RESUMO
BACKGROUND: Bacterial colonisation with Moraxella catarrhalis may partly sustain chronic inflammation in the lower airways of patients with chronic obstructive pulmonary disease (COPD). In addition, this bacterium causes infectious exacerbations of COPD, which often necessitate treatment with antibiotics. Antimicrobial peptides are the body's own antibiotic substances with bactericidal and bacteriostatic, as well as immunomodulatory function. In particular, human beta-defensin 3 (hBD-3) exerts an antimicrobial effect against an extraordinarily broad spectrum of pathogens. We therefore investigated the role of hBD-3 in infections of pulmonary epithelial cells with M. catarrhalis. METHODS: The antimicrobial activity of hBD-3 vs. M. catarrhalis was evaluated in an antimicrobial susceptibility assay. We analyzed hBD-3 secretion of M. catarrhalis-infected pulmonary epithelial cells using ELISA. The role of M. catarrhalis-specific virulence factors, toll-like receptors (TLR) 2 and 4, MAPK pathways, and transcription factors AP-1 and NF-κB in the induction and regulation of hBD-3 expression were explored with specific inhibitors, small interference RNA, Western Blot, and chromatin immunoprecipitation (ChIP) assays. RESULTS: HBD-3 exhibited a strong bactericidal effect against M. catarrhalis. M. catarrhalis induced hBD-3 expression in pulmonary epithelial cells, which was dependent on M. catarrhalis membranous lipoolygosaccharide (LOS), while the surface proteins UspA1 and UspA2 were not involved. Gene silencing of TLR2, but not TLR4, led to a reduced hBD-3 secretion after stimulation with M. catarrhalis or M. catarrhalis LOS. Inhibition of MAPKs ERK1/2 and JNK, but not p38, reduced hBD-3 secretion. HBD-3 expression was mediated through the recruitment of AP-1 to the hBD-3 gene promoter and was independent of NF-κB. CONCLUSION: The immune response of pulmonary epithelial cells towards M. catarrhalis involves secretion of hBD-3, which has a bactericidal effect against this pathogen. Binding of M. catarrhalis virulence factor LOS to TLR2 causes an ERK1/2- and JNK-dependent induction of AP-1-related transcription of the hBD-3 gene, resulting in the production and secretion of hBD-3.
Assuntos
Moraxella catarrhalis/patogenicidade , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologia , beta-Defensinas/metabolismo , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Regulação da Expressão Gênica , Humanos , Lipopolissacarídeos/imunologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Sistema de Sinalização das MAP Quinases , Moraxella catarrhalis/imunologia , Infecções por Moraxellaceae/complicações , NF-kappa B/metabolismo , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/microbiologia , Mucosa Respiratória/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição AP-1/metabolismo , beta-Defensinas/genéticaRESUMO
BACKGROUND: Chronic lower airway inflammation is considered to be a major cause of pathogenesis and disease progression in chronic obstructive pulmonary disease (COPD). Moraxella catarrhalis is a COPD-associated pathogen causing exacerbations and bacterial colonization in the lower airways of patients, which may contribute to chronic inflammation. Increasing evidence suggests that the epidermal growth factor receptor (EGFR) modulates inflammatory processes in the human airways. The goal of this study was to investigate the role of EGFR in the M. catarrhalis-induced pro-inflammatory immune response in airway epithelial cells. METHODS: The effects of inhibition and gene silencing of EGFR on M. catarrhalis-dependent pro-inflammatory cytokine expression in human primary bronchial epithelial cells (NHBEs), as well as the pulmonary epithelial cell lines BEAS-2B and A549 were analyzed. We also assessed the involvement of EGFR-dependent ERK and NF-κB signaling pathways. RESULTS: The M. catarrhalis-induced pro-inflammatory immune response depends, at least in part, on the phosphorylation and activation of the EGF receptor. Interaction of M. catarrhalis with EGFR increases the secretion of pro-inflammatory cytokines, which is mediated via ERK and NF-κB activation. CONCLUSION: The interaction between M. catarrhalis and EGFR increases airway inflammation caused by this pathogen. Our data suggest that the inhibition of EGFR signaling in COPD could be an interesting target for reducing M. catarrhalis-induced airway inflammation.
