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. 2012 May 11;149(4):847-59.
doi: 10.1016/j.cell.2012.03.036. Epub 2012 Apr 26.

DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88

Valeria Tarallo  1 , Yoshio Hirano, Bradley D Gelfand, Sami Dridi, Nagaraj Kerur, Younghee Kim, Won Gil Cho, Hiroki Kaneko, Benjamin J Fowler, Sasha Bogdanovich, Romulo J C Albuquerque, William W Hauswirth, Vince A Chiodo, Jennifer F Kugel, James A Goodrich, Steven L Ponicsan, Gautam Chaudhuri, Michael P Murphy, Joshua L Dunaief, Balamurali K Ambati, Yuichiro Ogura, Jae Wook Yoo, Dong-ki Lee, Patrick Provost, David R Hinton, Gabriel Núñez, Judit Z Baffi, Mark E Kleinman, Jayakrishna Ambati
Affiliations

DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88

Valeria Tarallo et al. Cell. .

Abstract

Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD, and IL18 and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.

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Figures

Figure 1
Figure 1. Alu RNA does not activate or function via toll-like receptors (TLRs)
(A–E) pAlu, but not pNull, induces RPE degeneration in WT (A), Tlr3−/− (B), Tlr7−/− (C), Unc93b1 mt mice, which are functionally deficient in TLRs-3,7,9 (D), and Tlr4−/− mice (E). Representative images shown. n = 8–12. Fundus photographs, top row; Flat mounts stained for zonula occludens-1 (ZO-1; red), bottom row. Degeneration outlined by blue arrowheads. Scale bars, 20 µm. (F) Stimulation of HEK293 cell lines expressing various TLRs with either of two different Alu RNA sequences does not elicit NF-κB activation. Positive (+) controls using TLR-specific ligands activated NF-κB. n = 3. Data are represented as mean +/− SEM. See also Figure S1.
Figure 2
Figure 2. Alu RNA induces RPE degeneration via MyD88
(A) pAlu does not induce RPE degeneration in Myd88−/− mice. (B) pAlu-induced RPE degeneration in WT mice is inhibited by a MyD88 homodimerization peptide inhibitor (MyD88i), but not by a control peptide. (C) pAlu-induced RPE degeneration in WT mice is inhibited by cholesterol-conjugated Myd88 siRNA but not control siRNA. (D and E) siRNA targeting MyD88 (siMyD88) reduces target gene (D) and protein (E) abundance in mouse RPE cells compared to control siRNA. n = 3, *p < 0.05 by Student t-test. (F) pAlu does not induce RPE degeneration in Myd88 heterozygous (het) mice. (G) Western blot of Alu RNA-induced IRAK1 and IRAK4 phosphorylation in human RPE cells. Image representative of 3 experiments. (H) pAlu reduces cell viability of WT but not Myd88−/− mouse RPE cells. (I) Loss of human RPE cell viability induced by pAlu is rescued by MyD88i. (J) AAV1-BEST1-Cre, but not AAV1-BEST1-GFP, protected Myd88f/f mice from pAlu-induced RPE degeneration. (K) pAlu induces IL-18 secretion from human RPE cells measured by ELISA. IL-1β secretion is barely detectable. n = 3, *p < 0.05 by Student t-test. (L) Recombinant IL-18 induces RPE degeneration in WT but not Myd88−/− mice. (M and N) pAlu-induced RPE degeneration in WT mice is rescued by IL-18 neutralizing antibody (N) but not by IL-1β neutralizing antibody (M). Representative images shown. n = 8–12. Fundus photographs, top row; ZO-1 stained (red) flat mounts, bottom row. Degeneration outlined by blue arrowheads. Scale bars, 20 µm (A–C,F,J,L–N). n = 3, *p < 0.05 by Student t-test. Data are represented as mean +/− SEM (D,E,H,I,K). See also Figure S2.
Figure 3
Figure 3. Alu RNA induces RPE degeneration via NLRP3 inflammasome
(A) Western blot of Caspase-1 activation (p20 subunit) by Alu RNA in human RPE cells. (B) Western blot of pAlu-induced IL-18 maturation in RPE cell lysates in wild-type mice impaired by Caspase-1 peptide inhibitor. (C) Caspase-1 peptide inhibitor protects WT mice from pAlu-induced RPE degeneration. (D and E) pAlu does not induce RPE degeneration in Casp1−/− mice or (E) cytotoxicity in Casp1−/− mouse RPE cells. (F) Alu RNA and LPS+ATP induce formation of PYCARD clusters in human RPE cells transfected with GFP-PYCARD. (G and H) pAlu does not induce RPE degeneration in Nlrp3−/− (G) or Pycard−/− (H) mice. (I) Nlrp3−/− and Pycard−/− mouse RPE cells are protected against pAlu-induced loss of cell viability. (J) siRNAs targeting NLRP3 or PYCARD rescued human RPE cells from pAlu-induced cytotoxicity, compared to control siRNA. n = 3–4, *p < 0.05 by Student t-test (A,B,E,F,I,J). Images representative of 3 experiments. Densitometry values normalized to Vinculin are shown in parentheses (A,B). Fundus photographs, top row; ZO-1 stained (red) flat mounts, bottom row. Degeneration outlined by blue arrowheads. n = 8–12. Scale bars, 20 µm (C,D,G,H). Representative images shown. See also Figure S3.
Figure 4
Figure 4. Alu RNA induces mitochondrial ROS production and NLRP3 priming
