Rac1 Regulates the NLRP3 Inflammasome Which Mediates IL-1beta Production in Chlamydophila pneumoniae Infected Human Mononuclear Cells

Chlamydophila pneumoniae causes acute respiratory tract infections and has been associated with development of asthma and atherosclerosis. The production of IL-1β, a key mediator of acute and chronic inflammation, is regulated on a transcriptional level and additionally on a posttranslational level by inflammasomes. In the present study we show that C. pneumoniae-infected human mononuclear cells produce IL-1β protein depending on an inflammasome consisting of NLRP3, the adapter protein ASC and caspase-1. We further found that the small GTPase Rac1 is activated in C. pneumoniae-infected cells. Importantly, studies with specific inhibitors as well as siRNA show that Rac1 regulates inflammasome activation in C. pneumoniae-infected cells. In conclusion, C. pneumoniae infection of mononuclear cells stimulates IL-1β production dependent on a NLRP3 inflammasome-mediated processing of proIL-1β which is controlled by Rac1

Molekulare Mechanismen der Interaktion von Legionella pneumophila mit dem pulmonalen Epithel

Legionella pneumophila is a major cause of sporadic and epidemic community- and hospital -acquired pneumonia (CAP and HAP) that leads to frequent deaths worldwide. To cause these diseases, L. pneumophila has to interact with the pulmonary epithelial surface of the lung, which is known not only to be a mechanical barrier against invading pathogens, but also to play an important role in innate immunity. Thus, it is essential to understand the molecular mechanisms underlying this interaction. The present work will give an overview on the pathogenesis of L. pneumophila-induced pulmonary infections. Focus will be put on mechanisms of target cell activation via putative host cell receptors. Furthermore activated signal transduction pathways in L. pneumophila-infected pulmonary epithelial cells will be presented in detail. In the studies addressed here, we demonstrate that L. pneumophila infects pulmonary epithelial cells. Identification of virulence factors of L. pneumophila by pattern recognition receptors such as Toll-Like Receptor (TLR)2, TLR5 and TLR9 strongly induce the expression of diverse cytokines, antimicrobial peptides, and prostaglandin E2 (PGE2). The expression of the above mentioned mediators depends on the activation of Mitogen-Activated Protein Kinases (MAPKs), Protein Kinase C (PKC), NF-kB (nuclear factor 'kappa- light-chainenhancer'of activated B-cells), and the Activator Protein 1 (AP-1). Furthermore, upon recognition of L. pneumophila by intracellular receptors such as Neuronal Apoptosis Inhibitory Protein (NAIP) and ICE protease- activating factor (Ipaf), the intracellular bacterial replication in pulmonary epithelial cells is strongly reduced. Taken together, we investigated relevant issues of innate immunity activation in L. pneumophila-infected pulmonary epithelial cells. These findings will contribute to the understanding of the pathogenesis of Legionnaires’disease.Legionella pneumophila ist ein wichtiger Erreger der sporadischen und endemischen ambulant erworbenen („community acquired pneumonia“, CAP) und nosokomialen Pneumonien („hospital acquired pneumonia“, HAP). Diese Erkrankungen führen weltweit zu einer erhöhten Sterblichkeit. Um eine Pneumonie auszulösen, muss L. pneumophila mit den Lungenepithelzellen interagieren. Diese stellen nicht nur eine physikalische Barriere für Atemwegserreger dar, sondern spielen auch eine wichtige Rolle in der angeborenen Immunität. Es ist daher von großer Bedeutung die molekularen Mechanismen dieser Interaktion zu verstehen. Die vorliegende Arbeit gibt eine Übersicht über die Pathogenese der durch L. pneumophila hervorgerufenen pulmonalen Infektionen und fokussiert hierbei insbesondere auf die Mechanismen der Zielzellinfektion über potentielle Wirtszellrezeptoren, sowie die nachfolgend aktivierten Signalübertragungswege in L. pneumophila-infizierten Lungenepithelzellen. Es konnte gezeigt werden, dass L. pneumophila Lungenepithelzellen infiziert. Die Erkennung der Virulenzfaktoren dieses Erregers durch Mustererkennungsrezeptoren wie Toll-Like Rezeptor (TLR)2, TLR5 und TLR9 führte zu einer starken Induktion unterschiedlicher Zytokine, sowie antimikrobieller Peptide und Prostaglandin E2 (PGE2). Der Produktion obengenannter Mediatoren ging eine Aktivierung der Mitogen-Aktivierten Protein Kinase (MAPK), Protein Kinase C (PKC), NF-kB (nuclear factor 'kappa-light- chain-enhancer' of activated B-cells), und dem Aktivator Protein 1 (AP-1) voraus. Darüber hinaus reduzierte die intrazelluläre Erkennung von L. pneumophila durch das Neuronal Apoptosis Inhibitory Protein (NAIP) und den ICE protease-activating factor (Ipaf) das Wachstum dieser Erreger in Lungenepithelzellen stark. Zusammenfassend, wurden wichtige und relevante Mechanismen der Aktivierung der angeborenen Immunität in L. pneumophila infizierten Lungenepithelzellen adressiert. Diese neuen Erkenntnisse werden zum Teil dazu beitragen, die komplexe Pathogenese der Legionärskrankheit zu verstehen

