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. Author manuscript; available in PMC: 2013 May 11.
Published in final edited form as: Cell. 2012 Apr 26;149(4):847–859. doi: 10.1016/j.cell.2012.03.036

DICER1 loss and Alu RNA Induce Age-Related Macular Degeneration via the NLRP3 Inflammasome and MyD88

Valeria Tarallo 1,16, Yoshio Hirano 1,16, Bradley D Gelfand 1,16, Sami Dridi 1, Nagaraj Kerur 1, Younghee Kim 1, Won Gil Cho 1,3, Hiroki Kaneko 1, Benjamin J Fowler 1, Sasha Bogdanovich 1, Romulo JC Albuquerque 1, William W Hauswirth 4, Vince A Chiodo 4, Jennifer F Kugel 5, James A Goodrich 5, Steven L Ponicsan 5, Gautam Chaudhuri 6, Michael P Murphy 7, Joshua L Dunaief 8, Balamurali K Ambati 9,10, Yuichiro Ogura 11, Jae Wook Yoo 12, Dong-ki Lee 12, Patrick Provost 13, David R Hinton 14, Gabriel Núñez 15, Judit Z Baffi 1, Mark E Kleinman 1, Jayakrishna Ambati 1,2,*
PMCID: PMC3351582  NIHMSID: NIHMS370679  PMID: 22541070

SUMMARY

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 IL-18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL-18 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 IL-18, 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.

INTRODUCTION

Age-related macular degeneration (AMD) affects the vision of millions of individuals (Smith et al., 2001). AMD is characterized by degeneration of the retinal pigmented epithelium (RPE), which is situated between the retinal photoreceptors and the choroidal capillaries (Ambati et al., 2003). RPE dysfunction disrupts both photoreceptors and choroidal vasculature (Blaauwgeers et al., 1999; Lopez et al., 1996; McLeod et al., 2009; Vogt et al., 2011). These tissue disruptions lead to atrophic or neovascular disease phenotypes. Although there are therapies for neovascular AMD, there is no effective treatment for the more common atrophic form. GA, the advanced stage of atrophic AMD, is characterized by degeneration of the RPE, and is the leading cause of untreatable vision loss.

Recently we showed that a dramatic and specific reduction of the RNase DICER1 leads to accumulation of Alu RNA transcripts in the RPE of human eyes with GA (Kaneko et al., 2011). These repetitive element transcripts, which are non-coding RNAs expressed by the highly abundant Alu retrotransposon (Batzer and Deininger, 2002), induce human RPE cell death and RPE degeneration in mice. DICER1 deficit in GA RPE was not a generic cell death response because DICER1 expression was not dysregulated in other retinal diseases. Likewise, Alu RNA accumulation did not represent generalized retrotransposon activation due to a stress response in dying cells because other retrotransposons were not elevated in GA RPE.

DICER1 is central to mature microRNA biogenesis (Bernstein et al., 2001). Yet following DICER1 deficit, the accumulation of Alu RNA and not the lack of mature microRNAs was the critical determinant of RPE cell viability (Kaneko et al., 2011). Moreover, 7SL RNA, transfer RNA, and primary microRNAs do not induce RPE degeneration (Kaneko et al., 2011), ruling out a nonspecific toxicity of excess, highly structured RNA. Still, the precise mechanisms of Alu RNA cytotoxicity are unknown.

Although the retina is exceptional for its immune privilege (Streilein, 2003), insults mediated by innate immune sensors can result in profound inflammation. The three major classes of innate immune receptors include the TLRs, RIG-I-like helicases, and NLR proteins (Akira et al., 2006). Numerous innate immune receptors are expressed in the RPE (Kumar et al., 2004), and several exogenous substances can induce retinal inflammation (Allensworth et al., 2011; Kleinman et al., 2012). However, it is not known whether this surveillance machinery recognizes or responds to host endogenous RNAs. We explored the concept that innate immune machinery, whose canonical function is the detection of pathogen associated molecular patterns and other moieties from foreign organisms, might also recognize Alu RNA.

