caspase-1 inhibitor VX-765 were as susceptible to LM infection as WT mice. Taken together, these results suggest that Mint3 depletion promotes pyroptosis in host macrophages, thereby preventing the spread of LM infection. Mint3 may serve as a target for treating severe listeriosis by inducing pyroptosis in LM-infected macrophages.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Listeria monocytogenes/metabolismo , Macrófagos/metabolismo , Estresse Oxidativo/fisiologia , Piroptose/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/fisiologia , Glicólise/fisiologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismoRESUMO
IL-1α serves as a pro-inflammatory cytokine. Although pro-IL-1α has cytokine activity, proteolytic maturation increases its potency and release from cells. IL-1α maturation occurs in a caspase-1-dependent manner following inflammasome activation. However, pro-IL-1α is not a substrate of caspase-1, and it remains unclear what mediates the maturation of this cytokine downstream of inflammasomes. Here, we show that gasdermin D (GSDMD), an executor of pyroptosis, is required for the rapid induction of IL-1α maturation by non-particulate inflammasome activators. Ablation of GSDMD abrogates the maturation of IL-1α, but not of IL-1ß. Inflammasome-induced maturation of IL-1α relies on extracellular Ca2+ and calpains. Ca2+ influx and calpain activation are induced in a GSDMD-dependent manner. Glycine, which inhibits cell lysis, but not GSDMD pore formation, does not affect IL-1α maturation. These results suggest that during inflammasome activation, GSDMD processed by caspase-1 forms plasma membrane pores that mediate Ca2+ influx, resulting in the calpain-dependent maturation of IL-1α.
Assuntos
Inflamassomos/metabolismo , Interleucina-1alfa/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteólise , Animais , Cálcio/metabolismo , Calpaína/metabolismo , Caspase 1/metabolismo , Forma Celular , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Fosfato/deficiência , Células RAW 264.7 , Células THP-1RESUMO
Pyroptosis is a necrotic form of regulated cell death. Gasdermines (GSDMs) are a family of intracellular proteins that execute pyroptosis. While GSDMs are expressed as inactive forms, certain proteases proteolytically activate them. The N-terminal fragments of GSDMs form pores in the plasma membrane, leading to osmotic cell lysis. Pyroptotic cells release pro-inflammatory molecules into the extracellular milieu, thereby eliciting inflammation and immune responses. Recent studies have significantly advanced our knowledge of the mechanisms and physiological roles of pyroptosis. GSDMs are activated by caspases and granzymes, most of which can also induce apoptosis in different situations, for example where the expression of GSDMs is too low to cause pyroptosis; that is, caspase/granzyme-induced apoptosis can be switched to pyroptosis by the expression of GSDMs. Pyroptosis appears to facilitate the killing of tumor cells by cytotoxic lymphocytes, and it may also reprogram the tumor microenvironment to an immunostimulatory state. Understanding pyroptosis may help the development of cancer immunotherapy. In this review article, recent findings on the mechanisms and roles of pyroptosis are introduced. The effectiveness and limitations of pyroptosis in inducing antitumor immunity are also discussed.
Assuntos
Apoptose , Inflamação/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias/patologia , Piroptose , Animais , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismoRESUMO
Interleukin-1ß (IL-1ß) plays pivotal roles in controlling bacterial infections and is produced after the processing of pro-IL-1ß by caspase-1, which is activated by the inflammasome. In addition, caspase-1 cleaves the cytosolic protein, gasdermin-D (GSDMD), whose N-terminal fragment subsequently forms a pore in the plasma membrane, leading to the pyroptic cell-death-mediated release of IL-1ß. Living cells can also release IL-1ß via GSDMD pores or other unconventional secretory pathways. However, the precise mechanisms are poorly defined. Here, we show that lipoproteins from Mycoplasma salivarium (MsLP) and Mycoplasma pneumoniae (MpLP) and an M. salivarium-derived lipopeptide (FSL-1), which are activators of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, induce IL-1ß release from mouse bone-marrow-derived macrophages (BMMs) without inducing cell death. The levels of IL-1ß release induced by MsLP, MpLP and FSL-1 were more than 100 times lower than those induced by the canonical NLRP3 activator nigericin. The IL-1ß release-inducing activities of MsLP, MpLP and FSL-1 were not attenuated in BMMs from GSDMD-deficient mice. Furthermore, both active caspase-1 and cleaved GSDMD were detected in response to transfection of FSL-1 into the cytosol of BMMs, but the release of IL-1ß was unaffected by GSDMD deficiency. Meanwhile, punicalagin, a membrane-stabilizing agent, drastically down-regulated the release of IL-1ß in response to FSL-1. These results suggest that mycoplasmal lipoprotein/lipopeptide-induced IL-1ß release by living macrophages is not mediated via GSDMD but rather through changes in membrane permeability.
