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Complement Component C5a Primes Retinal Pigment Epithelial Cells for Inflammasome Activation by Lipofuscin-mediated Photooxidative Damage*

  • Carolina Brandstetter
    Affiliations
    Department of Ophthalmology, University of Bonn, 53127 Bonn, Germany
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  • Frank G. Holz
    Affiliations
    Department of Ophthalmology, University of Bonn, 53127 Bonn, Germany
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  • Tim U. Krohne
    Correspondence
    To whom correspondence should be addressed: University of Bonn, Dept. of Ophthalmology, Ernst-Abbe-Str. 2, 53127 Bonn, Germany. Tel.: +49-228-287-15505; Fax: +49-228-287-11518
    Affiliations
    Department of Ophthalmology, University of Bonn, 53127 Bonn, Germany
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  • Author Footnotes
    * The study was supported by German Research Foundation (DFG), Bonn, Germany, Grant KR 2863/7-1; Pro Retina Foundation, Bonn, Germany; University of Bonn, BONFOR and SciMed Programs, Bonn, Germany; and Dr. Eberhard and Hilde Rüdiger Foundation, Bonn, Germany (all to T. U. K.). The paper was presented at the 2014 annual meeting of the Association of Research in Vision and Ophthalmology (ARVO) in Orlando, FL (Brandstetter, C., Holz, F. G., Krohne, T.U. (2014) Complement Component C5a Primes the NLRP3 Inflammasome in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 55: E-Abstract 3444). Conflict of interest: CB, none. FGH, research grants: Acucela, Alcon, Allergan, Bayer, Carl Zeiss Meditec, Genentech, Heidelberg Engineering, Novartis, Optos; consultancy honoraria, lecture fees, travel grants: Acucela, Alcon, Allergan, Bayer, Genentech, Heidelberg Engineering, Novartis, Roche. TUK, research grants: Alcon, Novartis; consultancy honoraria, lecture fees, travel grants: Bayer, Heidelberg Engineering, Novartis.
Open AccessPublished:November 12, 2015DOI:https://doi.org/10.1074/jbc.M115.671180
      Complement activation, oxidative damage, and activation of the NLRP3 inflammasome have been implicated in retinal pigment epithelium (RPE) pathology in age-related macular degeneration (AMD). Following priming of RPE cells, the NLRP3 inflammasome can be activated by various stimuli such as lipofuscin-mediated photooxidative damage to lysosomal membranes. We investigated whether products of complement activation are capable of providing the priming signal for inflammasome activation in RPE cells. We found that incubation of primary human RPE cells and ARPE-19 cells with complement-competent human serum resulted in up-regulation of C5a receptor, but not C3a receptor. Furthermore, human serum induced expression of pro-IL-1β and enabled IL-1β secretion in response to lipofuscin phototoxicity, thus indicating inflammasome priming. Complement heat-inactivation, C5 depletion, and C5a receptor inhibition suppressed the priming effect of human serum whereas recombinant C5a likewise induced priming. Conditioned medium of inflammasome-activated RPE cells provided an additional priming effect that was mediated by the IL-1 receptor. These results identify complement activation product C5a as a priming signal for RPE cells that allows for subsequent inflammasome activation by stimuli such as lipofuscin-mediated photooxidative damage. This molecular pathway provides a functional link between key factors of AMD pathogenesis including lipofuscin accumulation, photooxidative damage, complement activation, and RPE degeneration and may provide novel therapeutic targets in this disease.

      Introduction

      Age-related macular degeneration (AMD)
      The abbreviations used are: AMD
      age-related macular degeneration
      RPE
      retinal pigment epithelium
      HNE
      4-hydroxynonenal
      qPCR
      quantitative real-time PCR
      C5aR
      C5a receptor
      A2E
      N-retinylidene-N-retinyl-ethanolamine
      LDH
      lactate dehydrogenase
      MDA
      malondialdehyde.
      is the leading cause of blindness in all industrialized countries (
      • Resnikoff S.
      • Pascolini D.
      • Etya'ale D.
      • Kocur I.
      • Pararajasegaram R.
      • Pokharel G.P.
      • Mariotti S.P.
      Global data on visual impairment in the year 2002.
