Non‐transcriptional regulation of NLRP3 inflammasome signaling by IL‐4

Abstract

Th2 cytokine IL-4 has been previously shown to suppress the production of proinflammatory cytokines in monocytes. However, the underlying molecular mechanism by which IL-4 signaling antagonizes proinflammatory responses is poorly characterized. In particular, whether IL-4 can modulate inflammasome signaling is unknown. Here, we provide evidence that IL-4 suppresses NLRP3-dependent caspase-1 activation and the subsequent IL-1β secretion but does not inhibit AIM2- or NLRC4-dependent caspase-1 activation in THP-1 and mouse bone marrow-derived macrophages. Upon LPS or LPS/ATP stimulation, IL-4 markedly inhibited the assembly of NLRP3 inflammasome, including NLRP3-dependent ASC oligomerization, NLRP3-ASC interaction, and NLRP3 speck-like oligomeric structure formation. The negative regulation of NLRP3 inflammasome by IL-4 was not due to the impaired mRNA or protein production of NLRP3 and proinflammatory cytokines. Supporting this observation, IL-4 attenuated NLRP3 inflammasome activation even in reconstituted NLRP3-expressing macrophages in which NLRP3 expression is not transcriptionally regulated by TLR-NF-κB signaling. Furthermore, the IL-4-mediated suppression of NLRP3 inflammasome was independent of STAT6-dependent transcription and mitochondrial ROS. Instead, IL-4 inhibited subcellular redistribution of NLRP3 into mitochondria and microtubule polymerization upon NLRP3-activating stimulation. Our results collectively suggest that IL-4 could suppress NLRP3 inflammasome activation in a transcription-independent manner, thus providing an endogenous regulatory machinery to prevent excessive inflammasome activation.

Rotenone-induced Impairment of Mitochondrial Electron Transport Chain Confers a Selective Priming Signal for NLRP3 Inflammasome Activation

“…To determine inflammasome activity, the culture supernatants were precipitated as described previously and then immunoblotted with anti-caspase-1 or IL-1␤ antibody (23). To determine the oligomerization of NLRP3, confocal microscopy of NLRP3-GFP-expressing BMDMs was performed using a confocal microscope (Zeiss LSM 700), as described previously (22). To determine the oligomerization of ASC, a chemical cross-linking assay was performed using disuccinimidyl suberate as described previously (24).…”

“…To determine the oligomerization of ASC, a chemical cross-linking assay was performed using disuccinimidyl suberate as described previously (24). To visualize ASC specklike oligomeric structure, an immunofluorescence assay was performed using anti-ASC antibody as described previously (22).…”

“…Quantification of mRNA Expression-To measure mRNA expression of IL-1␤, Nlrp3, and Asc, a quantitative real-time PCR assay was performed as described previously (22). Primers were as follows: 5Ј-GCC CAT CCT CTG TGA CTC AT-3Ј and 5Ј-AGG CCA CAG GTA TTT TGT CG-3Ј (IL-1␤); 5Ј-ATG CTG CTT CGA CAT CTC CT-3Ј and 5Ј-AAC CAA TGC GAG ATC CTG AC-3Ј (Nlrp3); and 5Ј-CTT GTC AGG GGA TGA ACT CAA AA-3Ј and 5Ј-GCC ATA CGA CTC CAG ATA GTA GC-3Ј (Asc).…”

Targeting the inflammasome in rheumatic diseases

“…De-ubiquitination of NLRP3 has also been reported as an important intermediate step for inflammasome priming(7). Subcellular compartmentalization contributes to inflammasome regulation, and anti-inflammatory signals may block inflammasome assembly by preventing co-localization of inflammasome components(8). Other adaptor and signaling molecules, such as caspase 8, also participate in inflammasome regulation (reviewed in(9)).…”

“…The disease is manifested by recurrent fevers (typically occurring every 4–6 weeks), rashes, abdominal pain, diarrhea, vomiting, tender lymphadenopathy, nonerosive arthritis, and elevated inflammatory markers (8, 117). IgD levels are persistently elevated (118).…”