Krueppel-Like Factor 4 Expression in Phagocytes Regulates Early Inflammatory Response and Disease Severity in Pneumococcal Pneumonia.

The transcription factor Krueppel-like factor (KLF) 4 fosters the pro-inflammatory immune response in macrophages and polymorphonuclear neutrophils (PMNs) when stimulated with Streptococcus pneumoniae, the main causative pathogen of community-acquired pneumonia (CAP). Here, we investigated the impact of KLF4 expression in myeloid cells such as macrophages and PMNs on inflammatory response and disease severity in a pneumococcal pneumonia mouse model and in patients admitted to hospital with CAP. We found that mice with a myeloid-specific knockout of KLF4 mount an insufficient early immune response with reduced levels of pro-inflammatory cytokines and increased levels of the anti-inflammatory cytokine interleukin (IL) 10 in bronchoalveolar lavage fluid and plasma and an impaired bacterial clearance from the lungs 24 hours after infection with S. pneumoniae. This results in higher rates of bacteremia, increased lung tissue damage, more severe symptoms of infection and reduced survival. Higher KLF4 gene expression levels in the peripheral blood of patients with CAP at hospital admission correlate with a favourable clinical presentation (lower sequential organ failure assessment (SOFA) score), lower serum levels of IL-10 at admission, shorter hospital stay and lower mortality or requirement of intensive care unit treatment within 28 days after admission. Thus, KLF4 in myeloid cells such as macrophages and PMNs is an important regulator of the early pro-inflammatory immune response and, therefore, a potentially interesting target for therapeutic interventions in pneumococcal pneumonia.

Study on the additive protective effect of PGLYRP3 and Bifidobacterium adolescentis Reuter 1963 on severity of DSS-induced colitis in Pglyrp3 knockout (Pglyrp3 -/-) and wild-type (WT) mice.

BACKGROUND AND AIMS: Pglyrp3 is a bactericidal innate immunity protein known to sustain the habitual gut microbiome and protect against experimental colitis. Intestinal inflammation and metaflammation are commonly associated with a marked reduction of commensal bifidobacteria. Whether Pglyrp3 and bifidobacteria interact synergistically or additively to alleviate metaflammation is unknown. We investigated the extent to which Pglyrp3 and bifidobacteria regulate metaflammation and gut bacterial dysbiosis in DSS-induced mouse models of intestinal inflammation. MATERIAL & METHODS: 8-10 weeks old male mice were used. In both WT and Pglyrp3 -/- experiments, the mice were randomly divided into three groups of 16 mice per group: (1) a control group receiving sterile tap water, (2) an experimental group receiving sterile tap water supplemented with only 5% DSS, and (3) an experimental group receiving sterile tap water supplemented with 5% DSS and 1 × 109 CFU/ml of Bifidobacterium adolescentis (B.a.) for 7 days. Wild-type (WT) littermates of the respective gene (i.e. Pglyrp3) were used as controls throughout the study. Clinical signs of general health and inflammation were monitored daily. Faecal pellet samples were analysed by qRT-PCR for microbial composition. Histology of relevant organs was carried out on day 8. Metabolic parameters and liver inflammation were determined in serum samples. RESULTS: Intestinal inflammation in mice of group 2 were significantly increased compared to those of control group 1. There was a significant difference in mean scores for inflammation severity between DSS-treated WT and DSS-treated Pglyrp3 -/- mice. Buildup of key serum metabolic markers (cholesterol, triglyceride and glucose) was set off by colonic inflammation. qRT-PCR quantification showed that DSS significantly decreased the Clostridium coccoides and Bifidobacterium cell counts while increasing those of Bacteroides group in both WT and Pglyrp3 -/- mice. These manifestations of DSS-induced dysbiosis were significantly attenuated by feeding B.a. Both the local and systemic ill-being of the mice alleviated when they received B.a. DISCUSSION: This study shows that Pglyrp3 facilitates recognition of bifidobacterial cell wall-derived peptidoglycan, thus leading additively to a reduction of metaflammation through an increase in the number of bifidobacteria, which were able to mitigate intestinal immunopathology in the context of Pglyrp3 blockade.

Induction of Krüppel-Like Factor 4 Mediates Polymorphonuclear Neutrophil Activation in Streptococcus pneumoniae Infection.

