TLR priming licenses NAIP inflammasome activation by immunoevasive ligands.

ABSTRACT

NLR family, apoptosis inhibitory proteins (NAIPs) detect bacterial flagellin and structurally related components of bacterial type III secretion systems (T3SS), and recruit NLR family, CARD domain containing protein 4 (NLRC4) and caspase-1 into an inflammasome complex that induces pyroptosis. NAIP/NLRC4 inflammasome assembly is initiated by the binding of a single NAIP to its cognate ligand, but a subset of bacterial flagellins or T3SS structural proteins are thought to evade NAIP/NLRC4 inflammasome sensing by not binding to their cognate NAIPs. Unlike other inflammasome components such as NLRP3, AIM2, or some NAIPs, NLRC4 is constitutively present in resting macrophages, and not thought to be regulated by inflammatory signals. Here, we demonstrate that Toll-like receptor (TLR) stimulation upregulates NLRC4 transcription and protein expression in murine macrophages, which licenses NAIP detection of evasive ligands. TLR-induced NLRC4 upregulation and NAIP detection of evasive ligands required p38 MAPK signaling. In contrast, TLR priming in human macrophages did not upregulate NLRC4 expression, and human macrophages remained unable to detect NAIP-evasive ligands even following priming. Critically, ectopic expression of either murine or human NLRC4 was sufficient to induce pyroptosis in response to immunoevasive NAIP ligands, indicating that increased levels of NLRC4 enable the NAIP/NLRC4 inflammasome to detect these normally evasive ligands. Altogether, our data reveal that TLR priming tunes the threshold for NAIP/NLRC4 inflammasome activation and enables inflammasome responses against immunoevasive or suboptimal NAIP ligands.