DDX17 is an essential mediator of sterile NLRC4 inflammasome activation by retrotransposon RNAs

Abstract

Detection of microbial products by multiprotein complexes known as inflammasomes is pivotal to host defense against pathogens. Nucleotide-binding domain leucine-rich repeat (NLR) CARD domain containing 4 (NLRC4) forms an inflammasome in response to bacterial products; this requires their detection by NLR family apoptosis inhibitory proteins (NAIPs), with which NLRC4 physically associates. However, the mechanisms underlying sterile NLRC4 inflammasome activation, which is implicated in chronic noninfectious diseases, remain unknown. Here, we report that endogenous short interspersed nuclear element (SINE) RNAs, which promote atrophic macular degeneration (AMD) and systemic lupus erythematosus (SLE), induce NLRC4 inflammasome activation independent of NAIPs. We identify DDX17, a DExD/H box RNA helicase, as the sensor of SINE RNAs that licenses assembly of an inflammasome comprising NLRC4, NLR pyrin domain–containing protein 3, and apoptosis-associated speck-like protein–containing CARD and induces caspase-1 activation and cytokine release. Inhibiting DDX17-mediated NLRC4 inflammasome activation decreased interleukin-18 release in peripheral blood mononuclear cells of patients with SLE and prevented retinal degeneration in an animal model of AMD. Our findings uncover a previously unrecognized noncanonical NLRC4 inflammasome activated by endogenous retrotransposons and provide potential therapeutic targets for SINE RNA–driven diseases.

Fig. 1.. SINE RNAs activate NLRC4 inflammasome.

(A) Immunoblot analysis of indicated proteins in cell lysates and supernatant of Nlrc4^+/+, Nlrc4^−/− BMDMs transfected with various doses (25, 100, 250, 400 pmol) of B2 RNA or mock for 12 hours. “Mock” denotes the use of transfection reagents alone in all experiments. (B) ELISA quantification of IL-1β release from LPS-primed Nlrc4^+/+, Nlrc4^−/− BMDMs transfected with various doses of B2 RNA or mock for 12 hours. “Lipo” represents lipofectamine 3000. (C) Immunoblot analysis of indicated proteins in BMDMs transfected with 100 pmol Alu RNA or mock at the indicated time points. (D) Immunoblot analysis of NLRC4 and actin, under native PAGE or SDS PAGE conditions, in BMDMs transfected with 100 pmol Alu RNA or mock at the indicated time points. (E) Confocal images of ASC specks and NLRC4 aggregates in 100pmol Alu RNA- or mock-transfected BMDMs. Scale bars, 20 μm. (F) Immunoblot analysis of ASC, NLRC4, and actin in cell lysates (soluble) and of ASC in DSS-crosslinked pellets (insoluble) with BMDM cells treated with LPS/ATP or transfected with Alu RNA (100 pmol) or flagellin (3 μg/ml, Fla). o, oligomers; d, dimers; m, monomers. (G) Measurement of IL-1β in LPS-primed wild-type (WT), Pycard^−/− and Nlrc4^−/− BMDMs transfected with 100 pmol Alu RNA for 12 hours or 3 μg/ml of flagellin (Fla) for 6 hours. Shown is the mean ± SEM for independent experiments. ****P < 0.0001; two-way ANOVA with Sidak’s multiple comparisons. ns, not significant.

Fig. 2.. NLRC4 phosphorylation (S533A) is important for SINE RNAs induced inflammasome activation.

