Gene expression profiling of primary human type I alveolar epithelial cells exposed to Bacillus anthracis spores reveals induction of neutrophil and monocyte chemokines
Introduction
Anthrax, a virulent and zoonotic disease recognized since early human history, is caused by Bacillus anthracis, a Gram-positive, aerobic, spore-forming, rod-shaped bacterium. Anthrax disease manifests in three primary forms resulting from three different mechanisms of exposure to spores: Ingestion (gastrointestinal), skin contact (cutaneous), or inhalation (inhalational or pneumonic) [1]. Although all forms of disease can lead to fatal systemic infection, inhalation anthrax is the most life-threatening form, with significant mortality even with treatment, and nearly 100% mortality in the absence of early treatment [1,2].
The lung is the site of entry for B. anthracis spores in inhalation anthrax, however, the organism does not cause primary disease in the lung [[3], [4], [5]]. Following exposure to spores, there is a significant delay, as long as 43 days, before clinical disease. This implies that there is temporary containment of the pathogen, which is likely mediated by the innate immune system [2]. In order for disease to occur, spores must escape the alveolus and then pass through the lymphatic system to the mediastinal lymph nodes. Vegetative bacteria subsequently disseminate via lymphatic ducts with subsequent appearance in the bloodstream [1,6,7]. Thus, a critical step in disease pathogenesis is alveolar escape: overcoming local containment of the pathogen in the alveolus and the traversing the epithelial barrier.
Alveolar escape of the pathogen has been a focus of many studies, and has resulted in the formulation of several proposed mechanisms for how this occurs. Four mechanisms for alveolar escape have been postulated, and two of these pathways involve a lung carrier cell. It should be noted that AEC are stationary and not considered carrier cells, yet they are tacit players in all four pathways because they form the alveolar epithelial barrier through which the spores must pass.
The initial two proposed mechanisms of alveolar escape incorporate a migratory or “carrier” cell, often referred to as a “Trojan horse”, for spore uptake and subsequent dissemination. First, macrophages were proposed as the Trojan horse, because alveolar macrophages (AM) ingest spores and because studies suggested that B. anthracis virulence toxins damage or impair these cells [7,8]. However, other studies have shown spore dissemination from the lung in macrophage-depleted mice [4], and we have shown that primary human AM are not susceptible to toxins [9], indicating that macrophages may not play a key role in alveolar escape, assuming that the Trojan horse is sensitive to toxins. This work and the work of others has suggested that dendritic cells (DC) may be the Trojan horse. DC and other cells containing spores were observed in the thoracic lymph nodes of mice [3,10]. DC sample the airway luminal surface [11]. Human lung DC internalize B. anthracis spores [12].
A third possibility is that AEC alone are involved in alveolar escape and that a Trojan horse is not required. Isolated cell culture models suggest a transcellular route of spore passage through the lung epithelium [13]. Using the human lung epithelial cell line A549 and primary human small airway cells (SAEC), both of which internalize spores, Russell et al. [14] showed that spores survived and were translocated from the apical to basolateral side of the cell. Spore internalization in A549 cells involves interaction of the spore BclA protein with cell integrin α2β1 and complement C1q [15]. These results suggest that spores may cross the alveolar epithelial barrier and disseminate without the assistance of migratory cells [14]. Finally, in the “Jailbreak” model, it is proposed that the clustering and germination of spores within the alveolus followed by production of various B. anthracis virulence toxins causes epithelial damage which permits free spore passage not involving a Trojan horse [16].
All of these theories demonstrate that the interaction of spores with alveolar epithelium is important in disease pathogenesis. This interaction would occur between the pathogen and one of two alveolar epithelial cell types that line the alveolus. These cell types are Type I alveolar epithelial cells (AEC I) and type II AEC (AEC II). AEC II cells are classically described as the precursors of AEC I though there may be a common precursor to type I and type II AEC [17]. AEC II are cuboidal in shape, produce surfactant, and line approximately 5% of the surface area of the alveoli. AEC I have flattened 45–60 nm thick cytoplasm, are terminally differentiated, and cover the remaining 95% of the alveolar epithelial surface [18]. An AEC I-like cell can be derived from human lungs by specialized isolation and culture techniques facilitating investigation of the role of these cells in anthrax pathogenesis [19].
