Live Legionella pneumophila induces MUC5AC production by airway epithelial cells independently of intracellular invasion

The airway epithelium is the initial barrier against airborne pathogens, and it plays many roles in host airway defense. Legionella pneumophila is an intracellular pathogen that causes rapidly advancing pneumonia and is sometimes life-threatening. Here, we evaluated the role of the airway epithelial cells in the defense against L.?pneumophila by examining mucus production in vitro. The production of MUC5AC, a major mucin protein, was not induced by formalin- or ultraviolet-killed L.?pneumophila, but it was induced by live L.?pneumophila. Similarly, nuclear factor-kappaB (NF-κB) was activated only by live L.?pneumophila. Inhibitors of ERK and JNK, but not p38, dose-dependently inhibited the induction of MUC5AC by live L.?pneumophila. Inhibition of intracellular invasion by cytochalasin D did not affect MUC5AC production. Taken together, the results suggest that live L.?pneumophila induces MUC5AC production via the ERK-JNK and NF-κB pathways without internalization of bacteria and that the airway epithelium produces mucin as part of the immune response against L.?pneumophila.     

Fungal invasion of epithelial cells

Interaction between host cells and invasive Candida plays a large role in the pathogenicity of Candida species. Fungal-induced endocytosis and active penetration are the two distinct, yet complementary invasion mechanisms of invasive candidiasis. Induced endocytosis is a microorganism-triggered, epithelial-driven, clathrin-mediated and actin-dependent process. During the fundamental pathological process of induced endocytosis, invasins (Als3 and Ssa1), which mediate the binding of host epithelial surface proteins, are expressed by Candida species on the hyphal surface. Sequentially, the interaction between invasins and host epithelial surface proteins stimulates the recruitment of clathrin, dynamin and cortactin to the sites where Candida enters epithelial cells, which in turn induce the actin cytoskeleton reorganization. Actin cytoskeleton provides the force required for fungal internalization. Parallely, active penetration of Candida can directly pass through epithelial cells possibly due to progressive elongation of hyphae and physical forces. Several molecules, such as secreted hydrolases and Als3, can affect the protective barrier of the epithelium and make Candida actively penetrate into epithelial cells through intercellular gaps of epithelial layers.     

Infectivity of Legionella pneumophila mip mutant for alveolar epithelial cells

Legionella pneumophila can invade and grow within explanted alveolar epithelial cells. Given its potential clinical significance, an examination of the molecular basis of epithelial cell infection was initiated. The mip gene encodes a 24-kilodalton surface protein that promotes macrophage infection and virulence. To determine whether this gene is required for pneumocyte infection, we tested a strain bearing a mip null mutation for its ability to infect both explanted type II cells and type I-like cell lines. For infection of type II cells, the infective dose 50% for the Mip^- strain was 25-fold higher than an isogenic Mip⁺ strain. Type I cell monolayers infected with the mutant for 3 days yielded 50-fold fewer bacteria than did monolayers infected with the parental strain. These data indicate that Mip enhances infection of pneumocytes and that L. pneumophila employs some of the same genes (mechanisms) to infect epithelial cells and marcophages.     

Diversion of cytoskeletal processes by Shigella during invasion of epithelial cells

Shigella, the causative agent of bacillar dysentery, invades colonic epithelial cells and moves intracellularly to spread from cell to cell. The processes of Shigella entry, determined by the Ipa proteins, and of actin-based motility, dependent on the IcsA/VirG protein, represent different levels of bacterial manipulation of the cell cytoskeleton.     

Syndecans promote mycobacterial internalization by lung epithelial cells

Pulmonary tuberculosis (TB) is an airborne disease caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). Alveolar epithelial cells and macrophages are the first point of contact for Mtb in the respiratory tract. However, the mechanisms of mycobacterial attachment to, and internalization by, nonprofessional phagocytes, such as epithelial cells, remain incompletely understood. We identified syndecan 4 (Sdc4) as mycobacterial attachment receptor on alveolar epithelial cells. Sdc4 mRNA expression was increased in human and mouse alveolar epithelial cells after mycobacterial infection. Sdc4 knockdown in alveolar epithelial cells or blocking with anti‐Sdc4 antibody reduced mycobacterial attachment and internalization. At the molecular level, interactions between epithelial cells and mycobacteria involved host Sdc and the mycobacterial heparin‐binding hemagglutinin adhesin. In vivo, Sdc1/Sdc4 double‐knockout mice were more resistant to Mtb colonization of the lung. Our work reveals a role for distinct Sdcs in promoting mycobacterial entry into alveolar epithelial cells with impact on outcome of TB disease.     

Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA

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WAVE2 signaling mediates invasion of polarized epithelial cells by Salmonella typhimurium

The bacterial pathogen Salmonella penetrates the intestinal epithelium by inducing its own phagocytosis into epithelial cells. The dramatic reorganization of the actin cytoskeleton required for internalization is driven by bacterial manipulation of host signaling pathways, including activation of the Rho family GTPase Rac1 and subsequent activation of the Arp2/3 complex. However, the mechanisms linking these two events remain poorly understood. Rac1 is thought to promote activation of the Arp2/3 complex through its interaction with suppressor of cAMP receptor/WASP family verprolin-homologous (SCAR/WAVE) family proteins, but this interaction is apparently indirect. Two different Rac1 effectors have been shown to bind WAVE2: IRSp53, the SH3 domain of which binds the WAVE2 proline-rich domain, and PIR121/Sra-1, which forms a pentameric complex containing WAVE, Abi1, Nap1, and HSPC300. However, the extent to which each of these complexes contributes to Arp2/3 complex activation in the context of Salmonella infection is unclear. Here, we show that WAVE2 is necessary for efficient invasion of epithelial cells by Salmonella typhimurium. We found that although Salmonella infection strongly promotes the formation of an IRSp53/WAVE2 complex, IRSp53 is not necessary for bacterial internalization. In contrast, disruption of the PIR121/Nap1/Abi1/WAVE2/HSPC300 complex potently inhibits bacterial uptake. These results indicate that WAVE2 is an important component in signaling pathways leading to Salmonella invasion. Although infection leads to the formation of an IRSp53/WAVE2 complex, it is the association of WAVE2 with the Abi1/Nap1/PIR121/HSPC300 complex that regulates bacterial internalization.     

Expression of airway secretory epithelial functions by lung carcinoma cells

We examined 12 non-small cell lung carcinoma cell lines for expression of airway goblet, serous, and mucous cell characteristics. The cells expressed some ultrastructural traits of secretory epithelial cells but none contained secretory granules typical of the airway secretory cells. Using immunocytochemistry and cell-specific monoclonal antibodies, we identified heterogeneous expression of goblet, mucous, and serous cell markers among the cell lines. After metabolic radiolabeling, cells incorporated isotope into high molecular weight material. Incubation of pulse-radiolabeled cells with a number of known mucus secretogogues revealed that 5 of the 12 cell lines released radiolabeled material in response to the agonists. However, in each cell line only one of the receptor-activated pathways tested was intact. Although we did not identify a single cell line expressing a phenotype similar to normal airway secretory cells, particular functions retained by some of these cell lines may make them useful for specific studies of mucus production or secretion.     

Mechanotransduction of stretch-induced prostanoid release by fetal lung epithelial cells

Mechanical ventilation is the primary supportive treatment for infants and adults suffering from severe respiratory failure. Adverse mechanical ventilation (overdistension of the lung) triggers a proinflammatory response. Along with cytokines, inflammatory mediators such as bioactive lipids are involved in the regulation of the inflammatory response. The arachidonic acid pathway is a key source of bioactive lipid mediators, including prostanoids. Although ventilation has been shown to influence the production of prostanoids in the lung, the mechanotransduction pathways are unknown. Herein, we established that cyclic stretch of fetal lung epithelial cells, but not fibroblasts, can evoke an extremely sensitive, rapid alteration in eicosanoid metabolism through a cyclooxygenase (COX)-2 dependent mechanism. Cyclic stretch significantly increased PGI(2), PGF(2alpha), PGD(2), PGE(2), and thromboxane B(2) levels in the media of epithelial cells, but did not alter leukotriene B(4) or 12-hydroxyeicosatetraenoic acid levels. Inhibition of COX-2, but not COX-1, attenuated the cyclic stretch-induced PG increase in the media, suggesting that cyclic stretch primarily affected PG synthesis. Substrate (free arachidonic acid) availability for PG generation was increased because of a cyclic stretch-induced activation of cytosolic phospholipase A(2) (cPLA(2)) via an influx of extracellular calcium and phosphorylation by mitogen-activated protein kinase, p44/42MAPK. The data are compatible with cPLA(2) and COX-2 being intimately involved in regulating the injury response to adverse mechanical ventilation.     

