Anthrax poses a clear and present danger as an agent of biological terrorism. The major virulence factor of Bacillus anthracis is the anthrax toxin, which comprises 3 subunits: protective antigen (PA), edema factor (EF) and lethal factor (LF). LF and PA together form a toxin known as lethal toxin (LT), which appears to play exquisitely different immunomodulatory roles, depending on the dose of toxin used. At low concentrations, it cleaves components of the MAP-kinase pathway (MKK1,2 & 3), thereby rendering macrophages anergic to further stimulation by LPS. The consequences of this for adaptive immunity are not known. Moreover, the effects of LT on dendritic cells (DCs), the most efficient antigen-presenting cells in the body, are not known. At higher concentrations, LT elicits a radically different outcome - lysis of macrophages and systemic inflammation, followed by rapid death of the host. The reason(s) for these strikingly different effects are not known. These mechanisms and their pathophysiological relevance will be investigated in the following aims:
Aim 1 : To determine the effect of LT on routine DC,function and adaptive immunity in vitro Sub-Aim 1a) Activation and viability of DCs from spleens, lungs and skin exposed to different concentrations of LT, Sub-Aim 1b) Stimulation of antigen-specific T cells by DCs cultured with different doses of LT; Sub-Aim 1c) Effect of LT on antigen-specific naive and memory T cells.
Aim 2 : To determine the effect of LT on murine DC function and adaptive immunity in vivo Sub-Aim 2a) Does injection of sub-lethal doses of LT impair DC activation in vivo? Sub Aim 2b) Does injection of sub-lethal doses of LT impair immune stimulatory capacity of DCs in vivo? Sub Aim 2c) To determine the principal cell types and inflammatory mediators of toxicity elicited by high doses of LT Aim 3: To determine the effect of LT on distinct human DC subsets and adaptive immunity in vitro Sub Aim 3b) Activation, viability and function of human monocyte-derived DCs cultured with LT, Sub-Aim 3b) Effect of LT on distinct human DC subsets.
Aim 4 : To determine the pathophysiological relevance of LT-induced suppression during B.anthracis infection Sub Aim 4a) To determine the phenotype, function and microenvironmental localization of DCs in the lymphoid organs, various times after infection with LT-deficient or LT-sufficient strains of B.anthracis Sterne;
Sub Aim 4 b) To determine whether LT suppresses immune function during a B. anthracis infection in macaques. Thus, the overall goal of this proposal is to acquire a deeper, mechanistic understanding of anthrax pathogenesis, and to use this knowledge to devise novel therapeutic modalities, which may be optimally effective at different stages of the infection.
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