Early events during infection with anthrax

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DESCRIPTION (provided by applicant): Following inhalation, anthrax spores are phagocytosed by macrophages from which they germinate into a vegetative form that replicates, disseminates and produces potent virulence factors responsible for the clinical manifestations of the disease. Relatively little is known about an initial step in the innate immune response to anthrax: recognition of spores by host Toll-like receptors (TLR), and their role in the pathogenesis of anthrax infection. We will explore the initial contact of B. anthracis (BA) spores with TLRs expressed on human and murine cells. We hypothesize that TLR and/or FcgammaR activation is required for killing of the ingested spore, but in the absence of immune antibody, the exosporium shields underlying spore structures, particularly the peptidoglycan (PG) from TLR recognition and cell activation. Specific Aim I will add germination-deficient BA spore (to avoid vegetative form contamination) to human embryonal kidney cells transfected with specific human TLR constructs. Induction of IL-8 and NFkappaB activation will be measured following addition of intact BA spores, spores stripped of exosporium, purified PG and purified exosporium. Involvement of specific TLRs will be confirmed in primary non-alveolar and alveolar human and murine macrophages, including those of TLR2-, TLR4- and MyD88 knockout mice. The adaptor protein MyD88 is required for most TLR-mediated signaling. Specific Aim II will assess the potential role of TLRs in FcRgamma-mediated killing of Sterne strain (toxin+, capsule -) BA spores. Antisera raised to spores with and without exosporium will be used to opsonize Sterne BA spores, and the effect of these antisera on BA killing and cytokine induction will be determined in human alveolar and non-alveolar macrophages as well as those from wild type and TLR2-, TLR4- and MyD88 KO mice. These latter studies will address whether killing of ingested BA spores requires cooperativity between TLRs and FcRgammas. These studies using specific human TLR constructs, inhibition of expression/function of endogenous TLR proteins in primary human macrophages with siRNA and cell permeable TLR peptide inhibitors, TLR knockout mice and a unique strain of anthrax spore will define the initial interaction of macrophages with BA spores, and may provide data enabling intellingent manipulation of the cytokine milieu in favor of the host.