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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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 7

HERC6 promotes VSV-induced inflammation.

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HERC6 promotes VSV-induced inflammation. (A and B) Immunoblot analysis o...
(A and B) Immunoblot analysis of HERC6 and ISG15 expression in VSV-infected (A) or mIFN-β–stimulated (B) mouse PMs. (C) ELISA analysis of IL-1β in supernatants of PMs from Herc6+/+ or Herc6–/– mice after infection with VSV for 24 hours (2-tailed t test, Herc6+/+ vs. Herc6–/–, **P = 0.003646). (D) Immunoblot analysis of PMs from Herc6+/+ or Herc6–/– mice after VSV infection. (E) ELISA analysis of TNF-α and IL-6 in supernatants of PMs from Herc6+/+ or Herc6–/– mice after VSV infection for 24 hours (2-tailed t test, Herc6+/+ vs. Herc6–/–, ***P = 0.000399, **P = 0.004338). (F) ELISA analysis of IL-1β, TNF-α, and IL-6 in supernatants from mouse PMs transfected with Ctrl or Isg15 siRNA for 48 hours — following VSV infection for 12 or 24 hours (2-tailed t test, Ctrl vs. Isg15 siRNA, IL-1β: ***P = 0.000059, *P = 0.023051; TNF-α: *P = 0.011107 [12 h], *P = 0.026233 [24h]; IL-6: **P = 0.001473 [12h], **P = 0.001505 [24h]. (G) Immunoblot analysis of mouse PMs transfected with Ctrl or Isg15 siRNA for 48 hours, following VSV infection. (H) Immunoblot analysis of THP-1 cells transfected with Ctrl or HERC5 siRNA for 48 hours, following VSV infection. (I–K) ELISA analysis IL-1β in serum (I) and BALF (J), or TNF-α and IL-6 in serum (K) of Herc6+/+ or Herc6–/– mice after infection with VSV by i.p. injection for 12 hours (PBS n = 3, VSV n = 5 per group; 2-tailed t test, Herc6+/+ vs. Herc6–/–, I: ***P = 3.44 × 10–5, J: ***P = 0.000233, K: TNF-α ***P = 9.78 × 10–7, IL-6 ***P = 0.000241). All data are presented as mean ± SD in C, E, F, and I–K. Similar results were obtained from 3 independent experiments.