Anthrax Lethal Toxin Inhibits the Production of Proinflammatory Cytokines (original) (raw)
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Sublethal Doses of Anthrax Lethal Toxin on the Suppression of Macrophage Phagocytosis
PLOS One, 2010
Background: Lethal toxin (LT), the major virulence factor produced by Bacillus anthracis, has been shown to suppress the immune system, which is beneficial to the establishment of B. anthracis infections. It has been suggested that the suppression of MEK/MAPK signaling pathways of leukocytes contributes to LT-mediated immunosuppressive effects. However, the involvement of MAPK independent pathways has not been clearly elucidated; nor has the crucial role played by LT in the early stages of infection. Determining whether LT exerts any pathological effects before being enriched to an MEK inhibitory level is an important next step in the furtherance of this field.
Effect ofBacillus anthracislethal toxin on human peripheral blood mononuclear cells
FEBS Letters, 2002
Lethal toxin (LeTx) plays a central role in anthrax pathogenesis, however a cytotoxicity of LeTx has been di⁄cult to demonstrate in vitro. No cytolytic e¡ect has been reported for human cells, in contrast to murine cell lines, indicating that cell lysis can not be considered as a marker of LeTx activity. We have recently shown that murine macrophage-like RAW 264.7 cells treated with LeTx or infected with anthrax spores underwent changes typical of apoptotic death. Here we demonstrate that cells from human peripheral blood display a proapoptotic behavior similar to murine cells. TUNEL assay detected a nucleosomal degradation typical of apoptosis in peripheral blood mononuclear cells (PBMC) treated with LeTx. Membrane staining with apoptotic dyes was detected in macrophages derived from monocytes in presence of LeTx. The toxin inhibited production of proin£ammatory cytokines in PBMC stimulated with a preparation of Bacillus anthracis cell wall. Infection of PBMC with anthrax spores led to the appearance of a large population of cells stained positively for apoptosis, with a reduced capacity to eliminate spores and vegetative bacteria. The aminopeptidase inhibitor, bestatin, capable of protecting cells from LeTx, restored a bactericidal activity of infected cells. These ¢ndings may be explained by LeTx expression within phagocytes and support an important role of LeTx as an early intracellular virulence factor contributing to bacterial dissemination and disease progression.
Lethal toxin of Bacillus anthracis causes apoptosis of macrophages
Biochemical and Biophysical Research Communications, 2002
Lethal toxin is a major anthrax virulence factor, causing the rapid death of experimental animals. Lethal toxin can enter most cell types, but only certain macrophages and cell lines are susceptible to toxin-mediated cytolysis. We have shown that in murine RAW 264.7 cells, sublytic amounts of lethal toxin trigger intracellular signaling events typical for apoptosis, including changes in membrane permeability, loss of mitochondrial membrane potential, and DNA fragmentation. The cells were protected from the toxin by specific inhibitors of caspase-1, -2, -3, -4, -6, and -8. Phagocytic activity of macrophages was inhibited by sublytic concentrations of lethal toxin. Infection of cells with anthrax (Sterne) spores impaired their bactericidal capacity, which could be reversed by a lethal toxin inhibitor, bestatin. We suggest that apoptosis rather than direct lysis is biologically relevant to lethal toxin intracellular activity. Ó
Anthrax toxins: a paradigm of bacterial immune suppression
Trends in Immunology, 2006
Several species of microorganism have developed immune evasion and/or immunosuppression strategies. Bacillus anthracis secretes two toxins, edema toxin and lethal toxin, that enter the cytosol of almost every cell type, including the cells of the innate and adaptive immune systems, and subvert cell signaling. Edema toxin causes a consistent elevation of cyclic adenosine monophosphate, whereas lethal toxin cleaves most isoforms of mitogen-activated protein kinase kinases. In a concerted manner, these toxins alter major signaling pathways involved in the development of immune-cell effector functions, with the inhibition of bacterial clearance by phagocytes and of B. anthracis-specific responses. Thus, B. anthracis can invade the host, with ensuing massive bacteremia and toxemia. Here, we review the specific effects of B. anthracis on neutrophils, macrophages, dendritic cells, T-and B-lymphocytes.
