Vineet I. Patel

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.

Transcriptional Classification and Functional Characterization of Human Airway Macrophage and Dendritic Cell Subsets

The respiratory system is a complex network of many cell types, including subsets of macrophages and dendritic cells that work together to maintain steady-state respiration. Owing to limitations in acquiring cells from healthy human lung, these subsets remain poorly characterized transcriptionally and phenotypically. We set out to systematically identify these subsets in human airways by developing a schema of isolating large numbers of cells by whole-lung bronchoalveolar lavage. Six subsets of phagocytic APC (HLA-DR+) were consistently observed. Aside from alveolar macrophages, subsets of Langerin+, BDCA1−CD14+, BDCA1+CD14+, BDCA1+CD14−, and BDCA1−CD14− cells were identified. These subsets varied in their ability to internalize Escherichia coli, Staphylococcus aureus, and Bacillus anthracis particles. All subsets were more efficient at internalizing S. aureus and B. anthracis compared with E. coli. Alveolar macrophages and CD14+ cells were overall more efficient at particle internalization compared with the four other populations. Subsets were further separated into two groups based on their inherent capacities to upregulate surface CD83, CD86, and CCR7 expression levels. Whole-genome transcriptional profiling revealed a clade of “true dendritic cells” consisting of Langerin+, BDCA1+CD14+, and BDCA1+CD14− cells. The dendritic cell clade was distinct from a macrophage/monocyte clade, as supported by higher mRNA expression levels of several dendritic cell–associated genes, including CD1, FLT3, CX3CR1, and CCR6. Each clade, and each member of both clades, was discerned by specific upregulated genes, which can serve as markers for future studies in healthy and diseased states.

Identification and characterization of human dendritic cell subsets in the steady state: a review of our current knowledge

Dendritic cells (DC) are generally categorized as a group of rare antigen presenting cells that are to the crucial development of immune responses to pathogens and also of tolerance to self-antigens. Therefore, having the ability to identify DC in specific tissues and to test their functional abilities in the steady state are scientific gaps needing attention. Research on primary human DC is lacking due to their rarity and the difficulty of obtaining tissue samples. However, recent findings have shown that several different DC subsets exist, and that these subsets vary both by markers expressed and functions depending on their specific microenvironment. After discriminating from other cell types, DC can be split into myeloid and plasmacytoid fractions. While plasmacytoid DC express definite markers, CD123 and BDCA-2, myeloid DC encompass several different subsets with overlapping markers expressed. Such markers include the blood DC antigens BDCA-1 and BDCA-3, along with Langerin, CD1a and CD14. Marker specificity is further reduced when accounting for microenvironmental differences, as observed in the blood, primary lymphoid tissues, skin and lungs. The mixed leukocyte reaction (MLR) has been used to measure the strength of antigen presentation by specific DC subsets. Surface markers and MLR require standardization to enable consistent identification of and comparisons between DC subsets. To alleviate these issues, researchers have begun comparing DC subsets at the transcriptional level. This has allowed degrees of relatedness to be determined between DC in different microenvironments, and should be a continued area of focus in years to come.

Bacillus anthracis spore movement does not require a carrier cell and is not affected by lethal toxin in human lung models

The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores escape from the alveolus to regional lymph nodes, germinate and enter the circulatory system to cause disease. The roles of carrier cells and the effects of B. anthracis toxins in this process are unclear. We used a human lung organ culture model to measure spore uptake by antigen presenting cells (APC) and alveolar epithelial cells (AEC), spore partitioning between these cells, and the effects of B. anthracis lethal toxin and protective antigen. We repeated the study in a human A549 alveolar epithelial cell model. Most spores remained unassociated with cells, but the majority of cell-associated spores were in AEC, not in APC. Spore movement was not dependent on internalization, although the location of internalized spores changed in both cell types. Spores also internalized in a non-uniform pattern. Toxins affected neither transit of the spores nor the partitioning of spores into AEC and APC. Our results support a model of spore escape from the alveolus that involves spore clustering with transient passage through intact AEC. However, subsequent transport of spores by APC from the lung to the lymph nodes may occur.

Gene expression profiling of primary human type I alveolar epithelial cells exposed to Bacillus anthracis spores reveals induction of neutrophil and monocyte chemokines

The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores must escape through the alveolar epithelial cell (AEC) barrier and migrate to regional lymph nodes, germinate and enter the circulatory system to cause disease. Several mechanisms to explain alveolar escape have been postulated, and all these tacitly involve the AEC barrier. In this study, we incorporate our primary human type I AEC model, microarray and gene enrichment analysis, qRT-PCR, multiplex ELISA, and neutrophil and monocyte chemotaxis assays to study the response of AEC to B. anthracis, (Sterne) spores at 4 and 24 h post-exposure. Spore exposure altered gene expression in AEC after 4 and 24 h and differentially expressed genes (±1.3 fold, p ≤ 0.05) included CCL4/MIP-1β (4 h), CXCL8/IL-8 (4 and 24 h) and CXCL5/ENA-78 (24 h). Gene enrichment analysis revealed that pathways involving cytokine or chemokine activity, receptor binding, and innate immune responses to infection were prominent. Microarray results were confirmed by qRT-PCR and multiplex ELISA assays. Chemotaxis assays demonstrated that spores induced the release of biologically active neutrophil and monocyte chemokines, and that CXCL8/IL-8 was the major neutrophil chemokine. The small or sub-chemotactic doses of CXCL5/ENA-78, CXCL2/GROβ and CCL20/MIP-3α may contribute to chemotaxis by priming effects. These data provide the first whole transcriptomic description of the human type I AEC initial response to B. anthracis spore exposure. Taken together, our findings contribute to an increased understanding of the role of AEC in the pathogenesis of inhalational anthrax.

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.