RIPK3 Promotes Mefv Expression and Pyrin Inflammasome Activation via Modulation of mTOR Signaling

Abstract

Mutations in MEFV, the gene encoding pyrin in humans, are associated with the autoinflammatory disorder familial Mediterranean fever. Pyrin is an innate sensor that assembles into an inflammasome complex in response to Rho-modifying toxins, including Clostridium difficile toxins A and B. Cell death pathways have been shown to intersect with and modulate inflammasome activation, thereby affecting host defense. Using bone marrow-derived macrophages and a murine model of peritonitis, we show in this study that receptor-interacting protein kinase (RIPK) 3 impacts pyrin inflammasome activation independent of its role in necroptosis. RIPK3 was instead required for transcriptional upregulation of Mefv through negative control of the mechanistic target of rapamycin (mTOR) pathway and independent of alterations in MAPK and NF-κB signaling. RIPK3 did not affect pyrin dephosphorylation associated with inflammasome activation. We further demonstrate that inhibition of mTOR was sufficient to promote Mefv expression and pyrin inflammasome activation, highlighting the cross-talk between the mTOR pathway and regulation of the pyrin inflammasome. Our study reveals a novel interaction between molecules involved in cell death and the mTOR pathway to regulate the pyrin inflammasome, which can be harnessed for therapeutic interventions.

FIGURE 1.. Role of RIPK3 in pyrin inflammasome activation.

(A) Immunoblot analysis of caspase-1 processing in bone marrow-derived macrophages (BMDMs) from wild type (WT) and knockout mice treated with control or C. difficile toxin stimuli for 6 hours. Filled arrow designates the pro-form (p45), and open arrow designates cleaved caspase-1 (p20). (B) IL-18 release after stimulation of BMDMs with control or C. difficile toxin stimuli for 6 hours. (C) Percent of BMDMs exhibiting membrane permeability after stimulation with control or C. difficile toxin stimuli for 6 hours, assessed by lactate dehydrogenase (LDH) release. (D) Number of BMDMs exhibiting membrane permeability following stimulation with control or C. difficile toxin stimuli, assessed by Sytox staining. (D) Representative images of BMDMs stimulated with control or C. difficile toxin stimuli for 6 hours. The green demonstrates Sytox signal, and the pink mask outlines cells counted as dead in the analysis. Scale bar is 100 μM. Control refers to stimulation with supernatant from toxin-negative C. difficile cultures. Data are presented as mean ± s.e.m. (B and C) from technical replicates or as the average number from four fields of view (D), and data are representative of at least three independent repeats. ****P < 0.0001 using one-way ANOVA followed by Fischer’s LSD post-test.

FIGURE 2.. Activation of the pyrin inflammasome is independent of necroptosis.

(A, E) Immunoblot analysis of caspase-1 processing in bone marrow-derived macrophages (BMDMs) from wild type (WT) and knockout mice treated with control or C. difficile toxin stimuli for 6 hours. Filled arrow designates the pro-form (p45), and open arrow designates cleaved caspase-1 (p20). (B, F) IL-18 and (C, G) LDH release after stimulation of BMDMs with control or C. difficile toxin stimuli for 6 hours. (D, H) Representative images of BMDMs stimulated with control or C. difficile toxin stimuli for 6 hours obtained by brightfield microscopy. Blue arrows depict pyroptotic cell death. Scale bar is 20 μM. Control refers to stimulation with supernatant from toxin-negative C. difficile cultures. Data are presented as mean ± s.e.m. (B, E, and H) from technical replicates, and are representative of at least three independent repeats. ns, not significant; ****P < 0.0001 using one-way ANOVA followed by Fischer’s LSD post-test.

FIGURE 3.. RIPK3 modulates pyrin inflammasome activation independent of inflammatory signaling or pyrin dephosphorylation.

(A) Immunoblot analysis of the activation status of MAPK and NF-κB pathways in bone marrow-derived macrophages (BMDMs) from wild type (WT) and knockout mice. (B) Tnf and Pyrin induction in BMDMs stimulated with control or C. difficile toxin stimuli. Expression was normalized to set WT expression at baseline to 1. (C) Immunoblot analysis of caspase-1 processing in BMDMs treated with or without purified C. difficile toxin B (TcdB) for 6 hours. Filled arrow designates the pro-form (p45), and open arrow designates cleaved caspase-1 (p20). (D) IL-18 release after stimulation of BMDMs with TcdB for 6 hours. (E) Immunoblot analysis of pyrin dephosphorylation in response to TcdB. The top panel shows the phos-tag gel, and the bottom panel shows the western blot probed with anti-pyrin antibody. An increase in migration (shown as the lower band) on a phos-tag gel depicts dephosphorylation of pyrin. Data are presented as mean ± s.e.m. (B, D) from technical replicates, and are representative of at least three independent repeats. ns, not significant; *P < 0.05; ***P < 0.001; and ****P < 0.0001 using one-way ANOVA followed by Fischer’s LSD post-test.

FIGURE 4.. RIPK3 modulates pyrin inflammasome activation via mTOR signaling in response to C. difficile toxin stimuli.

(A and B) Immunoblot analysis of the activation status of mTOR and its downstream pathway in wild type (WT) and knockout (A) or WT (B) bone marrow-derived macrophages (BMDMs) stimulated with control or C. difficile toxin stimuli for the indicated time. (C and E) Immunoblot analysis of caspase-1 processing in BMDMs from WT or knockout mice treated with control or C. difficile toxin stimuli and either vehicle, rapamycin, PP242, or torin1 for 6 hours. Filled arrow designates the pro-form (p45), and open arrow designates cleaved caspase-1 (p20). (D and F) IL-18 release after stimulation of WT BMDMs with control or C. difficile toxin stimuli for 6 hours. (G) Percent of BMDMs exhibiting membrane permeability after stimulation with control or C. difficile toxin stimuli for 6 hours, assessed by lactate dehydrogenase (LDH) release. (B–G) BMDMs were incubated with mTOR inhibitors rapamycin, PP242, or torin1 (as indicated) for 30 minutes prior to addition of control or C. difficile toxin stimuli. Control refers to stimulation with supernatant from toxin-negative C. difficile cultures. Data are presented as mean ± s.e.m. (D, F, and G) from technical replicates, and are representative of at least three independent repeats. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001 using student’s t-test (D and F) and one-way ANOVA followed by Fischer’s LSD post-test (G).

FIGURE 5:. RIPK3 is required for pyrin inflammasome activation in vivo.

(A) Cytokine levels in the sera of wild type (WT) or knockout mice 4 hours post-intraperitoneal injection with supernatant from toxin-negative (control) or toxin-positive C. difficile cultures. (B) Cytokine levels in the sera of WT or knockout mice 4 hours post-intraperitoneal injection with supernatant from toxin-positive C. difficile cultures. Control refers to stimulation with supernatant from toxin-negative C. difficile cultures. Data are pooled from two to three independent repeats, and N = 7–15 for each genotype. ns, not significant; *P < 0.05; and ****P < 0.0001 using one-way ANOVA followed by Fischer’s LSD post-test or Kruskal-Wallis and Dunn’s post-test.