The Neutrophil Lineage in Inflammasomopathies

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Abstract The study of cryopyrin associated periodic syndromes (CAPS) has shaped our view of innate immunity, and led to the clinical translation of therapies for CAPS and other NLRP3-dependent inflammatory diseases. Our preliminary in vivo genetic data now suggest that the assumed central role of monocytes and macrophages in CAPS may be overstated. Rather, the neutrophil lineage alone can drive lethal autoinflammation in CAPS neonatal mice, and this is indistinguishable from systemic NLRP3 activation. The molecular regulation of NLRP3 inflammasome activation is distinct in neutrophils compared to macrophages, so this research will study biochemical regulation of NLRP3 activation in mouse and human cells of the neutrophil and monocyte lineages. We will investigate cells from patients with CAPS, and from mouse models with activating NLRP3 mutations expressed specifically in neutrophils (Nlrp3PMN mice). Neutrophil precursors are elevated in successfully-treated CAPS patients and in Nlrp3PMN mice, suggesting developmental abnormalities of neutrophils in Nlrp3PMN mice, or a reduced threshold for pyroptosis induction in the neutrophil lineage - hypotheses that will be formally tested in this study. Single cell Western and RNA-Seq data from purified neutrophil precursor populations have revealed constitutive IL-1b expression in neutrophil progenitors and immature neutrophils in healthy mice, suggesting that these precursors, but not mature neutrophils, are the dominant source of IL-1b in organs of CAPS patients. We now hypothesize that this developmentally-regulated expression of pro-IL-1b enables neutrophil precursors with NLRP3 activating mutations to release processed IL-1b independent of signal 1 or signal 2. We also hypothesize that the absence of mature neutrophils from all organs and tissues of neutrophil- specific CAPS mice is a consequence of pyroptosis. This project seeks to define: (a) the differences in organ pathology and morbidity of mice with monocyte- or neutrophil-specific NLRP3 activation; (b) the cell-intrinsic effects of NLRP3 activation on neutrophil differentiation and lifespan; and (c) the sensitivity of cells of the neutrophil lineage in CAPS patients and neonatal CAPS mice to canonical and non-canonical activators of the inflammasome and pyroptosis; Our specific aims are therefore to: (1) compare organ involvement and morbidity of mice with monocyte-specific and neutrophil-specific activation of NLRP3; (2) understand why mature neutrophils are absent in neonates of neutrophil-specific CAPS mice; and (3) investigate inflammasome activation and pyroptosis induction in neutrophil lineage cells from patients diagnosed with CAPS. Identification of the key cell types causing disease in CAPS will help us to understand the development of other NLRP3-driven inflammatory diseases, and may also improve disease management and highlight novel biomarkers for autoinflammation.