J. L. Booth

Adenovirus Type 7 Induces Interleukin-8 Production via Activation of Extracellular Regulated Kinase 1/2

ABSTRACT Infection with adenovirus serotype 7 (Ad7) frequently causes lower respiratory pneumonia and is associated with severe lung inflammation and neutrophil infiltration. Earlier studies indicated release of proinflammatory cytokines, specifically interleukin-8 (IL-8), by pulmonary epithelial cells following infection by Ad7. However, the mechanism of IL-8 induction by Ad7 is unclear. We have explored the role of the Ras/Raf/MEK/Erk pathway in the Ad7-associated induction of IL-8 using a model system of A549 epithelial cells. We found that Ad7 infection induced a rapid activation of epithelial cell-derived Erk. The MEK-specific inhibitors PD98059 and U0126 blocked Erk activation and release of IL-8 following infection with Ad7. Treatment with PD98059 is cytostatic and not cytotoxic, as treated cells regain the ability to phosphorylate Erk and secrete IL-8 after removal of the drug. The expression of a mutated form of Ras in A549 epithelial cells blocked the induction of IL-8 promoter activity, and MEK inhibitor blocked induction of IL-8 mRNA. These results suggest that the Ras/Raf/MEK/Erk pathway is necessary for the Ad7 induction of IL-8 and that induction occurs at the level of transcription. Further, the kinetics of Erk activation and IL-8 induction suggest that an early viral event, such as receptor binding, may be responsible for the observed inflammatory response.

Adenovirus Type 7 Induces Interleukin-8 in a Lung Slice Model and Requires Activation of Erk

ABSTRACT Adenovirus (Ad), particularly Ad type 7 (Ad7), causes severe lung infection and pneumonia. Initially, Ad causes neutrophilic inflammation of the distal airways and alveoli. Interleukin-8 (IL-8) is the major lung neutrophil chemotaxin, and we have shown that Ad7 induces IL-8 release from the A549 alveolar epithelial cell line. We sought to determine whether ex vivo human and bovine lung tissue containing primary pneumocytes could be used as a more accurate and relevant model to study Ad acute inflammation. We found that cultured lung tissue preserved normal lung architecture for more than 10 days. IL-8 was generated upon exposure of the lung organ culture to Ad7. IL-8 production required activation of the Ras/Erk pathway, since a pharmacological inhibitor blocked the appearance of IL-8 in the medium. Both human and bovine lung explants supported replication of Ad7, and immunohistochemistry experiments demonstrated the presence of the Ad hexon antigen within alveolar epithelial cells. These findings show that our novel human lung organ culture accurately reproduces the in vivo infectious disease process. Thus, this organ culture model represents a valuable tool for studying the acute innate immune response to respiratory infections.

Human lung innate immune cytokine response to adenovirus type 7.

Adenovirus (Ad) type 7 can cause severe infection, including pneumonia, in military recruits and children. The initial inflammation is a neutrophilic interstitial infiltration with neutrophilic alveolitis. Subsequently, monocytes become evident and, finally, there is a predominantly lymphocytic infiltrate. We have established that Ad7 infection of epithelial cells stimulates release of the neutrophil chemotaxin interleukin (IL)-8, and have extended these studies to a human lung tissue model. Here, we studied cytokine responses to Ad7 in human alveolar macrophages (HAM) and our human lung tissue model. Both ELISA and RNase-protection assay (RPA) data demonstrated that, upon Ad7 infection, IP-10 and MIP-1alpha/beta are released from HAM. IP-10 and MIP-1alpha/beta protein levels were induced 2- and 3-fold, respectively, in HAM 24 h after Ad7 infection. We then investigated induction of specific cytokines in human lung tissue by RPA and ELISA. The results showed that IL-8 and IL-6 were induced 8 h after infection and, by 24 h, levels of IL-8, IL-6, MIP-1alpha/beta and MCP-1 were all increased. IP-10, a monocyte and lymphocyte chemokine, was also induced 30-fold, but only 24 h after infection. Immunohistochemistry staining confirmed that IL-8 was only released from the epithelial cells of lung slices and not from macrophages. IP-10 was secreted from both macrophages and epithelial cells. Moreover, full induction of IP-10 is likely to require participation and cooperation of both epithelial cells and macrophages in intact lung. Understanding the cytokine and chemokine induction during Ad7 infection may lead to novel ways to modulate the response to this pathogen.