The recruitment and activation of polymorphonuclear neutrophils (PMNs) are of central importance for the elimination of pathogens in bacterial infections. We investigated the Streptococcus pneumoniae-dependent induction of the transcription factor Krüppel-like factor (KLF) 4 in PMNs as a potential regulator of PMN activation. We found that KLF4 expression is induced in human blood-derived PMNs in a time- and dose-dependent manner by wild-type S. pneumoniae and capsule knockout mutants. Unencapsulated knockout mutants induced stronger KLF4 expression than encapsulated wild types. The presence of autolysin LytA-competent (thus viable) pneumococci and LytA-mediated bacterial autolysis were required for KLF4 induction in human and murine PMNs. LyzMcre-mediated knockdown of KLF4 in murine blood-derived PMNs revealed that KLF4 influences pneumococci killing and increases the release of the proinflammatory cytokines tumor necrosis factor α and keratinocyte chemoattractant and decreases the release of the anti-inflammatory cytokine interleukin-10. Thus, S. pneumoniae induces KLF4 expression in PMNs, which contributes to PMN activation in S. pneumoniae infection.

Peptidoglycan Recognition Protein 4 Limits Bacterial Clearance and Inflammation in Lungs by Control of the Gut Microbiota.

Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. Endogenous host defense molecules such as peptidoglycan recognition protein 4 (PGLYRP4) might influence the course of this disease. To the best of our knowledge, there are no reports on the relevance of PGLYRP4 in pneumonia. Therefore, wild type (WT) and PGLYRP4-deficient (PGLYRP4KO) mice were analyzed in an in vivo and in vitro experimental setting to examine the influence of PGLYRP4 on the course of pneumococcal pneumonia. Furthermore, caecal 16S rRNA microbiome analysis was performed, and microbiota were transferred to germfree WT mice to assess the influence of microbiotal communities on the bacterial burden. Mice lacking PGLYRP4 displayed an enhanced bacterial clearance in the lungs, and fewer mice developed bacteremia. In addition, an increased recruitment of immune cells to the site of infection, and an enhanced bacterial killing by stronger activation of phagocytes could be shown. This may depend partly on the detected higher expression of complement factors, interferon-associated genes, and the higher pro-inflammatory cytokine response in isolated primary PGLYRP4KO vs. WT cells. This phenotype is underlined by changes in the complexity and composition of the caecal microbiota of PGLYRP4KO compared to WT mice. Strikingly, we provided evidence, by cohousing and stable transfer of the respective WT or PGLYRP4KO mice microbiota into germfree WT mice, that the changes of the microbiota are responsible for the improved clearance of S. pneumoniae lung infection. In conclusion, the deficiency of PGLYRP4, a known antibacterial protein, leads to changes in the gut microbiota. Thus, alterations in the microbiota can change the susceptibility to S. pneumoniae lung infection independently of the host genotype.

Peptidoglycan Recognition Protein 2 Regulates Neutrophil Recruitment Into the Lungs After Streptococcus pneumoniae Infection.

Peptidoglycan (PGN) recognition proteins (PGLYRPs) are a highly conserved group of host defense proteins in insects and mammals that sense bacterial cell wall PGN and act bactericidally or cleave PGN by amidase function. Streptococcus (S.) pneumoniae is one of the top five killers worldwide and causes, e.g., pneumonia, endocarditis, meningitis and sepsis. S. pneumoniae accounts for approximately 1.5-2 million deaths every year. The risk of antibiotic resistance and a general poor prognosis in young children and elderly people have led to the need for new treatment approaches. To the best of our knowledge, there is no report on the relevance of PGLYRP2 in lung infections. Therefore, we infected mice deficient for PGLYRP2 transnasally with S. pneumoniae and examined the innate immune response in comparison to WT animals. As expected, PGLYRP2-KO animals had to be sacrificed earlier than their WT counterparts, and this was due to higher bacteremia. The higher bacterial load in the PGLYRP2-KO mice was accomplished with lower amounts of proinflammatory cytokines in the lungs. This led to an abolished recruitment of neutrophils into the lungs, the spread of bacteria and the subsequent aggravated course of the disease and early mortality of the PGLYRP2-KO mice. These data suggest a substantial role of PGLYRP2 in the early defense against S. pneumoniae infection, and PGLYRP2 might also affect other infections in the lungs.