The role and regulation of Moraxella catarrhalis-induced human beta-defensin 3 expression in human pulmonary epithelial cells

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-kappa B 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-kappa B. 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. (C) 2015 Published by Elsevier Inc

Rac1 controls IL-1β production at a posttranscriptional level in <i>C. pneumoniae</i>-infected cells.

<p>PBMCs were transfected with control siRNA or siRNA specific for Rac1. After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs and knock down of Rac1 was assessed by reverse transcription PCR (A). Cell supernatants were subjected to IL-1β ELISA (B), and levels of pro-IL-1β mRNA were analyzed by Q-PCR (C). (D) THP-1 cells were incubated with the indicated concentrations of NSC23766 for 30 min and afterwards infected with <i>C. pneumoniae</i> (MOI 3) for 8 h. Cell lysates were assayed for pro-caspase-1 and caspase-1 p20 by Western blot. The western Blot is representative of three independent experiments. (E, F) THP-1 cells seeded on coverslips were treated or not treated with NSC23766, and infected with <i>C. pneumoniae</i> for 20 h. Bacteria (red) and ASC (green) were visualized by confocal laser scanning microscopy using specific antibodies. The arrowheads point to ASC foci. Images are representative of three independent experiments (original magnification 63×).</p

Role of Rac1 in the production of IL-1β in <i>C. pneumoniae</i>-infected cells.

<p>(A) PBMCs were incubated with different concentrations of the Rac1 inhibitor NSC23766 for 30 min and subsequently infected with <i>C. pneumoniae</i> (MOI 3), or (C) cells were first infected with <i>C. pneumoniae</i> (MOI 3) and NSC23766 was added 2.5 h post-infection. After incubating 16 hrs production of IL-1β was quantified by ELISA. Total RNA was harvested for quantification of chlamydial 16s rRNA production (B, D) using real-time PCR as indicated in the Materials and Methods section. (E) THP-1 cells were transfected with control siRNA or siRNA specific for Rac1. After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs and knock down of Rac1 was assessed by reverse transcription PCR. (F) HEp-2 reinfection assay in which siRNA-transfected THP-1 cells infected with <i>C. pneumoniae</i> (MOI 0.5; 72 h) were harvested and inoculated onto monolayers of HEp-2 cells. Infected cells were then stained for Chlamydia 48 h p.i. and clamydial inclusions were counted. Data shown are representative for at least three (A–D) or two (E, F) experiments performed in duplicates.</p

<i>C. pneumoniae</i> stimulates production of mature IL-1β in human PBMCs.

<p>(A) Human PBMCs were infected with different MOI of <i>C. pneumoniae</i> for 16 hrs and production of IL-1β was determined by ELISA. (B) Human PBMCs were infected with <i>C. pneumoniae</i> (MOI 3) for different time intervals and amounts of mature IL-1β (17 kDA) in the cell supernatant was visualized by Western Blot. The western blot is representative of three independent experiments. Results obtained from ELISAs represent mean ± SD of three independent experiments.</p

Caspase-1, ASC and NLRP3 mediate IL-1β production in <i>C. pneumoniae</i>-infected cells.

<p>(A) THP-1 monocytes were infected with <i>C. pneumoniae</i> (MOI 3) for different time intervalls. Cell lysates were harvested and assayed for procaspase-1 and caspase-1 p20. (B, C) PBMCs were transfected with control siRNA or siRNA specific for caspase-1. After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs. Expression of caspase-1 was examined by reverse transcription PCR, and supernatants were subjected to IL-1β ELISA. (D–G) PBMCs were transfected with control siRNA or siRNA specific for ASC (D, E) or NLRP3 (F, G). After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3), expression of ASC (D) and NLRP3 (F) was examined by reverse transcription PCR, and supernatants were subjected to IL-1β ELISA (E, G). (H) Cells were transfected with siRNA as indicated and, after 48 h, infected with <i>C. pneumoniae</i> (MOI 3). Cell lysates were assayed for procaspase-1 and caspase-1 p20. (I) Mouse BMMs obtained from wildtype and Nlrp3−/− mice were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs. Production of mIL-1β was quantified by ELISA. Western Blots are representative for at least three independent experiments. Results obtained from ELISAs represent mean ± SD of three independent experiments.</p