      ). For the majority of patients, in particular those affected by the intermediate stage and the atrophic late stage of the disease, there is currently no effective treatment available. Elucidating the still unresolved pathogenesis of this multifactorial, complex disease will help to identify potential targets for therapeutic intervention. The retinal pigment epithelium (RPE), a monolayer of post-mitotic support cells essential for photoreceptor function, is primarily affected by AMD. Oxidative/photooxidative damage to the RPE contributes to AMD, and antioxidative treatment has been demonstrated to slow disease progression in clinical trials (
      • Age-Related Eye Disease Study Research Group
      A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8.
      ). This damage is believed to be mediated at least in part by the photoreactive properties of lipofuscin and lipofuscin component A2E that accumulate in the macular RPE in large amounts over a lifetime (
      • Rózanowska M.
      • Jarvis-Evans J.
      • Korytowski W.
      • Boulton M.E.
      • Burke J.M.
      • Sarna T.
      Blue light-induced reactivity of retinal age pigment. In vitro generation of oxygen-reactive species.
      ,
      • Sparrow J.R.
      • Nakanishi K.
      • Parish C.A.
      The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells.
      ).
      In addition, several lines of evidence indicate that processes of the innate immune system play a critical role in the pathogenesis of AMD. Activated components of the complement system such as C3a and C5a are detectable both locally in the sub-RPE space and systemically in plasma of AMD patients (
      • Anderson D.H.
      • Mullins R.F.
      • Hageman G.S.
      • Johnson L.V.
      A role for local inflammation in the formation of drusen in the aging eye.
      ,
      • Scholl H.P.N.
      • Charbel Issa P.
      • Walier M.
      • Janzer S.
      • Pollok-Kopp B.
      • Börncke F.
      • Fritsche L.G.
      • Chong N.V.
      • Fimmers R.
      • Wienker T.
      • Holz F.G.
      • Weber B.H.F.
      • Oppermann M.
      Systemic complement activation in age-related macular degeneration.
      ). Genetic polymorphisms in several complement components and regulators such as CFH, C2, C3, and CFB are strongly associated with AMD (
      • Lim L.S.
      • Mitchell P.
      • Seddon J.M.
      • Holz F.G.
      • Wong T.Y.
      Age-related macular degeneration.
      ). Another part of the innate immune system, the NLRP3 inflammasome, has recently been proposed to also contribute to AMD pathogenesis. Activation of the NLRP3 inflammasome in RPE cells was demonstrated in both atrophic and neovascular AMD (
      • Tarallo V.
      • Hirano Y.
      • Gelfand B.D.
      • Dridi S.
      • Kerur N.
      • Kim Y.
      • Cho W.G.
      • Kaneko H.
      • Fowler B.J.
      • Bogdanovich S.
      • Albuquerque R.J.
      • Hauswirth W.W.
      • Chiodo V.A.
      • Kugel J.F.
      • Goodrich J.A.
      • Ponicsan S.L.
      • Chaudhuri G.
      • Murphy M.P.
      • Dunaief J.L.
      • Ambati B.K.
      • Ogura Y.
      • Yoo J.W.
      • Lee D.K.
      • Provost P.
      • Hinton D.R.
      • Nuñez G.
      • Baffi J.Z.
      • Kleinman M.E.
      • Ambati J.
      DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88.
      ,
      • Tseng W.A.
      • Thein T.
      • Kinnunen K.
      • Lashkari K.
      • Gregory M.S.
      • D'Amore P.A.
      • Ksander B.R.
      NLRP3 Inflammasome Activation in Retinal Pigment Epithelial Cells by Lysosomal Destabilization: Implications for Age-Related Macular Degeneration.
      ), and increased intravitreal and systemic levels of the inflammasome activation products IL-1β and IL-18 have been reported in AMD patients (
      • Zhao M.
      • Bai Y.
      • Xie W.
      • Shi X.
      • Li F.
      • Yang F.
      • Sun Y.
      • Huang L.
      • Li X.
      Interleukin-1β Level Is Increased in Vitreous of Patients with Neovascular Age-Related Macular Degeneration (nAMD) and Polypoidal Choroidal Vasculopathy (PCV).
      ,
      • Ijima R.
      • Kaneko H.
      • Ye F.
      • Nagasaka Y.
      • Takayama K.
      • Kataoka K.
      • Kachi S.
      • Iwase T.
      • Terasaki H.
      Interleukin-18 induces retinal pigment epithelium degeneration in mice.
