Caspase-8 scaffolding function and MLKL regulate NLRP3 inflammasome activation downstream of TLR3

Abstract

TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA.

Figure 1. RIPK1, FADD and caspase-8 are required for activation of the NLRP3 inflammasome by dsRNA.

Immunoblots of caspase-1 in the culture supernatants (Sup) or cell lysates (Lys) of C57BL/6 mouse macrophages derived from WT (a) RIPK1-KO (b) TRIF-KO (c) RIPK3-KO (d) FADD/RIPK3-DKO (e) caspase-8/RIPK3-DKO (f) Caspase-8/RIPK1/RIPK3-TKO (g) mice, treated with Pam3CSK4 (Pam), poly(I:C) or LPS for the indicated times (min) followed by stimulation with ATP for an additional 45 min. Results are representative of at least three independent experiments.

Figure 2. dsRNA priming promotes NLRP3-induced pyroptosis.

LDH release in the culture supernatants of macrophages from WT and the indicated mouse knockout strains, treated with poly(I:C) (a) or Pam3CSK4 (Pam) (b) for the indicated times (min) followed by stimulation with ATP for an additional 45 min. DKO, caspase-8/RIPK3-DKO; TKO, caspase-8/RIPK1/RIPK3-TKO. Results are representative of at least three independent experiments. Error bars represent s.d. (c) Immunoblots of caspase-1 in the culture supernatants (Sup) or cell lysates (Lys) of mouse macrophages derived from WT, MyD88-KO or NLRP3-KO mice treated with poly(I:C) for the indicated times (min) followed by infection with Listeria (multiplicity of infection (MOI): 100) for an additional 45 min. Caspase-1 activation in macrophages infected with Listeria alone without prior priming with poly(I:C) is shown in lanes 2 and 7 of the left and right panels. (d) LDH release in the culture supernatants of macrophages derived from WT, MyD88-KO or NLRP3-KO mice treated with poly(I:C) for the indicated times (min) followed by infection with Listeria (MOI: 100) for an additional 45 min. LDH release in macrophages infected with Listeria alone without prior priming with poly(I:C) is shown in the second columns from left. Error bars represent s.d.

Figure 3. Caspase-8 enzymatic activity is not required for dsRNA-induced ASC polymerization.

(a–c) (upper panels) Immunoblots of disuccinimidyl suberate (DSS) cross-linked ASC in the NP40-insoluble pellets (Pel) of WT (a), RIPK3-KO (b) and RIPK1-KO (c) macrophages after stimulation with poly(I:C) (I and II) for the indicated times (min) in the absence (I) or presence (II) of zVAD followed by stimulation with ATP for an additional 45 min as indicated. Immunoblots of caspase-1 in the culture supernatants (Sup) of the corresponding samples are shown underneath the ASC panels. Immunoblots of total ASC in the cell lysates (Lys) of all samples is shown at the bottom of (a–c) panels. The ASC aggregates present in the NP40-insoluble pellets are labelled ASC polymers. These fractionate as monomeric and dimeric ASC species following cross-linking with DSS, solubilization in SDS sample buffer and subsequent fractionation on SDS–PAGE. Results are representative of at least three independent experiments.

Figure 4. dsRNA and zVAD induce transcription- and signal 2-independent inflammasome activation.

(a) Immunoblots of disuccinimidyl suberate (DSS) cross-linked ASC in the NP40-insoluble pellets of WT macrophages after stimulation with poly(I:C) for the indicated times (min) in the presence of zVAD (left panel), zVAD followed by stimulation with ATP for an additional 45 min (middle panel) or zVAD plus actinomycin D (ActD, right panel) as indicated. (b) Immunoblots of DSS cross-linked ASC in the NP40-insoluble pellets of WT (first panel), RIPK3-KO (second panel), TRIF-KO (third panel) or NLRP3-KO (fourth panel) macrophages after stimulation with poly(I:C) for the indicated times (min) in the presence of zVAD as indicated. Immunoblots of total ASC in the cell lysates (Lys) of all samples is shown at the bottom of a, b panels. Results are representative of at least three independent experiments.

Figure 5. Kinase activity of RIPK3 but not of RIPK1 is required in the late pathway.

(a–c) Immunoblots of disuccinimidyl suberate cross-linked ASC in the NP40-insoluble pellets of WT, kinase-dead RIPK1 (RIPK1-KD) or RIPK3 (RIPK3-KD) macrophages after stimulation with poly(I:C) for the indicated times (min) in the presence of zVAD (left panel) or zVAD followed by stimulation with ATP for 45 min (middle panel), or in the absence of zVAD followed by stimulation with ATP for an additional 45 min (right panel) as indicated. Immunoblots of caspase-1 in the culture supernatants (Sup) of the corresponding samples are shown underneath the ASC panels. Immunoblots of total ASC in the cell lysates (Lys) of all samples is shown at the bottom of a–c panels. Results are representative of at least three independent experiments.

Figure 6. MLKL activity is critical in the late pathway.

