TNF superfamily receptor OX40 triggers invariant NKT cell pyroptosis and liver injury
Peixiang Lan, … , Xiang Xiao, Xian Chang Li
Peixiang Lan, … , Xiang Xiao, Xian Chang Li
Published April 24, 2017
Citation Information: J Clin Invest. 2017;127(6):2222-2234. https://doi.org/10.1172/JCI91075.
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Research Article Immunology Inflammation

TNF superfamily receptor OX40 triggers invariant NKT cell pyroptosis and liver injury

Abstract

Tissue-resident immune cells play a key role in local and systemic immune responses. The liver, in particular, hosts a large number of invariant natural killer T (iNKT) cells, which are involved in diverse immune responses. However, the mechanisms that regulate survival and homeostasis of liver iNKT cells are poorly defined. Here we have found that liver iNKT cells constitutively express the costimulatory TNF superfamily receptor OX40 and that OX40 stimulation results in massive pyroptotic death of iNKT cells, characterized by the release of potent proinflammatory cytokines that induce liver injury. This OX40/NKT pyroptosis pathway also plays a key role in concanavalin A–induced murine hepatitis. Mechanistically, we demonstrated that liver iNKT cells express high levels of caspase 1 and that OX40 stimulation activates caspase 1 via TNF receptor–associated factor 6–mediated recruitment of the paracaspase MALT1. We also found that activation of caspase 1 in iNKT cells results in processing of pro–IL-1β to mature IL-1β as well as cleavage of the pyroptotic protein gasdermin D, which generates a membrane pore–forming fragment to produce pyroptotic cell death. Thus, our study has identified OX40 as a death receptor for iNKT cells and uncovered a molecular mechanism of pyroptotic cell death. These findings may have important clinical implications in the development of OX40-directed therapies.

Authors

Peixiang Lan, Yihui Fan, Yue Zhao, Xiaohua Lou, Howard P. Monsour, Xiaolong Zhang, Yongwon Choi, Yaling Dou, Naoto Ishii, Rafik M. Ghobrial, Xiang Xiao, Xian Chang Li

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Figure 3

Involvement of GSDMD cleavage in OX40-triggered pyroptosis of iNKT cells.

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Involvement of GSDMD cleavage in OX40-triggered pyroptosis of iNKT cells...
(A) WT B6 mice were treated with OX86 (200 μg, i.p.), and 24 hours later iNKT cells in the treated mice were assessed for IL-1β production by intracellular IL-1β staining. Data shown are cells gated on CD1d-αGalCer and CD3 double-positive iNKT cells. The FACS plot shown is representative of one of 6 experiments. (B) ELISA analysis of IL-1β and IL-18 levels in the serum of B6 mice injected with control IgG or OX86 (200 μg, i.p.). (C) FACS-sorted iNKT cells from WT B6 and caspase 1–KO mice were stimulated with OX86 for 24 hours, and cell survival was determined by FACS by staining with the vital dye SYTOX green. Data are from one representative experiment of 6 independent experiments. (D) FACS-sorted iNKT cells from WT B6 mice were stimulated with OX86 for 24 hours, and LDH in the culture supernatant was assessed using the LDH assay kit. Data represent mean ± SD of 6 experiments. (E) FACS-sorted iNKT cells from WT B6 mice and caspase 1–KO mice were stimulated with OX86 for 24 hours, and cleavage of caspase 1 and GSDMD was determined by immunoblotting. β-Actin was used as a loading control. The blot shown is one of 6 individual experiments. (F) FACS-sorted iNKT cells from WT B6 mice were transduced with a shRNA viral vector targeting GSDMD or a scrambled RNA vector as a control; cells were stimulated with OX86 for 24 hours, and cell survival was determined by FACS following staining with the vital dye SYTOX green. Data shown are from one of 6 independent experiments. (G) The summary graphs represent cell death in relative percentage of gated cells from experiments as described in F. The data shown are mean ± SD of 6 experiments. The P value was calculated by unpaired 2-tailed Student’s t test (B, D, and G), *P < 0.05.