(A) pAlu induces NLRP3 and IL18 mRNAs in WT and Myd88−/− mouse RPE cells. (B) pAlu induces generation of reactive oxygen species (ROS) in human RPE cells as monitored with the fluorescent probe H2DCFDA (A.U, arbitrary units). (C) DPI blocks pAlu-induced NLRP3 and IL18 mRNAs in human RPE cells. (D) DPI protects WT mice from pAlu-induced RPE degeneration. (E) pAlu induces generation of mitochondrial reactive oxygen species in human RPE cells as detected by the fluorescence of MitoSOX Red (green pseudocolor), colocalized with respiring mitochondria labeled by MitoTracker Deep Red (red). (F) PMA, but not pAlu, induces phagosomal ROS generation, as assessed by fluorescent Fc OXYBURST Green assay in human RPE cells. (A.U, arbitrary units). (G) MitoTempo and MitoQ, but not vehicle or dTPP controls, prevent Alu RNA-induced RPE degeneration in WT mice. (H) NADPH oxidase inhibitor gp91ds-tat or a scrambled peptide do not prevent Alu RNA-induced RPE degeneration in WT mice. (I) Alu RNA induces RPE degeneration mice deficient in Cybb (which encodes the gp91phox subunit of NADPH oxidase). (J and K) siRNAs targeting VDAC1 and VDAC2, but not VDAC3 or scrambled control, prevent pAlu-induced mROS generation (J) and upregulation of NLRP3 and IL18 mRNAs (K) in human RPE cells. mROS visualized with MitoSox Red dye and cell nuclei with Hoechst stain. n = 3–4, *p < 0.05 by Student t-test (A–C, K), NS, not significant by Student t-test (F). Representative images shown. n = 8–12. ZO-1 stained (red) flat mounts. Scale bars, 20 µm (D, E, G–I), n = 3–4. Scale bar, 100 µm (J). See also Figure S4.
Figure 5
Figure 5. RPE degeneration does not occur via pyroptosis
(A and B) Glycine inhibits human RPE cell death induced by LPS+ATP (A) but not by pAlu (B). (C) Recombinant IL-18 induces RPE degeneration in Casp1−/− mice. n = 3–4 (A,B), *p < 0.05 by Student t-test. Representative images shown. n = 8–12. Fundus photographs, top row; ZO-1 stained (red) flat mounts, bottom row. Degeneration outlined by blue arrowheads. Scale bars, 20 µm (C).
Figure 6
Figure 6. DICER1 loss induces cell death via inflammasome
(A) Western blot of Alu RNA-induced Caspase-1 cleavage (p20) inhibited by DICER1 overexpression in human RPE cells. (B and C) DICER1 overexpression reduces Alu RNA-induced Caspase-1 activation in human RPE cells (measured by cleavage (B left panel, green) of Caspalux®1 fluorescent substrate). Fluorescence quantification shown in right panel. (C) Western blot of increased Caspase-1 activation (p20 subunit) in RPE cell lysates of BEST1-Cre; Dicer1f/f mice compared to BEST1-Cre or Dicer1f/f mice. (D) Western blot of increased Caspase-1 activation (p20 subunit) and IL-18 maturation in RPE cell lysates of Dicer1f/f mice treated with AAV1-BEST1-Cre. (E and F) RPE degeneration induced by AAV1-BEST1-Cre in Dicer1f/f mice is rescued by peptide inhibitors of either Caspase-1 (E) or MyD88 (F). (G) MyD88 inhibitor rescues loss of human RPE cell viability induced by DICER1 antisense (AS) treatment. (H) DICER1 antisense (AS) treatment of human RPE cells reduces DICER1 and increases IRAK1 and IRAK4 phosphorylation. (I) MyD88 inhibitor rescues loss of cell viability in Dicer1f/f mouse RPE cells treated with adenoviral vector coding for Cre recombinase (Ad-Cre). (J) Ad-Cre induced global miRNA expression deficits in Dicer1f/f mouse RPE cells compared to Ad-Null. No significant difference in miRNA abundance between MyD88 inhibitor and control peptide-treated Dicer1 depleted cells. n = 3 (A,B,F–H). Densitometry values normalized to Vinculin are shown in parentheses (A,C). Degeneration outlined by blue arrowheads. n = 8 (E,F). *p < 0.05 by Student t-test (G,I). Images representative of 3 experiments (A,B,C,D,H). See also Figure S5
Figure 7
Figure 7. NLRP3 Inflammasome and MyD88 activation in human GA
(A) NLRP3 and IL18 abundance was significantly elevated in macular GA RPE (n = 13) compared to normal age-matched controls (n = 12). *p < 0.05 by Mann-Whitney U-test. There was no significant difference between groups (p = 0.32 by Mann-Whitney U-test) in IL1B abundance. (B–D) Increased immunolocalization of NLRP3 (B), PYCARD (C) and Caspase-1 (D) in macular GA RPE compared to age-matched normal controls. Scale bar, 20 µm. (E) Western blots of macular RPE lysates from individual human donor eyes show that abundance of NLRP3, PYCARD, and phosphorylated IRAK1/4, normalized to the levels of the housekeeping protein Vinculin, is reduced in geographic atrophy (GA) compared to age-matched normal controls. Data are represented as mean +/− SEM (A). Representative images shown. n = 6 (B–E). See also Figure S6.
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