Assuntos
Células Epiteliais/imunologia , Receptores ErbB/metabolismo , Moraxella catarrhalis/imunologia , Mucosa Respiratória/imunologia , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inativação Gênica , Humanos , Interleucina-8/metabolismo , Viabilidade Microbiana , NF-kappa B/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologia , Transdução de Sinais , Especificidade da EspécieRESUMO
Streptococcus pneumoniae is an important causative agent of pneumonia in humans. Pulmonary epithelial surfaces constitutes not only a mechanical barrier against invading pathogens but also essentially contribute to innate immunity by producing antimicrobial peptides such as human ß-defensin-2 (hBD-2) and -3 (hBD-3). In this study the authors demonstrated that pneumococci induced hBD-2 and hBD-3 expression in human pulmonary epithelial cells. Further analysis indicated an essential role of Toll-like receptor 2 (TLR2) for the expression of both peptides in infected pulmonary epithelial cells. Whereas the hBD-2 release was controlled by the phosphoinositide 3-kinase (PI3K) and the transcription factor nuclear factor kappa B (NF-κB), hBD-3 was triggered via the c-Jun N-terminal kinase (JNK)-activator protein 1 (AP-1) pathway. Additionally, the authors showed that exogenous hBD-2 as well as hBD-3 elicited a strong antimicrobial effect on S. pneumoniae. Thus, differential regulation of the expression of hBD-2 and hBD-3 might play an important role in pneumococci pneumonia.
Assuntos
Células Epiteliais Alveolares/microbiologia , Pulmão/microbiologia , Streptococcus pneumoniae/patogenicidade , beta-Defensinas/biossíntese , Células Epiteliais Alveolares/imunologia , Células Epiteliais Alveolares/metabolismo , Humanos , Imunidade Inata , Pulmão/imunologia , Pulmão/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Streptococcus pneumoniae/imunologia , Receptor 2 Toll-Like/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
Legionella pneumophila causes severe pneumonia. Acetylation of histones is thought to be an important regulator of gene transcription, but its impact on L. pneumophila-induced expression of proinflammatory cytokines is unknown. L. pneumophila strain 130b induced the expression of the important chemoattractant IL-8 and genome-wide histone modifications in human lung epithelial A549 cells. We analyzed the IL-8-promoter and found that histone H4 was acetylated and H3 was phosphorylated at Ser(10) and acetylated at Lys(14), followed by transcription factor NF-kappaB. Recruitment of RNA polymerase II to the IL-8 promoter corresponded with increases in gene transcription. Histone modification and IL-8 release were dependent on p38 kinase and NF-kappaB pathways. Legionella-induced IL-8 expression was decreased by histone acetylase (HAT) inhibitor anacardic acid and enhanced by histone deacetylase (HDAC) inhibitor trichostatin A. After Legionella infection, HATs p300 and CREB-binding protein were time-dependently recruited to the IL-8 promoter, whereas HDAC1 and HDAC5 first decreased and later reappeared at the promoter. Legionella specifically induced expression of HDAC5 but not of other HDACs in lung epithelial cells, but knockdown of HDAC1 or 5 did not alter IL-8 release. Furthermore, Legionella-induced cytokine release, promoter-specific histone modifications, and RNA polymerase II recruitment were reduced in infection with flagellin-deletion mutants. Legionella-induced histone modification as well as HAT-/HDAC-dependent IL-8 release could also be shown in primary lung epithelial cells. In summary, histone acetylation seems to be important for the regulation of proinflammatory gene expression in L. pneumophila infected lung epithelial cells. These pathways may contribute to the host response in Legionnaires' disease.