Indeed, we show that Alu transcripts can hijack innate immunity machinery to induce RPE cell death. Surprisingly, our data show that DICER1 deficit or Alu RNA activates the NLRP3 inflammasome in a MyD88-dependent, but TLR-independent manner. NLRP3 inflammasome activation in vivo has been largely restricted to immune cells, although our data open the possibility that NLRP3 activity may be more widespread, as reflected by examples in cell culture studies of keratinocytes (Feldmeyer et al., 2007; Keller et al., 2008). Our data also broaden the scope of DICER1 function beyond microRNA biogenesis, and identify it as a guardian against aberrant accumulation of toxic retrotransposon elements that comprise roughly 50% of the human genome (Lander et al., 2001). In sum, our findings present a novel self-recognition immune response, whereby endogenous non-coding RNA-induced NLRP3 inflammasome activation results from DICER1 deficiency in a non-immune cell.

RESULTS

Alu RNA does not activate a variety of TLRs or RNA sensors

Alu RNA has single-stranded (ss) RNA and double-stranded (ds) RNA motifs (Sinnett et al., 1991). Thus we tested whether Alu RNA induced RPE degeneration in mice deficient in toll-like receptor-3 (TLR3), a dsRNA sensor (Alexopoulou et al., 2001), or TLR7, a ssRNA sensor (Diebold et al., 2004; Heil et al., 2004). Subretinal delivery of a plasmid coding for Alu RNA (pAlu) induced RPE degeneration in Tlr3−/− and Tlr7−/− mice just as in wild-type (WT) mice (Figures 1A–C). We previously showed that ≥21-nucleotide fully complementary siRNAs activate TLR3 on RPE cells (Kleinman et al., 2011). Lack of TLR3 activation by Alu RNA is likely due to its complex structure containing multiple hairpins and bulges that might preclude TLR3 binding. Neither 7SL RNA, the evolutionary precursor of Alu RNA, nor p7SL induced RPE degeneration in WT mice (Figures S1A and S1B), suggesting that Alu RNA cytotoxicity might be due to as yet unclear structural features. pAlu induced RPE degeneration in Unc93b1 mice (Figure 1D), which lack TLR3, TLR7, and TLR9 signaling (Tabeta et al., 2006), indicating that these nucleic acid sensors are not activated by Alu RNA redundantly. pAlu induced RPE degeneration in Tlr4−/− mice (Figure 1E), and the TLR4 antagonist Rhodobacter sphaeroides LPS (Qureshi et al., 1991) did not inhibit pAlu-induced RPE degeneration in WT mice (Figure S1C). Thus the observed RPE cell death is not due to lipopolysaccharide contamination. Further, two different in vitro transcribed Alu RNAs (Kaneko et al., 2011) did not activate multiple TLRs (Figure 1F).

Figure 1. Alu RNA does not activate or function via toll-like receptors (TLRs).

Figure 1

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(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.

Next we tested whether other dsRNA sensors such as MDA5 (Kato et al., 2006) or PKR (encoded by Prkr, (Yang et al., 1995)) might mediate Alu RNA toxicity. However, pAlu induced RPE degeneration in Mda5−/− and Prkr−/− mice (Figure S1D and S1E). We tested whether the 5´-triphosphate on in vitro transcribed Alu RNA, which could activate RIG-I or IFIT-1 that sense this moiety (Hornung et al., 2006; Pichlmair et al., 2011), was responsible for RPE degeneration. Dephosphorylated Alu RNA induced RPE degeneration in WT mice just as well as Alu RNA not subjected to dephosphorylation (Figure S1F), indicating that this chemical group is not responsible for the observed cell death. Indeed a 5´-triphosphate ssRNA that activates RIG-I does not induce RPE degeneration in mice (Kleinman et al., 2011). Further, pAlu induced RPE degeneration in mice deficient in MAVS (Figure S1G), through which RIG-I and MDA-5 signal (Kumar et al., 2006; Sun et al., 2006). Collectively these data pointed to a novel mechanism of Alu RNA-induced RPE degeneration not mediated by a wide range of canonical RNA sensors.