Assuntos
Proteínas de Bactérias/metabolismo , Interleucina-1beta/metabolismo , Lipoproteínas/metabolismo , Macrófagos/imunologia , Infecções por Mycoplasma/imunologia , Mycoplasma pneumoniae/metabolismo , Mycoplasma salivarium/metabolismo , Proteínas de Neoplasias/metabolismo , Peptídeos/metabolismo , Animais , Permeabilidade da Membrana Celular , Células Cultivadas , Taninos Hidrolisáveis/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Ligação a FosfatoRESUMO
Inflammasomes are innate immune mechanisms that promote inflammation by activating the protease caspase-1. Active caspase-1 induces pyroptosis, a necrotic form of regulated cell death, which facilitates the release of intracellular proinflammatory molecules, including IL-1 family cytokines. Recent studies identified mediators of inflammasome-associated cell death and suggested that inflammasomes induce not only pyroptosis, but also apoptosis. Caspase-1 has the potential to induce pyroptosis and apoptosis in a manner that is dependent on the expression of the pyroptosis mediator gasdermin D. Caspase-1-induced apoptosis is mediated by Bid and caspase-7. Caspase-8 is also activated following the formation of inflammasomes and may induce apoptosis. Because inflammasomes contribute to the pathogenesis of inflammatory disorders and host defenses against microbial pathogens, a more detailed understanding of the mechanisms underlying inflammasome-associated cell death may contribute to the development of novel therapeutic strategies for inflammasome-related diseases. Pyroptosis has been implicated in inflammasome-related diseases, and compounds that inhibit this process have been reported. The molecular mechanisms of inflammasome-associated cell death and its physiological implications are discussed herein.
Assuntos
Caspase 1/imunologia , Inflamassomos/imunologia , Inflamação/imunologia , Piroptose , Animais , Apoptose , Linhagem Celular , Humanos , CamundongosRESUMO
Inflammasomes are innate immune mechanisms that activate caspase-1 in response to a variety of stimuli, including Salmonella infection. Active caspase-1 has a potential to induce two different types of cell death, depending on the expression of the pyroptosis mediator gasdermin D (GSDMD); following caspase-1 activation, GSDMD-sufficient and GSDMD-null/low cells undergo pyroptosis and apoptosis, respectively. Although Bid, a caspase-1 substrate, plays a critical role in caspase-1 induction of apoptosis in GSDMD-null/low cells, an additional mechanism that mediates this cell death independently of Bid has also been suggested. This study investigated the Bid-independent pathway of caspase-1-induced apoptosis. Caspase-1 has been reported to process caspase-6 and caspase-7. Silencing of caspase-7, but not caspase-6, significantly reduced the activation of caspase-3 induced by caspase-1, which was activated by chemical dimerization, in GSDMD/Bid-deficient cells. CRISPR/Cas9-mediated depletion of caspase-7 had the same effect on the caspase-3 activation. Moreover, in the absence of GSDMD and Bid, caspase-7 depletion reduced apoptosis induced by caspase-1 activation. Caspase-7 was activated following caspase-1 activation independently of caspase-3, suggesting that caspase-7 acts downstream of caspase-1 and upstream of caspase-3. Salmonella induced the activation of caspase-3 in GSDMD-deficient macrophages, which relied partly on Bid and largely on caspase-1. The caspase-3 activation and apoptotic morphological changes seen in Salmonella-infected GSDMD/Bid-deficient macrophages were attenuated by caspase-7 knockdown. These results suggest that in addition to Bid, caspase-7 can also mediate caspase-1-induced apoptosis and provide mechanistic insights into inflammasome-associated cell death that is one major effector mechanism of inflammasomes.
