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. 2015 Feb 15;194(4):1938-44.
doi: 10.4049/jimmunol.1402167. Epub 2015 Jan 7.

A RIPK3-caspase 8 complex mediates atypical pro-IL-1β processing

Kenta Moriwaki  1 , John Bertin  2 , Peter J Gough  2 , Francis Ka-Ming Chan  3
Affiliations

"VSports在线直播" A RIPK3-caspase 8 complex mediates atypical pro-IL-1β processing

Kenta Moriwaki et al. J Immunol. .

Abstract

Caspase 8, the initiator caspase for death receptor-induced apoptosis, functions as a negative regulator of receptor interacting protein kinase 3 (RIPK3), an essential factor for TNF-, TLR3-, and TLR4-induced necroptosis. In certain situations, caspase 8 can also participate in pro-IL-1β processing. However, the biochemical complex that mediates caspase 8-mediated processing is not defined. In this study, we show that RIPK3 is crucial for caspase 1- and caspase 8-mediated pro-IL-1β and pro-IL-18 processing in bone marrow-derived dendritic cells (BMDCs) in response to LPS stimulation. Caspase 8-mediated pro-IL-1β processing requires intact RIPK1, RIPK3, TRIF, and FADD. In response to LPS, a complex that contains RIPK1, RIPK3, FADD, and caspase 8 is formed. Surprisingly, RIPK3-specific kinase inhibitors strongly enhanced caspase 8 activation and pro-IL-1β processing in LPS-stimulated BMDCs VSports手机版. However, studies in BMDCs expressing the kinase-inactive RIPK3-K51A mutant or RIPK1-K45A mutant showed that the kinase activity of neither RIPK1 nor RIPK3 is required for LPS-induced caspase 8 activation and IL-1β secretion. Hence, RIPK3 is an unexpected positive regulator of caspase 8 activity that promotes IL-1β maturation in BMDCs. .

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FIGURE 1
FIGURE 1
RIPK3 promotes caspase 1- and caspase 8-dependent IL-1β secretion in BMDCs. (A) RIPK3 is required for LPS-induced IL-1β secretion in BMDCs. IL-1β secretion by WT and Ripk3−/− BMDCs stimulated with LPS for 6 hours was determined by ELISA (n=4). (B) Caspase 1 is partially responsible for RIPK3-dependent IL-1β secretion. WT BMDCs were pretreated with 10 µM z-YVAD-fmk (YVAD) for 1 hour and stimulated with LPS for 4 hours (n=4). IL-1β secretion was determined by ELISA. (C) RIPK3 is critical for LPS-induced caspase 8 activation. Cell lysates from WT and Ripk3−/− BMDCs stimulated with LPS for 1 hour were subjected to western blot analysis. (D) Caspase 1 and caspase 8 cooperate to mediate optimal IL-1β secretion. WT BMDCs pretreated with 10 µM YVAD and/or 10 µM z-IETD-fmk (IETD) for 1 hour were stimulated with 50 ng/ml LPS for 6 hours (n=4). IL-1β secretion was determined by ELISA. (E) Inhibition of protein synthesis converts the LPS-induced RIPK3 signal to one that causes apoptosis. WT and Ripk3−/− BMDCs pretreated with CHX for 1 hour were stimulated with LPS for 14 hours. Cell death was determined by measuring intracellular ATP level (n=3). (F) RIPK3-dependent caspase 8 activation requires an intact TRIF. Cell lysates from BMDCs of the indicated genotypes stimulated with LPS for 1 hour were subjected to western blot analysis. Results shown are mean ± SEM. Asterisks: p < 0.05.
FIGURE 2
FIGURE 2
RIPK3 promotes formation of an alternative caspase 8 activating complex. (A) RIPK3 mediates caspase 8 activation independent of ROS production. WT BMDCs pretreated with NAC for 1 hour were stimulated with LPS for 1 hour. Cell lysates were subjected to western blot analysis. (B and C) LPS induces assembly of a complex between RIPK3, FADD and caspase 8. Cell lysates from WT and Ripk3−/− BMDCs stimulated with LPS for 1 hour were subjected to immunoprecipitation with (B) anti-caspase 8 or (C) anti-RIPK3 antibodies followed by western blot analysis. WCE: whole cell extract. IP: immunoprecipitate. (D) An intact RIPK1, but not its kinase activity, is required for LPS-induced caspase 8 activation. WT and Ripk1K45A-KD BMDCs were transduced with lentivirus expressing GFP or Cre and subsequently stimulated with LPS for 1 hour. Cell lysates were subjected to western blot analysis.
FIGURE 3
FIGURE 3
RIPK3 kinase inhibitor enhances LPS-induced IL-1β secretion. (A) RIPK3 kinase inhibitors protect BMDCs against necroptosis. WT and Ripk3−/− BMDCs pretreated with indicated concentration (µM) of RIPK3 kinase inhibitors for 1 hour were stimulated with 10 µM z-VAD-fmk (zVAD) and 0.1 µM LBW242 for 14 hours to induce necroptosis (n=3). (B and C) RIPK3 kinase inhibitors increase LPS-induced IL-1β secretion. WT or Ripk3−/− BMDCs pretreated with the indicated RIPK3 inhibitors (µM) for 1 hour were stimulated with 50 ng/ml LPS for 6 hours (n=3). IL-1β secretion was determined by ELISA. (D) The effect of RIPK3 inhibitor on IL-1β secretion is not mediated by caspase 1 or caspase 11. WT and Casp1−/−Casp11−/− BMDCs pretreated with GSK’872 for 1 hour were stimulated with LPS for 6 hours (n=4). IL-1β secretion was determined by ELISA. Results shown are mean ± SEM. Asterisks: p < 0.05.
FIGURE 4
FIGURE 4
RIPK3 kinase activity is dispensable for LPS-induced caspase 8 activation. (A and B) FADD is required for RIPK3-dependent caspase 8 activation. BMDCs generated from Faddfl/fl and Faddfl/fl:Cd11c-Cre (dcFadd−/−) BM cells were pretreated with GSK’872 for 1 hour and then stimulated with LPS for (A) 6 hours or (B) 1 hour. (A) IL-1β secretion was determined by ELISA (n=2). (B) Cell lysates were subjected to western blot analysis. (C and D) RIPK3 kinase inhibitor specifically enhances caspase 8, but not caspase 1 activation. WT BMDCs pretreated with GSK’872 for 1 hour were stimulated with LPS for 1 hour. Cell lysates were subjected to (C) western blot analysis or (D) immunoprecipitation with anti-RIPK3 antibody followed by western blot analysis. WCE: whole cell extract. IP: immunoprecipitate. (E) RIPK3 kinase inhibitor enhances caspase 8 activation through RIPK1. WT and Ripk1K45A-KD BMDCs were transduced with lentivirus expressing GFP or Cre and subsequently treated with GSK’872 for 1 hour followed by LPS for 1 hour. Cell lysates were subjected to western blot analysis. (F–G) RIPK3 kinase activity is essential for necroptosis, but not IL-1β secretion. (F) WT and Ripk3K51A-KD BMDCs were treated with 10 µM z-VAD-fmk (zVAD) and 0.1 µM LBW242 for 14 hours to induce necroptosis (n=3). Cell death was determined by measuring intracellular ATP level. The upper panel shows protein expression level of RIPK3. (G) WT and Ripk3K51A-KD BMDCs pretreated with GSK’872 for 1 hour were stimulated with LPS for 6 hours (n=3). IL-1β secretion was determined by ELISA. Results shown are mean ± SEM.
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