      ). The inflammasome protein complex serves as an intracellular sensor for various signals of cell damage (
      • Stutz A.
      • Golenbock D.T.
      • Latz E.
      Inflammasomes: too big to miss.
      ). Its activation results in the secretion of highly pro-inflammatory cytokines such as IL-1β and IL-18 and eventually in cell death by pyroptosis.
      Activation of the NLRP3 inflammasome is a two-step process that requires an initial priming signal and a subsequent activation signal (
      • Stutz A.
      • Golenbock D.T.
      • Latz E.
      Inflammasomes: too big to miss.
      ). The priming signal results in NF-κB-dependent transcriptional induction of NLRP3 and pro-IL-1β. The activation signal subsequently triggers assembly of NLRP3 and other protein components into the active inflammasome protein complex that results in caspase-1-mediated cleavage of pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18. Several substances have been suggested to provide the inflammasome activation signal in AMD including drusen components such as C1q (
      • Doyle S.L.
      • Campbell M.
      • Ozaki E.
      • Salomon R.G.
      • Mori A.
      • Kenna P.F.
      • Farrar G.J.
      • Kiang A.S.
      • Humphries M.M.
      • Lavelle E.C.
      • O'Neill L.A.
      • Hollyfield J.G.
      • Humphries P.
      NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components.
      ) and amyloid-β (
      • Liu R.T.
      • Wang A.
      • To E.
      • Gao J.
      • Cao S.
      • Cui J.Z.
      • Matsubara J.A.
      Vinpocetine inhibits amyloid-beta induced activation of NF-κB, NLRP3 inflammasome and cytokine production in retinal pigment epithelial cells.
      ), Alu RNA accumulation secondary to DICER1 deficiency (
      • Tarallo V.
      • Hirano Y.
      • Gelfand B.D.
      • Dridi S.
      • Kerur N.
      • Kim Y.
      • Cho W.G.
      • Kaneko H.
      • Fowler B.J.
      • Bogdanovich S.
      • Albuquerque R.J.
      • Hauswirth W.W.
      • Chiodo V.A.
      • Kugel J.F.
      • Goodrich J.A.
      • Ponicsan S.L.
      • Chaudhuri G.
      • Murphy M.P.
      • Dunaief J.L.
      • Ambati B.K.
      • Ogura Y.
      • Yoo J.W.
      • Lee D.K.
      • Provost P.
      • Hinton D.R.
      • Nuñez G.
      • Baffi J.Z.
      • Kleinman M.E.
      • Ambati J.
      DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88.
      ), the lipofuscin component N-retinylidene-N-retinyl-ethanolamine (A2E) (
      • Anderson O.A.
      • Finkelstein A.
      • Shima D.T.
      A2E induces IL-1β production in retinal pigment epithelial cells via the NLRP3 inflammasome.
      ), and the lipid peroxidation product 4-hydroxynonenal (HNE) (
      • Kauppinen A.
      • Niskanen H.
      • Suuronen T.
      • Kinnunen K.
      • Salminen A.
      • Kaarniranta K.
      Oxidative stress activates NLRP3 inflammasomes in ARPE-19 cells–implications for age-related macular degeneration (AMD).
      ). We have recently suggested an additional mechanism by demonstrating that photooxidative damage to the RPE, enhanced by accumulated lipofuscin, can activate the NLRP3 inflammasome by inducing lysosomal membrane permeabilization and cytosolic leakage of lysosomal enzymes (
      • Brandstetter C.
      • Mohr L.K.M.
      • Latz E.
      • Holz F.G.
      • Krohne T.U.
      Light induces NLRP3 inflammasome activation in retinal pigment epithelial cells via lipofuscin-mediated photooxidative damage.
      ,
      • Mohr L.K.M.
      • Hoffmann A.V.
      • Brandstetter C.
      • Holz F.G.
      • Krohne T.U.
      Effects of Inflammasome Activation on Secretion of Inflammatory Cytokines and Vascular Endothelial Growth Factor by Retinal Pigment Epithelial Cells.
      ).
      In contrast to inflammasome activation, the mechanism of inflammasome priming in AMD has been little investigated so far. Interestingly, a recent study in patients with early or intermediate AMD demonstrated the CFH risk genotype to be associated with significantly increased plasma levels of the inflammasome-regulated cytokine IL-18, suggesting a role for activated complement components like C3a and C5a in inflammasome activation in AMD (
      • Cao S.
