K. M. Coggeshall

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.

The sepsis model: an emerging hypothesis for the lethality of inhalation anthrax

Inhalation anthrax is often described as a toxin‐mediated disease. However, the toxaemia model does not account for the high mortality of inhalation anthrax relative to other forms of the disease or for the pathology present in inhalation anthrax. Patients with inhalation anthrax consistently show extreme bacteraemia and, in contrast to animals challenged with toxin, signs of sepsis. Rather than toxaemia, we propose that death in inhalation anthrax results from an overwhelming bacteraemia that leads to severe sepsis. According to our model, the central role of anthrax toxin is to permit the vegetative bacteria to escape immune detection. Other forms of B. anthracis infection have lower mortality because their overt symptoms early in the course of disease cause patients to seek medical care at a time when the infection and its sequelae can still be reversed by antibiotics. Thus, the sepsis model explains key features of inhalation anthrax and may offer a more complete understanding of disease pathology for researchers as well as those involved in the care of patients.

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.