Nociceptive Sensory Neurons Mediate Inflammation Induced by Bacillus Anthracis Edema Toxin

Bacterial products are able to act on nociceptive neurons during pathogenic infection. Neurogenic inflammation is an active part of pain signaling and has recently been shown to impact host-pathogen defense. Bacillus anthracis Edema Toxin (ET) produces striking edema in peripheral tissues, but the cellular mechanisms involved in tissue swelling are not completely understood. Here, we find that nociceptive neurons play a role in ET-induced edema and inflammation in mice. Subcutaneous footpad infection of B. anthracis Sterne caused ET-dependent local mechanical allodynia, paw swelling and body weight gain. Subcutaneous administration of ET induced paw swelling and vascular leakage, the early phases of which were attenuated in the absence of Trpv1+ or Nav1.8+ nociceptive neurons. Nociceptive neurons express the anthrax toxin receptor ANTXR2, but this did not mediate ET-induced edema. ET induced local cytokine expression and neutrophil recruitment, which were dependent in part on Trpv1+ nociceptive neurons. Ablation of Trpv1+ or Nav1.8+ nociceptive neurons also attenuated early increases in paw swelling and body weight gain during live B. anthracis infection. Our findings indicate that nociceptive neurons play an active role in inflammation caused by B. anthracis and Edema Toxin to potentially influence bacterial pathogenesis.

A Review of the Efficacy of FDA-Approved B. anthracis Anti-Toxin Agents When Combined with Antibiotic or Hemodynamic Support in Infection- or Toxin-Challenged Preclinical Models

Anti-toxin agents for severe B. anthracis infection will only be effective if they add to the benefit of the two mainstays of septic shock management, antibiotic therapy and titrated hemodynamic support. Both of these standard therapies could negate benefits related to anti-toxin treatment. At present, three anthrax anti-toxin antibody preparations have received US Food and Drug Administration (FDA) approval: Raxibacumab, Anthrax Immune Globulin Intravenous (AIGIV) and ETI-204. Each agent is directed at the protective antigen component of lethal and edema toxin. All three agents were compared to placebo in antibiotic-treated animal models of live B. anthracis infection, and Raxibacumab and AIGIV were compared to placebo when combined with standard hemodynamic support in a 96 h canine model of anthrax toxin-associated shock. However, only AIG has actually been administered to a group of infected patients, and this experience was not controlled and offers little insight into the efficacy of the agents. To provide a broader view of the potential effectiveness of these agents, this review examines the controlled preclinical experience either in antibiotic-treated B. anthracis models or in titrated hemodynamic-supported toxin-challenged canines. The strength and weaknesses of these preclinical experiences are discussed.

Bacillus anthracis edema toxin inhibits hypoxic pulmonary vasoconstriction via edema factor and cAMP-mediated mechanisms in isolated perfused rat lungs

The most important findings here are edema toxin’s potent adenyl cyclase activity can interfere with hypoxic pulmonary vasoconstriction, an action that could worsen hypoxemia during invasive anthrax infection with lung involvement. These findings, coupled with other studies showing that lethal toxin can disrupt pulmonary vascular integrity, indicate that both toxins can contribute to pulmonary pathophysiology during infection. In combination, these investigations provide a further basis for the use of antitoxin therapies in patients with worsening invasive anthrax disease.

Bacillus anthracis lethal toxin, but not edema toxin, increases pulmonary artery pressure and permeability in isolated perfused rat lungs

Although lethal toxin (LT) and edema toxin (ET) contribute to lethality during Bacillus anthracis infection, whether they increase vascular permeability and the extravascular fluid accumulation characterizing this infection is unclear. We employed an isolated perfused Sprague-Dawley rat lung model to investigate LT and ET effects on pulmonary vascular permeability. Lungs ( n ≥ 6 per experimental group) were isolated, ventilated, suspended from a force transducer, and perfused. Lung weight and pulmonary artery (Ppa) and left atrial pressures were measured over 4 h, after which pulmonary capillary filtration coefficients (Kf.c) and lung wet-to-dry weight ratios (W/D) were determined. When compared with controls, LT increased Ppa over 4 h and Kf.c and W/D at 4 h ( P < 0.0001). ET decreased Ppa in a significant trend ( P = 0.09) but did not significantly alter Kf.c or W/D ( P ≥ 0.29). Edema toxin actually blocked LT increases in Ppa but not LT increases in Kf.c and W/D. When Ppa was maintained at control levels, LT still increased Kf.c and W/D ( P ≤ 0.004). Increasing the dose of each toxin five times significantly increased and a toxin-directed monoclonal antibody decreased the effects of each toxin ( P ≤ 0.05). Two rho-kinase inhibitors (GSK269962 and Y27632) decreased LT increases in Ppa ( P ≤ 0.02) but actually increased Kf.c and W/D in LT and control lungs ( P ≤ 0.05). A vascular endothelial growth factor receptor inhibitor (ZM323881) had no significant effect ( P ≥ 0.63) with LT. Thus, LT but not ET can increase pulmonary vascular permeability independent of increased Ppa and could contribute to pulmonary fluid accumulation during anthrax infection. However, pulmonary vascular dilation with ET could disrupt protective hypoxic vasoconstriction. NEW & NOTEWORTHY The most important findings from the present study are that Bacillus anthracis lethal toxin increases pulmonary artery pressure and pulmonary permeability independently in the isolated rat lung, whereas edema toxin decreases the former and does not increase permeability. Each effect could be a basis for organ dysfunction in patients with this lethal infection. These findings further support the need for adjunctive therapies that limit the effects of both toxins during infection.