DNA-release by Streptococcus pneumoniae autolysin LytA induced Krueppel-like factor 4 expression in macrophages.

The recruitment of myeloid cells to the lung is of utmost importance for the elimination of invading pathogens. We investigated the Streptococcus pneumoniae-dependent induction mechanism of KLF4 in macrophages as a potential regulator of the macrophage immune response. We demonstrated that only viable pneumococci, which have direct contact to the host cells and release LytA-dependent DNA, induced KLF4. Exogenous supplementation of pneumococcal, other bacterial, eukaryotic foreign (human) or self (mouse) DNA to autolysis-deficient pneumococci restored (at least in part) pneumococci-related KLF4 induction. Experiments using TLR9, TRIF and MyD88 knockout macrophages revealed that TLR9, TRIF and MyD88 were partly involved in the S. pneumoniae-induced KLF4 expression. BMMs missing important DNA receptor related molecules (ASC-/-, STING-/-) showed no differences in pneumococci-related KLF4 expression. Similar results were observed with IFNAR-/- BMMs and Type I IFN stimulated cells. LyzMcre mediated knockdown of KLF4 in BMMs resulted in a decreased secretion of proinflammatory cytokines and enhanced IL-10 release. In summary, we showed that pneumococci-related KLF4 induction in macrophages is mediated via a PAMP-DAMP induction mechanism involving a hitherto unknown host cell DNA sensor leading to a more proinflammatory macrophage phenotype.

Peptidoglycan Recognition Protein 3 Does Not Alter the Outcome of Pneumococcal Pneumonia in Mice.

Pneumococci frequently cause community-acquired pneumonia, a disease with high mortality rates, particularly in young children and in the elderly. Endogenous antimicrobial peptides and proteins such as PGLYRP3 may contribute to the progression and outcome of this disease. Since increasing antibiotic resistant strains occur all over the world, these endogenous antimicrobial molecules are interesting new targets for future therapies. In this study, the expression pattern of PGLYRP3 was analyzed in alveolar epithelial cells, alveolar macrophages and neutrophils. Additionally, the function of PGLYRP3 during Streptococcus pneumoniae-induced pneumonia was investigated in a murine pneumococcal pneumonia model using PGLYRP3KO mice. PGLYRP3 is expressed in all selected cell types but pneumococcus-dependent induction of PGLYRP3 was observed only in neutrophils and alveolar macrophages. Interestingly, there were no significant differences in the bacterial loads within the lungs, the blood or the spleens, in the cytokine response, the composition of immune cells and the histopathology between wild type and PGLYRP3KO mice. Finally, we could neither observe significant differences in the clinical symptoms nor in the overall survival. Collectively, PGLYRP3 seems to be dispensable for the antibacterial defense during pneumococcal pneumonia.

Role of Pneumococcal Autolysin for KLF4 Expression and Chemokine Secretion in Lung Epithelium.

In severe pneumococcal pneumonia, the delicate balance between a robust inflammatory response necessary to kill bacteria and the loss of organ function determines the outcome of disease. In this study, we tested the hypothesis that Krueppel-like factor (KLF) 4 may counter-regulate Streptococcus pneumoniae-related human lung epithelial cell activation using the potent proinflammatory chemokine IL-8 as a model molecule. Pneumococci induced KLF4 expression in human lung, in primary human bronchial epithelial cells, and in the lung epithelial cell line BEAS-2B. Whereas proinflammatory cell activation depends mainly on the classical Toll-like receptor 2-mitogen-activated protein kinase or phosphatidylinositide 3-kinase and NF-κB pathways, the induction of KLF4 occurred independently of these molecules but relied, in general, on tyrosine kinase activation and, in part, on the src kinase family member yamaguchi sarcoma viral oncogene homolog (yes) 1. The up-regulation of KLF4 depended on the activity of the main pneumococcal autolysin LytA. KLF4 overexpression suppressed S. pneumoniae-induced NF-κB and IL-8 reporter gene activation and release, whereas small interfering RNA-mediated silencing of KLF4 or yes1 kinase led to an increase in IL-8 release. The KLF4-dependent down-regulation of NF-κB luciferase activity could be rescued by the overexpression of the histone acetylase p300/cAMP response element-binding protein-associated factor. In conclusion, KLF4 acts as a counter-regulatory transcription factor in pneumococci-related proinflammatory activation of lung epithelial cells, thereby potentially preventing lung hyperinflammation and subsequent organ failure.