Assuntos
Células Epiteliais/imunologia , Flagelina/metabolismo , Histonas/metabolismo , Interleucina-8/metabolismo , Legionella pneumophila/imunologia , NF-kappa B/metabolismo , Alvéolos Pulmonares/imunologia , Acetilação , Ácidos Anacárdicos/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Interleucina-8/genética , Interleucina-8/imunologia , Legionella pneumophila/efeitos dos fármacos , Legionella pneumophila/metabolismo , NF-kappa B/imunologia , Regiões Promotoras Genéticas , Inibidores da Síntese de Proteínas/farmacologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/microbiologia , RNA Polimerase II/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In mice, different alleles of the mNAIP5 (murine neuronal apoptosis inhibitory protein-5)/mBirc1e gene determine whether macrophages restrict or support intracellular replication of Legionella pneumophila, and whether a mouse is resistant or (moderately) susceptible to Legionella infection. In the resistant mice strains, the nucleotide-binding oligomerization domain (Nod)-like receptor (NLR) family member mNAIP5/mBirc1e, as well as the NLR protein mIpaf (murine ICE protease-activating factor), are involved in recognition of Legionella flagellin and in restriction of bacterial replication. Human macrophages and lung epithelial cells support L. pneumophila growth, and humans can develop severe pneumonia (Legionnaires disease) after Legionella infection. The role of human orthologs to mNAIP5/mBirc1e and mIpaf in this bacterial infection has not been elucidated. Herein we demonstrate that flagellin-deficient L. pneumophila replicate more efficiently in human THP-1 macrophages, primary monocyte-derived macrophages, and alveolar macrophages, and in A549 lung epithelial cells compared with wild-type bacteria. Additionally, we note expression of the mNAIP5 ortholog hNAIP in all cell types examined, and expression of hIpaf in human macrophages. Gene silencing of hNAIP or hIpaf in macrophages or of hNAIP in lung epithelial cells leads to an enhanced bacterial growth, and overexpression of both molecules strongly reduces Legionella replication. In contrast to experiments with wild-type L. pneumophila, hNAIP or hIpaf knock-down affects the (enhanced) replication of flagellin-deficient Legionella only marginally. In conclusion, hNAIP and hIpaf mediate innate intracellular defense against flagellated Legionella in human cells.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células Epiteliais/microbiologia , Legionella pneumophila/crescimento & desenvolvimento , Doença dos Legionários/imunologia , Macrófagos/microbiologia , Proteína Inibidora de Apoptose Neuronal/metabolismo , Western Blotting , Linhagem Celular , Proliferação de Células , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Flagelina/genética , Flagelina/metabolismo , Humanos , Imunidade Inata , Macrófagos/imunologia , Macrófagos/metabolismo , Microscopia Confocal , Reação em Cadeia da Polimerase , Interferência de RNA , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologiaRESUMO
Moraxella catarrhalis is a major cause of exacerbations of chronic obstructive pulmonary disease (COPD) and emphysema. M. catarrhalis-specific UspA1 and the epithelial carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) were required to induce apoptosis. M. catarrhalis-induced apoptosis was significantly enhanced in HeLa cells stably transfected with CEACAM1, compared with HeLa cells not expressing CEACAM1. Infected cells showed increased activity of caspases 3, 6, and 9 but not of caspase 8. Reduced expression of Bcl-2, translocation of Bax into the mitochondria, and cytosolic increase of apoptosis-inducing factor in M. catarrhalis-infected cells implicated the involvement of mitochondrial death pathways. In conclusion, M. catarrhalis induced apoptosis in pulmonary epithelial cells--a process that was triggered by interaction between CEACAM1 and UspA1. Thus, M. catarrhalis-induced apoptosis of pulmonary epithelial cells may contribute to the development of COPD and emphysema.
Assuntos
Antígenos CD/metabolismo , Apoptose/fisiologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Epiteliais/microbiologia , Moraxella catarrhalis/patogenicidade , Alvéolos Pulmonares/microbiologia , Linhagem Celular , Células HeLa , Humanos , Moraxella catarrhalis/metabolismo , Alvéolos Pulmonares/citologiaRESUMO
Legionella pneumophila causes community- and hospital-acquired pneumonia. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX) and microsomal PGE(2) synthase-1 (mPGES-1)-derived prostaglandins like prostaglandin E(2) (PGE(2)) are considered as important regulators of lung function. Herein we tested the hypothesis that L. pneumophila induced COX-2 and mPGES-1-dependent PGE(2) production in pulmonary epithelial cells. Legionella induced the release of PGE(2) in primary human small airway epithelial cells and A549 cells. This was accompanied by an increased expression of COX-2 and mPGES-1 as well as an increased PLA(2) activity in infected cells. Deletion of the type IV secretion system Dot/Icm did not impair Legionella-related COX-2 expression or PGE(2) release in A549 cells. L. pneumophila induced the degradation of IkappaBalpha and activated NF-kappaB. Inhibition of IKK blocked L. pneumophila-induced PGE(2) release and COX-2 expression. We noted activation of p38 and p42/44 MAP kinase in Legionella-infected A549 cells. Moreover, membrane translocation and activation of PKCalpha was observed in infected cells. PKCalpha and p38 and p42/44 MAP kinase inhibitors reduced PGE(2) release and COX-2 expression. In summary, PKCalpha and p38 and p42/44 MAP kinase controlled COX-2 expression and subsequent PGE(2) release by Legionella-infected lung epithelial cells. These pathways may significantly contribute to the host response in Legionnaires' disease.