(A) Primary BMDMs were isolated from Nlrc4^+/+, Nlrc4^S533A/S533A and Nlrc4^−/− mice, and transfected with 100 pmol Alu RNA or mock for 12 hours. The cell lysates were used for detecting phosphorylated NLRC4 (p-NLRC4) and actin; supernatant for pro-CASP1 (p45), cleaved CASP1 (p20) by immunoblotting. L+S, lysates and supernatant. (B) ELISA measurement of IL-1β release in LPS-primed Nlrc4^+/+, Nlrc4^S533A/S533A and Nlrc4^−/− BMDMs transfected with 100 pmol Alu RNA or mock for 12 hours. (C) Immunoblot analysis of indicated proteins in wild-type (Prkcd^+/+), Prkcd^+/−, and Prkcd^−/− BMDMs transfected with 100 pmol of Alu RNA or mock. Sup (supernatant); WCL (whole cell lysates). (D) Measurement of IL-1β in LPS-primed wild-type (Prkcd^+/+), Prkcd^+/−, and Prkcd^−/− BMDMs transfected with 100 pmol of Alu RNA or mock for 12 hours. (E) Immunoblot analysis of indicated proteins in wild-type (WT), Nlrc4^−/−, or Naip1-6^Δ/Δ BMDMs transfected with flagellin (3 μg/ml) using DOTAP or the transfection reagent DOTAP alone. Sup (supernatant). (F) ELISA measurement of IL-1β in LPS-primed wild-type (WT), Nlrc4^−/−, or Naip1-6^Δ/Δ BMDMs transfected with flagellin (3 μg/ml) using DOTAP. (G) Immunoblot analysis of pro-CASP1 (p45) and cleaved CASP1 (p20) in WT and Naip1-6^Δ/Δ BMDMs transfected with 100 pmol of Alu RNA or mock. L+S, lysates and supernatant. (H) ELISA measurement of IL-1β in LPS-primed WT and Naip1-6^Δ/Δ BMDMs transfected with 100 pmol Alu RNA, B2 RNA, or mock for 12 hours. Shown is the mean ± SEM for independent experiments. *P < 0.05; **P < 0.01; ****P < 0.0001; two-way ANOVA with Sidak’s multiple comparisons (B, D, H); or one-way ANOVA with Dunnett’s multiple comparisons (F). ns, not significant.

Fig. 3.. DDX17 acts as the sensor of SINE RNA-induced NLRC4 inflammasome activation.

(A) Streptavidin affinity pull down of lysates of Myc-tagged DDX17-expressing HEK293T cells treated with biotin-labeled Alu RNA or label-free competitor RNAs as indicated, followed by immunoblot analysis of Myc, DDX17, and actin. (B) Immunoblot analysis of DDX17, histone H3, GAPDH and actin in cell fractions (N, nuclear; C, cytosol) and total lysates of THP-1 cells transfected with Alu RNA. WCL, whole cell lysate. (C) Immunoblot analysis of pro-CASP1 (p45), cleaved CASP1 (p20), DDX17, and actin in Alu RNA- or Mock-transfected Ddx17^+/+ and Ddx17^−/− iBMDMs. (D) ELISA measurement of IL-1β secreted from LPS primed Ddx17^+/+ and Ddx17^−/− iBMDMs transfected with 100 pmol of Alu RNA or mock for 12 hours. (E) Immunoblot analysis of pro-CASP1 (p45), cleaved CASP1 (p20), DDX17, DDX5, and actin in cell lysates from Alu RNA- or mock-transfected THP-1 cells pre-transfected with siRNA. Sup (supernatant). (F) ELISA measurement of IL-1β secreted from LPS primed THP-1 cells pre-transfected with siRNAs targeting DDX17, DDX5, or both, and then transfected with 100 pmol of Alu RNA or mock. Shown is the mean ± SEM for independent experiments. ****P < 0.0001; two-way ANOVA with Sidak’s multiple comparisons.

Fig. 4.. DDX17 is required for sterile NLRC4/NLRP3 inflammasome assembly.

(A) Immunoblot analysis of indicated proteins in WT and Nlrp3^−/− BMDMs transfected with 100 pmol of Alu RNA, B2 RNA, or mock. L+S, lysates and supernatant. (B). Measurement of IL-1β in LPS primed WT and Nlrp3^−/− BMDMs transfected with 100 pmol of Alu RNA or mock. Shown is the mean ± SEM for independent experiments. ****P < 0.0001; two-way ANOVA with Sidak’s multiple comparisons. (C) Immunoblot analysis of ASC and NLRP3 in cell lysates (soluble) and of ASC in DSS-crosslinked pellets (insoluble) in WT, Nlrp3^−/−, and Pycard^−/− BMDMs transfected with 100 pmol of Alu RNA or mock. o, oligomers; d, dimers; m, monomers. (D) Immunoblot analysis of cleaved CASP1 (p20), NLRC4, NLRP3 and actin in cell lysates or supernatant of wild-type (WT), Nlrc4^−/−, Nlrp3^−/−, and Nlrc4^−/− Nlrp3^−/− primary BMDMs transfected with 100 pmol of B2 RNA or mock for 12 hours. (E) Co-immunoprecipitation (IP) of ASC and NLRP3 in WT and Nlrc4^−/− BMDMs transfected with 100 pmol of Alu RNA or mock. * denotes the NLRC4 protein bands. (F) Mass spectrometry analysis of NLRC4 and NLRP3 peptides in purified DDX17-associated proteins. M, mock; Alu, Alu RNA. (G) Co-immunoprecipitation (IP) of NLRC4 and NLRP3 in Ddx17^+/+ and Ddx17^−/− iBMDMs transfected with B2 RNA or Mock. (H) Immunoblot analysis of ASC, DDX5, DDX17, and actin in cell lysates (soluble) and of ASC in DSS-crosslinked pellets (insoluble) in THP-1 cells transfected with siRNAs targeting DDX17, DDX5, or both and followed with transfection of 100 pmol of Alu RNA or mock for 12 hours.

