NLRP3 is a thermosensor that is negatively regulated by high temperature

Inflammation is an essential response to infection and injury, but unregulated inflammation is damaging and must be limited by negative feedback signalling. Inflammasome signalling drives local inflammation and systemic responses like fever. However, our understanding of how inflammasome signalling is negatively regulated is limited. NLRP3 is activated by a vast number of stimuli and senses perturbations of cytoplasmic homeostasis. As temperature is a fundamental environmental stressor, we hypothesised that NLRP3 inflammasome signalling would be sensitive to increased temperatures and so we investigated the effects of high temperatures on NLRP3 in macrophages. Short-term incubation at high fever range temperatures significantly inhibits NLRP3 activation, while secretion of the inflammasome-independent cytokines TNF and IL-6 are much less affected. High temperature blocks NLRP3 inflammasome formation in a transcription-independent manner, and NLRP3 is highly sensitive to temperature-mediated inhibition relative to the NLRC4, AIM2, and NLRP1 inflammasomes. Using cellular assays and molecular simulations we show that the effect of high temperature on NLRP3 is protein intrinsic. NLRP3 activation is associated with a decrease in the thermal stability of the protein and multiscale molecular dynamics simulations identified a peptide in the C-terminal of the FISNA domain (COFI) that is highly flexible and undergoes a significant conformational shift at high temperature. Cellular assays demonstrate that the COFI regulates NLRP3 stability and is required for activation. Furthermore, mice exposed to high temperature display attenuated inflammatory cytokine production upon in vivo LPS challenge. Our studies thus reveal that high temperatures associated with fever limit NLRP3 activity and identify a novel role for NLRP3 as a protein thermosensor.