      • Ko A.
      • Partanen M.
      • Pakzad-Vaezi K.
      • Merkur A.B.
      • Albiani D.A.
      • Kirker A.W.
      • Wang A.
      • Cui J.Z.
      • Forooghian F.
      • Matsubara J.A.
      Relationship between systemic cytokines and complement factor H Y402H polymorphism in patients with dry age-related macular degeneration.
      ). Inflammasome priming by complement activation products has also been proposed in the context of other diseases such as atherosclerosis and gout (
      • Samstad E.O.
      • Niyonzima N.
      • Nymo S.
      • Aune M.H.
      • Ryan L.
      • Bakke S.S.
      • Lappegård K.T.
      • Brekke O.-L.
      • Lambris J.D.
      • Damås J.K.
      • Latz E.
      • Mollnes T.E.
      • Espevik T.
      Cholesterol Crystals Induce Complement-Dependent Inflammasome Activation and Cytokine Release.
      ,
      • An L.-L.
      • Mehta P.
      • Xu L.
      • Turman S.
      • Reimer T.
      • Naiman B.
      • Connor J.
      • Sanjuan M.
      • Kolbeck R.
      • Fung M.
      Complement C5a potentiates uric acid crystal-induced IL-1β production.
      ). In this study, we investigated the capacity of activated complement components to prime human RPE cells for inflammasome activation by lipofuscin-mediated photooxidative damage.

      Discussion

      Blue light irradiation of RPE cells in the presence of oxygen results in generation of reactive oxygen species in a lipofuscin-dependent manner (
      • Rózanowska M.
      • Jarvis-Evans J.
      • Korytowski W.
      • Boulton M.E.
      • Burke J.M.
      • Sarna T.
      Blue light-induced reactivity of retinal age pigment. In vitro generation of oxygen-reactive species.
      ) and subsequent permeabilization of lysosomal membranes by oxidative damage (
      • Davies S.
      • Elliott M.H.
      • Floor E.
      • Truscott T.G.
      • Zareba M.
      • Sarna T.
      • Shamsi F.A.
      • Boulton M.E.
      Photocytotoxicity of lipofuscin in human retinal pigment epithelial cells.
      ,
      • Schütt F.
      • Davies S.
      • Kopitz J.
      • Holz F.G.
      • Boulton M.E.
      Photodamage to human RPE cells by A2-E, a retinoid component of lipofuscin.
      ). We have previously shown that lysosomal membrane permeabilization by lipofuscin-mediated photooxidative damage activates the NLRP3 inflammasome in primed RPE cells (
      • Brandstetter C.
      • Mohr L.K.M.
      • Latz E.
      • Holz F.G.
      • Krohne T.U.
      Light induces NLRP3 inflammasome activation in retinal pigment epithelial cells via lipofuscin-mediated photooxidative damage.
      ,
      • Mohr L.K.M.
      • Hoffmann A.V.
      • Brandstetter C.
      • Holz F.G.
      • Krohne T.U.
      Effects of Inflammasome Activation on Secretion of Inflammatory Cytokines and Vascular Endothelial Growth Factor by Retinal Pigment Epithelial Cells.
      ). This mechanism may underlie the inflammasome activation observed in the RPE of AMD patients (
      • Tarallo V.
      • Hirano Y.
      • Gelfand B.D.
      • Dridi S.
      • Kerur N.
      • Kim Y.
      • Cho W.G.
      • Kaneko H.
      • Fowler B.J.
      • Bogdanovich S.
      • Albuquerque R.J.
      • Hauswirth W.W.
      • Chiodo V.A.
      • Kugel J.F.
      • Goodrich J.A.
      • Ponicsan S.L.
      • Chaudhuri G.
      • Murphy M.P.
      • Dunaief J.L.
      • Ambati B.K.
      • Ogura Y.
      • Yoo J.W.
      • Lee D.K.
      • Provost P.
      • Hinton D.R.
      • Nuñez G.
      • Baffi J.Z.
      • Kleinman M.E.
      • Ambati J.
      DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88.
      ,
      • Tseng W.A.
      • Thein T.
      • Kinnunen K.
      • Lashkari K.
      • Gregory M.S.
      • D'Amore P.A.
      • Ksander B.R.
