Posttranslational ISGylation of NLRP3 by HERC enzymes facilitates inflammasome activation in models of inflammation
Ying Qin, … , Chunyuan Zhao, Wei Zhao
Ying Qin, … , Chunyuan Zhao, Wei Zhao
Published August 31, 2023
Citation Information: J Clin Invest. 2023;133(20):e161935. https://doi.org/10.1172/JCI161935.
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Research Article Immunology Inflammation

Posttranslational ISGylation of NLRP3 by HERC enzymes facilitates inflammasome activation in models of inflammation

Abstract

The NOD-, LRR-, and pyrin domain–containing protein 3 (NLRP3) inflammasome is a crucial component of the innate immune system that initiates inflammatory responses. Posttranslational modifications (PTMs) of NLRP3, including ubiquitination and phosphorylation, control inflammasome activation and determine the intensity of inflammation. However, the role of other PTMs in controlling NLRP3 inflammasome activation remains unclear. This study found that TLR priming induced NLRP3 ISGylation (a type of PTM in which ISG15 covalently binds to the target protein) to stabilize the NLRP3 protein. Viral infection, represented by SARS-COV-2 infection, and type I IFNs induced expression of ISG15 and the predominant E3 ISGylation ligases HECT domain- and RCC1-like domain–containing proteins (HERCs; HERC5 in humans and HERC6 in mice). HERCs promoted NLRP3 ISGylation and inhibited K48-linked ubiquitination and proteasomal degradation, resulting in the enhancement of NLRP3 inflammasome activation. Concordantly, Herc6 deficiency ameliorated NLRP3-dependent inflammation as well as hyperinflammation caused by viral infection. The results illustrate the mechanism by which type I IFNs responses control inflammasome activation and viral infection–induced aberrant NLRP3 activation. This work identifies ISGylation as a PTM of NLRP3, revealing a priming target that modulates NLRP3-dependent immunopathology.

Authors

Ying Qin, Xintong Meng, Mengge Wang, Wenbo Liang, Rong Xu, Jingchunyu Chen, Hui Song, Yue Fu, Jingxin Li, Chengjiang Gao, Mutian Jia, Chunyuan Zhao, Wei Zhao

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Figure 2

HERC6 selectively promotes NLRP3 inflammasome activation.

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HERC6 selectively promotes NLRP3 inflammasome activation. (A and B) ELIS...
(A and B) ELISA analysis of IL-1β, TNF-α, and IL-6 in supernatants of PMs from Herc6+/+ or Herc6–/– mice following priming with LPS for 7 hours and stimulation with ATP or Nig for 1 hour (A: 2-tailed t test, Herc6+/+ vs. Herc6–/–, *P = 0.012074, ***P = 0.000040). (C) ELISA analysis of IL-1β in supernatants of PMs from Herc6+/+ or Herc6–/– mice following LPS priming for 7 hours and stimulation with poly(dA:dT) or flagellin for 1 hour. (D) Immunoblot analysis of supernatants (SN) and cell lysates (CL) of PMs from Herc6+/+ or Herc6–/– mice, following LPS priming and subsequent ATP stimulation for 1 hour. (E) ELISA analysis of IL-1β in supernatants from PMs transfected with control (Ctrl) siRNA or Herc6 siRNA for 48 hours, followed by priming with LPS for 7 hours and stimulation with ATP, Nig, or poly(dA:dT) for 1 hour (2-tailed t test, Ctrl siRNA vs. Herc6 siRNA, **P = 0.009794, *P = 0.018808). (F) ELISA analysis of TNF-α and IL-6 in supernatants from PMs transfected with Ctrl or Herc6 siRNA for 48 hours, followed by priming with LPS for 7 hours and stimulation with ATP or Nig for 1 hour. (G) Immunoblot analysis of SN and CL of mouse PMs transfected with Ctrl siRNA or Herc6 siRNA for 48 hours, followed by LPS priming and ATP stimulation for 1 hour. (H and I) Immunoblot analysis of p-IκBα in LPS-stimulated PMs from Herc6+/+ or Herc6–/– mice (H), or PMs transfected with Ctrl or Herc6 siRNA (I). (J and K) RT-PCR analysis of Nlrp3 or Il1b, Tnfa, and Il6 mRNA in LPS-stimulated PMs from Herc6+/+ or Herc6–/– mice. All data are presented as the mean ± SD in A–C, E, F, J, and K. Similar results were obtained from 3 independent experiments.