Bacillus anthracis Lethal Toxin Reduces Human Alveolar Epithelial Barrier Function

ABSTRACT The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness.

ID: 106: ALVEOLAR ESCAPE BY BACILLUS ANTHRACIS SPORES DOES NOT REQUIRE A CARRIER CELL AND IS NOT ALTERED BY LETHAL TOXIN

Rationale The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. B. anthracis spores must escape from the alveolus, pass to the regional lymph nodes, germinate and enter the circulatory system as vegetative bacteria to cause systemic disease. Of the resident lung cells, three have been reported to take up B. anthracis spores: the antigen presenting cells (APC) alveolar macrophages and dendritic cells, and alveolar epithelial cells (AEC). Also, B. anthracis produces the exotoxins lethal factor and protective antigen (PA) which combine to form lethal toxin (LT), a metalloproteinase important in pathogenicity. The roles of carrier cells and the effects of B. anthracis toxins in escape of spores from the alveolus are unclear, especially in humans. Methods We employed a human lung organ culture model and a human A549 alveolar epithelial cell culture model, along with fluorescent confocal imaging to quantitate spore partitioning between APC and AEC, and the effects of B. anthracis LT and PA on this process. Cell types were distinguished by positive staining for HLA-DR (APC) and cytokeratin (AEC). Results We found that spores progressed through the lung slice over time, and that spore movement was not dependent on cell internalization. Both free and cell-associated spores moved through slices between 2 and 48 hrs of incubation. However, partitioning of spores between AEC, APC, and the extracellular space did not significantly change over this time. After 2 hrs, 4.7% of spores were in APC; 13.8% in AEC; and 81.5% were not cell-associated. By 48 hrs, 2.9% were in APC; 12.7% were in AEC; and 84.4% were not cell-associated. Spores also internalized in a non-uniform manner, with more variable spore internalization into AEC than into APC. At all incubation times, the majority of cell-associated spores were in AEC, not in APC. PA and LT did not affect transit of the spores through the lung tissue or the distribution of spores into AEC and APC. In A549 cells, spore internalization increased significantly after 24 hrs incubation. However, there was no statistically consistent effects of PA or LT on spore internalization in A549 cells. Conclusions Overall, our results support a “Jailbreak”-like model of spore escape from the alveolus that involves transient passage of spores, although this occurs through intact AEC. However, subsequent transport of spores by APC from the lung to the lymph nodes may occur.

ID: 141: ANTIVIRAL INNATE IMMUNE RESPONSES IN HUMAN BRONCHIAL EPITHELIAL CELLS (HBECS) OF SMOKERS ARE IMPAIRED THROUGH EPIGENETIC MODIFICATIONS

Rationale Chronic obstructive pulmonary disease (COPD) is a leading global cause of morbidity and mortality. Cigarette smoking (CS) is the main risk factor for the development of COPD and most COPD exacerbations are caused by respiratory infections including influenza. Influenza A virus (IAV) infections are more severe in smokers with increased morbidity and mortality. The mechanism of increased susceptibility to infections in smokers is likely due to alteration of immunologic host defenses. Our group has shown that Retinoic acid-inducible protein I (RIG-I) induction in the lung is inhibited by CS in our human lung organ culture model and two animal models. Methods We investigated RIG-I, TLR3 and interferon (IFN) induction by IAV in human bronchial epithelial cells (HBEC) isolated from smokers or non-smokers. Subcultured HBECs were infected with A/Puerto Rico/8/1934 (PR8) influenza virus at an MOI of 1. Virus-free diluents (mock) were used as negative controls. After 24 h infection, cells and supernatants were collected for qRT-PCR, immunoblot or ELISA to determine RIG-I, TLR3 and IFN expression levels. Results As expected, we found IAV PR8 exposure induced a vigorous IFN-β, IFN-λ 1 and IFN-λ 2/3 antiviral response in HBECs from nonsmokers as well as high RIG-I and TLR3 induction by virus. In cells from smokers, viral RIG-I and TLR3 mRNA induction was reduced 87% and 79% compared to the response from nonsmokers. CS inhibited viral induction of the IFN-β, IFN-λ1 and IFN-λ 2/3 mRNA response 85%, 96% and 95%, respectively, from that seen in HBEC from nonsmokers. However, preincubation with the demethylating agent 5-Aza-2-deoxycytidine reversed the immunosuppressive effects of CS exposure in HBEC since normal induction of all three IFNs was restored. We also demonstrated, even without virus infection, that IFN-β-mediated induction of RIG-I and TLR3 was also suppressed in cells from smokers. Conclusions Our results suggest that active smoking reduces expression of antiviral cytokines in primary HBECs. This effect likely occurs via downregulation of RIG-I and TLR3 through epigenetic modifications. Reduction in lung epithelial cell RIG-I and TLR3 responses may be one major mechanism contributing to the increased incidence of viral respiratory infections in smokers and to viral induction of acute exacerbations of COPD.