      NLRP3 Inflammasome Activation in Retinal Pigment Epithelial Cells by Lysosomal Destabilization: Implications for Age-Related Macular Degeneration.
      ) and may contribute to RPE pathology in this disease.
      Activation of the NLRP3 inflammasome is a posttranscriptionally regulated event mediated by assembly of inflammasome components and subsequent proteolytic maturation of interleukin precursors. In most cells, however, inflammasome component NLRP3 and interleukin precursor pro-IL-1β are not expressed constitutively or only to low amounts. Therefore, inflammasome activation requires a prior priming signal to induce expression of these proteins. Most previous studies including our own investigated the mechanisms of inflammasome activation in RPE cells by utilizing well-established priming agents such as LPS and IL-1α (
      • Tseng W.A.
      • Thein T.
      • Kinnunen K.
      • Lashkari K.
      • Gregory M.S.
      • D'Amore P.A.
      • Ksander B.R.
      NLRP3 Inflammasome Activation in Retinal Pigment Epithelial Cells by Lysosomal Destabilization: Implications for Age-Related Macular Degeneration.
      ,
      • Anderson O.A.
      • Finkelstein A.
      • Shima D.T.
      A2E induces IL-1β production in retinal pigment epithelial cells via the NLRP3 inflammasome.
      • Kauppinen A.
      • Niskanen H.
      • Suuronen T.
      • Kinnunen K.
      • Salminen A.
      • Kaarniranta K.
      Oxidative stress activates NLRP3 inflammasomes in ARPE-19 cells–implications for age-related macular degeneration (AMD).
      ,
      • Brandstetter C.
      • Mohr L.K.M.
      • Latz E.
      • Holz F.G.
      • Krohne T.U.
      Light induces NLRP3 inflammasome activation in retinal pigment epithelial cells via lipofuscin-mediated photooxidative damage.
      • Mohr L.K.M.
      • Hoffmann A.V.
      • Brandstetter C.
      • Holz F.G.
      • Krohne T.U.
      Effects of Inflammasome Activation on Secretion of Inflammatory Cytokines and Vascular Endothelial Growth Factor by Retinal Pigment Epithelial Cells.
      ). However, the relevance of these substances as priming agents of the RPE in vivo in the context of AMD is unclear. We therefore investigate activated complement components as potential priming agents in RPE cells.
      Chronic complement activation is associated with AMD, and activated complement components like C3a and C5a are deposited in the sub-RPE space in AMD (
      • Anderson D.H.
      • Mullins R.F.
      • Hageman G.S.
      • Johnson L.V.
      A role for local inflammation in the formation of drusen in the aging eye.
      ). Thus, RPE cells are in constant, direct contact with these bioactive substances that, therefore, represent candidates for the inflammasome priming signal in AMD via anaphylatoxin receptors such as C5aR that is expressed on the basolateral side of the RPE (
      • Skeie J.M.
      • Fingert J.H.
      • Russell S.R.
      • Stone E.M.
      • Mullins R.F.
      Complement component C5a activates ICAM-1 expression on human choroidal endothelial cells.
      ). Indeed, AMD patients with the CFH risk genotype exhibit significantly increased systemic levels of the inflammasome-regulated cytokine IL-18 as compared with AMD patients without the CFH risk genotype, supporting a role for activated complement components in inflammasome activation in AMD (
      • Cao S.
      • Ko A.
      • Partanen M.
      • Pakzad-Vaezi K.
      • Merkur A.B.
      • Albiani D.A.
      • Kirker A.W.
      • Wang A.
      • Cui J.Z.
      • Forooghian F.
      • Matsubara J.A.
      Relationship between systemic cytokines and complement factor H Y402H polymorphism in patients with dry age-related macular degeneration.
      ). In other autoinflammatory diseases such as atherosclerosis and gout, inflammasome priming by complement activation products has likewise been proposed (
      • Samstad E.O.
      • Niyonzima N.
      • Nymo S.
      • Aune M.H.
      • Ryan L.
      • Bakke S.S.
      • Lappegård K.T.
      • Brekke O.-L.
      • Lambris J.D.
      • Damås J.K.
      • Latz E.
      • Mollnes T.E.
      • Espevik T.
      Cholesterol Crystals Induce Complement-Dependent Inflammasome Activation and Cytokine Release.
      ,
      • An L.-L.
      • Mehta P.
      • Xu L.
