Structure-function Analysis of Nlrp1b Reveals a Link between Metabolism and Inflammation: Energy Stress Triggers Inflammasome Activation

Immune cells have evolved an array of pattern recognition receptors (PRRs) to initiate immune responses upon detecting pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs). Nlrp1b, a cytosolic PRR, forms a protein complex termed an inflammasome that activates pro-caspase-1, which leads to processing of inflammatory cytokines, such as pro-IL-1β, and to cell death. Nlrp1b has been shown to mediate death of murine macrophages by anthrax lethal toxin. Lethal toxin is a binary toxin that comprises lethal factor (LF), a zinc metalloprotease, and protective antigen (PA), which translocates LF into the cytosol. In this thesis, the murine Nlrp1b inflammasome was reconstituted in a human fibroblast cell line and shown to be responsive to the enzymatic activity of LF. Activation of Nlrp1b by LF was allele specific in the reconstituted system since an allele derived from macrophages that are killed by the toxin facilitated processing of pro-caspase-1, whereas an allele from macrophages that are resistant to the toxin did not mediate processing of pro-caspase-1. That the reconstituted system recapitulates the cellular events in intoxicated macrophages was further supported by the observation that a proteasome inhibitor that inhibits macrophage intoxication also prevented pro-caspase-1 activation in the fibroblasts. Next, I identified energy stress as a danger signal that activates the Nlrp1b inflammasome: reduction of intracellular ATP by 2-deoxy-glucose and sodium azide caused Nlrp1b-dependent processing of pro-IL-1β. AMP-activated protein kinase (AMPK), a major energy sensor in cells, facilitated Nlrp1b activation after ATP-depletion whereas it was not involved in lethal toxin-stimulated Nlrp1b signaling. However, signals derived from lethal toxin and ATP-depletion seemed to converge upstream of Nlrp1b activation because pharmacological inhibition of the proteasome blocked both triggers. Lastly, mutations that disrupted the nucleotide-binding site of Nlrp1b caused Nlrp1b activation, which is consistent with the finding that Nlrp1b was activated when the cytosolic ATP level decreased.Ph