Gene expression profiling of human alveolar macrophages infected by B. anthracis spores demonstrates TNF-α and NF-κb are key components of the innate immune response to the pathogen (original) (raw)
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Microbial pathogenesis, 2018
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 t...
Germination of Bacillus anthracis spores within alveolar macrophages
Molecular Microbiology, 1999
The fatal character of the infection caused by inhalation of Bacillus anthracis spores results from a complex pathogenic cycle involving the synthesis of toxins by the bacterium. We have shown using immunofluorescent staining, confocal scanning laser microscopy and image cytometry analysis that the alveolar macrophage was the primary site of B. anthracis germination in a murine inhalation infection model. Bacillus anthracis germinated inside murine macrophage-like RAW264.7 cells and murine alveolar macrophages. Germination occurred in vesicles derived from the phagosomal compartment. We have also demonstrated that the toxin genes and their trans-activator, AtxA, were expressed within the macrophages after germination.
Infection and Immunity, 2006
The development of new approaches to combat anthrax requires that the pathogenesis and host response to Bacillus anthracis spores be better understood. We investigated the roles that macrophages and neutrophils play in the progression of infection by B. anthracis in a mouse model. Mice were treated with a macrophage depletion agent (liposome-encapsulated clodronate) or with a neutrophil depletion agent (cyclophosphamide or the rat anti-mouse granulocyte monoclonal antibody RB6-8C5), and the animals were then infected intraperitoneally or by aerosol challenge with fully virulent, ungerminated B. anthracis strain Ames spores. The macrophage-depleted mice were significantly more susceptible to the ensuing infection than the salinepretreated mice, whereas the differences observed between the neutropenic mice and the saline-pretreated controls were generally not significant. We also found that augmenting peritoneal neutrophil populations before spore challenge did not increase resistance of the mice to infection. In addition, the bacterial load in macrophage-depleted mice was significantly greater and appeared significantly sooner than that observed with the saline-pretreated mice. However, the bacterial load in the neutropenic mice was comparable to that of the saline-pretreated mice. These data suggest that, in our model, neutrophils play a relatively minor role in the early host response to spores, whereas macrophages play a more dominant role in early host defenses against infection by B. anthracis spores.
Role of Bacillus anthracis Spore Structures in Macrophage Cytokine Responses
Infection and Immunity, 2007
The innate immune response of macrophages (M) to spores, the environmentally acquired form of Bacillus anthracis, is poorly characterized. We therefore examined the early M cytokine response to B. anthracis spores, before germination. M were exposed to bacilli and spores of Sterne strain 34F2 and its congenic nongerminating mutant (⌬gerH), and cytokine expression was measured by real-time PCR and an enzymelinked immunosorbent assay. The exosporium spore layer was retained (exo ؉ ) or removed by sonication (exo ؊ ). Spores consistently induced a strong cytokine response, with the exo ؊ spores eliciting a two-to threefold-higher response than exo ؉ spores. The threshold for interleukin-1 (IL-1) production by wild-type M was significantly lower than that required for tumor necrosis factor alpha expression. Cytokine production was largely dependent on MyD88, suggesting Toll-like receptor involvement; however, the expression of beta interferon in MyD88 ؊/؊ M suggests involvement of a MyD88-independent pathway. We conclude that (i) the B. anthracis spore is not immunologically inert, (ii) the exosporium masks epitopes recognized by the M, (iii) the M cytokine response to B. anthracis involves multiple pattern recognition receptors and signaling pathways, and (iv) compared to other cytokines, IL-1 is expressed at a lower spore concentration.
Bacillus anthracis Lethal Toxin Reduces Human Alveolar Epithelial Barrier Function
Infection and Immunity, 2012
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 alveo...
Differential susceptibility of macrophage cell lines to Bacillus anthracis–Vollum 1B
Toxicology in Vitro, 2005
Bacillus anthracis (BA) is a spore forming bacterium and the causative agent of anthrax disease. Macrophages (M/s) play a central role in anthrax disease. An important step in disease progression is the ability of BA to secrete lethal toxin (LeTx) that kills M/s. LeTx is a heterodimer composed of protective antigen (PA) and lethal factor (LF). Researchers have shown that M/ cell lines demonstrate differential susceptibility to purified LeTx; for example RAW264.7 and J774A.1 M/s are sensitive to LeTx whereas IC-21 M/s are resistant. Research has also suggested that exogenous factors, including other BA proteins, can influence the activity of LeTx. For this reason, the objective of the current work was to examine if RAW264.7, J774A.1, and IC-21 M/s demonstrated differential susceptibility when cultured with a LeTx-producing strain of BA. Here, we co-cultured M/s with LeTx + Vollum 1B (V1B) spores for >15 h and assayed for M/ cell death by morphology, trypan blue (TB) staining, neutral red (NR) activity, and lactate dehydrogenase (LDH) activity in the culture media. Following the addition of V1B spores, necrosis (%50% mortality) was observed in RAW264.7 and J774A.1 M/s at 7.5 and 10 h, respectively. By 15 h, both RAW264.7 and J774A.1 M/s demonstrated 100% mortality. In contrast, IC-21 M/s, under identical culture conditions, remained viable (98%) and activated throughout the course of the experiment (>24 h). The mechanism of RAW264.7 cell death appeared to involve LeTx because the V1B-induced cytotoxicity was dose-dependently reversed by the addition of anti-PA antibody to the culture media. These observations suggest there is differential susceptibility of M/ cell lines to the LeTx + V1B strain of BA. Further development of this in vitro model may be useful to further characterize the interactions between M/s and BA spores. Published by Elsevier Ltd.