Assuntos
Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 1/metabolismo , Caspase 7/metabolismo , Inflamassomos/metabolismo , Animais , Apoptose/fisiologia , Caspase 3/metabolismo , Linhagem Celular , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Fosfato/metabolismo , Piroptose/fisiologiaRESUMO
It is widely accepted that inflammasomes protect the host from microbial pathogens by inducing inflammatory responses through caspase-1 activation. Here, we show that the inflammasome components ASC and NLRP3 are required for resistance to pneumococcal pneumonia, whereas caspase-1 and caspase-11 are dispensable. In the lung of S. pneumoniae-infected mice, ASC and NLRP3, but not caspase-1/11, were required for optimal expression of several mucosal innate immune proteins. Among them, TFF2 and intelectin-1 appeared to be protective against pneumococcal pneumonia. During infection, ASC and NLRP3 maintained the expression of the transcription factor SPDEF, which can facilitate the expression of the mucosal defense factor genes. Moreover, activation of STAT6, a key regulator of Spdef expression, depended on ASC and NLRP3. Overexpression of these inflammasome proteins sustained STAT6 phosphorylation induced by type 2 cytokines. Collectively, this study suggests that ASC and NLRP3 promote airway mucosal innate immunity by an inflammasome-independent mechanism involving the STAT6-SPDEF pathway.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Homeostase , Imunidade Inata , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Caspases Iniciadoras/metabolismo , Citocinas/metabolismo , Citometria de Fluxo , Genes Reporter , Imunidade nas Mucosas , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Infiltração de Neutrófilos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Streptococcus pneumoniae/imunologiaRESUMO
Caspase-1 activated in inflammasomes triggers a programmed necrosis called pyroptosis, which is mediated by gasdermin D (GSDMD). However, GSDMD-deficient cells are still susceptible to caspase-1-mediated cell death. Therefore, here, we investigate the mechanism of caspase-1-initiated cell death in GSDMD-deficient cells. Inflammasome stimuli induce apoptosis accompanied by caspase-3 activation in GSDMD-deficient macrophages, which largely relies on caspase-1. Chemical dimerization of caspase-1 induces pyroptosis in GSDMD-sufficient cells, but apoptosis in GSDMD-deficient cells. Caspase-1-induced apoptosis involves the Bid-caspase-9-caspase-3 axis, which can be followed by GSDME-dependent secondary necrosis/pyroptosis. However, Bid ablation does not completely abolish the cell death, suggesting the existence of an additional mechanism. Furthermore, cortical neurons and mast cells exhibit little or low GSDMD expression and undergo apoptosis after oxygen glucose deprivation and nigericin stimulation, respectively, in a caspase-1- and Bid-dependent manner. This study clarifies the molecular mechanism and biological roles of caspase-1-induced apoptosis in GSDMD-low/null cell types.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Caspase 1/fisiologia , Inflamassomos/imunologia , Piroptose/imunologia , Receptores de Estrogênio/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Córtex Cerebral/citologia , Embrião de Mamíferos , Técnicas de Inativação de Genes , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Nigericina/farmacologia , Proteínas de Ligação a Fosfato , Cultura Primária de Células , Piroptose/efeitos dos fármacos , Células RAW 264.7 , Salmonella typhimurium/imunologiaRESUMO
PYNOD (also called NLRP10) is a member of the nucleotide-binding domain and leucine-rich repeat containing family. Many members of this family play important roles in the activation and/or regulation of immune and inflammatory responses. We previously showed that PYNOD inhibits the IL-1ß secretion in response to microbial infection in PYNOD-transgenic mice. In this study, we generated PYNOD-knockout (KO) mice and further investigated PYNOD's role in the innate and adaptive immune responses. Similar to wild-type macrophages, PYNOD-KO macrophages produced IL-1ß and induced pyroptosis, a caspase-1-dependent programmed cell death, in response to various inflammasome activators and microbial infection. In addition, the PYNOD deficiency did not significantly affect the proliferation or cytokine production of T cells, the delayed-type hypersensitivity responses, the anti-tumor immunity, the Ag-specific Ab production, the cytotoxicity of NK cells, or the maturation, Ag-presenting capacity, or elicited migration of dendritic cells. Furthermore, the steady-state skin self-antigen transport to regional lymph nodes was not impaired in PYNOD-KO mice, suggesting that PYNOD is dispensable for steady-state dendritic cell migration. These results suggested that PYNOD is dispensable for the regulation of innate and adaptive immune responses in mice, unless PYNOD's expression is highly induced under certain conditions.