Induction of p53 by urokinase in lung epithelial cells

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. The plasminogen/plasmin system includes the uPA, its receptor, and its inhibitor (plasminogen activator inhibitor-1). Interactions between these molecules regulate cellular proteolysis as well as adhesion, cellular proliferation, and migration, processes germane to the pathogenesis of lung injury and neoplasia. In previous studies, we found that uPA regulates cell surface fibrinolysis by regulating its own expression as well as that of the uPA receptor and plasminogen activator inhibitor-1. In this study, we found that uPA alters expression of the tumor suppressor protein p53 in Beas2B airway epithelial cells in both a time- and concentration-dependent manner. These effects do not require uPA catalytic activity because the amino-terminal fragment of uPA lacking catalytic activity was as potent as two chain active uPA. Single chain uPA also enhanced p53 expression to the same extent as intact two chain active uPA and the amino-terminal fragment. Pretreatment of cells with anti-beta1 integrin antibody blocked uPA-induced p53 expression. uPA-induced p53 expression occurs without increased p53 mRNA expression. However, uPA induced oncoprotein MDM2 in a concentration-dependent manner. uPA-induced p53 expression does not require activation of tyrosine kinases. Inactivation of protein-tyrosine phosphatase SHP-2 inhibits both basal and uPA-induced p53 expression. Plasmin did not alter uPA-mediated p53 expression. The induction of p53 expression by exposure of lung epithelial cells to uPA is a newly recognized pathway by which urokinase may influence the proliferation of lung epithelial cells. This pathway could regulate pathophysiologic alterations of p53 expression in the setting of lung inflammation or neoplasia.     

Type 1 pilus-mediated bacterial invasion of bladder epithelial cells

Most strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili. We have determined that the type 1 pilus adhesin, FimH, mediates not only bacterial adherence, but also invasion of human bladder epithelial cells. In contrast, adherence mediated by another pilus adhesin, PapG, did not initiate bacterial internalization. FimH-mediated invasion required localized host actin reorganization, phosphoinositide 3-kinase (PI 3-kinase) activation and host protein tyrosine phosphorylation, but not activation of Src-family tyrosine kinases. Phosphorylation of focal adhesin kinase (FAK) at Tyr397 and the formation of complexes between FAK and PI 3-kinase and between alpha-actinin and vinculin were found to correlate with type 1 pilus-mediated bacterial invasion. Inhibitors that prevented bacterial invasion also blocked the formation of these complexes. Our results demonstrate that UPEC strains are not strictly extracellular pathogens and that the type 1 pilus adhesin FimH can directly trigger host cell signaling cascades that lead to bacterial internalization.     

Legionella pneumophila induces IFNbeta in lung epithelial cells via IPS-1 and IRF3, which also control bacterial replication

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.     

Collagen binding protein Mip enables Legionella pneumophila to transmigrate through a barrier of NCI-H292 lung epithelial cells and extracellular matrix

Guinea pigs are highly susceptible to Legionella pneumophila infection and therefore have been the preferred animal model for studies of legionellosis. In this study guinea pig infections revealed that the Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to the bacterial dissemination within the lung tissue and the spread of Legionella to the spleen. Histopathology of infected animals, binding assays with components of the extracellular matrix (ECM), bacterial transmigration experiments across an artificial lung epithelium barrier, inhibitor studies and ECM degradation assays were used to elucidate the underlying mechanism of the in vivo observation. The Mip protein, which belongs to the enzyme family of FK506-binding proteins (FKBP), was shown to bind to the ECM protein collagen (type I, II, III, IV, V, VI). Transwell assays with L. pneumophila and recombinant Escherichia coli HB101 strains revealed that Mip enables these bacteria to transmigrate across a barrier of NCI-H292 lung epithelial cells and ECM (NCI-H292/ECM barrier). Mip-specific monoclonal antibodies and the immunosuppressants rapamycin and FK506, which inhibit the peptidyl prolyl cis/trans isomerase (PPIase) activity of Mip, were able to inhibit this transmigration. By using protease inhibitors we found that the penetration of the NCI-H292/ECM barrier additionally requires a serine protease activity. Degradation assays with (35)S-labelled ECM proteins supported the finding of a concerted action of Mip and a serine protease. The described synergism between the activity of the collagen binding Mip protein and the serine protease activity represents an entirely new mechanism for bacterial penetration of the lung epithelial barrier and has implications for other prokaryotic and eukaryotic pathogens.     