Alu RNA cytotoxicity is mediated via MyD88 and IL-18

We then tested the involvement of TRIF (encoded by Ticam1), an adaptor for TLR3 and TLR4 (Hoebe et al., 2003; Yamamoto et al., 2003), and MyD88, an adaptor for all TLRs except TLR3 (Akira et al., 2006; Alexopoulou et al., 2001; Suzuki et al., 2003). Alu RNA induced RPE degeneration in Ticam1−/− mice (Figure S2A), consistent with findings in Tlr3−/− and Tlr4−/− mice. Unexpectedly, neither Alu RNA nor two different pAlu plasmids induced RPE degeneration in Myd88−/− mice (Figures 2A, S2B, and S2C). Intravitreous delivery of a peptide inhibitor of MyD88 homodimerization (Loiarro et al., 2005) prevented RPE degeneration induced by Alu RNA in WT mice, whereas a control peptide did not do so (Figure 2B). A MyD88-targeting short interfering RNA (siRNA), which was shorter than 21 nucleotides in length to prevent TLR3 activation and conjugated to cholesterol to enable cell permeation (Kleinman et al., 2008), but not a control siRNA, inhibited RPE degeneration induced by pAlu in WT mice (Figures 2C–2E). Myd88+/− heterozygous mice were protected against Alu RNA-induced RPE degeneration (Figures 2F and S2D), corroborating the siRNA studies that partial knockdown of MyD88 is therapeutically sufficient.

Figure 2. Alu RNA induces RPE degeneration via MyD88.

Figure 2

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(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.

MyD88-mediated signal transduction induced by interleukins leads to recruitment and phosphorylation of IRAK1 and IRAK4 (Cao et al., 1996; Kanakaraj et al., 1999; Suzuki et al., 2003; Suzuki et al., 2002). Alu RNA increased IRAK1/4 phosphorylation in human RPE cells (Figure 2G), supporting the concept that Alu RNA triggers MyD88 signaling. The MyD88 inhibitory peptide reduced Alu RNA-induced IRAK1/4 phosphorylation in human RPE cells (Figure S2E), confirming its mode of action.

Next we assessed whether MyD88 activation mediates Alu RNA-induced cell death in human and mouse RPE cell culture systems. Consonant with the in vivo data, pAlu reduced cell viability in WT but not Myd88−/− mouse RPE cells (Figure 2H). The MyD88-inhibitory peptide, but not a control peptide, inhibited cell death in human RPE cells transfected with pAlu (Figure 2I). Together, these data indicate that MyD88 is a critical mediator of Alu RNA-induced RPE degeneration.

MyD88 is generally considered an adaptor of immune cells (O'Neill and Bowie, 2007). However, Alu RNA induced cell death via MyD88 in RPE monoculture. Thus, we tested whether Alu RNA-induced RPE degeneration in mice was also dependent solely on MyD88 activation in RPE cells. Conditional ablation of MyD88 in the RPE by subretinal injection of AAV1-BEST1-Cre in Myd88f/f mice protected against Alu RNA-induced RPE degeneration (Figures 2J and S2F). Consistent with this finding, Alu RNA induced RPE degeneration in WT mice receiving Myd88−/− bone marrow but did not do so in Myd88−/− mice receiving WT bone marrow (Figure S2G). Collectively, these results indicate that MyD88 expression in the RPE, and not in circulating immune cells, is critical for Alu RNA-induced RPE degeneration. These findings comport with histopathological studies of human GA tissue that show no infiltration of immune cells in the area of pathology (personal communication, C.A. Curcio, H.E. Grossniklaus, G.S. Hageman, L.V. Johnson).

Although MyD88 is critical in TLR signaling (O'Neill and Bowie, 2007), MyD88 activation by Alu RNA was independent of TLR activation. Thus, we examined other mechanisms of MyD88 involvement. MyD88 can regulate IFN-γ signaling by interacting with IFN-γ receptor 1 (encoded by Ifngr1) (Sun and Ding, 2006). However, pAlu induced RPE degeneration in both Ifng−/− and Ifngr1−/− mice (Figures S2H and S2I). MyD88 is also essential in interleukin-1 signaling (Muzio et al., 1997). Thus, we tested whether IL-1β and the related cytokine IL-18, both of which activate MyD88 (Adachi et al., 1998), mediated Alu RNA cytotoxicity. Interestingly, whereas Alu RNA overexpression in human RPE cells increased IL-18 secretion, IL-1β secretion was barely detectable (Figure 2K).

Recombinant IL-18 induced RPE degeneration in WT but not Myd88−/− mice (Figure 2L). IL-18 neutralization protected against pAlu-induced RPE degeneration in WT mice, but IL-1β did not (Figures 2M and 2N). Also, pAlu induced RPE degeneration in Il1r1−/− mice but not Il18r1−/− mice (Figures S2J and S2K). These data indicate that IL-18 is an effector of Alu RNA-induced cytotoxicity.