Assuntos
Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/imunologia , Imunidade Adaptativa , Proteínas Adaptadoras de Transdução de Sinal , Animais , Formação de Anticorpos/imunologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Dermatite de Contato/imunologia , Feminino , Hipersensibilidade Tardia/imunologia , Imunidade Inata , Infecções/imunologia , Inflamassomos/efeitos dos fármacos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interleucina-1beta , Células Matadoras Naturais/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Piroptose/imunologia , Linfócitos T/imunologiaRESUMO
Pneumolysin (PLY), a major virulence factor of Streptococcus pneumoniae, is a pore-forming cytolysin that modulates host innate responses contributing to host defense against and pathogenesis of pneumococcal infections. Interleukin-1α (IL-1α) has been shown to be involved in tissue damage in a pneumococcal pneumonia model; however, the mechanism by which this cytokine is produced during S. pneumoniae infection remains unclear. In this study, we examined the role of PLY in IL-1α production. Although the strains induced similar levels of pro-IL-1α expression, wild-type S. pneumoniae D39, but not a deletion mutant of the ply gene (Δply), induced the secretion of mature IL-1α from host macrophages, suggesting that PLY is critical for the maturation and secretion of IL-1α during S. pneumoniae infection. Further experiments with calcium chelators and calpain inhibitors indicated that extracellular calcium ions and calpains (calcium-dependent proteases) facilitated the maturation and secretion of IL-1α from D39-infected macrophages. Moreover, we found that PLY plays a critical role in calcium influx and calpain activation, as elevated intracellular calcium levels and the degradation of the calpain substrate α-fodrin were detected in macrophages infected with D39 but not the Δply strain. These results suggested that PLY induces the influx of calcium in S. pneumoniae-infected macrophages, followed by calpain activation and subsequent IL-1α maturation and secretion.
Assuntos
Calpaína/metabolismo , Interações Hospedeiro-Patógeno , Interleucina-1alfa/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Streptococcus pneumoniae/crescimento & desenvolvimento , Estreptolisinas/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Células Cultivadas , Feminino , Camundongos Endogâmicos C57BLRESUMO
Vitamin B6 includes six water-soluble vitamers: pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), and their phosphorylated forms. Pyridoxal 5'-phosphate (PLP) is an important cofactor for many metabolic enzymes. Several lines of evidence demonstrate that blood levels of PLP are significantly lower in patients with inflammation than in control subjects and that vitamin B6 has anti-inflammatory effects, with therapeutic potential for a variety of inflammatory diseases. Although one of our group previously demonstrated that PL inhibits the NF-κB pathway, the molecular mechanism by which vitamin B6 suppresses inflammation is not well understood. Here, we showed that both PL and PLP suppressed the expression of cytokine genes in macrophages by inhibiting Toll-like receptor (TLR)-mediated TAK1 phosphorylation and the subsequent NF-κB and JNK activation. Furthermore, PL and PLP abolished NLRP3-dependent caspase-1 processing and the subsequent secretion of mature IL-1ß and IL-18 in LPS-primed macrophages. In contrast, PM and PN had little effect on IL-1ß production. PLP, but not PL, markedly reduced the production of mitochondrial reactive oxygen species (ROS) in peritoneal macrophages. Importantly, PL and PLP reduced IL-1ß production induced by LPS and ATP, or by LPS alone, in mice. Moreover, PL and PLP protected mice from lethal endotoxic shock. Collectively, these findings reveal novel anti-inflammatory activities for vitamin B6 and suggest its potential for preventing inflammatory diseases driven by the NLRP3 inflammasome.