Effects of enamel matrix derivative and transforming growth factor-ß1 on connective tissue growth factor in human periodontal ligament fibroblasts.

BACKGROUND: Enamel matrix derivative (EMD) is suggested to stimulate transforming growth factor-ß (TGF-ß) production. Connective tissue growth factor (CTGF) is a downstream mediator of TGF-ß. This study explores the effects of EMD and TGF-ß1 on CTGF in periodontal ligament (PDL) fibroblasts and their interactions in PDL proliferation and development. METHODS: Human PDL cells were stimulated with EMD. To explore the effects of EMD and TGF-ß1 on CTGF expression, cells were treated with and without TGF-ß inhibitor, and CTGF protein levels were assayed by Western blot analysis. To study the role of CTGF in PDL development, cells were treated with CTGF inhibitor. DNA synthesis was analyzed by bromodeoxyuridine enzyme-linked immunosorbent assay. Reverse-transcription polymerase chain reaction was performed to examine messenger RNA expression of PDL osteoblastic differentiation markers: type I collagen, alkaline phosphatase, and osteocalcin. RESULTS: EMD induced a concentration-dependent increase of CTGF protein expression in PDL cells. EMD- and TGF-ß1-stimulated CTGF expression was significantly reduced in the presence of TGF-ß inhibitor. CTGF inhibition downregulated both EMD- and TGF-ß1-induced DNA synthesis. The effect of CTGF and EMD on osteoblastic mRNA expression in PDL cells is not obvious. CONCLUSIONS: EMD stimulates CTGF expression in human PDL cells, a process modulated by the TGF-ß pathway. CTGF can affect EMD- and TGF-ß1-induced PDL cell proliferation, but its effects on PDL with regard to osteoblastic differentiation remain inconclusive. The results provide novel insights into EMD-CTGF interaction in PDL cells.

Streptococcus pneumoniae-Induced Oxidative Stress in Lung Epithelial Cells Depends on Pneumococcal Autolysis and Is Reversible by Resveratrol.

BACKGROUND: Streptococcus pneumoniae is the most common cause of community-acquired pneumonia worldwide. During pneumococcal pneumonia, the human airway epithelium is exposed to large amounts of H2O2 as a product of host and pathogen oxidative metabolism. Airway cells are known to be highly vulnerable to oxidant damage, but the pathophysiology of oxidative stress induced by S. pneumoniae and the role of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant systems of the host are not well characterized. METHODS: For gluthation/gluthathion disulfide analysis BEAS-2B cells, primary broncho-epithelial cells (pBEC), explanted human lung tissue and mouse lungs were infected with different S. pneumoniae strains (D39, A66, R6x, H2O2/pneumolysin/LytA- deficient mutants of R6x). Cell death was proven by LDH assay and cell viability by IL-8 ELISA. The translocation of Nrf2 and the expression of catalase were shown via Western blot. The binding of Nrf2 at the catalase promoter was analyzed by ChIP. RESULTS: We observed a significant induction of oxidative stress induced by S. pneumoniae in vivo, ex vivo, and in vitro. Upon stimulation, the oxidant-responsive transcription factor Nrf2 was activated, and catalase was upregulated via Nrf2. The pneumococci-induced oxidative stress was independent of S. pneumoniae-derived H2O2 and pneumolysin but depended on the pneumococcal autolysin LytA. The Nrf2 inducer resveratrol, as opposed to catalase, reversed oxidative stress in lung epithelial cells. CONCLUSIONS: These observations indicate a H2O2-independent induction of oxidative stress in lung epithelial cells via the release of bacterial factors of S. pneumoniae. Resveratrol might be an option for prevention of acute lung injury and inflammatory responses observed in pneumococcal pneumonia.

TLR9- and Src-dependent expression of Krueppel-like factor 4 controls interleukin-10 expression in pneumonia.

