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Age-related increases in amyloid beta and membrane attack complex: evidence of inflammasome activation… Zhao, Tom; Gao, Jiangyuan; Van, Jenifer; To, Eleanor; Wang, Aikun; Cao, Sijia; Cui, Jing Z; Guo, Jian-Ping; Lee, Moonhee; McGeer, Patrick L; Matsubara, Joanne A Jun 24, 2015

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RESEARCH Open AccessAge-related increases in amyloid beta andmembrane attack complex: evidence ofinflammasome activation in the rodent eyeKeywords: Age-related macular degeneration, Membrane attack complex, Amyloid beta, NLRP3 inflammasome,JOURNAL OF NEUROINFLAMMATIONZhao et al. Journal of Neuroinflammation  (2015) 12:121 DOI 10.1186/s12974-015-0337-1CanadaFull list of author information is available at the end of the article†Equal contributors1Department of Ophthalmology and Visual Sciences, Faculty of Medicine,University of British Columbia, 2550 Willow Street, Vancouver V5Z 3N9BC,* Correspondence: jms@mail.ubc.caNF-κB, RPE/choroidlow-grade inflammation associated with IL-18 and IL-1βTom Zhao1†, Jiangyuan Gao1†, Jenifer Van1, Eleanor To1, Aikun Wang1, Sijia Cao1, Jing Z. Cui1, Jian-Ping Guo2,Moonhee Lee2, Patrick L. McGeer2 and Joanne A. Matsubara1*AbstractBackground: The membrane attack complex (MAC) is a key player in the pathogenesis of age-related maculardegeneration (AMD) and is a putative activator of the NLRP3 inflammasome. Amyloid beta (Aβ), a component ofdrusen deposits, has also been implicated in inflammasome activation by our work and those of others. However,the interactions of MAC and Aβ are still poorly understood, especially their roles in aging and retinal degenerativepathologies. Since inflammasome activation may represent a key cellular pathway underlying age-related chronicinflammation in the eye, the purpose of this study is to identify the effects associated with MAC and inflammasomeactivation in the retinal pigment epithelium (RPE)/choroid and to evaluate the therapeutic merits of MAC suppression.Methods: Adult Long-Evans rats were divided into treatment and control groups. Treatment groups received oral aurintricarboxylic acid complex (ATAC), a MAC inhibitor, in drinking-water, and control groups received drinking-water alone(No ATAC). Groups were sacrificed at 7.5 or 11.5 months, after approximately 40 days of ATAC treatment. To studyage-related changes of Aβ and MAC in RPE/choroid, naive animals were sacrificed at 2.5, 7.5, and 11.5 months. Eyetissues underwent immunohistochemistry and western blot analysis for MAC, Aβ, NF-κB activation, as well as cleavedcaspase-1 and IL-18. Vitreal samples were collected and assessed by multiplex assays for secreted levels of IL-18 andIL-1β. Statistical analyses were performed, and significance level was set at p≤ 0.05.Results: In vivo studies demonstrated an age-dependent increase in MAC, Aβ, and NF-κB activation in the RPE/choroid.Systemic ATAC resulted in a prominent reduction in MAC formation and a concomitant reduction in inflammasomeactivation measured by cleaved caspase-1 and secreted levels of IL-18 and IL-1β, but not in NF-κB activation. In vitrostudies demonstrated Aβ-induced MAC formation on RPE cells.Conclusions: Age-dependent increases in Aβ and MAC are present in the rodent outer retina. Our results suggest thatsuppressing MAC formation and subsequent inflammasome activation in the RPE/choroid may reduce chronicin the outer retina.© 2015 Zhao et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Zhao et al. Journal of Neuroinflammation  (2015) 12:121 Page 2 of 14IntroductionAge-related macular degeneration (AMD), the leadingcause of irreversible blindness in the elderly in developedcountries, is a condition of the central retina, character-ized by retinal pigment epithelium (RPE) atrophy andphotoreceptor loss. Advanced chronological age is animportant risk factor for AMD. In North America, theprevalence of AMD increases dramatically with age from10 % in individuals at 55 to 65 years of age, to 30 % at75 to 85 years of age [1]. Key to understanding the ef-fects of aging on the pathogenesis of retinal degenerativediseases are the cellular pathways that become dysregu-lated with age [2–4].For example, aging is associated with the dysregulationin the complement cascade, part of the innate immuneresponse. The complement cascade causes opsonizationand agglutination, as well as cell lysis by the formationof the membrane attack complex (MAC) [5]. Geneticstudies showed that certain variants of the complementfactor H (CFH) gene, an inhibitor of the alternativepathway, can increase the risk of AMD by up to