Alu RNA activates the NLRP3 inflammasome

We explored whether Caspase-1 (encoded by Casp1), a protease that induces maturation of interleukins into biologically active forms (Ghayur et al., 1997; Gu et al., 1997; Thornberry et al., 1992), was involved in Alu RNA-induced RPE degeneration. Alu RNA treatment of human RPE cells led to Caspase-1 activation as measured by western blotting and by a fluorescent reporter of substrate cleavage (Figures 3A and S3A). Indeed, Alu RNA induced Caspase-1 activation in other cell types such as HeLa and THP-1 monocytic cells (Figure S3B), suggesting that Alu RNA cytotoxicity has potentially broad implications in many systems. Intravitreous delivery of the Caspase-1-inhibitory peptide Z-WEHD-FMK, but not a control peptide Z-FA-FMK, blocked IL-18 maturation and pAlu-induced RPE degeneration in WT mice (Figures 3B and 3C). The Caspase-1-inhibitory peptide blocked Alu RNA-induced substrate cleavage in human RPE cells (Figure S3C), confirming its mode of action. Similarly, Casp1−/− mice treated with Alu RNA or pAlu did not exhibit RPE degeneration (Figures 3D and S3D). Also, pAlu did not induce cell death in Casp1−/− mouse RPE cells (Figure 3E).

Figure 3. Alu RNA induces RPE degeneration via NLRP3 inflammasome.

Figure 3

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(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.

Caspase-1 can be activated within a multiprotein innate immune complex termed the inflammasome (Tschopp et al., 2003). The best-characterized inflammasome pathway is one that is activated by binding of NLRP3 to the caspase-1 adaptor ASC (encoded by PYCARD). One hallmark of inflammasome assembly is spatial clustering of PYCARD (Fernandes-Alnemri et al., 2007). In human RPE cells transfected with fluorescent tagged PYCARD (GFP-PYCARD), Alu RNA induced the appearance of a brightly fluorescent cytoplasmic cluster similar to treatment with LPS and ATP, which activates the NLRP3 inflammasome (Figures 3F and S3E) (Mariathasan et al., 2006).

Next we tested the functional relevance of NLRP3 and PYCARD to Alu RNA cytotoxicity. Neither pAlu nor Alu RNA induced RPE degeneration in either Nlrp3−/− or Pycard−/− mice (Figures 3G, 3H, S3F and S3G), demonstrating the critical importance of the inflammasome in Alu RNA cytotoxicity. Also, pAlu did not induce cell death in Nlrp3−/− or Pycard−/− mouse RPE cells (Figure 3I). Moreover, knockdown of NLRP3 or PYCARD by siRNAs rescued pAlu-induced human RPE cell death (Figures 3J and S3H). These findings provide direct evidence that NLRP3 activation in response to Alu RNA occurs in RPE cells and does not require the presence of other immune cells.

We determined that IL-18 and MyD88 activation indeed were downstream of Caspase-1 activation by showing (1) that whereas MyD88 inhibition reduced Alu RNA-induced IRAK1/4 phosphorylation in human RPE cells (Figure S2E), it did not reduce Alu RNA-induced Caspase-1 cleavage or fluorescent substrate cleavage (Figures S3I and S3J); (2) that IL-18 neutralization did not inhibit Alu RNA-induced Caspase-1 cleavage (Figures S3K); and (3) that Caspase-1 inhibition reduced Alu RNA-induced phosphorylation of IRAK1/4 (Figure S3L).

Alu RNA induces mitochondrial ROS and NLRP3 priming

NLRP3 inflammasome function requires two signals, the first of which is termed priming. pAlu induced inflammasome priming as it upregulated both NLRP3 and IL18 mRNAs. This priming occurred equivalently in both WT and Myd88−/− mouse RPE cells (Figure 4A), further corroborating that MyD88 functions downstream of NLRP3 in this system. Akin to other inflammasome agonists that do not directly interact with NLRP3 (Tschopp and Schroder, 2010), we did not observe a physical interaction between Alu RNA and NLRP3 (Figure S4A). To determine how Alu RNA primed the inflammasome, we studied whether it induced reactive oxygen species (ROS) production, a signal for priming (Bauernfeind et al., 2011; Nakahira et al., 2011). pAlu induced ROS generation in human RPE cells (Figure 4B), and the ROS inhibitor diphenyliodonium (DPI) blocked pAlu-induced NLRP3 and IL18 mRNA upregulation and Alu RNA-induced RPE degeneration in WT mice (Figures 4C and 4D). As DPI blocks mitochondrial ROS and phagosomal ROS (Li and Trush, 1998), we tested which pathway was triggered because there is controversy surrounding the source of ROS contributing to NLRP3 responses (Latz, 2010).

