UC Berkeley

To provide the first line of defense against pathogenic microbes, the innate immune system detects infection using pattern recognition receptors, such as Toll-like receptors (TLRs) and cytosolic nucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) which form multi-protein complexes called inflammasomes. Once activated inflammasomes cause lytic cell death (termed pyroptosis) and secretion of interluken-1β (IL-1β) and IL-18. Most in vivo studies of the inflammasome have been conducted in the presence of TLR ligands; thus, it has been difficult to determine whether inflammasome activation is sufficient to induce immunity and inflammation in vivo. Moreover, the question of whether inflammasomes exhibit unique functions in distinct cell types has been greatly ignored.

In this dissertation, I will begin with an overview of basic background on inflammasomes and then expand on human autoinflammatory conditions in patients with various inflammasome mutations. In chapter 2, I will describe a novel, knock-in mouse model, iOvaFla. When Cre recombinase is present, the iOvaFla fusion gene, which is the C-terminal 166 amino acids of Legionella pneumophila flagellin fused to ovalbumin, is expressed. The portion of flagellin expressed selectively activates a specific inflammasome called NAIP/NLRC4. NLRC4 activation in Lysozyme2+ cells (monocytes, macrophages, neutrophils via LysM-Cre) in vivo caused a severe systemic inflammatory disease, characterized by systemic neutrophilia, weight loss, and hind limb joint swelling. Disease was entirely NLRC4-dependent and was nearly fully ameliorated on a genetic background that leads to decreased cytokine production, suggesting a dominant role for cytokines and a minimal role for pyroptosis. Consistent with this postulation, neutrophil levels and disease symptoms decreased after therapeutic blockade of the IL-1 receptor. Interestingly, disease was recapitulated by NLRC4 activation selectively in neutrophils (using MRP8-Cre) but the same severe disease symptoms were not induced upon selective inflammasome activation in dendritic cells/tissue macrophages (using CD11c-Cre). Disease that arose after neutrophil-specific NLRC4 activation was similarly ameliorated by therapeutic anti-IL-1 receptor blockade.

In chapter 3, I present two more available iOvaFla mouse models: iOvaFla; ER-CreT2+/– and iOvaFla; Vil-Cre+/– mice. iOvaFla; ER-CreT2+/– mice are an inducible, ubiquitous way to activate endogenous NAIP/NLRC4 in any cell type. I developed a protocol to induce iOvaFla expression via tamoxifen without introducing contaminating TLR ligands. This will allow the mice to be used in experiments to study the specific roles of inflammasomes. Next, I describe the function of inflammasomes in intestinal epithelial cells with iOvaFla; Vil-Cre+/– mice. The mice are embryonic lethal. When I crossed them on a genetic background where pyroptosis still occurs but cytokine maturation decreases, the lethality was not rescued. Unlike the iOvaFla; LysM-Cre+/– phenotype caused by cytokines, intestinal epithelial cell pyroptosis appears to be the primary cause for lethality.

Lastly in chapter 4, I conclude with a discussion on the lingering questions and future directions of how the iOvaFla mouse models could be used. There are several important mechanistic studies to complete to fully verify consequences of inflammasome activation in neutrophils. I also explore how iOvaFla; ER-CreT2+/– mice can be used to systematically uncover if inflammasomes are sufficient to stimulate the creation of adaptive immune responses. I conclude with a final reexamination of conclusions presented throughout my dissertation.