Dave Boucher

NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase‐4 and caspase‐5

Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase‐4 and caspase‐5. When activated, these trigger pyroptotic cell death and caspase‐1‐dependent IL‐1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase‐4/5‐dependent IL‐1β production elicited by transfected LPS. Given that both caspase‐4 and caspase‐5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase‐4 and caspase‐5 were genetically deleted either individually or together. We found that the deletion of caspase‐4 suppressed cell death and IL‐1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase‐5 did not confer protection against transfected LPS, cell death and IL‐1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase‐4 and caspase‐5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL‐1β maturation, downstream of cytoplasmic LPS detection by caspase‐4/5. We also show that both caspase‐4 and caspase‐5 are functionally important for appropriate responses to intracellular Gram‐negative bacteria.

Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis

During apoptosis, hundreds of proteins are cleaved by caspases, most of them by the executioner caspase-3. However, caspase-7, which shares the same substrate primary sequence preference as caspase-3, is better at cleaving poly(ADP ribose) polymerase 1 (PARP) and Hsp90 cochaperone p23, despite a lower intrinsic activity. Here, we identified key lysine residues (K38KKK) within the N-terminal domain of caspase-7 as critical elements for the efficient proteolysis of these two substrates. Caspase-7s N-terminal domain binds PARP and improves its cleavage by a chimeric caspase-3 by ∼30-fold. Cellular expression of caspase-7 lacking the critical lysine residues resulted in less-efficient PARP and p23 cleavage compared with cells expressing the wild-type peptidase. We further showed, using a series of caspase chimeras, the positioning of p23 on the enzyme providing us with a mechanistic insight into the binding of the exosite. In summary, we have uncovered a role for the N-terminal domain (NTD) and the N-terminal peptide of caspase-7 in promoting key substrate proteolysis.

Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity.

Host-protective caspase-1 activity must be tightly regulated to prevent pathology, but mechanisms controlling the duration of cellular caspase-1 activity are unknown. Caspase-1 is activated on inflammasomes, signaling platforms that facilitate caspase-1 dimerization and autoprocessing. Previous studies with recombinant protein identified a caspase-1 tetramer composed of two p20 and two p10 subunits (p20/p10) as an active species. In this study, we report that in the cell, the dominant species of active caspase-1 dimers elicited by inflammasomes are in fact full-length p46 and a transient species, p33/p10. Further p33/p10 autoprocessing occurs with kinetics specified by inflammasome size and cell type, and this releases p20/p10 from the inflammasome, whereupon the tetramer becomes unstable in cells and protease activity is terminated. The inflammasome–caspase-1 complex thus functions as a holoenzyme that directs the location of caspase-1 activity but also incorporates an intrinsic self-limiting mechanism that ensures timely caspase-1 deactivation. This intrinsic mechanism of inflammasome signal shutdown offers a molecular basis for the transient nature, and coordinated timing, of inflammasome-dependent inflammatory responses.

Molecular determinants involved in activation of caspase 7

During apoptosis, initiator caspases (8, 9 and 10) activate downstream executioner caspases (3, 6 and 7) by cleaving the IDC (interdomain connector) at two sites. Here, we demonstrate that both activation sites, site 1 and site 2, of caspase 7 are suboptimal for activation by initiator caspases 8 and 9 in cellulo, and in vitro using recombinant proteins and activation kinetics. Indeed, when both sites are replaced with the preferred motifs recognized by either caspase 8 or 9, we found an up to 36-fold improvement in activation. Moreover, cleavage at site 1 is preferred to site 2 because of its location within the IDC, since swapping sites does not lead to a more efficient activation. We also demonstrate the important role of Ile195 of site 1 involved in maintaining a network of contacts that preserves the proper conformation of the active enzyme. Finally, we show that the length of the IDC plays a crucial role in maintaining the necessity of proteolysis for activation. In fact, although we were unable to generate a caspase 7 that does not require proteolysis for activity, shortening the IDC of the initiator caspase 8 by four residues was sufficient to confer a requirement for proteolysis, a key feature of executioner caspases. Altogether, the results demonstrate the critical role of the primary structure of caspase 7s IDC for its activation and proteolytic activity.