Assuntos
Ciclo-Oxigenase 2/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Legionella pneumophila/patogenicidade , Pulmão/metabolismo , Pulmão/microbiologia , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Proteína Quinase C-alfa/metabolismo , Sequência de Bases , Linhagem Celular , Células Cultivadas , DNA Complementar/genética , Dinoprostona/biossíntese , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Oxirredutases Intramoleculares/genética , Doença dos Legionários/etiologia , Doença dos Legionários/metabolismo , Pulmão/citologia , Fosfolipases A/metabolismo , Prostaglandina-E SintasesRESUMO
Legionella pneumophila, a Gram-negative facultative intracellular bacterium, causes severe pneumonia (Legionnaires' disease). Type I interferons (IFNs) were so far associated with antiviral immunity, but recent studies also indicated a role of these cytokines in immune responses against (intracellular) bacteria. Here we show that wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, or heat-inactivated Legionella induced IFNbeta expression in human lung epithelial cells. We found that factor (IRF)-3 and NF-kappaB-p65 translocated into the nucleus and bound to the IFNbeta gene enhancer after L. pneumophila infection of lung epithelial cells. RNA interference demonstrated that in addition to IRF3, the caspase recruitment domain (CARD)-containing adapter molecule IPS-1 (interferon-beta promoter stimulator 1) is crucial for L. pneumophila-induced IFNbeta expression, whereas other CARD-possessing molecules, such as RIG-I (retinoic acid-inducible protein I), MDA5 (melanoma differentiation-associated gene 5), Nod27 (nucleotide-binding oligomerization domain protein 27), and ASC (apoptosis-associated speck-like protein containing a CARD) seemed not to be involved. Finally, bacterial multiplication assays in small interfering RNA-treated cells indicated that IPS-1, IRF3, and IFNbeta were essential for the control of intracellular replication of L. pneumophila in lung epithelial cells. In conclusion, we demonstrated a critical role of IPS-1, IRF3, and IFNbeta in Legionella infection of lung epithelium.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Epiteliais/citologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Legionella pneumophila/fisiologia , Pulmão/citologia , Pulmão/microbiologia , Transporte Ativo do Núcleo Celular , Diferenciação Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Células Epiteliais/metabolismo , Humanos , Legionella pneumophila/metabolismo , Pulmão/metabolismo , NF-kappa B/metabolismo , RNA/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Although pneumococcal pneumonia is one of the most common causes of death due to infectious diseases, little is known about pneumococci-lung cell interaction. Herein we tested the hypothesis that pneumococci activated pulmonary epithelial cell cytokine release by c-Jun-NH2-terminal kinase (JNK) METHODS: Human bronchial epithelial cells (BEAS-2B) or epithelial HEK293 cells were infected with S. pneumoniae R6x and cytokine induction was measured by RT-PCR, ELISA and Bioplex assay. JNK-phosphorylation was detected by Western blot and nuclear signaling was assessed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). JNK was modulated by the small molecule inhibitor SP600125 and AP1 by transfection of a dominant negative mutant. RESULTS: S. pneumoniae induced the release of distinct CC and CXC, as well as Th1 and Th2 cytokines and growth factors by human lung epithelial cell line BEAS-2B. Furthermore, pneumococci infection resulted in JNK phosphorylation in BEAS-2B cells. Inhibition of JNK by small molecule inhibitor SP600125 reduced pneumococci-induced IL-8 mRNA expression and release of IL-8 and IL-6. One regulator of the il8 promoter is JNK-phosphorylated activator protein 1 (AP-1). We showed that S. pneumoniae time-dependently induced DNA binding of AP-1 and its phosphorylated subunit c-Jun with a maximum at 3 to 5 h after infection. Recruitment of Ser63/73-phosphorylated c-Jun and RNA polymerase II to the endogenous il8 promoter was found 2 h after S. pneumoniae infection by chromatin immunoprecipitation. AP-1 repressor A-Fos reduced IL-8 release by TLR2-overexpressing HEK293 cells induced by pneumococci but not by TNFalpha. Antisense-constructs targeting the AP-1 subunits Fra1 and Fra2 had no inhibitory effect on pneumococci-induced IL-8 release. CONCLUSION: S. pneumoniae-induced IL-8 expression by human epithelial BEAS-2B cells depended on activation of JNK and recruitment of phosphorylated c-Jun to the il8 promoter.
