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. 2012 May;18(5):791-8.
doi: 10.1038/nm.2717.

NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components

Sarah L Doyle  1 , Matthew Campbell, Ema Ozaki, Robert G Salomon, Andres Mori, Paul F Kenna, Gwyneth Jane Farrar, Anna-Sophia Kiang, Marian M Humphries, Ed C Lavelle, Luke A J O'Neill, Joe G Hollyfield, Peter Humphries
Affiliations

NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components

Sarah L Doyle et al. Nat Med. 2012 May.

Abstract

Age-related macular degeneration (AMD) is the leading cause of central vision loss worldwide. Drusen accumulation is the major pathological hallmark common to both dry and wet AMD. Although activation of the immune system has been implicated in disease progression, the pathways involved are unclear. Here we show that drusen isolated from donor AMD eyes activates the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, causing secretion of interleukin-1b (IL-1b) and IL-18. Drusen component C1Q also activates the NLRP3 inflammasome. Moreover, the oxidative-stress-related protein-modification carboxyethylpyrrole (CEP), a biomarker of AMD, primes the inflammasome. We found cleaved caspase-1 and NLRP3 in activated macrophages in the retinas of mice immunized with CEP-adducted mouse serum albumin, modeling a dry-AMD–like pathology. We show that laser-induced choroidal neovascularization (CNV), a mouse model of wet AMD, is exacerbated in Nlrp3(-/-) but not Il1r1(-/-) mice, directly implicating IL-18 in the regulation of CNV development. These findings indicate a protective role for NLRP3 and IL-18 in the progression of AMD.