Bacillus anthracis Edema Toxin Inhibits Efferocytosis in Human Macrophages and Alters Efferocytic Receptor Signaling

The Bacillus anthracis Edema Toxin (ET), composed of a Protective Antigen (PA) and the Edema Factor (EF), is a cellular adenylate cyclase that alters host responses by elevating cyclic adenosine monophosphate (cAMP) to supraphysiologic levels. However, the role of ET in systemic anthrax is unclear. Efferocytosis is a cAMP-sensitive, anti-inflammatory process of apoptotic cell engulfment, the inhibition of which may promote sepsis in systemic anthrax. Here, we tested the hypothesis that ET inhibits efferocytosis by primary human macrophages and evaluated the mechanisms of altered efferocytic signaling. ET, but not PA or EF alone, inhibited the efferocytosis of early apoptotic neutrophils (PMN) by primary human M2 macrophages (polarized with IL-4, IL-10, and/or dexamethasone) at concentrations relevant to those encountered in systemic infection. ET inhibited Protein S- and MFGE8-dependent efferocytosis initiated by signaling through MerTK and αVβ5 receptors, respectively. ET inhibited Rac1 activation as well as the phosphorylation of Rac1 and key activating sites of calcium calmodulin-dependent kinases CamK1α, CamK4, and vasodilator-stimulated phosphoprotein, that were induced by the exposure of M2(Dex) macrophages to Protein S-opsonized apoptotic PMN. These results show that ET impairs macrophage efferocytosis and alters efferocytic receptor signaling.

Shock and lethality with anthrax edema toxin in rats are associated with reduced arterial responsiveness to phenylephrine and are reversed with adefovir

Although edema toxin (ETx) and lethal toxin (LTx) contribute to Bacillus anthracis shock and lethality, the mechanisms underlying their cardiovascular effects are unclear. We have previously shown that ETx but not LTx inhibited phenylephrine-stimulated contraction of aortic rings prepared from healthy rats and that adefovir, a selective inhibitor of ETx cAMP production, blocked this effect. Here, we examined arterial function in rats that received 24-h ETx or LTx infusions. Compared with control rats, ETx reduced mean arterial pressure (MAP) and survival over 48 h ( P ≤ 0.0003) and increased plasma cAMP at 4, 24, and 48 h ( P < 0.0001) and nitric oxide (NO) at 24 and 48 h ( P ≤ 0.01). Compared with control animals, at 24- and 48-h phenylephrine stimulation of aortic rings from ETx animals produced decreased maximal contractile force (MCF; P = 0.05 and 0.006) and in vivo phenylephrine infusion in ETx animals produced decreased proportional increases in MAP ( P < 0.0001 and P = 0.05). In ETx-treated animals, compared with placebo-treated animals, adefovir treatment prevented all lethality ( P = 0.01), increased MAP ( P ≤ 0.0001), decreased plasma and aortic tissue cAMP at 24 and 48 h, respectively ( P ≤ 0.03), and plasma NO at both times ( P ≤ 0.004), and increased phenylephrine-stimulated increases in MCF in aortic rings and MAP in vivo at 48 h ( P = 0.02). LTx decreased MAP and survival also, but it did not alter the response to phenylephrine of MCF in aortic rings prepared from LTx animals or of MAP in vivo. In conclusion, in rats, hypotension and lethality are associated with reduced arterial contractile function with ETx but not LTx and adefovir improves ETx-induced hypotension and lethality. NEW & NOTEWORTHY The most important aspects of the present study are the findings that 1) in vivo challenge with anthrax edema but not lethal toxin depresses arterial contractile function measured both ex vivo and in vivo and 2) adefovir inhibits the effects of edema toxin on arterial hypotension and improves survival with lethal dose of edema toxin challenge.