Mutant NLRP3 causing Cryopyrin-Associated Periodic Syndromes forms a constitutively active inflammasome and results in alteration in monocyte immunometabolism to limit IL-1β production

Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory condition resulting from monoallelic NLRP3 variants that exacerbate IL-1 production. Although these are gain-of-function variants, the exact regulatory mechanism of the NLRP3-inflammasome in CAPS is not yet well understood. Despite being considered a hypersensitive inflammasome triggered by cell priming, patients with CAPS and animal models of the disease may present inflammatory flares even in the absence of identifiable external triggers. Herein, we found that CAPS-associated variants result in constitutively active NLRP3-inflammasome, which induce an increased basal cleavage of gasdermin D, IL-18 release and pyroptosis, with a concurrent basal pro-inflammatory gene expression signature, including the induction of nuclear receptors 4A. The constitutively active NLRP3-inflammasome was blocked by MCC950 and was dependent on NLRP3 expression level, further regulated by deubiquitination. Additionally, we determined that the activation of the NF-B pathway with lipopolysaccharide or other endogenous molecules (palmitate, S100A9, IL-6) further modulated the activation of the NLRP3-inflammasome in CAPS, thus expanding the repertoire of molecules secreted from patients’ macrophages involved in disease pathogenesis. NLRP3-inflammasomes with CAPS-associated variants mainly affected the immunometabolism of the myeloid compartment, leading to disruptions in lipids and amino acid pathways and impaired glycolysis, limiting IL-1β production. These findings demonstrate that NLRP3 variants causing CAPS form a constitutively active inflammasome that induces basal pyroptosis and IL-18 release without cell priming, favouring the host's innate defense against pathogens while also tempering the onset of IL-1β–dependent inflammatory episodes through immunometabolism modulation. Nlrp3−/− immortalized macrophages (iMos) with a doxycyclyne inducible system were used. iMos were treated for 16 h with or without doxycycline (1 µg/ml) to induce the expression of the human NLRP3 wild-type or p.D303N variant, in the absence or presence of MCC950 (10 µM). Total RNA was extracted from Nlrp3−/− immortalized mouse macrophages expressing NLRP3 wild type or NLRP3 p.D303N using the RNeasy kit (74104, Qiagen) following manufacturer instructions. Upon extraction, total RNA was quantified with Qubit RNA BR Assay (Invitrogen), and RNA integrity was analysed with TapeStation 4200 (Agilent Technologies). All samples showed RIN >8. cDNA synthesis and library generation were performed using Stranded mRNA Prep Kit (Illumina) following the manufacturer’s instructions.RNA-seq libraries were sequenced using Paired-End 200 bases (PE200) sequencing chemistry on NextSeq 2000 Sequencing Systems (Illumina).