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Figures

Figure 1
Figure 1
Drusen activates the NLRP3 inflammasome. (a) Fundus photography from a nonsmoking unaffected individual, an individual with dry AMD and an individual with wet AMD. (b) Drusen fragments (arrows) in a range of sizes from just under 500 µm to submicroscopic-sized particles. (c) SDS-PAGE analysis of a Bruch’s membrane and drusen (BM/drusen) preparation. MW, molecular weight. (d) Live-cell imaging of immortalized C57BL/6 BMDMs stably expressing YFP-ASC. Poly(dA-dT) was used as a positive control. Oligomerization of ASC-YFP was observed by speck formation at an original magnification of ×60. (e,f) Production of IL-1β and IL-18, as measured by ELISA in human PBMCs primed overnight with LPS and subsequently treated with increasing doses of the drusen preparation. *P ≤ 0.05, ***P ≤ 0.0001 by analysis of variance (ANOVA) with Tukey post test compared to LPS-treated cells. (g) Western blot of the cleavage products of caspase-1 after treatment of THP1 cells with drusen. The arrow points to the caspase-1 p10 band. (h) Production of IL-1β (left) and IL-6 (right), as measured by ELISA in WT and Nlrp3−/− BMDMs after treatment with increasing doses of drusen. *P ≤ 0.05, ***P ≤ 0.0001 by ANOVA with Tukey post test compared to LPS-treated cells. (i) Production of IL-1β (left) and TNF-α (right), as measured by ELISA in WT and Nlrp3−/− BMDCs after treatment with increasing doses of drusen. ***P ≤ 0.0001 by ANOVA with Tukey post test compared to LPS-treated cells. All ELISA data are representative of a minimum of three separate experiments carried out in triplicate, and all data are means ± s.e.m.
Figure 2
Figure 2
CEP (CEP-HSA), a component of drusen, can prime the NLRP3 inflammasome. (a) Production of IL-1β in human PBMCs primed with LPS, CEP-HSA or HSA alone in increasing doses and subsequently treated with ATP. *P ≤ 0.05, ***P ≤ 0.0001 by ANOVA with Tukey post test compared to LPS-treated cells. (b) IL-1β production in WT and Nlrp3−/− BMDMs primed with CEP-HSA and then activated with either ATP or poly(dA-dT). ***P ≤ 0.0001 by ANOVA with Tukey post test compared to Nlrp3−/− cells. (c,d) IL-1β and IL-6 production in WT or Tlr2−/− BMDMs primed with HSA or CEP-HSA and activated with ATP or left untreated. ***P ≤ 0.0001 by ANOVA with Tukey post test for WT BMDMs treated with CEP-HSA and ATP compared to TLR2−/− BMDMs treated with CEP-HSA and ATP. (e,f) IL-1β and TNF-α production measured in C3H/HeN BMDMs or C3H/HeJ BMDMs primed with either LPS or CEP and activated with ATP. (g) Live-cell imaging of immortalized C57BL/6 BMDMs stably expressing YFP-ASC. Shown are cells primed with CEP-HSA (top) or HSA (bottom), followed by treatment with drusen. Oligomerization of ASC-YFP was observed by speck formation at an original magnification of ×60. All ELISA data are representative of a minimum of three separate experiments carried out in triplicate, and all data are means ± s.e.m.
Figure 3
Figure 3
Complement factor C1Q, a component of drusen, activates the NLRP3 inflammasome. (a) Production of IL-1β (top) and TNF-α (bottom) in WT BMDMs primed with CEP-HSA and activated with increasing doses of C1Q. *P ≤ 0.05 by ANOVA with Tukey post test compared to CEP-HSA treatment alone. (b) Western blot of caspase-1 cleavage products in THP1 cells primed with LPS and treated with increasing doses of C1Q. The arrow points to the caspase-1 p10 band. (c) Live-cell imaging of immortalized C57BL/6 BMDMs stably expressing YFP-ASC. Shown are cells primed with either LPS (top) or CEP-HSA, followed by treatment with C1Q (right). Oligomerization of ASC-YFP was observed by speck formation at an original magnification of ×60. (d) IL-1β (left) and TNF-α (right) production in WT and Nlrp3−/− BMDCs primed with LPS and activated with increasing doses of C1Q. ***P ≤ 0.0001 by ANOVA with Tukey post test for WT BMDCs compared to NLRP3−/− BMDCs for each given treatment. (e) IL-1β, IL-18 and IL-6 production in human PBMCs primed with LPS overnight and activated with C1Q, with the addition of increasing doses of Z-VAD before C1Q treatment. All ELISA data are representative of a minimum of three separate experiments carried out in triplicate, and all data are means ± s.e.m.
Figure 4
Figure 4
Cleaved caspase-1 p10 colocalizes with activated macrophages in CEP-MSA–immunized mice. (a–c) Immunostaining of retinal cryosections of CEP-MSA–immunized mice showing localization of F4/80+ macrophages to regions of the choroid (a), extending from the choroid toward the Bruch’s membrane (b) and present above the RPE in the outer segments (OS) and outer nuclear layer (ONL) of the retina (c). Differential contrast images are shown at the top in a and b and on the left in c, and fluorescent images (with F4/80 in red and DAPI in blue) are shown at the bottom in a and b and on the right in c. (d,e) Colabeling of retinal cryosections of CEP-MSA–immunized mice, with caspase-1 p10 (red) and F4/80 (green) showing colocalization in a macrophage present within and transcending the choroid and the Bruch’s membrane (d) and a macrophage protrusion in the outer segment of the retina (e). (f) Colabeling of retinal cryosections of CEP-MSA–immunized mice showing colocalization of NLRP3 (red) and F4/80 (green). (g) A higher magnification of the NLRP3 and F4/80 staining in f.
Figure 5
Figure 5
NLRP3 is protective against laser-induced CNV lesion formation in an IL-1β–independent manner. (a) Laser-induced CNV in WT (top left), Nlrp3−/− (top middle) and Il1r1−/− (top right) mice showing CNV development after laser burn. Three-dimensional reconstructed images of confocal z-stacks from WT (bottom left), Nlrp3−/− (bottom middle) and Il1r1−/− (bottom right) mice. Also shown is a CNV volume rendering (bar chart). *P ≤ 0.0496 by ANOVA followed by Tukey post-hoc test. Data are means ± s.e.m. (b) ERG analysis of rod and cone function in Nlrp3−/− and Il1r1−/− mice. The a wave represents hyperpolarization of photoreceptors, and the b wave represents the second-order neuron response. (c) Immunostaining showing localization of activated macrophages (F4/80, green) to the site of laser-induced injury in Nlrp3−/− mice. (d) Immunostaining for cleaved caspase-1 (red) of WT (left) or Nlrp3−/− (right) retinal cryosections after injury.
Figure 6
Figure 6
NLRP3 confers its protection against CNV lesion formation through its role in IL-18 production, which in turn regulates VEGF concentrations. (a) ERG analysis of rod and cone function in Il18−/− mice. (b) Laser induced CNV in Il18−/− mice showing CNV development after laser burn (left). Three-dimensional (3D) reconstructed images of confocal z-stacks (right). (c) CNV volumes in Il18−/− and WT mice (Fig. 5). *P = 0.0292 by Student’s t test. (d,e) The production of VEGF, as assayed by ELISA in ARPE-19 cells (d) and bEnd.3 cells (e) treated with increasing doses of IL-18 or left untreated. *P ≤ 0.05, ***P ≤ 0.0001 by ANOVA with Tukey post test compared to untreated cells. ELISA data are representative of a minimum of three separate experiments carried out in triplicate, and all data are means ± s.e.m.
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