Systemic cytokine response in murine anthrax
Cellular Microbiology, 2004
Systemic pro-inflammatory cytokine release has been previously implicated as a major death-causing factor in anthrax, however, direct data have been absent. We determined the levels of IL-1 β β β β , IL-6 and TNF-α α α α in serum of mice challenged with virulent (Ames) or attenuated (Sterne) strains of Bacillus anthracis . More than 10-fold increase in the IL-1 β β β β levels was detected in Ames-challenged Balb/c mice, in contrast to more susceptible C57BL/6 mice, which showed no IL-1 β β β β response. Balb/c mice have also responded with higher levels of IL-6. The A/J mice demonstrated IL-1 β β β β and IL-6 systemic response to either Ames or Sterne strain of B. anthracis , whereas no increase in TNF-α α α α was detected in any murine strain. We used RT-PCR for gene expression analyses in the liver which often is a major source of cytokines and one of the main targets in infectious diseases. A/J mice challenged with B. anthracis (Sterne) showed increased gene expression for Fas, FasL, Bax, IL-1 β β β β , TNF-α α α α , TGFβ β β β , MIP-1 α α α α , KC and RANTES. These data favour the hypothesis that apoptotic cell death during anthrax infection causes chemokine-induced transmigration of inflammatory cells to vitally important organs such as liver. Administration of caspase inhibitors z-VADfmk and ac-YVAD-cmk improved survival in Sternechallenged mice indicating a pathogenic role of apoptosis in anthrax.
2003
Macrophages from different inbred mouse strains exhibit striking differences in their sensitivity to anthrax lethal toxin (LeTx)-induced cytolysis. Although LeTxinduced cytolysis of macrophages plays an important role in the outcome of anthrax infection, the sensitivity of macrophages in vitro does not correlate with in vivo susceptibility to infection of Bacillus anthracis. This divergence suggests that additional factors other than LeTx are involved in the cytolysis of LeTx-resistant macrophages in vivo. We found that LeTx-resistant macrophages became sensitive to LeTx-induced cytolysis when these cells were activated by bacterial components. Tumor necrosis factor-␣ induced by bacterial components was a key factor that cooperated with LeTx in inducing LeTx-resistant macrophage death. Tumor necrosis factor-␣/LeTx-induced death of LeTx-resistant macrophages was dependent on mTor (mammalian target of rapamycin), but independent of caspases. Our data indicate that host responses to anthrax infection contribute to cytolysis of LeTx-resistant 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.
Infection and Immunity, 2004
We investigated the ability of using monoclonal antibodies (MAbs) against anthrax protective antigen (PA), an anthrax exotoxin component, to modulate exotoxin cytotoxic activity on target macrophage cell lines. Anthrax PA plays a critical role in the pathogenesis of Bacillus anthracis infection. PA is the cell-binding component of the two anthrax exotoxins: lethal toxin (LeTx) and edema toxin. Several MAbs that bind the PA component of LeTx are known to neutralize LeTx-mediated killing of target macrophages. Here we describe for the first time an overlooked population of anti-PA MAbs that, in contrast, function to increase the potency of LeTx against murine macrophage cell lines. The results support a possible mechanism of enhancement: binding of MAb to PA on the macrophage cell surface stabilizes the PA by interaction of MAb with macrophage Fc␥ receptors. This results in an increase in the amount of PA bound to the cell surface, which in turn leads to an enhancement in cell killing, most likely due to increased internalization of LF. Blocking of PA-receptor binding eliminates enhancement by MAb, demonstrating the importance of this step for the observed enhancement. The additional significance of these results is that, at least in mice, immunization with PA appears to elicit a polyclonal response that has a significant prevalence of MAbs that enhance LeTx-mediated killing in macrophages. on September 15, 2013 by guest http://iai.asm.org/ Downloaded from MATERIALS AND METHODS Cell lines. Murine macrophage cell lines J774.1, RAW 264.7, and P388D 1 , human macrophage cell lines U937 and THP-1, and Chinese hamster ovary (CHO) cells were obtained from the American Type Culture Collection (ATCC) and were maintained according to ATCC protocols. The Fc␥R-transfected CHO cell line mRG1-1, a CHO cell line transfected with the binding chain of the mouse Fc␥RI , was a gift from Daniel Conrad, Virginia Commonwealth University. Early-passage cells were frozen down to create working cell banks. Murine macrophage cell lines J774A.1, RAW 264.7, and P388D 1 were grown in Dulbecco's modified Eagle medium containing 10% fetal bovine serum (FBS). Human macrophage cell lines U937 and THP-1 were grown in RPMI containing 10% FBS. CHO cells and mRG1-1 cells were grown in F12K containing 5% FBS. To ensure consistency in bioassay results, cells were discarded after 12 to 13 passages and a new working cell bank vial was thawed for use.