Fig. 5.. Endogenous Alu RNA accumulation and sterile NLRC4 inflammasome activation in SLE PBMCs.

(A) Northern blot analysis of Alu RNA and 5S rRNA in primary PBMCs isolated from healthy control (Ctrl) and SLE patients. (B) Quantification of DDX17 and NLRC4 mRNA levels in primary PBMCs by RT-qPCR. (C to E) Immunoblot analysis of DDX17, NLRC4, p-PKCδ (Tyr311), total PKCδ (t-PKCδ), cleaved CASP1 (p20), and actin in primary PBMCs isolated from healthy control and SLE patients. Quantification of increased DDX17 expression (D) and PKCδ phosphorylation (E). (F and G) Immunofluorescence analysis and quantification of DDX17 and Alu RNA co-localization puncta in SLE PBMCs. Scale bars, 10 μm. (H) ELISA measurement of IL-18 secreted by primary PBMCs pre-treated with the PKC-δ inhibitor Rottlerin (5.0 μM) or vehicle, with or without LPS (250 ng/ml) priming. Shown is the mean ± SEM for independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, two-way ANOVA with Sidak’s multiple comparisons (B and H) or Unpaired t test (D, E, G). ns, not significant.

Fig. 6.. SINE RNAs-induced RPE degeneration requires NLRC4.

(A) Confocal microscopy of RPE cells transfected with 100 pmol of Alu RNA or mock for 12 hours following proximity ligation assay (PLA) between NLRC4-NLRP3; NLRP3-ASC; NLRC4-ASC. The PLA labeled protein interaction sites (gray puncta); F-actin is labeled with Alexa Fluor™ 488 Phalloidin to visualize cell morphology. Scale bars, 20 μm. (B) Quantification of PLA puncta in human RPE cells transfected with 100 pmol of Alu RNA or mock. Shown is the mean ± SEM for independent experiments. **P < 0.01; ***P < 0.001, two-way ANOVA with Sidak’s multiple comparisons. (C) Immunoblot analysis of ASC and NLRC4 in cell lysates (soluble) and of ASC in DSS-crosslinked pellets (insoluble) in human RPE cells transfected with siRNAs (48 h) targeting NLRC4 or NAIP and transfected with 100 pmol of Alu RNA or mock. o, oligomers; d, dimers; m, monomers. (D) Fundus photographs (top) and ZO-1–stained RPE flat mounts (bottom) of Nlrc4^+/+ and Nlrc4^−/− mice treated with subretinal injection of vehicle (Ctrl), Alu RNA, or B2 RNA. Scale bars, 10 μm. Loss of regular hexagonal cellular boundaries represents degenerated RPE (outlined by white arrowheads). n = 5–6 (Nlrc4^+/+), n = 4–6 (Nlrc4^−/−) mice. (E) Immunoblot analysis of DDX17 and actin in primary mouse retinal pigment epithelium (mRPE) cells isolated from wild-type (WT) mice, transfected with Ddx17 siRNA or control siRNA, followed by transfection with Alu RNA or mock. (F) Fundus photographs (top) and ZO-1-stained RPE flat mounts (bottom) of WT mice treated with subretinal injection of Alu RNA and intravitreous administration of Ddx17 siRNA or control siRNA. Scale bars, 10 μm. Binary and morphometric quantification of RPE degeneration is shown (*P < 0.05; **P < 0.005). PM, polymegethism (mean (SEM)).