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamassomos/metabolismo , Interleucina-1beta/biossíntese , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Vitamina B 6/farmacologia , Animais , Interleucina-18/biossíntese , Lipopolissacarídeos/toxicidade , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Choque Séptico/induzido quimicamente , Choque Séptico/metabolismo , Choque Séptico/prevenção & controleRESUMO
Invertebrate animal species that can withstand temperatures as high as 37°C, the human body temperature, are limited. In the present study, we utilized the two-spotted cricket, Gryllus bimaculatus, which lives in tropical and subtropical regions, as an animal model of human pathogenic bacterial infection. Injection of Pseudomonas aeruginosa or Staphylococcus aureus into the hemolymph killed crickets. Injected P. aeruginosa or S. aureus proliferated in the hemolymph until the cricket died. The ability of these pathogenic bacteria to kill the crickets was blocked by the administration of antibiotics. S. aureus gene-knockout mutants of virulence factors, including cvfA, agr and srtA, exhibited decreased killing ability compared with the parent strain. The dose at which 50% of crickets were killed by P. aeruginosa or S. aureus was not decreased at 37°C compared with that at 27°C. Injection of Listeria monocytogenes, which upregulates toxin expression at 37°C, killed crickets, and the dose at which 50% of crickets were killed was decreased at 37°C compared with that at 27°C. These findings suggest that the two-spotted cricket is a useful model animal for evaluating the virulence properties of various human pathogenic bacteria at variable temperature including 37°C.
Assuntos
Infecções Bacterianas/microbiologia , Gryllidae/microbiologia , Listeria monocytogenes/patogenicidade , Modelos Animais , Pseudomonas aeruginosa/patogenicidade , Staphylococcus aureus/patogenicidade , Aminoaciltransferases/deficiência , Aminoaciltransferases/genética , Animais , Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/deficiência , Cisteína Endopeptidases/genética , Técnicas de Inativação de Genes , Hemolinfa/microbiologia , Humanos , Infecções por Pseudomonas/tratamento farmacológico , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/genética , Temperatura , Transativadores/genética , Transativadores/metabolismoRESUMO
Listeria monocytogenes has a well-characterized ability to cross the placental barrier, resulting in spontaneous abortion and fetal infections. However, the mechanisms resulting in infection-associated abortion are not fully understood. In this study, we demonstrate that the dephosphorylation of MAPK family proteins caused by L. monocytogenes infection of trophoblast giant (TG) cells, which are placental immune cells, contributes to infectious abortion. Dephosphorylation of c-Jun, p38, and ERK1/2 was observed in infected TG cells, causing the downregulation of cytoprotective heme oxygenase (HO)-1. Blocking the dephosphorylation of proteins, including MAPK family proteins, inhibited the decrease in HO-1 expression. Treatment with MAPK inhibitors inhibited bacterial internalization into TG cells. Moreover, Toll-like receptor 2 involved in the expression of MAPK family proteins. Infection with a listeriolysin O-deleted mutant impaired dephosphorylation of MAPK family proteins in TG cells and did not induce infectious abortion in a mouse model. These results suggest that inactivation of the MAPK pathway by L. monocytogenes induces TG cell death and causes infectious abortion.
