TLR priming licenses NAIP inflammasome activation by immunoevasive ligands

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 known to be induced by inflammatory signals. Here, we demonstrate that Toll-like receptor (TLR)-dependent p38 mitogen-activated protein kinase signaling up-regulates NLRC4 transcription and protein expression in murine macrophages, which licenses NAIP detection of evasive ligands. In contrast, TLR priming in human macrophages did not up-regulate 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 the murine NAIP/NLRC4 inflammasome to enable inflammasome responses against immunoevasive or suboptimal NAIP ligands. These findings provide insights into species-specific TLR regulation of NAIP/NLRC4 inflammasome activation.

Methods
LDH cytotoxicity assays

After infection, cells were spun at 250g for 10 minutes to pellet cellular debris. Supernatants were removed and used to assess cytotoxicity via lactate dehydrogenase (LDH) activity. LDH release was quantified using an LDH Cytotoxicity Detection Kit (Roche). Samples were incubated for 25 minutes at room temperature and absorbance at 490 nm was assessed using a spectrophotometer. Percent cytotoxicity was calculated after normalizing to uninfected controls and 100% cell death, which is based on 1% triton X-100-treated cells. 

ELISA

Supernatants from in vitro infections and murine serum from in vivo infected mice were used to assess IL-1β and IL-18 levels, respectively. For murine IL-1β and IL-18, ELISAs were performed as described in Supplemental Materials and Methods. For measuring human IL-1β, an ELISA kit from BD Biosciences was used.

Quantitative RT-PCR

RNA was isolated using TRIzol reagent (ThermoFisher) from either 5 x 106 BMDMs or THP-1s following the manufacturer’s protocol. cDNA was prepared from the RNA samples using the high-capacity cDNA reverse transcription kit (Applied Biosystems) per manufacturer’s protocol. Quantitative PCR was conducted with the QuantBio Studio 6 Flex Real-Time PCR system using the PerfeCTa SYBR Green SuperMix (QuantaBio). For analysis, mRNA levels of siRNA-treated cells were normalized to housekeeping gene GAPDH (murine) or HPRT (human) and fold induction was determined using the 2−ΔΔCT (cycle threshold) method

Graphing and statistical analyses of data were performed using Prism 9 software (GraphPad). Statistical significance was determined using the statistical tests indicated in each figure legend. Differences were considered statistically significant if the P value was less than or equal to 0.05