Monocytes promote the early host response to infection releasing key pro-inflammatory cytokines, such as IL-1β. The biologically inactive IL-1β precursor is processed to active form by inflammasomes, multi-protein complexes activating caspase-1. Human monocytes exhibit an unconventional one-step pathway of inflammasome activation in response to lipopolysaccharide (LPS) alone. Although this lineage-restricted mechanism is likely to contribute to the pathology of endotoxin shock, signalling pathways regulating this mechanism are currently unknown. Here we report that caspase-4 and caspase-5 mediate IL-1α and IL-1β release from human monocytes after LPS stimulation. Although caspase-4 remains uncleaved, caspase-5 undergoes rapid processing upon LPS treatment. We also identify an additional caspase-5 cleavage product in LPS-stimulated monocytes, which correlates with IL-1 secretion. This one-step pathway requires Syk activity and Ca²⁺ flux instigated by CD14/TLR4-mediated LPS internalization. Identification of caspase-4/5 as the key determinants of one-step inflammasome activation in human monocytes provides potential targets for therapeutic intervention in endotoxin shock.

The inflammasome is a critical molecular complex that activates IL-1 driven inflammation in response to pathogen- and danger-associated signals. Germline mutations in the inflammasome sensor, NLRP1 cause Mendelian systemic autoimmunity and skin cancer susceptibility, but its endogenous regulation remains less understood. Here we use a proteomics screen to uncover dipeptidyl dipeptidase, DPP9 as a novel interacting partner with human NLRP1 and a related inflammasome regulator, CARD8. DPP9 functions as an endogenous inhibitor of NLRP1 inflammasome in diverse primary cell types from human and mice. DPP8/9 inhibition via small molecule drugs and CRISPR/Cas9-mediated genetic deletion specifically activate human NLRP1 inflammasome, leading to ASC speck formation, pyroptotic cell death and secretion of cleaved IL-1β. Mechanistically, DPP9 interacts with a unique auto-proteolytic domain (FIIND) found in NLRP1 and CARD8. This scaffolding function of DPP9 and its catalytic activity act synergistically to maintain NLRP1 in its inactive state and repress downstream inflammasome activation. We further identified a single patient-derived germline missense mutation in NLRP1 FIIND domain that abrogates DPP9 binding, leading to inflammasome hyper-activation seen in the Mendelian auto-inflammatory disease AIADK. These results unite recent findings on the regulation of murine Nlrp1b by Dpp8/9 and uncover a new regulatory mechanism for the NLRP1 inflammasome in primary human cells. Our results further suggest that DPP9 could be a multi-functional inflammasome regulator involved in human auto-inflammatory diseases.

Taking the heat together: Many of the processes in living cells are mediated by protein complexes that dynamically assemble and dissociate depending on cellular needs. Tan et al. developed a method called thermal proximity coaggregation (TPCA) to monitor the dynamics of native protein complexes inside cells (see the Perspective by Li et al. ). The method is based on the idea that proteins within a complex will coaggregate upon heat denaturation. It uses a previously described cellular shift assay to determine melting curves for thousands of proteins and assigns a TPCA signature on the basis of similarity between the curves. The method was validated by detection of many known protein complexes. It identified cell-specific interactions in six cell lines, highlighting the potential for identifying protein complexes that are modulated by disease. Science , this issue p. 1170 ; see also p. 1105

A cellular thermal shift assay (CETSA) protocol identifies and resolves antimalarial drug targets in P. falciparum .