General In Vitro Caspase Assay Procedures

One of the most valuable tools that have been developed for the study of apoptosis is the availability of recombinant active caspases. The determination of caspase substrate preference, the design of sensitive substrates and potent inhibitors, the resolution of caspase structures, the elucidation of their activation mechanisms, and the identification of their substrates were made possible by the availability of sufficient amounts of enzymatically pure caspases. The current chapter describes at length the expression, purification, and basic enzymatic characterization of apoptotic caspases.

Noncanonical inflammasome signaling elicits gasdermin D–dependent neutrophil extracellular traps

Neutrophils form gasdermin D pores and expel antimicrobial neutrophil extracellular traps to defend against cytosolic bacteria. Casting NETs Gasdermin D (GSDMD), a pore-forming protein, has emerged a key downstream effector in pyroptosis, a form of cell death induced by intracellular lipopolysaccharide (LPS). Here, by examining the role of GSDMD in the neutrophil response to LPS and cytosolic Gram-negative bacteria, Chen et al. have uncovered an important role for GSDMD in the generation of neutrophil extracellular traps (NETs). NETs are composed of chromatin and antimicrobial proteins and are cast by dying neutrophils in a process termed NETosis. The authors report that GSDMD is directly cleaved by caspase-11 and that intracellular LPS–induced NETosis is dependent on both caspase-11 and GSDMD. In the same issue, Sollberger et al. also report a role for GSDMD in NETosis. Neutrophil extrusion of neutrophil extracellular traps (NETs) and concomitant cell death (NETosis) provides host defense against extracellular pathogens, whereas macrophage death by pyroptosis enables defense against intracellular pathogens. We report the unexpected discovery that gasdermin D (GSDMD) connects these cell death modalities. We show that neutrophil exposure to cytosolic lipopolysaccharide or cytosolic Gram-negative bacteria (Salmonella ΔsifA and Citrobacter rodentium) activates noncanonical (caspase-4/11) inflammasome signaling and triggers GSDMD-dependent neutrophil death. GSDMD-dependent death induces neutrophils to extrude antimicrobial NETs. Caspase-11 and GSDMD are required for neutrophil plasma membrane rupture during the final stage of NET extrusion. Unexpectedly, caspase-11 and GSDMD are also required for early features of NETosis, including nuclear delobulation and DNA expansion; this is mediated by the coordinate actions of caspase-11 and GSDMD in mediating nuclear membrane permeabilization and histone degradation. In vivo application of deoxyribonuclease I to dissolve NETs during murine Salmonella ΔsifA challenge increases bacterial burden in wild-type but not in Casp11−/− and Gsdmd −/− mice. Our studies reveal that neutrophils use an inflammasome- and GSDMD-dependent mechanism to activate NETosis as a defense response against cytosolic bacteria.

Quantifying Caspase-1 Activity in Murine Macrophages

The caspase-1 protease is a core component of multiprotein inflammasome complexes, which play a critical role in regulating the secretion of mature, bioactive pro-inflammatory cytokines interleukin (IL)-1β and IL-18. The activity of caspase-1 is often measured indirectly, by monitoring cleavage of cellular caspase-1 substrates, processing of caspase-1 itself, or by quantifying cell death. Here we describe methods for eliciting caspase-1 activity in murine macrophages, via activation of the NLRP3, NAIP/NLRC4 or AIM2 inflammasomes. We then describe a simple fluorogenic assay for directly quantifying cellular caspase-1 activity.

Cutting Edge: Blockade of Inhibitor of Apoptosis Proteins Sensitizes Neutrophils to TNF- but Not Lipopolysaccharide-Mediated Cell Death and IL-1β Secretion

The mammalian inhibitor of apoptosis proteins (IAPs) are key regulators of cell death and inflammation. A major function of IAPs is to block the formation of a cell death–inducing complex, termed the ripoptosome, which can trigger caspase-8–dependent apoptosis or caspase-independent necroptosis. Recent studies report that upon TLR4 or TNF receptor 1 (TNFR1) signaling in macrophages, the ripoptosome can also induce NLRP3 inflammasome formation and IL-1β maturation. Whether neutrophils have the capacity to assemble a ripoptosome to induce cell death and inflammasome activation during TLR4 and TNFR1 signaling is unclear. In this study, we demonstrate that murine neutrophils can signal via TNFR1-driven ripoptosome assembly to induce both cell death and IL-1β maturation. However, unlike macrophages, neutrophils suppress TLR4-dependent cell death and NLRP3 inflammasome activation during IAP inhibition via deficiencies in the CD14/TRIF arm of TLR4 signaling.