(a) Immunoblots of caspase-1 in the culture supernatants (Sup) or cell lysates (Lys) of mouse macrophages derived from MLKL knockout (MLKL-KO) mice treated with Pam3CSK4 (Pam), poly(I:C) or LPS for the indicated times (min) followed by stimulation with ATP for 45 min. (b) Immunoblots of disuccinimidyl suberate cross-linked ASC in the NP40-insoluble pellets of WT and MLKL-KO macrophages after stimulation with poly(I:C) for the indicated times (min) in the presence of zVAD (first and second panels) or zVAD followed by stimulation with ATP for an additional 45 min (third and fourth panels) as indicated. Immunoblots of total ASC in the cell lysates (Lys) of all samples is shown at the bottom. Results are representative of at least three independent experiments.

Figure 7. Caspase-8 scaffolding function is required for inflammasome activation and RIPK3 aggregation.

(a) Immunoblot of disuccinimidyl suberate cross-linked ASC in the NP40-insoluble pellets (upper panel) of stable RIPK3-GFP-reconstituted RIPK3-KO (RIPK3-KO+RIPK3-GFP) or caspase-8-RIPK3-DKO (Casp8/RIPK3-DKO+RIPK3-GFP) macrophages after stimulation with poly(I:C) for the indicated times (min) in the presence of zVAD. The lower panels show immunoblots of RIPK3-GFP, caspase-8 and MLKL in the lysates of the same samples. (b) Confocal images of unstimulated (Un, upper panels) or poly(I:C) plus zVAD-stimulated (180 min; lower panels) stable RIPK3-GFP-reconstituted RIPK3-KO (RIPK3-KO+RIPK3-GFP) or caspase-8-RIPK3-DKO (Casp8/RIPK3-DKO+RIPK3-GFP) macrophages. Scale bar, 10 μm. (c) RIPK3 or RIPK3-ΔRHIM fused to two copies of FKBP^F36V (RIPK3-2xFV or RIPK3-ΔRHIM-2xFV, respectively; upper diagram), were ectopically expressed in 293T-CAN cell line, which is stably reconstituted with the human NLRP3 inflammasome components procaspase-1, ASC and NLRP3, or 293T-CA cell line stably reconstituted with only procaspase-1 and ASC. Caspase-1 immunoblots of cell lysates (lower panels) show caspase-1 p20 band only in RIPK3-2xFV-transfected 293T-CAN, but not in 293T-CA cells after stimulation with the oligomerization drug AP20187. Results are representative of at least three independent experiments.

Figure 8. Cytoplasmic dsRNA requires FADD and caspase-8 but not DRP1 for inflammasome activation.

Immunoblots of caspase-1 in the culture supernatants (Sup) or cell lysates (Lys) of WT or the indicated knockout primary macrophages (a–c,e) or stable RIPK3-GFP-reconstituted RIPK3-KO (RIPK3-KO+RIPK3-GFP) or caspase-8-RIPK3-DKO (Casp8/RIPK3-DKO+RIPK3-GFP) immortalized macrophages (d) transfected with poly(I:C) in the presence or absence of GSK'872 (GSK) for 5 h as indicated. The lower two panels in d show immunoblots with anti-caspase-8 or anti-RIPK3 antibodies. The decrease in RIPK3-GFP in the lysates (third lane) is due to GSK'872-induced RIPK3-GFP aggregation in the insoluble fraction. Results are representative of at least three independent experiments.

Figure 9. VSV-induced NLRP3 activation is partially dependent on RIPK3 but not on DRP1 or RIPK1.

(a–h) Immunoblots of caspase-1 (first panels from top) and mature IL-1β p17 (second panels from top) in the culture supernatants (Sup) of macrophages derived from WT or the indicated knockout mice after infection with the indicated doses of VSV (plaque forming units (p.f.u.)) for 16 h. Immunoblots of procaspase-1, pro-IL-1β and knocked out proteins in the total cell lysates are shown underneath the supernatants blots. Results are representative of at least three independent experiments.

Figure 10. Schematic representations of the TLR and cytoplasmic dsRNA pathways that regulate NLRP3 activation.

(a) Three pathways downstream of TLRs are involved in priming and activation of the NLRP3 inflammasome. The early MyD88-dependent pathway provides a priming signal 1 downstream of TLR2, whereas the intermediate TRIF-dependent pathway provides a priming signal 1 downstream of TLR3. Both of these pathways require exogenous signal 2 from purinergic receptors or pore-forming toxins for activation of NLRP3. Signal 2 is required to induce potassium efflux. The late TRIF-dependent pathway can provide both signal 1 and an endogenous signal 2 by recruiting RIPK3 and MLKL when caspase-8 is inhibited. (b) In WT macrophages, FADD/caspase-8 complex is required to remove RIPK3 inhibition of the cytoplasmic dsRNA sensor. In RIPK3-deficient macrophages such as Casp8/RIPK3-DKO macrophages, caspase-8 is not required for activation of NLRP3 by dsRNA sensor.