      • Turman S.
      • Reimer T.
      • Naiman B.
      • Connor J.
      • Sanjuan M.
      • Kolbeck R.
      • Fung M.
      Complement C5a potentiates uric acid crystal-induced IL-1β production.
      ). To elucidate the role of complement activation products in inflammasome activation in AMD, we studied the capacity of activated complement components to provide the priming signal in human RPE cells for subsequent NLRP3 inflammasome activation by lipofuscin-mediated photooxidative damage.
      Our experiments were performed in the human RPE cell line ARPE-19 and primary fetal human RPE cells. In both cell types be detected constitutive expression of the anaphylatoxin receptors C5aR, C3aR, and C5L2. Employing inflammasome-regulated IL-1β secretion and inflammasome-mediated pyroptotic cell death as measures for inflammasome activation, we demonstrated distinct priming effects for activated complement in human serum as well as for recombinant C5a. Complement heat-inactivation, C5 depletion, and C5a receptor inhibition suppressed the priming effect of human serum, indicating that C5a represents the active priming agent in complement-activated human serum. Priming by C5a enabled subsequent inflammasome activation by lipofuscin-mediated photooxidative damage. Inflammasome activation was dependent on activity of caspase-1 and cathepsin B. Unlike the priming signal, complement-activated serum and C5a were found to be unable to provide the activation signal and, thus, to directly induce inflammasome activation in RPE cells which is consistent with previous reports (
      • Laudisi F.
      • Spreafico R.
      • Evrard M.
      • Hughes T.R.
      • Mandriani B.
      • Kandasamy M.
      • Morgan B.P.
      • Sivasankar B.
      • Mortellaro A.
      Cutting Edge: The NLRP3 Inflammasome Links Complement-Mediated Inflammation and IL-1? Release.
      ).
      Proteins covalently modified with the lipid peroxidation product carboxyethylpyrrole (CEP) have been reported to prime the NLRP3 inflammasome via TLR2 (
      • Doyle S.L.
      • Campbell M.
      • Ozaki E.
      • Salomon R.G.
      • Mori A.
      • Kenna P.F.
      • Farrar G.J.
      • Kiang A.S.
      • Humphries M.M.
      • Lavelle E.C.
      • O'Neill L.A.
      • Hollyfield J.G.
      • Humphries P.
      NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components.
      ). However, subsequent reports have questioned the priming ability of CEP-adducted proteins and rather found it to potentiate inflammasome priming by other signals (
      • Saeed A.M.
      • Duffort S.
      • Ivanov D.
      • Wang H.
      • Laird J.M.
      • Salomon R.G.
      • Cruz-Guilloty F.
      • Perez V.L.
      The oxidative stress product carboxyethylpyrrole potentiates TLR2/TLR1 inflammatory signaling in macrophages.
      ). In our experiments, we did not observe a priming effect of HNE-adducted POS in RPE cells. For example, inflammasome activation was not inducible in cells incubated with HNE-POS alone (Fig. 3, unprimed group) but only in cells co-incubated with the priming agent IL-1α (Fig. 3, IL-1α group). In similar experiments, we determined that POS modified by malondialdehyde (MDA) likewise did not induce inflammasome priming in RPE cells (data not shown). Thus, proteins modified by the lipid peroxidation products HNE and MDA do not seem to represent inflammasome priming signals for the RPE.
      While immunological and inflammatory processes are usually believed to contribute to AMD pathogenesis in a detrimental way, the role of inflammasome activation in AMD is still controversial and may vary depending on disease stage and subtype. Inflammasome activation in retinal microglial cells and macrophages has been suggested to reduce choroidal neovascularization via IL-18 and thus to be protective in neovascular AMD (
      • Doyle S.L.
      • Campbell M.
      • Ozaki E.
      • Salomon R.G.
      • Mori A.
      • Kenna P.F.
      • Farrar G.J.
      • Kiang A.S.
      • Humphries M.M.
      • Lavelle E.C.
      • O'Neill L.A.
      • Hollyfield J.G.
      • Humphries P.
      NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components.
      ,
      • Doyle S.L.
      • Ozaki E.
      • Brennan K.
      • Humphries M.M.
      • Mulfaul K.
      • Keaney J.
      • Kenna P.F.
      • Maminishkis A.
      • Kiang A.-S.
      • Saunders S.P.
      • Hams E.