Murine Macrophages Kill the Vegetative Form of Bacillus anthracis
Infection and Immunity, 2005
Anti-protective antigen antibody was reported to enhance macrophage killing of ingested Bacillus anthracis spores, but it was unclear whether the antibody-mediated macrophage killing mechanism was directed against the spore itself or the vegetative form emerging from the ingested and germinating spore. To address this question, we compared the killing of germination-proficient (gp) and germination-deficient (⌬gerH) Sterne 34F2 strain spores by murine peritoneal macrophages. While macrophages similarly ingested both spores, only gp Sterne was killed at 5 h (0.37 log kill). Pretreatment of macrophages with gamma interferon (IFN-␥) or opsonization with immunoglobulin G (IgG) isolated from a subject immunized with an anthrax vaccine enhanced the killing of Sterne to 0.49 and 0.73 log, respectively, but the combination of IFN-␥ and IgG was no better than either treatment alone. Under no condition was there killing of ⌬gerH spores. To examine the ability of the exosporium to protect spores from macrophages, we compared the macrophage-mediated killing of nonsonicated (exosporium ؉ ) and sonicated (exosporium ؊ ) Sterne 34F2 spores. More sonicated spores than nonsonicated spores were killed at 5 h (0.98 versus 0.37 log kill, respectively). Pretreatment with IFN-␥ increased the sonicated spore killing to 1.39 log. However, the opsonization with IgG was no better than no treatment or pretreatment with IFN-␥. We conclude that macrophages appear unable to kill the spore form of B. anthracis and that the exosporium may play a role in the protection of spores from macrophages.
Infection and Immunity, 2006
Alveolar macrophages (AM) are very important for pulmonary innate immune responses against invading inhaled pathogens because they directly kill the organisms and initiate a cascade of innate and adaptive immune responses. Although several factors contribute to inhalational anthrax, we hypothesized that unimpeded infection of Bacillus anthracis is directly linked to disabling the innate immune functions contributed by AM. Here, we investigated the effects of lethal toxin (LT), one of the binary complex virulence factors produced by B. anthracis, on freshly isolated nonhuman primate AM. Exposure of AM to doses of LT that killed susceptible macrophages had no effect on the viability of AM, despite complete MEK1 cleavage. Intoxicated AM remained fully capable of B. anthracis spore phagocytosis. However, pretreatment of AM with LT resulted in a significant decrease in the clearance of both the Sterne strain and the fully virulent Ames strain of B. anthracis, which may have been a result of impaired AM secretion of proinflammatory cytokines. Our data imply that cytolysis does not correlate with MEK1 cleavage, and this is the first report of LT-mediated impairment of nonhuman primate AM bactericidal activity against B. anthracis.
Dendritic Cells Endocytose Bacillus anthracis Spores: Implications for Anthrax Pathogenesis
The Journal of Immunology, 2005
Phagocytosis of inhaled Bacillus anthracis spores and subsequent trafficking to lymph nodes are decisive events in the progression of inhalational anthrax because they initiate germination and dissemination of spores. Found in high frequency throughout the respiratory track, dendritic cells (DCs) routinely take up foreign particles and migrate to lymph nodes. However, the participation of DCs in phagocytosis and dissemination of spores has not been investigated previously. We found that human DCs readily engulfed fully pathogenic Ames and attenuated B. anthracis spores predominately by coiling phagocytosis. Spores provoked a loss of tissue-retaining chemokine receptors (CCR2, CCR5) with a concurrent increase in lymph node homing receptors (CCR7, CD11c) on the membrane of DCs. After spore infection, immature DCs displayed a mature phenotype (CD83 bright , HLA-DR bright , CD80 bright , CD86 bright , CD40 bright ) and enhanced costimulatory activity. Surprisingly, spores activated the MAPK cascade (ERK, p38) within 30 min and stimulated expression of several inflammatory response genes by 2 h. MAPK signaling was extinguished by 6 h infection, and there was a dramatic reduction of secreted TNF-␣, IL-6, and IL-8 in the absence of DC death. This corresponded temporally with enzymatic cleavage of proximal MAPK signaling proteins (MEK-1, MEK-3, and MAP kinase kinase-4) and may indicate activity of anthrax lethal toxin. Taken together, these results suggest that B. anthracis may exploit DCs to facilitate infection. The Journal of Immunology, 2005, 174: 5545-5552.