Figure 4. Alu RNA induces mitochondrial ROS production and NLRP3 priming.

Figure 4

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(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.

We used MitoSOX Red, which labels ROS-generating mitochondria, in combination with MitoTracker Deep Red, which labels respiring mitochondria. To monitor phagosomal ROS generation, we used Fc OxyBURST Green, which measures activation of NADPH oxidase within the phagosome. A marked increase in ROS-generating mitochondria was observed in human RPE cells transfected with pAlu (Figure 4E). In contrast, whereas phorbol myristate acetate (PMA) induced phagosomal ROS as expected (Savina et al., 2006), pAlu did not do so (Figure 4F). These data are consistent with the findings that NLRP3 responses are impaired by mitochondrial ROS inhibitors (Nakahira et al., 2011; Zhou et al., 2011) but are preserved in cells carrying genetic mutations that impair NADPH-oxidase-dependent ROS production (Meissner et al., 2010; van Bruggen et al., 2010).

Consonant with these reports and the observation that the principal source of cellular ROS is mitochondria (Murphy, 2009), we found that the mitochondria-targeted antioxidants Mito-TEMPO and MitoQ (Murphy and Smith, 2007; Nakahira et al., 2011) both blocked Alu RNA-induced RPE degeneration in WT mice, whereas dTPP, a structural analog of MitoQ that does not scavenge mitochondrial ROS, did not do so (Figure 4G). In contrast, gp91ds-tat, a cell-permeable peptide that inhibits association of two essential NADPH oxidase subunits (gp91phox and p47phox) (Rey et al., 2001), did not do so (Figure 4H). Corroborating these data, Alu RNA induced RPE degeneration in mice deficient in Cybb (which encodes gp91phox) just as in WT mice (Figure 4I). Next we studied the voltage-dependent anion channels (VDAC) because VDAC1 and VDAC2, but not VDAC3, are important in mitochondrial ROS produced by NLRP3 activators in macrophages (Zhou et al., 2011). Consistent with these observations, siRNA knockdown of VDAC1 and VDAC2, but not VDAC3, impaired pAlu-induced mitochondrial ROS (Figures 4J and S4B) and NLRP3 and IL18 mRNA induction in human RPE cells (Figure 4K). Collectively, these data implicate mitochondrial ROS in Alu RNA-induced NLRP3 inflammasome-mediated RPE degeneration.

Alu RNA does not induce RPE degeneration via pyroptosis

Alu RNA activates Caspase-1, which can trigger pyroptosis, a form of cell death characterized by formation of membrane pores and osmotic lysis (Fink and Cookson, 2006). The cytoprotective agent glycine, which attenuates pyroptosis (Fink et al., 2008; Fink and Cookson, 2006; Verhoef et al., 2005), inhibited human RPE cells death induced by LPS+ATP but not by Alu RNA (Figure 5A and 5B). Pyroptosis requires Caspase-1 but can proceed independent of IL-18 (Miao et al., 2010). Thus, our finding that IL-18 induced RPE degeneration in Casp1−/− mice (Figure 5C), coupled with the lack of rescue by glycine, suggests that Alu RNA-induced RPE degeneration does not occur via pyroptosis.

Figure 5. RPE degeneration does not occur via pyroptosis.

Figure 5

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(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).

DICER1 loss induces cell death via inflammasome

We previously demonstrated the key role of DICER1 in maintaining RPE cell health (Kaneko et al., 2011): DICER1-cleaved Alu RNA did not induce RPE degeneration in vivo; DICER1 overexpression protected against Alu RNA-induced RPE degeneration; and DICER1 loss-induced RPE degeneration was blocked by antagonizing Alu RNA (Kaneko et al., 2011). Also, rescue of DICER1 knockdown-induced RPE degeneration by Alu RNA inhibition was not accompanied by restoration of microRNA deficits (Kaneko et al., 2011). Therefore, we tested whether DICER1 also prevented NLRP3 inflammasome activation by Alu-RNA. Alu RNA-induced Caspase-1 activation in human RPE cells was inhibited by DICER1 overexpression (Figures 6A and 6B). Conversely, Caspase-1 cleavage induced by DICER1 knockdown in human RPE cells was inhibited by simultaneous antisense knockdown of Alu RNA (Figures S5A and S5B).