The release of potent pro-inflammatory mediators is crucial to mounting an efficient host response during infection. However, excessive inflammation may lead to deleterious tissue damage. This is highlighted in severe pneumococcal pneumonia, in which the delicate balance between a robust inflammatory response necessary to kill pneumococci and the loss of organ function determines the outcome of the disease. We assessed the regulation of the potent anti-inflammatory cytokine interleukin (IL)-10 in pneumococcal infection via Western blot, ELISA and chromatin immunoprecipitation analysis. Streptococcus pneumoniae induced IL-10 expression in mouse lungs and human lung epithelial cells. Pneumococcal infection resulted in a strong induction of Krueppel-like factor (KLF)4 expression in vivo and in vitro. The induction of both IL-10 and KLF4 is mediated by a pathway involving bacterial DNA, Toll-like receptor (TLR)9, MyD88 and Src kinase. KLF4 is recruited to the il10 promoter, and small-interfering RNA-mediated knockdown of KLF4 expression blocked IL-10 expression during pneumococcal infection. In conclusion, KLF4 is induced in a bacterial DNA-TLR9-Src-dependent manner and regulates IL-10 expression, linking the detection of bacterial DNA by TLR9 to the control of an inflammatory response.

Streptococcus pneumoniae induces human ß-defensin-2 and -3 in human lung epithelium.

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.

The impact of Porphyromonas gingivalis lipids on apoptosis of primary human chondrocytes.

The role of oral bacterial infections including periodontal disease in the pathogenesis of rheumatoid arthritis (RA) has gained increasing interest. Among the major periodontal pathogens, Porphyromonas gingivalis has been mostly associated with RA pathogenesis. The aim of this study was to analyze the effect of P. gingivalis total lipid (TL) fraction and dihydroceramides, as potent virulence factors, on human primary chondrocytes. Primary chondrocyte cultures were incubated with P. gingivalis phosphoglycerol dihydroceramide (PG DHC) lipids, the TL fraction or phosphoethanolamine dihydroceramide. Cell morphology changes were determined by phase contrast light microscopy. Early and late apoptosis cell analysis was performed by Annexin-V, active caspases, and 7-Aminoactinomycin D staining, and examined by flow cytometry, and cell necrosis was evaluated by lactate dehydrogenase release. Procaspase-3 activation was determined by Western blot analysis. Microscopic analysis showed altered cell morphology and cell shrinkage following incubation with P. gingivalis TLs and PG DHC lipids. Flow cytometry demonstrated an increase of Annexin-V positive and active caspases positive chondrocytes after incubation with TL and PG DHC fractions but not after phosphoethanolamine dihydroceramide (control lipid) treatment or in untreated control cells. Furthermore, Western blot analysis showed an early cleavage of procaspase-3 after 1 hr. Significant lactate dehydrogenase release following incubation with P. gingivalis lipids was demonstrated. The present data demonstrate that P. gingivalis lipids promote apoptosis in primary human chondrocytes, and thereby may contribute to the joint damage seen in the pathogenesis of RA.

TLR2- and nucleotide-binding oligomerization domain 2-dependent Krüppel-like factor 2 expression downregulates NF-kappa B-related gene expression.

The release of potent proinflammatory mediators is not only central for mounting an efficient host response, but also bears the risk for deleterious excessive tissue-damaging inflammation. This is highlighted in severe pneumococcal pneumonia, in which the delicate balance between a robust inflammatory response to kill pneumococci and loss of organ function determines the outcome of disease. In this study, we tested the hypothesis that Krüppel-like factor (KLF)2 counterregulates pneumococci- and pattern recognition receptor-related human lung cell activation. Pneumococci induced KLF2 expression in vitro and in a murine pneumonia model. Activation of TLR2- and nucleotide-binding oligomerization domain protein 2-related signaling induced KLF2 expression in a PI3K-dependent manner. Overexpression of KLF2 downregulated pneumococci-, TLR2-, and nucleotide-binding oligomerization domain protein 2-related NF-kappaB-dependent gene expression and IL-8 release, whereas small interfering RNA-based silencing of KLF2 provoked an enhanced inflammatory response. KLF2-dependent downregulation of NF-kappaB activity is partly reversible by overexpression of the histone acetylase p300/CREB-binding protein-associated factor. In conclusion, KLF2 may act as a counterregulatory transcription factor in pneumococci- and pattern recognition receptor-related proinflammatory activation of lung cells, thereby preventing lung hyperinflammation and subsequent organ failure.

