Bastian Opitz

Differential requirement for the activation of the inflammasome for processing and release of IL-1beta in monocytes and macrophages.

The processing of pro-interleukin-1beta depends on activation of caspase-1. Controversy has arisen whether Toll-like receptor (TLR) ligands alone can activate caspase-1 for release of interleukin-1beta (IL-1beta). Here we demonstrate that human blood monocytes release processed IL-1beta after a one-time stimulation with either TLR2 or TLR4 ligands, resulting from constitutively activated caspase-1 and release of endogenous adenosine triphosphate. The constitutive activation of caspase-1 depends on the inflammasome components, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and NALP3, but in monocytes caspase-1 activation is uncoupled from pathogen-associated molecular pattern recognition. In contrast, macrophages are unable to process and release IL-1beta solely by TLR ligands and require a second adenosine triphosphate stimulation. We conclude that IL-1beta production is differentially regulated in monocytes and macrophages, and this reflects their separate functions in host defense and inflammation.

IFNβ induction by influenza A virus is mediated by RIG-I which is regulated by the viral NS1 protein

Influenza A virus causes epidemics of respiratory diseases in humans leading to thousands of death annually. One of its major virulence factors, the non‐structural protein 1 (NS1), exhibits interferon‐antagonistic properties. While epithelial cells of the respiratory tract are the primary targets of influenza virus, the virus‐sensing mechanisms in these cells eventually leading to IFNβ production are incompletely understood. Here we show that infection of epithelial cells with NS1‐deficient influenza A virus upregulated expression of two molecules that have been previously implicated in sensing of RNA viruses, the retinoic acid‐inducible gene I (RIG‐I) and the melanoma differentiation‐associated gene 5 (MDA5). Gene silencing and overexpression experiments demonstrated that RIG‐I, its adapter interferon‐beta promoter stimulator 1 (IPS‐1) and interferon‐regulated factor 3 (IRF3) were involved in influenza A virus‐mediated production of the antiviral IFNβ. In addition, we showed that the NS1 protein is capable to inhibit the RIG‐I‐induced signalling, a mechanism which corresponded to the observation that only NS1‐deficient but not the wild‐type virus induced high‐level production of IFNβ. In conclusion, we demonstrated a critical involvement of RIG‐I, IPS‐1 and IRF3 in influenza A virus infection of epithelial cells.

Nod1-Mediated Endothelial Cell Activation by Chlamydophila pneumoniae

Seroepidemiological and animal studies, as well as demonstration of viable bacteria in atherosclerotic plaques, have linked Chlamydophila pneumoniae infection to development of chronic vascular lesions and coronary heart disease. Inflammation and immune responses are dependent on host recognition of invading pathogens. The recently identified cytosolic Nod proteins are candidates for intracellular recognition of bacteria, such as the obligate intracellular chlamydia. In the present study, mechanisms of endothelial cell activation by C. pneumoniae via Nod proteins were examined. Viable, but not heat-inactivated, chlamydia activated human endothelial cells, suggesting that invasion of these cells is necessary for their profound activation. Endothelial cells express Nod1. Nod1 gene silencing by small interfering RNA reduced C pneumoniae–induced IL-8 release markedly. Moreover, in HEK293 cells, overexpressed Nod1 or Nod2 amplified the capacity of C pneumoniae to induce nuclear factor &kgr;B (NF-&kgr;B) activation. Interestingly, heat-inactivated bacteria were still able to induced a NF-&kgr;B reporter gene activity via Nod proteins when transfected intracellularly, but not when provided from the extracellular side. In contrast, TLR2 sensed extracellular heat-inactivated chlamydia. In conclusion, we demonstrated that C pneumoniae induced a Nod1-mediated and Nod2-mediated NF-&kgr;B activation in HEK293 cells. In endothelial cells, Nod1 played a dominant role in triggering a chlamydia-mediated inflammatory process.

The NLRP3 Inflammasome Is Differentially Activated by Pneumolysin Variants and Contributes to Host Defense in Pneumococcal Pneumonia

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1β. This IL-1β production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1β production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.