We used an unbiased “top-down” transcriptomic approach to evaluate the transcriptional response to the initial interaction of B. anthracis spores with AEC I-like cells. There were no prerequisites or biases towards particular functions or pathways. This differs from kits and platforms that assess individual processes (e.g., apoptosis or inflammation) based on previous knowledge, and therefore, introduce preexisting bias by exclusion of other genes. The method of microarray data analysis we employed used not only statistical comparisons, but also considered biological properties of the data [20] in order to find unbiased, but reliable, results. The approach was to identify stable differentially expressed genes, i.e. genes whose expression was consistently low among microarrays in one case and consistently high in another. This increased the specificity of analysis and allowed us to identify the most robust “beacons” driving response to the pathogen. The results provided us with information about the major processes affected. Examination of the whole dataset based on this knowledge identified additional highly dynamic co-players that were selected by strict statistical criteria.
Our earlier studies examined the interaction of anthrax spores with intact lung epithelia in a human lung slice model. We studied the initial events up to 48 h after exposing intact lung tissue slices to B. anthracis spores. Spore exposure caused transcriptional activation of cytokines and chemokine genes and the data was confirmed at the level of translation. Immuno-staining for IL-6 and IL-8 in spore-exposed lung slices revealed that alveolar epithelial cells and macrophages and a few interstitial cells were the source of the cytokines and chemokines [21].
In the current study, we infected human type I-like AEC with B. anthracis (Sterne) spores and performed transcriptome analysis to provide a comprehensive view of the innate immune response of the type I-like AEC to the pathogen. In spore-exposed AEC, cytokine and chemokine transcripts were more highly expressed compared to mock-infected cells. We also found that pathways involved in these processes were identified by functional and pathway enrichment analysis. We then confirmed this response at the transcriptional and translational level, and assessed the relative contribution of select chemokines on monocyte and neutrophil chemotaxis.
Section snippets
Isolation of primary human type I alveolar epithelial cells
The isolation and culture of human AEC, type II, have been described previously [19]. Briefly, three human lungs rejected for transplant were obtained from the International Institute for the Advancement of Medicine (IIAM; Jessup, PA, USA) under a protocol approved by the University of Oklahoma Health Sciences Institutional Review Board. Following perfusion of 1 lobe with PBS without Ca2+ or Mg2+ and lavage with physiological (150 mM) NaCl containing 100 Kunitz units (KU) per mg protein of
B. anthracis spore exposure alters gene expression in primary human AEC I-like cells
Initial interactions of the B. anthracis spore with the alveolar epithelia are likely important in the pathogenesis of inhalational anthrax, the disease caused by this agent. We, therefore, sought to take an unbiased approach to examine the human AEC response to B. anthracis (Sterne) spores. Primary human AEC were purified from three normal human donor lungs, seeded on collagen and fibronectin-coated tissue plates at 25,000 cells/cm2, and grown for 10–14 days until confluence [19]. Culturing
Discussion
Escape of B. anthracis from the alveolar space of the lung is required for the pathogen to produce the fatal disease known as inhalation anthrax [1]. The time between spore exposure and symptoms can vary from days to weeks and the incubation time is inversely dependent on the dose of spores [1]. This latency period offers an opportunity to block progression of the disease. Therefore, understanding the initial interactions of the B. anthracis spore with the alveolar epithelium is important in
Acknowledgments
The research described in this work was partially supported by the Federal Aviation Administration, Office of Aviation Medicine, by a Clinical Innovator Award from the Flight Attendant Medical Research Institute, by the Merit Review Program of the Department of Veterans Affairs, the National Institutes of Health [grant number 1 I01 BX001937], and by the National Institutes of Health, [grant numbers U19AI62629 to K.M.C and GM103648 to J.P.M.].
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J.L.B. and E.S.D. contributed equally to this work.