      • Lavelle E.C.
      • Gardiner C.
      • Fallon P.G.
      • Adamson P.
      • Humphries P.
      • Campbell M.
      IL-18 attenuates experimental choroidal neovascularization as a potential therapy for wet age-related macular degeneration.
      ). In contrast, inflammasome activation in RPE cells has been reported to result in RPE degeneration which may contribute to the development of atrophic AMD (
      • Tarallo V.
      • Hirano Y.
      • Gelfand B.D.
      • Dridi S.
      • Kerur N.
      • Kim Y.
      • Cho W.G.
      • Kaneko H.
      • Fowler B.J.
      • Bogdanovich S.
      • Albuquerque R.J.
      • Hauswirth W.W.
      • Chiodo V.A.
      • Kugel J.F.
      • Goodrich J.A.
      • Ponicsan S.L.
      • Chaudhuri G.
      • Murphy M.P.
      • Dunaief J.L.
      • Ambati B.K.
      • Ogura Y.
      • Yoo J.W.
      • Lee D.K.
      • Provost P.
      • Hinton D.R.
      • Nuñez G.
      • Baffi J.Z.
      • Kleinman M.E.
      • Ambati J.
      DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88.
      ,
      • Kim Y.
      • Tarallo V.
      • Kerur N.
      • Yasuma T.
      • Gelfand B.D.
      • Bastos-Carvalho A.
      • Hirano Y.
      • Yasuma R.
      • Mizutani T.
      • Fowler B.J.
      • Li S.
      • Kaneko H.
      • Bogdanovich S.
      • Ambati B.K.
      • Hinton D.R.
      • Hauswirth W.W.
      • Hakem R.
      • Wright C.
      • Ambati J.
      DICER1/Alu RNA dysmetabolism induces Caspase-8-mediated cell death in age-related macular degeneration.
      ). While patients with neovascular AMD can be effectively treated with VEGF blocking drugs, no effective therapeutic options are currently available for atrophic AMD. Therefore, the unmed need for identification of potential pharmaceutical targets in atrophic AMD is of crucial importance for clinical ophthalmology.
      Inhibitors of C5 and C5a as well as of complement components upstream of C5a generation such as CFD and C3 are currently evaluated in clinical studies in patients with AMD. In our study, inhibition of the C5a/C5aR axis reduced inflammasome activation by lipofuscin phototoxicity in RPE cells. This result supports the rational for therapeutic complement inhibition in atrophic AMD. Moreover, our results suggest the presence of a paracrine amplification loop of inflammasome priming in RPE cells via IL1R. Treatment by the IL1R-inhibitory drug anakinra significantly reduced inflammasome activation in our in vitro experiments, thus providing another potential treatment strategy. Finally, direct therapeutic interference with inflammasome activation has been demonstrated to be effective in vivo, for example using small molecules that provide specific inhibition of NLRP3 (
      • Coll R.C.
      • Robertson A.A.B.
      • Chae J.J.
      • Higgins S.C.
      • Muñoz-Planillo R.
      • Inserra M.C.
      • Vetter I.
      • Dungan L.S.
      • Monks B.G.
      • Stutz A.
      • Croker D.E.
      • Butler M.S.
      • Haneklaus M.
      • Sutton C.E.
      • Núñez G.
      • Latz E.
      • Kastner D.L.
      • Mills K.H.G.
      • Masters S.L.
      • Schroder K.
      • Cooper M.A.
      • O'Neill L.A.J.
      A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases.
      ), and could be tested in future clinical trials for AMD.
      In summary, our study identifies complement component C5a as a priming agent for the inflammasome in RPE cells that enables subsequent NLRP3 inflammasome activation by stimuli such as lipofuscin-mediated photooxidative damage. This molecular pathway links hallmark events of AMD pathogenesis including complement activation, lipofuscin accumulation, oxidative damage, and RPE degeneration and may provide novel treatment targets. Inflammasome-inhibiting therapeutics may serve as a potential future means of prevention and treatment of atrophic AMD.

      Author Contributions

      F. G. H. and T. U. K. coordinated the study and provided funding. C. B. and T. U. K. designed the experiments. C. B. performed the experiments. C. B. and T. U. K. analyzed the data and wrote the manuscript. F. G. H. revised the manuscript. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Claudine Strack, BTA, for expert technical assistance.

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