Chemical genetic screening identifies critical pathways in anthrax lethal toxin-induced pathogenesis.

Anthrax lethal toxin (LT)-induced cell death via mitogen-activated protein kinase kinase (MAPKK) cleavage remains questionable. Here, a chemical genetics approach was used to investigate what pathways mediate LT-induced cell death. Several small molecules were found to protect macrophages from anthrax LT cytotoxicity and MAPKK from cleavage by lethal factor (LF), without inhibiting LF enzymatic activity or cellular proteasome activity. Interestingly, the compounds activated MAPK-signaling molecules, induced proinflammatory cytokine production, and inhibited LT-induced macrophage apoptosis in a concentration-dependent manner. We propose that induction of antiapoptotic responses by MAPK-dependent or -independent pathways and activation of host innate responses may protect macrophages from anthrax LT-induced cell death. Altering host responses through a chemical genetics approach can help identify critical cellular pathways involved in the pathogenesis of anthrax and can be exploited to further explore host-pathogen interactions.

Transcriptome analysis of human immune responses following live vaccine strain (LVS) Francisella tularensis vaccination.

The live vaccine strain (LVS) of Francisella tularensis is the only vaccine against tularemia available for humans, yet its mechanism of protection remains unclear. We probed human immunological responses to LVS vaccination with transcriptome analysis using PBMC samples from volunteers at time points pre- and post-vaccination. Gene modulation was highly uniform across all time points, implying commonality of vaccine responses. Principal components analysis revealed three highly distinct principal groupings: pre-vaccination (-144 h), early (+18 and +48 h), and late post-vaccination (+192 and +336 h). The most significant changes in gene expression occurred at early post-vaccination time points (

Deficiency of fatty acid-binding proteins in mice confers protection from development of experimental autoimmune encephalomyelitis.

Fatty acid-binding proteins (FABPs) act as intracellular receptors for a variety of hydrophobic compounds, enabling their diffusion within the cytoplasmic compartment. Recent studies have demonstrated the ability of FABPs to simultaneously regulate metabolic and inflammatory pathways. We investigated the role of adipocyte FABP and epithelial FABP in the development of experimental autoimmune encephalomyelitis to test the hypothesis that these FABPs impact adaptive immune responses and contribute to the pathogenesis of autoimmune disease. FABP-deficient mice exhibited a lower incidence of disease, reduced clinical symptoms of experimental autoimmune encephalomyelitis and dramatically lower levels of proinflammatory cytokine mRNA expression in CNS tissue as compared with wild-type mice. In vitro Ag recall responses of myelin oligodendrocyte glycoprotein 35-55-immunized FABP(-/-) mice showed reduced proliferation and impaired IFN-gamma production. Dendritic cells deficient for FABPs were found to be poor producers of proinflammatory cytokines and Ag presentation by FABP(-/-) dendritic cells did not promote proinflammatory T cell responses. This study reveals that metabolic-inflammatory pathway cross-regulation by FABPs contributes to adaptive immune responses and subsequent autoimmune inflammation.

Anthrax lethal toxin impairs innate immune functions of alveolar macrophages and facilitates Bacillus anthracis survival.

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.

The fatty acid-binding protein, aP2, coordinates macrophage cholesterol trafficking and inflammatory activity. Macrophage expression of aP2 impacts peroxisome proliferator-activated receptor gamma and IkappaB kinase activities.

Fatty acid-binding proteins are cytosolic fatty acid chaperones, and the adipocyte isoform, aP2, plays an important role in obesity and glucose metabolism. Recently, this protein has been detected in macrophages where it strongly contributes to the development of atherosclerosis. Here, we investigated the role of aP2 in macrophage biology and the molecular mechanisms underlying its actions. We demonstrate that aP2-deficient macrophages display defects in cholesterol accumulation and alterations in pro-inflammatory responsiveness. Deficiency of aP2 alters the lipid composition in macrophages and enhances peroxisome proliferator-activated receptor gamma activity, leading to elevated CD36 expression and enhanced uptake of modified low density lipoprotein. The increased peroxisome proliferator-activated receptor gamma activity in aP2-deficient macrophages is also accompanied by a significant stimulation of the liver X receptor alpha-ATP-binding cassette transporter A1-mediated cholesterol efflux pathway. In parallel, aP2-deficient macrophages display reduced IkappaB kinase and NF-kappaB activity, resulting in suppression of inflammatory function including reduced cyclooxygenase-2 and inducible nitric-oxide synthase expression and impaired production of inflammatory cytokines. Our results demonstrate that aP2 regulates two central molecular pathways to coordinate macrophage cholesterol trafficking and inflammatory activity.

Dendritic cells endocytose Bacillus anthracis spores: implications for anthrax pathogenesis.

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-alpha, 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.