Microrheology of human lung epithelial cells measured by atomic force microscopy

Lung epithelial cells are subjected to large cyclic forces from breathing. However, their response to dynamic stresses is poorly defined. We measured the complex shear modulus (G(*)(omega)) of human alveolar (A549) and bronchial (BEAS-2B) epithelial cells over three frequency decades (0.1-100 Hz) and at different loading forces (0.1-0.9 nN) with atomic force microscopy. G(*)(omega) was computed by correcting force-indentation oscillatory data for the tip-cell contact geometry and for the hydrodynamic viscous drag. Both cell types displayed similar viscoelastic properties. The storage modulus G'(omega) increased with frequency following a power law with exponent approximately 0.2. The loss modulus G"(omega) was approximately 2/3 lower and increased similarly to G'(omega) up to approximately 10 Hz, but exhibited a steeper rise at higher frequencies. The cells showed a weak force dependence of G'(omega) and G"(omega). G(*)(omega) conformed to the power-law model with a structural damping coefficient of approximately 0.3, indicating a coupling of elastic and dissipative processes within the cell. Power-law behavior implies a continuum distribution of stress relaxation time constants. This complex dynamics is consistent with the rheology of soft glassy materials close to a glass transition, thereby suggesting that structural disorder and metastability may be fundamental features of cell architecture.     

Induction of COX-2 by acrolein in rat lung epithelial cells

Acrolein is a highly reactive alpha, beta-unsaturated aldehyde, and a product of lipid peroxidation reactions. Acrolein is also an environmental pollutant and a key component of cigarette smoke, and has been implicated in multiple respiratory diseases. Lung tissue is a primary target for acrolein toxicity in smokers and may lead to chronic lung inflammation and lung cancer. Chronic inflammation, associated with expression of cyclooxygenase-2 (COX-2) and prostaglandins, are predisposing factors for malignancy. In this study, we investigated the induction of COX-2 by acrolein in rat lung epithelial cells and its related signaling cascade. Induction of COX-2 by acrolein was significant at 6 h post-treatment and was dependent upon NFκB activation. The activation of NFκB by acrolein was induced as a result of degradation of IκBα over the time of treatment. In addition, the upstream signaling cascade involved Raf-1/ERK activation by acrolein in the COX-2 induction and was inhibited by GW5074 (a Ras/Raf-1/ERK inhibitor), thereby providing evidence for the role of this cascade in this process. The results of these studies offer an explanation for the mechanism of COX-2 induction by acrolein in rat lung epithelial cells.     

Cathelicidin LL-37 increases lung epithelial cell stiffness, decreases transepithelial permeability, and prevents epithelial invasion by Pseudomonas aeruginosa

In addition to its antibacterial activity, the cathelicidin-derived LL-37 peptide induces multiple immunomodulatory effects on host cells. Atomic force microscopy, F-actin staining with phalloidin, passage of FITC-conjugated dextran through a monolayer of lung epithelial cells, and assessment of bacterial outgrowth from cells subjected to Pseudomonas aeruginosa infection were used to determine LL-37's effect on epithelial cell mechanical properties, permeability, and bacteria uptake. A concentration-dependent increase in stiffness and F-actin content in the cortical region of A549 cells and primary human lung epithelial cells was observed after treatment with LL-37 (0.5-5 μM), sphingosine 1-phosphate (1 μM), or LPS (1 μg/ml) or infection with PAO1 bacteria. Other cationic peptides, such as RK-31, KR-20, or WLBU2, and the antibacterial cationic steroid CSA-13 did not reproduce the effect of LL-37. A549 cell pretreatment with WRW4, an antagonist of the transmembrane formyl peptide receptor-like 1 protein attenuated LL-37's ability to increase cell stiffness. The LL-37-mediated increase in cell stiffness was accompanied by a decrease in permeability and P. aeruginosa uptake by a confluent monolayer of polarized normal human bronchial epithelial cells. These results suggested that the antibacterial effect of LL-37 involves an LL-37-dependent increase in cell stiffness that prevents epithelial invasion by bacteria.