RESUMO
BACKGROUND: Syndecan-4 is a transmembrane heparan sulfate proteoglycan expressed in a variety of cells, and glycosaminoglycan side chains of syndecan-4 bind to several proteins, suggesting several biological functions. However, the role of syndecan-4 in acute bacterial pneumonia has not yet been elucidated. METHODS: Serum syndecan-4 levels were measured in patients with acute pneumonia, and the relationships between serum syndecan-4 levels and clinical parameters were analyzed. Next, we treated wild-type and syndecan-4-deficient mice with Streptococcus pneumoniae intranasally and analyzed the phenotype of syndecan-4-deficient mice. RESULTS: In the patients with acute pneumonia, serum syndecan-4 levels were significantly higher than in the healthy volunteers and correlated negatively with the pneumonia severity score. In addition, in patients who improved with short-term antibiotic therapy, serum syndecan-4 levels were higher on admission and gradually increased during antibiotic therapy. Furthermore, in syndecan-4-deficient mice, the survival rate was significantly worse, and total neutrophil counts in bronchoalveolar lavage fluid, bacterial counts in blood, and plasma levels of inflammatory cytokines were significantly higher than in wild-type mice. CONCLUSIONS: These results suggest that syndecan-4 has an anti-inflammatory function in acute pneumonia and could serve as a useful biomarker in these patients.
Assuntos
Biomarcadores/sangue , Pneumonia Bacteriana/sangue , Sindecana-4/sangue , Doença Aguda , Idoso , Animais , Antibacterianos/uso terapêutico , Anti-Inflamatórios/sangue , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/sangue , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Neutrófilos/citologia , Neutrófilos/imunologia , Pneumonia Bacteriana/tratamento farmacológico , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/metabolismo , Sindecana-4/deficiênciaRESUMO
Listeria monocytogenes induces the formation of inflammasomes and subsequent caspase-1 activation, and the adaptor apoptosis-associated speck-like protein containing a CARD (ASC) is crucial for this response. However, the role of ASC in L. monocytogenes infection in vivo is unclear. In this study, we demonstrate that ASC has a detrimental effect on host defense against L. monocytogenes infection at a lethal dose (10(6) CFU), but not at a sublethal dose (10(3) CFU). During lethal L. monocytogenes infection, serum levels of IL-18 and IL-10 were markedly elevated in WT mice, but not in ASC KO mice. IL-18 KO mice were more resistant to lethal L. monocytogenes infection than WT mice and had lower levels of serum IL-10. Furthermore, blockade of IL-10 receptor resulted in a reduction in bacterial counts, suggesting that ASC and IL-18 might exacerbate L. monocytogenes infection through induction of IL-10. We noticed that maturation of IL-18 during lethal infection was partially independent of caspase-1, but was critically dependent on ASC. ASC was required for the elevation of serum neutrophil serine protease activity, which correlated with caspase-1-independent IL-18 maturation and IL-10 production. Collectively, these results suggest that ASC plays a detrimental role in lethal L. monocytogenes infection through IL-18 production in an inflammasome-dependent and -independent manner.
Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Inflamassomos/imunologia , Interleucina-10/imunologia , Interleucina-18/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Adaptadoras de Sinalização CARD , Inflamassomos/genética , Interleucina-10/genética , Interleucina-18/genética , Listeriose/genética , Listeriose/patologia , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/patologia , Receptores de Interleucina-10/genética , Receptores de Interleucina-10/imunologia , Serina Proteases/genética , Serina Proteases/imunologiaRESUMO
Inflammasomes, multiprotein platforms of caspase-1 activation, are assembled in response to a number of exogenous and endogenous danger signals, leading to the production of pro-inflammatory cytokines and induction of inflammatory cell death through the activation of caspase-1. Inflammasomes have been implicated in a wide range of physiological and pathological processes, including host defense against microbial pathogens, maintenance of intestinal homeostasis, and even development of inflammatory disorders. Thus, inflammasomes can be both beneficial and detrimental, and understanding the mechanisms involved in inflammasome activation may provide a better approach to prevent the harmful effects of the inflammatory response. Although inflammasome complexes are formed via protein-protein interactions between their components, accumulating evidence suggests that inflammasome activation is positively and negatively regulated by ligand-binding receptors, accessory proteins, other caspases, cytokines, kinases/phosphatases, redox sensors, ion homeostasis, second messengers, organelles, cytoskeleton, and autophagy, among others. Moreover, inflammasome activation can result in the formation of another caspase-1-activating protein complex, the ASC speck/pyroptosome, which is also tightly controlled. In this review, we discuss how the assembly of inflammasomes and ASC speck is regulated by complex mechanisms. Recent findings on effector functions and biological roles of inflammasomes also are summarized.