Figure 6. DICER1 loss induces cell death via inflammasome.

Figure 6

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(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

Next we tested the relevance of these pathways in the context of DICER1 loss in vivo. Caspase-1 cleavage was increased in the RPE of BEST1 Cre; Dicer1f/f mice (Figure 6C), which lose DICER1 expression in the RPE during development and exhibit RPE degeneration (Kaneko et al., 2011). Subretinal delivery of AAV1-BEST1-Cre in Dicer1f/f mice induced Caspase-1 activation and IL-18 maturation in the RPE (Figure 6D). This treatment also induced RPE degeneration, which was blocked by intravitreous delivery of the Caspase-1-inhibitory peptide but not the control peptide (Figure 6E). AAV1-BEST1-Cre-induced RPE degeneration in Dicer1f/f mice was also blocked by intravitreous delivery of the MyD88-inhibitory peptide but not a control peptide (Figure 6F). In addition, MyD88 inhibition prevented cell death in human RPE cells treated with antisense oligonucleotides targeting DICER1 (Figure 6G). DICER1 knockdown in human RPE cells increased IRAK1/4 phosphorylation, providing further evidence of MyD88 activation upon loss of DICER1 (Figure 6H). MyD88 inhibition also prevented cell death in Dicer1f/f mouse RPE cells treated with an adenoviral vector coding for Cre recombinase (Figure 6I). MyD88 inhibition blocked RPE cell death without restoring the microRNA expression deficits induced by Dicer1 knockdown (Figure 6J). These findings demonstrate that DICER1 is an essential endogenous negative regulator of NLRP3 inflammasome activation, and that DICER1 deficiency leads to Alu RNA-mediated, MyD88-dependent, microRNA-independent RPE degeneration.

Inflammasome and MyD88 activation in human GA

Next we tested whether human eyes with GA, which exhibit loss of DICER1 and accumulation of Alu RNA in their RPE (Kaneko et al., 2011), also display evidence of inflammasome activation. The abundance of NLRP3 mRNA in the RPE of human eyes with GA was markedly increased compared to control eyes (Figure 7A). IL18 and IL1B mRNA abundance also was increased in GA RPE; however, only the disparity in IL18 levels reached statistical significance (Figure 7A). Immunolocalization studies showed that the expression of NLRP3, PYCARD, and Caspase-1 proteins was also increased in GA RPE (Figures 7B–D). Western blot analyses corroborated the increased abundance of NLRP3 and PYCARD in GA RPE, and revealed greatly increased levels of the enzymatically active cleaved Caspase-1 p20 subunit in GA RPE (Figure 7E). There was also an increase in the abundance of phosphorylated IRAK1 and IRAK4 in GA RPE, indicative of increased MyD88 signal transduction (Figure 7E). Collectively, these data provide evidence of NLRP3 inflammasome and MyD88 activation in situ in human GA, mirroring the functional data in human RPE cell culture and mice in vivo.

Figure 7. NLRP3 Inflammasome and MyD88 activation in human GA.

Figure 7

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(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.

DISCUSSION

Our data establish a functional role for the subversion of innate immune sensing pathways by Alu RNA in the pathogenesis of GA. Collectively, our findings demonstrate that the NLRP3 inflammasome senses GA-associated Alu RNA danger signals, contributes to RPE degeneration, and potentially vision loss in AMD (Figure S6). To date, the function of the NLRP3 inflammasome has been largely restricted to immune cells in vivo. Our finding that it plays a critical function in RPE cell survival broadens the cellular scope of this inflammasome and raises the possibility that other non-immune cells could employ this platform.

The NLRP3 inflammasome was originally recognized as a sensor of external danger signals such as microbial toxins (Kanneganti et al., 2006; Mariathasan et al., 2006; Muruve et al., 2008). Subsequently, endogenous crystals, polypeptides, and lipids were reported to activate it in diseases such as gout, atherogenesis, Alzheimer disease, and Type 2 diabetes (Halle et al., 2008; Masters et al., 2010; Muruve et al., 2008; Wen et al., 2011). To our knowledge, Alu RNA is the first endogenous nucleic acid known to activate this immune platform. Our findings expand the diversity of endogenous danger signals in chronic human diseases, and comport with the concept that this inflammasome is a sensor of metabolic danger (Schroder et al., 2010).