36 BACILLUS ANTHRACIS SPORES STIMULATE CYTOKINE AND CHEMOKINE INNATE IMMUNE RESPONSES IN HUMAN ALVEOLAR MACROPHAGES THROUGH MULTIPLE MAPK PATHWAYS.

Contact with the human alveolar macrophage plays a key role in the innate immune response to Bacillus anthracis spores. Because there is a significant delay between the initial contact of the spore with the host and clinical evidence of disease, there appears to be temporary containment of the pathogen by the innate immune system. Therefore, the early macrophage response to anthrax exposure is important in understanding the pathogenesis of this disease. We examined the initial events after exposure of human alveolar macrophages obtained by bronchoscopy to Bacillus anthracis (Sterne) spores. Spores were rapidly internalized as determined by confocal microscopy. Spore exposure also rapidly activated the MAPK signaling pathways ERK, JNK, and P38. This was followed by transcriptional activation of cytokine and primarily monocyte chemokine genes as determined by RNase protection assays. Transcriptional induction was reflected at the translational level as IL-1a and b, IL-6, and TNF-a cytokine protein levels were markedly elevated as determined by ELISA. Induction of IL-6 and TNF-a, and to a lesser extent IL-1a and -b, was partially inhibited by blockade of individual mitogen-activated protein kinases, while complete inhibition of cytokine induction was achieved when multiple signaling pathway inhibitors were used. Taken together, these data clearly show activation of the innate immune system in human alveolar macrophages by Bacillus anthracis spores. The data also show that multiple signaling pathways are involved in this cytokine response.

73 CALCIUM DEPLETION PREVENTS ADENOVIRUS INDUCTION OF INTERLEUKIN-8 AND CELL ENTRY, BUT NOT ERK ACTIVATION

The host response to adenovirus (Ad) infection involves induction of cytokines in both hemopoietic cells and lung epithelia. This is accompanied by an initial neutrophilic alveolitis. We have demonstrated induction of the lung neutrophil chemokine IL-8 by Ad7, a major lung pathogen, in A549 lung epithelial cells and lung tissue in an Erk (extracellular-signal-regulated-kinase)-dependent manner. We wished to determine the mechanism of chemokine induction from the standpoint of the Ad infectious cycle in the epithelial cell and whether Erk induction is sufficient for this process. A549 cells were exposed to Ad treated with psoralen/UV irradiation, which terminates the infectious cycle prior to viral gene expression. This treatment significantly diminished viral gene expression but not IL-8 gene induction as measured by semiquantitative RT-PCR. We then inhibited internalization of Ad by treatment of cells with EGTA in the presence of calcium-free media. The treatment inhibited internalization by 81% as determined by FACS for Ad hexon in the presence or absence of trypsin, and as visually determined by confocal microscopy. We next measured the effect of inhibition of internalization by EGTA + calcium free conditions on IL-8 induction and Erk induction. PMA (100 ng/mL) was used as a positive control, virus-free buffer as a negative control. Ad7 and PMA induced IL-8 release by 3-4 fold and 12-22 fold respectively over buffer-exposed cells. Conditions preventing Ad internalization inhibited IL-8 induction 70-80% by Ad7, but only 8-37% by PMA. The same conditions transiently delayed, but did not significantly inhibit Erk induction by Ad7. The results demonstrate that inhibition of Ad internalization, but not viral gene expression, prevents the cellular response of IL-8 induction by Ad. The data also show that Erk induction occurs prior to internalization and is required for IL-8 induction. Thus the site of induction of IL-8 by Ad is internal to the cell membrane, but external to the nucleus. In addition, Erk induction, while necessary, is not sufficient for IL-8 induction by Ad.