Listeria monocytogenes activated p38 MAPK and induced IL-8 secretion in a nucleotide-binding oligomerization domain 1-dependent manner in endothelial cells.

Nucleotide-binding oligomerization domain (Nod) proteins serve as intracellular pattern recognition molecules recognizing peptidoglycans. To further examine intracellular immune recognition, we used Listeria monocytogenes as an organism particularly amenable for studying innate immunity to intracellular pathogens. In contrast to wild-type L. monocytogenes, the nonpathogenic Listeria innocua, or L. monocytogenes mutants lacking internalin B or listeriolysin O, poorly invaded host cells and escaped into host cell cytoplasm, respectively, and were therefore used as controls. In this study, we show that only the invasive wild-type L. monocytogenes, but not the listeriolysin O- or internalin B-negative L. monocytogenes mutants or L. innocua, substantially induced IL-8 production in HUVEC. RNA interference and Nod1-overexpression experiments demonstrated that Nod1 is critically involved in chemokine secretion and NF-κB activation initiated by L. monocytogenes in human endothelial cells. Moreover, we show for the first time that Nod1 mediated activation of p38 MAPK signaling induced by L. monocytogenes. Finally, L. monocytogenes- and Nod1-induced IL-8 production was blocked by a specific p38 inhibitor. In conclusion, L. monocytogenes induced a Nod1-dependent activation of p38 MAPK signaling and NF-κB which resulted in IL-8 production in endothelial cells. Thus, Nod1 is an important component of a cytoplasmic surveillance pathway.

Listeria monocytogenes-infected human peripheral blood mononuclear cells produce IL-1beta, depending on listeriolysin O and NLRP3.

Different NOD-like receptors, including NLRP1, NLRP3, and NLRC4, as well as the recently identified HIN-200 protein, AIM2, form multiprotein complexes called inflammasomes, which mediate caspase-1–dependent processing of pro-IL-1β. Listeria monocytogenes is an intracellular pathogen that is actively phagocytosed by monocytes/macrophages and subsequently escapes from the phagosome into the host cell cytosol, depending on its pore-forming toxin listeriolysin O (LLO). In this study, we demonstrate that human PBMCs produced mature IL-1β when infected with wild-type L. monocytogenes or when treated with purified LLO. L. monocytogenes mutants lacking LLO or expressing a noncytolytic LLO as well as the avirulent Listeria innocua induced strongly impaired IL-1β production. RNA interference and inhibitor experiments in human PBMCs as well as experiments in Nlrp3 and Rip2 knockout bone marrow-derived macrophages demonstrated that the Listeria-induced IL-1β release was dependent on ASC, caspase-1, and NLRP3, whereas NOD2, Rip2, NLRP1, NLRP6, NLRP12, NLRC4, and AIM2 appeared to be dispensable. We found that L. monocytogenes-induced IL-1β production was largely dependent on phagosomal acidification and cathepsin B release, whereas purified LLO activated an IL-1β production independently of these mechanisms. Our results indicate that L. monocytogenes-infected human PBMCs produced IL-1β, largely depending on an LLO-mediated phagosomal rupture and cathepsin B release, which is sensed by Nlrp3. In addition, an LLO-dependent but cathepsin B-independent NLRP3 activation might contribute to some extent to the IL-1β production in L. monocytogenes-infected cells.

Innate Immune Recognition in Infectious and Noninfectious Diseases of the Lung

Diseases of the respiratory tract are among the leading causes of death in the world population. Increasing evidence points to a key role of the innate immune system with its pattern recognition receptors (PRRs) in both infectious and noninfectious lung diseases, which include pneumonia, chronic obstructive pulmonary disease, acute lung injury, pneumoconioses, and asthma. PRRs are capable of sensing different microbes as well as endogenous molecules that are released after cell damage. This PRR engagement is the prerequisite for the initiation of immune responses to infections and tissue injuries which can be beneficial or detrimental to the host. PRRs include the Toll-like receptors, NOD-like receptors, RIG-I-like receptors, and cytosolic DNA sensors. The PRRs and their signaling pathways represent promising targets for prophylactic and therapeutic interventions in various lung diseases.