Dampening inflammasome activation can be essential to limiting the inflammatory response. Pathogens have evolved many strategies to inhibit inflammasome activation (Martinon et al., 2009). Likewise, host autophagy proteins (Nakahira et al., 2011), Type I interferon (Guarda et al., 2011), and T cell contact with macrophages can inhibit this process (Guarda et al., 2009). Our finding that DICER1, through its cleavage of Alu RNA, prevents activation of NLRP3 adds to the repertoire of host inflammasome modulation capabilities and reveals a new facet of how dysregulation of homeostatic anti-inflammatory mechanisms can promote AMD (Ambati et al., 2003; Takeda et al., 2009).

Added to its recently described anti-apoptotic and tumor-related functions, DICER1 emerges as a multifaceted protein. It remains to be determined how this functional versatility is channeled in various states. As DICER1 dysregulation is increasingly recognized in several human diseases, it is reasonable to imagine that Alu RNA might be an inflammasome activating danger signal in those conditions too. It is also interesting that, at least in adult mice and in a variety of mouse and human cells, the microRNA biogenesis function of DICER1 is not critical for cell survival, at least in a MyD88-deficient environment (data not shown).

Our data that mitochondrial ROS production is involved in Alu RNA-induced RPE degeneration comport with observations of mitochondrial DNA damage (Lin et al., 2011), downregulation of proteins involved in mitochondrial energy production and trafficking (Nordgaard et al., 2008), and reduction in the number and size of mitochondria (Feher et al., 2006) in the RPE of human eyes with AMD. Jointly, these findings suggest a potential therapeutic benefit to interfering with mitochondrial ROS generation.

Current clinical programs targeting the inflammasome largely focus on IL-1β; presently there are no IL-18 inhibitors in registered clinical trials. However, our data indicate that IL-18 is more important than IL-1β in mediating RPE cell death in GA (similar to selective IL-18 involvement in a colitis model (Zaki et al., 2010)), pointing to the existence of regulatory mechanisms by which inflammasome activation bifurcates at the level of or just preceding the interleukin effectors. Although Caspase-1 inhibition could be an attractive local therapeutic strategy, caspase inhibitors can promote alternative cell death pathways, possibly limiting their utility (Vandenabeele et al., 2006).

MyD88 is best known for transducing TLR signaling initiated by pathogen associated molecular patterns (O'Neill and Bowie, 2007), although recently it has been implicated in human cancers (Ngo et al., 2011; Puente et al., 2011). Our findings introduce an unexpected new function for MyD88 in effecting death signals from mobile element transcripts that can lead to retinal degeneration and blindness, and raise the possibility that MyD88 could be a central integrator of signals from other non-NLRP3 inflammasomes that also employ Caspase-1 (Schroder and Tschopp, 2010). Since non-canonical activation of MyD88 is a critical checkpoint in RPE degeneration in GA (Figure S6), it represents an enticing therapeutic target. A potential concern is its important anti-microbial function in mice (O'Neill and Bowie, 2007). However, in contrast to Myd88−/− mice, adult humans with MyD88 deficiency are described to be generally healthy and resistant to a wide variety of microbial pathogens (von Bernuth et al., 2008). MyD88-deficient humans have a narrow susceptibility range to pyogenic bacterial infections, and that too only in early childhood and not adult life (Picard et al., 2010). Moreover, as evident from the siRNA and Myd88+/− studies, partial inhibition of MyD88 is sufficient to protect against Alu RNA. Localized intraocular therapy, the current standard of care in most retinal diseases, would further limit the likelihood of adverse infectious outcomes. It is reasonable to foresee development of MyD88 inhibitors for prevention or treatment of GA.

EXPERIMENTAL PROCEDURES

A detailed description of materials and methods can be found in Supplemental Information.

Subretinal injection and imaging

Subretinal injections (1 µL) were performed using a Pico-Injector (PLI-100, Harvard Apparatus). Plasmids were transfected in vivo using 10% Neuroporter (Genlantis). Fundus imaging was performed on a TRC-50 IX camera (Topcon) linked to a digital imaging system (Sony). RPE flat mounts were immunolabeled using antibodies against zonula occludens-1 (Invitrogen).