Systemic use of the endolysin Cpl-1 rescues mice with fatal pneumococcal pneumonia.

OBJECTIVES: Community-acquired pneumonia is a very common infectious disease associated with significant morbidity and mortality. Streptococcus pneumoniae is the predominant pathogen in this disease, and pneumococcal resistance to multiple antibiotics is increasing. The recently purified bacteriophage endolysin Cpl-1 rapidly and specifically kills pneumococci on contact. The aim of this study was to determine the therapeutic potential of Cpl-1 in a mouse model of severe pneumococcal pneumonia. DESIGN: Controlled, in vivo laboratory study. SUBJECTS: Female C57/Bl6 mice, 8-12 weeks old. INTERVENTIONS: Mice were transnasally infected with pneumococci and therapeutically treated with Cpl-1 or amoxicillin by intraperitoneal injections starting 24 or 48 hours after infection. MEASUREMENTS AND MAIN RESULTS: Judged from clinical appearance, decreased body weight, reduced dynamic lung compliance and Pao2/Fio2 ratio, and morphologic changes in the lungs, mice suffered from severe pneumonia at the onset of therapy. When treatment was commenced 24 hours after infection, 100% Cpl-1-treated and 86% amoxicillin-treated mice survived otherwise fatal pneumonia and showed rapid recovery. When treatment was started 48 hours after infection, mice had developed bacteremia, and three of seven (42%) Cpl-1-treated and five of seven (71%) amoxicillin-treated animals survived. Cpl-1 dramatically reduced pulmonary bacterial counts, and prevented bacteremia, systemic hypotension, and lactate increase when treatment commenced at 24 hours. In vivo, treatment with Cpl-1 or amoxicillin effectively reduced counts of penicillin-susceptible pneumococci. The inflammatory response in Cpl-1-and amoxicillin-treated mice was lower than in untreated mice, as determined by multiplex cytokine assay of lung and blood samples. In human epithelial cell cultures, lysed bacteria evoked less proinflammatory cytokine release and cell death, as compared with viable bacteria. CONCLUSIONS: Cpl-1 may provide a new therapeutic option in the treatment of pneumococcal pneumonia.

Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

The nucleotide-binding domain and leucine-rich repeat containing protein NOD2 serves as a cytoplasmic pattern recognition molecule sensing bacterial muramyl dipeptide (MDP), whereas TLR2 mediates cell surface recognition of bacterial lipopeptides. In this study, we show that NOD2 stimulation activated Rac1 in human THP-1 cells and primary human monocytes. Rac1 inhibition or knock-down, or actin cytoskeleton disruption increased MDP-stimulated IL-8 secretion and NF-kappaB activation, whereas TLR2-dependent cell activation was suppressed by Rac1 inhibition. p21-activated kinase [Pak]-interacting exchange factor (beta-PIX) plays a role in this negative regulation, because knock-down of beta-PIX also led to increased NOD2-mediated but not TLR2-mediated IL-8 secretion, and coimmunoprecipitation experiments demonstrated that NOD2 interacted with beta-PIX as well as Rac1 upon MDP stimulation. Moreover, knock-down of beta-PIX or Rac1 abrogated membrane recruitment of NOD2, and interaction of NOD2 with its negative regulator Erbin. Overall, our data indicate that beta-PIX and Rac1 mediate trafficking and negative regulation of NOD2-dependent signaling which is different from Rac1's positive regulatory role in TLR2 signaling.

Histone acetylation and flagellin are essential for Legionella pneumophila-induced cytokine expression.

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.

NAIP and Ipaf control Legionella pneumophila replication in human cells.

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.

Streptococcus pneumoniae induced c-Jun-N-terminal kinase- and AP-1 -dependent IL-8 release by lung epithelial BEAS-2B cells.

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.

Moraxella catarrhalis induces inflammatory response of bronchial epithelial cells via MAPK and NF-kappaB activation and histone deacetylase activity reduction.

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.