Lung epithelium as a sentinel and effector system in pneumonia - molecular mechanisms of pathogen recognition and signal transduction

Pneumonia, a common disease caused by a great diversity of infectious agents is responsible for enormous morbidity and mortality worldwide. The bronchial and lung epithelium comprises a large surface between host and environment and is attacked as a primary target during lung infection. Besides acting as a mechanical barrier, recent evidence suggests that the lung epithelium functions as an important sentinel system against pathogens. Equipped with transmembranous and cytosolic pathogen-sensing pattern recognition receptors the epithelium detects invading pathogens. A complex signalling results in epithelial cell activation, which essentially participates in initiation and orchestration of the subsequent innate and adaptive immune response. In this review we summarize recent progress in research focussing on molecular mechanisms of pathogen detection, host cell signal transduction, and subsequent activation of lung epithelial cells by pathogens and their virulence factors and point to open questions. The analysis of lung epithelial function in the host response in pneumonia may pave the way to the development of innovative highly needed therapeutics in pneumonia in addition to antibiotics.

Streptococcus pneumoniae-induced p38 MAPK-dependent Phosphorylation of RelA at the Interleukin-8 Promotor

Streptococcus pneumoniae is the major cause of community-acquired pneumonia and one of the most common causes of death by infectious disease in industrialized countries. Little is known concerning the mechanisms of target cell activation in this disease. The present study shows that NF-κB and p38 MAPK signaling pathways contribute to chemokine synthesis by lung epithelial cells in response to pneumococci. In infected lungs of mice pneumococci stimulate expression of the interleukin (IL)-8 homolog keratinocyte-derived chemokine and granulocyte-macrophage colony-stimulating factor, as well as activate p38 MAPK. Human bronchial epithelium was chosen as a cellular model, because it establishes the first barrier against pathogens, and little is known about its function in innate immunity. Pneumococci infection induces expression of IL-8 and granulocyte-macrophage colony-stimulating factor as well as activation of p38 MAPK in human bronchial epithelial cells (BEAS-2B). Inhibition of p38 MAPK activity by SB202190 and SB203580 blocks pneumococci-induced cytokine release. In mouse lungs in vivo as well as in cultured cells, pneumococci activate NF-κBinanIκB kinase-dependent manner. Inhibition of p38 MAPK by chemical inhibitors or by RNA interference targeting p38α reduces pneumococci-induced NF-κB-dependent gene transcription. Blockade of p38 activity did not affect inducible nuclear translocation and recruitment of NF-κB/RelA to the IL-8 promotor but did reduce the level of phosphorylated RelA (serine 536) at IL-8 promotor and inhibited pneumococci-mediated recruitment of RNA polymerase II to IL-8 promotor. Thus, p38 MAPK contributes to pneumococci-induced chemokine transcription by modulating p65 NF-κB-mediated transactivation.

Recognition of Streptococcus pneumoniae by the innate immune system

Streptococcus pneumoniae is both a frequent colonizer of the upper respiratory tract and a leading cause of life‐threatening infections such as pneumonia, meningitis and sepsis. The innate immune system is critical for the control of colonization and for defence during invasive disease. Initially, pneumococci are recognized by different sensors of the innate immune system called pattern recognition receptors (PRRs), which control most subsequent host defence pathways. These PRRs include the transmembrane Toll‐like receptors (TLRs) as well as the cytosolic NOD‐like receptors (NLRs) and DNA sensors. Recognition of S. pneumoniae by members of these PRR families regulates the production of inflammatory mediators that orchestrate the following immune response of infected as well as neighbouring non‐infected cells, stimulates the recruitment of immune cells such as neutrophils and macrophages, and shapes the adaptive immunity. This review summarizes the current knowledge of the function of different PRRs in S. pneumoniae infection.