Infections due to the encapsulated fungus Cryptococcus neoformans are a major cause of morbidity and mortality in individuals with depressed T cell function, particularly those with AIDS. Prevention of cryptococcosis requires an immune response composed initially of innate defenses and then, if unsuccessful, acquired immunity. Recently, a key component of innate immunity, Toll-like receptors (TLRs), was discovered and found to function as pattern-recognition receptors for bacterial pathogens. TLR engagement leads to binding of a cytoplasmic adapter protein (MyD88) and the triggering of signaling cascades culminating in activation of transcription factors including NF-KB and the initiation of gene transcription. The central hypothesis of this proposal is that TLRs are critical for intracellular signaling in response to whole C. neoformans and its 2 major shed products; capsule glucuronoxylomannan (GXM) and mannoproteins (MP). The proposal will utilize transfected cell lines, primary macrophages and knockout mice to determine the mechanism(s) by which TLRs activate (or in some cases inhibit) cells stimulated by C. neoformans. There are 3 specific aims:
Aim 1. To use transfected cell lines to determine the role of TLRs in signaling responses to whole C. neoformans, GXM and MP. Cell lines will be transfected with TLR2 and/or TLR4 alone and in combination with receptors for LPS (CD14), mannose and complement. Activation of cells will be measured using NF-KB-dependent reporter constructs. The ability of dominant negative (DN) TLR and MyD88 constructs to inhibit NF-KB activation will also be studied.
Aim 2. To examine intracellular events triggered in macrophages following stimulation with whole C. neoformans, GXM and MP. The signaling mechanisms and the nature of the inflammatory response stimulated by whole C. neoformans, GXM and MP will be studied. Cryptococcal activation of NF-KB and MAP kinase pathways will be determined and its functional significance explored using assays for cytokine gene expression and release, respiratory burst products and microbicidal activity.
Aim 3. To determine the role of TLRs in host defenses against cryptococcosis in vivo and ex vivo using TLR2, TLR4 and MyD88 knockout (KO) mice. TLR2, TLR4, and MyD88 KO mice will be challenged with C. neoformans in vivo, and mortality, tissue CFU, and histopathology determined. Ex vivo experiments with macrophages from the KO mice will be used to complement data obtained in aims 1 and 2. Completion of these aims will add significantly to our understanding of the immunopathogenesis of cryptococcosis and may suggest novel approaches to the prevention and treatment of this life-threatening mycosis.
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