Molecular and evolutionary aspects of inflammatory caspase-mediated innate immunity

Abstract

The ability of the immune system to mount an appropriate inflammatory response is critical for our bodies’ defense against infection. To effectively coordinate this response, infected cells and immune cells communicate through the secretion of soluble signaling molecules called cytokines. Cytokines of the interleukin (IL)-1 family are particularly potent mediators of pro-inflammatory signaling. Unlike other cytokines, members of the IL-1 family are synthesized as inactive cytoplasmic pro-proteins whose N-termini must be proteolytically processed to achieve or enhance their bioactivity. The maturation and release of IL-1 family cytokines is controlled by a group of proteases called inflammatory caspases, which includes caspase-1, as well as caspase-4 and caspase-5 in humans and caspase-11 in mice. Despite the importance of these enzymes, the molecular basis of how caspases select their substrates and how these substrates mediate inflammation post-cleavage is not well understood. A long-held dogma in the field was that caspase-1 is the only inflammatory caspase capable of directly cleaving IL-1 cytokines. Two observations presented in this thesis challenge this view: We found that caspase-4 homologs present in mammals of the order Carnivora (such as dogs, cats, seals, bears) cleave the IL-1 family cytokine IL-1β, and human caspase-4 and caspase-5, but not murine caspase-11, cleave IL-18. Through comparative biology analyses, coupled with biochemical and structural biology approaches, we identified molecular determinants of IL-1β and IL-18 cleavage in these caspase-4 homologs. Many of the described principles extended to other inflammatory caspases, including caspase-1 and caspase-11, which allowed for the engineering of caspase variants with user-defined catalytic specificities. In cells, the ability to directly cleave IL-1β and/or IL-18 together with their established functions as sensors of bacterial lipopolysaccharide (LPS) and inducers of lytic cell death enabled select caspase-4 proteins to act as one-protein signaling pathways, which directly link cytosolic LPS detection to IL-1 cytokine release, independent of caspase-1 and inflammasomes. Our studies also revealed why cleavage of an IL-1 cytokine is necessary to activate its signaling capacity. Structural analyses showed that uncleaved pro-IL-18 (in its apo form or in complex with a caspase) exhibits extensive conformational differences to mature IL-18. Specifically, the receptor binding site is only formed following cleavage-induced rearrangements within IL-18. This finding explains why IL-18 but not pro-IL-18 can engage and signal through the cognate IL-18 receptor complex on target cells to induce inflammation, Before IL-1 cytokines can bind to target cells to induce inflammation, they are released from cells through the actions of another inflammatory caspase substrate, gasdermin D (GSDMD), whose N-terminal domain forms pores in the plasma membrane post-cleavage. In the final study of this thesis, we sought to identify cell biological mechanisms regulating this process. Using a combination of forward genetic screening, biochemical and cell biological analyses, we identified an unexpected link between cellular metabolism and GSDMD function. We found that reactive oxygen species produced downstream of the Ragulator-Rag complex (a regulator of lysosomal metabolism), detection of signs of microbial encounter, or mitochondrial dysfunction enhanced GSDMD activities through direct oxidation of a specific cysteine (C192) within GSDMD. We thus identified cellular redox state as a key determinant of GSDMD activities. Taken together, the work presented in this thesis uncovered an unappreciated functional diversity in caspase-4 proteins from different mammalian species and revealed important insights into caspase-mediated IL-1 cytokine maturation and signalling and novel regulatory mechanisms of GSDMD pore formation.