Anthrax toxin protective antigen protein (PA) binds to receptors on the surface of mammalian cells, is cleaved by cellular proteases, forms an oligomer, and transports two other toxin proteins, lethal factor (LF) or edema factor (EF) to the cytosol. EF is a potent calmodulin-dependent adenylyl cyclase that causes large increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs) and the N-terminus of the inflammasome sensor NLRP1. The inflammasomes are intracellular complexes that play a role in innate immune sensing for defense against pathogens. The cleavage of NLRP1 in macrophages and dendritic cells leads to caspase-1 activation and a rapid cell death termed pyroptosis. Caspase-1 activation, which is the resultant effect following activation of many other inflammasome sensors, including the NLRP3, NAIP/NLRC4 and AIM2 sensors, also leads to maturation and release of the pro-inflammatory cytokines IL-1β and IL-18. The screening for and characterization of inhibitors of LT-induced pyroptosis allows for identification of novel anti-inflammatory therapeutics that target multiple other inflammasomes responsible for detecting a variety of intracellular microbial ligands and endogenous danger signals. Inflammasome activation is an important part of the innate immune response, but is also linked to pathology of many inflammatory and autoimmune disorders. In the 2015 reporting period, we identified the natural isothiocyanate, sulforaphane, present in broccoli sprouts and available as a dietary supplement, as an inhibitor of the NLRP1, NLRP3, NAIP/NLRC4 and AIM2 inflammasomes. We found that sulforaphane inhibits autoproteolytic cleavage of caspase-1 in a manner independent of the actions it normally has on transcription factor Nrf2 and the antioxidant response-element pathway, to which many of the anti-inflammatory effects of sulforaphane have been previously attributed. In a separate study, we discovered that the eicosanoid 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2) and related cyclopentenone PGs which have been studied as NF-κB inhibitors can also inhibit caspase-1 activation by multiple inflammasome pathways. This inhibition is independent of the well-characterized role of 15d-PGJ2 as a peroxisome proliferator receptor-γ agonist, its activation of Nrf2, or any anti-inflammatory functions as an inhibitor of NF-κB. Instead, 15d-PGJ2 prevents the autoproteolytic activation of caspase-1 and the maturation of IL-1β through induction of a cellular state inhibitory to caspase-1 proteolytic function. Similar to sulforaphane, the drug effect is not manifested through direct modification or inactivation of the caspase-1 enzyme. But in a manner different from sulforaphane, the effects of 15d-PGJ2 depend on de novo protein synthesis of an unknown inhibitor or protective protein, through actions of the eicosanoid on a yet to be identified transcription pathway. Testing of both sulforaphane and 15d-PGJ2 in vivo, in a mouse model of gout where monosodium urate crystals are used to activate the NLRP3 inflammasome, showed that both chemicals inhibit inflammatory cell recruitment and IL-1β release. Furthermore, in our murine anthrax infection model, both drugs could reverse NLRP1-mediated murine resistance to Bacillus anthracis spore infection. The above two studies report on novel mechanisms of anti-inflammatory action for two NF-kB inhibitors which have been included in clinical trials on the basis of their impact on inflammation. The findings were published this year in the Journal of Leukocyte Biology and the Journal of Immunology. When injected in animals, both ET and LT induce vascular collapse and host death. These toxins are considered the primary virulence factors of B. anthracis, and play roles in different stages of infection. In the early stages of infection, both toxins work together to impair the innate immune response. At later stages, the induction of localized and systemic vascular dysfunction results in host death. The targeting of the common component of both toxins, PA, is the basis for the current vaccination against anthrax and most developed monoclonal antibody-based therapeutics. In collaborative work published in 2015 using the approved anti-PA monoclonal raxibacumab, the effects of the antibody on 24 h edema toxin and lethal toxin challenges in a canine model were studied. Raxibacumab was found to augment fluid and norepinephrine therapy to improve survival in toxin challenged animals. In a separate collaborative work, we identified novel antibodies for targeting PA. Variable domains of camelid heavy chain-only antibodies (VHHs) are small single chain, heat- and pH- stable entities that can access epitopes that large standard antibodies cannot. Furthermore, they can be produced in recombinant form and purified rapidly once their sequences are known. VHHs with high affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified, with one similar to most identified anti-PA monoclonal antibodies, in that it inhibited the toxin binding to its cellular receptor. A second novel neutralizing VHH was found to inhibit endocytosis of the PA oligomer while not preventing PA cleavage by cell surface proteases. Both VHHs, as well as a heterodimer of the two displayed neutralizing potency in cell assays and protected mice from anthrax toxin challenge and Bacillus anthracis spore infection. These studies showed the usefulness of VHHs as novel anti-PA agents, and introduced a novel mechanism for neutralization of anthrax toxin.
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