Assuntos
Células Epiteliais/microbiologia , Interleucina-8/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Pulmão/microbiologia , Streptococcus pneumoniae , Fator de Transcrição AP-1/metabolismo , Antracenos/farmacologia , Linhagem Celular , DNA/genética , DNA/metabolismo , Ativação Enzimática , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Humanos , Interleucina-8/genética , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Pulmão/imunologia , Pulmão/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismoRESUMO
Moraxella catarrhalis is a major cause of infectious exacerbations of chronic obstructive lung disease (COPD) and may also contribute to the pathogenesis of COPD. Little is known about M. catarrhalis-bronchial epithelium interaction. We investigated activation of M. catarrhalis infected bronchial epithelial cells and characterized the signal transduction pathways. Moreover, we tested the hypothesis that the M. catarrhalis-induced cytokine expression is regulated by acetylation of histone residues and controlled by histone deacetylase activity (HDAC). We demonstrated that M. catarrhalis induced a strong time- and dose-dependent inflammatory response in the bronchial epithelial cell line (BEAS-2B), characterized by the release of IL-8 and GM-CSF. For this cytokine liberation activation of the ERK and p38 mitogen-activated protein (MAP) kinases and transcription factor NF-kappaB was required. Furthermore, M. catarrhalis-infected bronchial epithelial cells showed an enhanced acetylation of histone H3 and H4 globally and at the promoter of the il8 gene. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A augmented the M. catarrhalis-induced IL-8 response. After exposure to M. catarrhalis, we found a decrease in global histone deacetylase expression and activity. Our findings suggest that M. catarrhalis-induced activation of il8 gene transcription was caused by interference with epigenetic mechanisms regulating il8 gene accessibility. Our findings provide insight into important molecular and cellular mechanisms of M. catarrhalis-induced activation of human bronchial epithelium.
Assuntos
Brônquios/microbiologia , Brônquios/fisiopatologia , Histona Desacetilases/metabolismo , Inflamação/microbiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Moraxella catarrhalis/fisiologia , NF-kappa B/metabolismo , Mucosa Respiratória/fisiopatologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Inibidores de Histona Desacetilases , Humanos , Interleucina-8/fisiologiaRESUMO
Streptococcus pneumoniae is the major pathogen of community-acquired pneumonia and a common cause of otitis, meningitis and sepsis. During pneumococci infection accompanied with bacterial invasion and hematogenous spreading, the endothelium is directly targeted by pneumococci and their virulence factors. Therefore, we tested the hypothesis that pneumococci induced endothelial apoptosis. Unencapsulated R6x pneumococci strongly induced apoptosis of human endothelial cells both from lung microvasculature and umbilical vein, whereas an encapsulated strain D39 mainly led to necrotic cell death. Deletion of the gene coding for pneumolysin reduced pneumococci-induced apoptosis in HUVEC. Furthermore, N-acetyl-L-cysteine, an antioxidant thiol, significantly reduced apoptosis caused by R6x, and LDH release induced by D39, pointing to a role for reactive oxygen species in the pathogenesis. Apoptotic cells showed increased cleavage and activity of caspases 6 and 9 but only late activation of caspase 3. Programmed cell death could be strongly reduced by pan-caspase inhibitor zVAD. Reduced levels of Bcl2 and cytosolic increase of apoptosis-inducing factor in pneumococci-infected cells implicated involvement of mitochondrial death pathways. Caspase activation and apoptosis were abolished by cAMP elevation. Moreover, p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase were activated in pneumococci-infected cells and inhibitors of both kinases strongly reduced pneumococci-induced caspase activation and apoptosis. Hence, kinase- and caspase-dependence of pneumococci-induced endothelial apoptosis may bear relevance to novel therapeutic approaches to pneumococci-related disease.