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Gene Expression of Primary Human Type I Alveolar Epithelial Cells Exposed to Bacillus anthracis, Sterne endospores

ABSTRACT: 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 gene profiling and gene enrichment analysis to study the response of AEC to B. anthracis, (Sterne) spores at 4 and 24 hours post-exposure. Spore exposure altered gene expression in AEC after 4 and 24 hours and differentially expressed genes (±1.3 fold, p ≤ 0.05) included CCL4/MIP-1β (4 hours), CXCL8/IL-8 (4 and 24 hours) and CXCL5/ENA-78 (24 hours). 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, CXCL3/GROββ 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, and contribute to an increased understanding of the role of AEC in the pathogenesis of inhalational anthrax. We used microarrays to create a whole transcriptomic description of the response of primary human type I alveolar epithelial cells to B. anthracis spore exposure and demonstrated that several of the most upregulated differentially expressed genes included those for neutrophil and monocyte chemokines.

ORGANISM(S): Homo sapiens

PROVIDER: GSE102106 | GEO | 2018/08/03

REPOSITORIES: GEO

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Project description:Bacillus anthracis is a gram-positive, aerobic, spore-forming, rod-shaped bacterium which has recently been used as an agent of bioterrorism. 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. Contact with the human alveolar macrophage (HAM) plays a key role in the innate immune response to B. anthracis spores. Therefore, the early macrophage response to anthrax exposure is important in understanding the pathogenesis of this disease. The majority of genes modulated by spores were upregulated, and a lesser number were downregulated. The data was subjected to Ingenuity Pathway analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis, and the Promoter Analysis and Interaction Network Toolset (PAINT). Among the upregulated genes, we identified a group of chemokine ligands, apoptosis genes and, interestingly, keratin filament genes. Central hubs regulating the activated genes were TNF-ï¡, NF-ÎºB and their ligands/receptors. Other activated genes included IL-1ï¡ and IL-18. RNA for these, and several additional cytokines including IL-6, IL-1ï¢, IP-10 and GM-CSF, were differentially expressed from 1.6- to 27-fold. The microarray cytokine data is consistent with our previously published findings which demonstrated that there was 4- to 43-fold induction of these cytokines at the transcriptional and translational levels as determined by RNase protection assays and ELISA. The PAINT analysis revealed that the majority of the genes affected by spores contain the binding site for c-Rel, a member of the NF-ÎºB family of transcription factors. Other transcription regulatory elements contained in many of the upregulated genes were c-Myb, CP2, Barbie Box, E2F and CRE-BP1. This study is the first detailed microarray analysis to describe the HAM response to B. anthracis. Experiment Overall Design: In this paper, we exposed HAM to B. anthracis Sterne spores at an MOI of 1 for 6 hours. RNA was extracted from HAM and analyzed by the Affymetrix Human Genome U133 Plus 2.0 Array. The transcriptional profile of Bacillus anthracis spore-treated HAM was compared with mock infected cells, and differentially expressed genes were identified.

Project description:Bacillus anthracis is a gram-positive, aerobic, spore-forming, rod-shaped bacterium which has recently been used as an agent of bioterrorism. 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. Contact with the human alveolar macrophage (HAM) plays a key role in the innate immune response to B. anthracis spores. Therefore, the early macrophage response to anthrax exposure is important in understanding the pathogenesis of this disease. The majority of genes modulated by spores were upregulated, and a lesser number were downregulated. The data was subjected to Ingenuity Pathway analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis, and the Promoter Analysis and Interaction Network Toolset (PAINT). Among the upregulated genes, we identified a group of chemokine ligands, apoptosis genes and, interestingly, keratin filament genes. Central hubs regulating the activated genes were TNF-alpha, NF-κB and their ligands/receptors. Other activated genes included IL-1alpha and IL-18. RNA for these, and several additional cytokines including IL-6, IL-1gamma, IP-10 and GM-CSF, were differentially expressed from 1.6- to 27-fold. The microarray cytokine data is consistent with our previously published findings which demonstrated that there was 4- to 43-fold induction of these cytokines at the transcriptional and translational levels as determined by RNase protection assays and ELISA. The PAINT analysis revealed that the majority of the genes affected by spores contain the binding site for c-Rel, a member of the NF-κB family of transcription factors. Other transcription regulatory elements contained in many of the upregulated genes were c-Myb, CP2, Barbie Box, E2F and CRE-BP1. This study is the first detailed microarray analysis to describe the HAM response to B. anthracis.

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Gene Expression of Primary Human Type I Alveolar Epithelial Cells Exposed to Bacillus anthracis, Sterne endospores

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