Cell viability

Cell viability was assessed using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) according to manufacturer’s instructions.

mRNA abundance

Transcript abundance was quantified by real-time RT-PCR using an Applied Biosystems 7900 HT Fast Real-Time PCR system by the 2−ΔΔCt method.

Protein abundance and activity

Protein abundance was assessed by Western blot analysis using antibodies against Caspase-1 (1:500; Invitrogen), pIRAK1 (1:500; Thermo Scientific), pIRAK4 (1:500, Abbomax), PYCARD (1:200, Santa Cruz Biotechnology), NLRP3 (1:500, Enzo Life Sciences) and Vinculin (1:1,000; Sigma-Aldrich). Caspase-1 activity was visualized using Caspalux1 E1D2 (OncoImmunin) according to manufacturer’s instructions.

Statistical Analysis

Results are expressed as mean ± SEM, with p values < 0.05 considered statistically significant. Differences between groups were compared by Mann–Whitney U test or Student t-test, as appropriate, and 2-tailed p values are reported.

Research Highlights.

  • ·

    Alu RNA accumulation due to DICER1 deficiency activates NLRP3 inflammasome in RPE

  • ·

    Pharmacological inhibition of the inflammasome, MyD88, or IL-18 prevents degeneration

  • ·

    Alu RNA induced RPE degeneration via mitochondrial ROS production, IL-18, and MyD88

  • ·

    RPE in human geographic atrophy eyes display evidence of NLRP3 and MyD88 activation

Supplementary Material

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02

ACKNOWLEDGMENTS

We thank S. Akira, Z. Chen, M. Chrenek, J. Garcia-Perez, T. Heidmann, and J.V Moran for providing mice, reagents, or tissues; R. King, L. Xu, M. McConnell, C. Payne, D. Robertson, G. Botzet, G.R. Pattison, and C. Spee for technical assistance; and S. Bondada, M.E. Boulton, R.A. Brekken, R. Kannan, K. Karikó, T.S. Khurana, R. Mohan, M.L. Peterson, V. Rangnekar, A. Sinai, A.M. Rao, G.S. Rao and K. Ambati for discussions. J.A. was supported by NEI/NIH grants R01EY018350, R01EY018836, R01EY020672, R01EY022238, R21EY019778, RC1EY020442, Doris Duke Distinguished Clinical Scientist Award, Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, Dr. E. Vernon Smith and Eloise C. Smith Macular Degeneration Endowed Chair, and Senior Scientist Investigator Award (Research to Prevent Blindness, RPB); J.Z.B. by NIH K08EY021521, International Retinal Research Foundation, and American Health Assistance Foundation; M.E.K. by NIH K08EY021757; B.J.F., S.B., and M.E.K. by NIH T32HL091812 and UL1RR033173; G.N. by NIH R01AI063331 and R01AR052756; Y.H. by Alcon Japan Research award; W.W.H. by NIH P30EY021721; B.K.A. by NIH R01EY017182 and R01EY017950, VA Merit Award and Department of Defense; D.R.H. by NIH P30EY003040 and R01EY001545; J.F.K. and J.A.G. by NIH R01GM068414; J.W.Y. and D-K.L. by Global Research Laboratory grant from MEST, Korea. Departmental unrestricted grants from RPB supported J.A. and W.W.H. University of Kentucky Physician Scientist Awards supported J.Z.B. and M.E.K. P.P. is a Senior Scholar from the Fonds de la Recherche en Santé du Québec (FRSQ). Several authors are named as inventors on patent applications filed by their university relating to described technologies. V.T., Y.H., B.D.G., S.D., Y.K., W.C., J.Z.B., H.K., N.K., B.J.F., S.B., and M.E.K. performed experiments. W.W.H., V.A.C, S.L.P., J.K., J.A.G., M.P.M., J.W.Y., D-K. L, D.R.H., P.P., and G.N. provided reagents. J.A. conceived and directed the project, and wrote the paper with assistance from B.K.A., B.J.F., and B.D.G. All authors had the opportunity to discuss the results and comment on the manuscript.

Footnotes

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SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures, 6 figures, and associated references.

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Supplementary Materials

01
NIHMS370679-supplement-01.pdf (1.4MB, pdf)
02
NIHMS370679-supplement-02.pdf (240.2KB, pdf)

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