Lysosomal cathepsins act in concert with Gasdermin-D during NAIP/NLRC4-dependent IL-1β secretion

Abstract

The NAIP/NLRC4 inflammasome is classically associated with the detection of bacterial invasion to the cytosol. However, recent studies have demonstrated that NAIP/NLRC4 is also activated in non-bacterial infections, and in sterile inflammation. Moreover, in addition to the well-established model for the detection of bacterial proteins by NAIP proteins, the participation of other cytosolic pathways in the regulation of NAIP/NLRC4-mediated responses has been reported in distinct contexts. Using pharmacological inhibition and genetic deletion, we demonstrate here that cathepsins, well known for their involvement in NLRP3 activation, also regulate NAIP/NLRC4 responses to cytosolic flagellin in murine and human macrophages. In contrast to that observed for NLRP3 agonists, cathepsins inhibition did not reduce ASC speck formation or caspase-1 maturation in response to flagellin, ruling out their participation in the effector phase of NAIP/NLRC4 activation. Moreover, cathepsins had no impact on NF-κB-mediated priming of pro-IL-1β, thus suggesting these proteases act downstream of the NAIP/NLRC4 inflammasome activation. IL-1β levels secreted in response to flagellin were reduced in the absence of either cathepsins or Gasdermin-D (GSDMD), a molecule involved in the induction of pyroptosis and cytokines release. Notably, IL-1β secretion was abrogated in the absence of both GSDMD and cathepsins, demonstrating their non-redundant roles for the optimal IL-1β release in response to cytosolic flagellin. Given the central role of NAIP/NLRC4 inflammasomes in controlling infection and, also, induction of inflammatory pathologies, many efforts have been made to uncover novel molecules involved in their regulation. Thus, our findings bring together a relevant contribution by describing the role of cathepsins as players in the NAIP/NLRC4-mediated responses.

NAIP/NLRC4 inflammasome participates in macrophage responses to Trypanosoma cruzi by a mechanism that relies on cathepsin-dependent caspase-1 cleavage

“…While K + efflux and ROS inhibition, known pathways associated with NLRP3 activation ( 5 , 34 ), did not exert any discernible influence on IL-1β ( Figure 3B ) or NO ( Figure 3C ) release in T. cruzi -infected macrophages, a distinct outcome was observed upon cathepsins inhibition. The inhibition of lysosomal cathepsins, protease enzymes implicated in both NLRP3 ( 35 ) and NLRC4 ( 36 , 37 ) activation, abrogated IL-1β and NO secretion in response to T. cruzi infection ( Figures 3B, C ). As expected, CA-074Me and KCl, but not NaCl or Apocynin, significantly reduced IL-1β secretion by nigericin-stimulated macrophages ( Supplementary Figure 2C ).…”

“…Lysosomal membrane permeabilization (LMP) and the consequent leakage of cathepsins into the cytosol are known pathways associated with NLRP3 activation in response to different stimuli ( 35 , 61 , 62 ), including protozoan infections ( 7 , 9 , 11 ). Recent studies have further highlighted the role of cathepsins in regulating NAIP/NLRC4-dependent IL-1β secretion by murine and human macrophages in response to cytosolic flagellin ( 37 ).…”

“…Cathepsins can influence ASC speck formation, caspase-1 maturation, pro-IL-1β transcription, and IL-1β secretion in response to classical NLRP3 agonists like particulate material, ATP, or toxins ( 35 , 63 , 64 ). In response to flagellin, a well-known NLRC4 agonist, cathepsins seem to collaborate with gasdermin D (GSDMD) to enhance IL-1β secretion ( 37 ). This study, however, suggests that cathepsins likely do not play a role during the priming and assembly stages of NLRP3 and NAIP/NLRC4 inflammasomes in response to T. cruzi .…”

Gasdermins assemble; recent developments in bacteriology and pharmacology

“…In both cases, GSDMD emerged as a key player in the lipopolysaccharide (LPS)-induced non-canonical inflammasome model deployed by both studies. Since, the family (except GSDMF) were shown to possess N-terminal pore-forming and C-terminal autoinhibitory functions ( 10 ), and GSDMD specifically has been shown to be activated by other caspases, in both activation and inhibition, as well as by other proteases ( 19 – 21 , 24 ). It should be noted that GSDMB can engage lipid binding in vitro , even in the presence of the C-terminal domain, indicating a possible divergent role associated with lipid interactions for full length GSDMB ( 40 ).…”

“…Apoptotic caspases such as caspase-3 and caspase-8 have emerged as inflammasome-independent contributors to gasdermin activation ( 19 ). Caspase independent contributions to gasdermin function also exist in the form of cathepsins ( 20 , 21 ), granzymes ( 22 , 23 ) and elastase, neutrophil expressed (ELANE) ( 24 , 25 ). These different drivers have both distinct and overlapping biological consequences dependent on the cellular context and/or sensed DAMP/pathogen, including switches between pyroptosis to apoptosis and necroptosis.…”