NAIP and CARD-only proteins in inflammasome signaling

The primary role of the innate immune system is to recognize and respond to harmful external and internal insults including microbial pathogens, harmful environmental factors and host-derived stress signals. Protection of the organism relies on the ability of the innate immune system to discriminate deleterious infectious agents and stress factors from innocuous selfcomponents and the commensal micro-organisms that inhabit mucosal interfaces. Germlineencoded Pattern Recognition Receptors (PRRs) are key components of the innate immune system that enable such adequate responses. PRRs sense unique, evolutionarily conserved pathogen-associated molecular patterns (PAMPs) that are frequently essential for establishing infection in its hosts and for survival of the pathogen in this hostile environment. In addition, PRRs scan the presence of self-molecules at extra- and intracellular locations that normally should be devoid of such factors, which serves as an indication of significant cellular stress. Such proxy molecules are referred to as danger-associated molecular patterns (DAMPs) or alarmins, and include ATP, HMGB1, interleukin (IL)-1α and hyaluronic acid. Although the innate immune system was long held to mount a non-discriminatory response to pathogens (as opposed to the tailored response of the adaptive immune system), it is now well-established that PRRs engage specific signaling cascades, the combination of which results in adequate inflammatory and host defense responses to fight the infectious agent. Many PRRs control signaling pathways that engage transcription factors to produce acute phase proteins, inflammatory cytokines and chemokines that attract and instruct other immune and regulatory cells. In contrast, a subset of PRRs of the Nucleotide-binding domain (NBD) and Leucine-rich Repeat (LRR)-containing (NLR), AIM2-like receptor (ALR) families and the protein Pyrin drive post-translational mechanisms that orchestrate assembly of inflammasomes - intracellular multiprotein platforms that promote autocatalytic activation of procaspase-1. Activated caspase-1 drives proteolytic maturation of pro-IL-1β and pro-IL-18 into their bioactive forms, as well as their subsequent release into the extracellular environment. IL-1β and IL-18 exert their downstream inflammatory and immune effects through their cognate receptors on both hematopoietic and stromal cells1. In addition, caspase-1 promotes an inflammatory mode of cell death termed pyroptosis, which is characterized by cell swelling, nuclear condensation and rapid cell membrane rapture and extracellular release of the cellular content. Pyroptosis is thought to contribute to host defense by eliminating intracellular replicative niches of pathogens and by preventing the further spread of the infectious agents to neighboring tissues2. These (and potentially other effector mechanisms) render inflammasome-driven responses a rapid and powerful response mechanism that itself may become a potential threat to the organism if their activation, strength and resolution are not carefully controlled. Consistently, multiple checkpoints balance inflammasome signaling in order to preserve or return to homeostasis after a microbial or other threat has been resolved. In addition, multiple gain-of-function mutations in inflammasome genes have been reported that cause excessive or uncontrolled inflammasome activation, which underlies destructive autoinflammatory and autoimmune diseases in man. Current understanding of the molecular mechanisms that control and modulate activation of the distinct inflammasomes is only starting to emerge, and further clarification of these processes is bound to illuminate novel disease mechanisms and highlight new anchor points for diagnosis and therapy of inflammatory diseases. The work described in this thesis contributed to these efforts. Part I of the Introduction reviews contemporary understanding of the regulatory checkpoints of inflammasome activation and modulation. In Part II of the Introduction, I focus on describing dominant-negative and decoy inhibitors and their roles in inflammasome biology, with an emphasis on members of the caspase recruitment domain (CARD)-only proteins (COP) and pyrin (PYD)-only proteins (POP). Part I of the Results section of this thesis reports our work on characterizing the roles of Nlrc4 phosphorylation and the NLR member Naip5 in activation of the Nlrc4 inflammasome in murine macrophages. In Part II of the Results section, I discuss our work detailing the regulatory roles of the human COP protein Inhibitory CARD (INCA) on the distinct known inflammasome pathways. Finally, Part III of the Results section provides an overview of our experiments suggesting the existence of a caspase-1 activityinduced processing of Nlrc4 independently of the proteasome. The thesis is completed with a General Discussion and Future Perspectives chapter in which I discuss our results from a broader perspective of the field, and highlight new questions that emerged from this work as a foundation for potentially interesting novel discoveries in the future