sixfoldin patients with the at-risk variant [6]. Postmortem eyesgenotyped for CFH at-risk variants have increased MAClevels in the RPE/choroid [7, 8]. Moreover, in dry AMDpatients, those with a CFH Y402H variant have elevatedsystemic levels of interleukin-6 (IL-6), tumor necrosisfactor (TNF-α), and IL-18 [9]. In addition to genetic influ-ences, aging also contributes to the dysregulation of thecomplement system, as evidenced by the association of ac-tivated complement products and increased MAC depos-ition in the RPE/choroid with advanced age [10–13].While MAC deposition may cause cell lysis, it may alsooccur at sublytic levels that promote chronic, low-gradepro-inflammation. This type of chronic, local inflamma-tion in the outer retina has been hypothesized to lead todegenerative changes in RPE function [14]. Recent studieshave suggested that MAC may mediate activation of theNLRP3 inflammasome [15, 16]. The inflammasome is amulti-protein complex that activates caspase-1 and pro-duces the pro-inflammatory cytokines IL-1β and IL-18,which in turn have been linked to RPE atrophy [17, 18].Whether MAC promotes inflammasome activation in theRPE/choroid is not known and is the premise of thisstudy. We postulate that sublytic MAC increases with age,promotes activation of the inflammasome, and thereby,dysregulates RPE function. To ameliorate MAC-inducedinflammasome activation on RPE, we also explore the ef-fects of an agent, aurin tricarboxylic acid complex(ATAC), which has been shown to inhibit MAC formationin mice and humans. Specifically, ATAC acts both to in-hibit the formation of the C3 convertase and to block theaddition of C9 to the C5b-8 complex and thereby inhibitsMAC [19, 20]. Here, we examine the age-related increasein MAC, the efficacy of ATAC in lowering levels ofsublytic MAC, and a subsequent, corresponding reductionin inflammasome activation in the rat RPE/choroid.MethodsIn vivo studiesThe animal procedures were approved by the AnimalCare Committee of the University of British Columbia,conformed to the guidelines of the Canadian Council onAnimal Care and were in accordance with the Reso-lution on the Use of Animals in Research of the Associ-ation of Research in Vision and Ophthalmology. AdultLong-Evans rats (Charles River, Wilmington MA) weredivided into four groups. Group 1 (N = 6) comprised 6-month-old rats treated with oral administration ofATAC for 40 days and sacrificed at the age of 7.5 months.A dosage of 60 mg/100 mL (in drinking-water) waschosen based on efficacy observed in an earlier study[19]. Group 2 (N = 6) comprised untreated 6-month-oldrats (controls) sacrificed at the age of 7.5 months. Group3 (N = 6) comprised 10-month-old rats treated withATAC in drinking-water (60 mg/100 mL) for 40 days andsacrificed at the age of 11.5 months. Group 4 (N = 6) com-prised untreated 10-month-old rats (controls) sacrificed at11.5 months. Additional untreated naive animals weresacrificed at 2.5, 6, 7.5, or 11.5 months, and retinal tissueswere used to demonstrate age-related changes in MAC,Aβ, and NF-κB activation. At the study endpoints, animalswere anesthetized, whole blood drawn for serum analysis,and then euthanized. Eyes were immediately enucleatedand frozen (western blot and ELISA) or fixed in 4 % para-formaldehyde in Dulbecco’s phosphate-buffered saline(Invitrogen, Carlsbad CA) for 48–72 h prior to embeddingin paraffin.Fibrillar Aβ1–40 preparationThe lyophilized, synthetic Aβ1–40 peptide in its HCl saltform was purchased from American Peptide (Sunnyvale,CA). We chose Aβ1–40 peptide over its structurally simi-lar but more toxic, Alzheimer’s disease-specific, form ofAβ1–42 peptide based on earlier studies that demon-strated the presence of Aβ1–40 in drusen deposits inpostmortem human eyes and its effects on upregulationof complement genes in RPE cells in vitro [21, 22].Fibrillar Aβ1–40 was prepared according to publishedprotocols [23, 24]. Briefly, the synthetic Aβ1–40 peptidewas reconstituted in sterile distilled H2O and incubatedat room temperature for 30 min, then evaporated byspeed vacuum for 1.5 h resulting in thin transparentAβ1–40 peptide film. Aβ1–40 peptide film was reconsti-tuted in 100 % dimethyl sulfoxide to a concentration of1 mM, and further diluted in Tris-buffered saline (TBS;20 mM Tris-HCl, 100 mM NaCl, pH 7.4, 37 °C) to pro-duce the Aβ1–40 stock solution of 100 μM. A longitudinalincubation assay consisting of six sampling time points at37 °C (24 h to 2 weeks) was set up to optimize the condi-tions for fibrillar Aβ1–40. Based on the protein separation,the sample at 1-week incubation time was selected, due toits higher fibrillar and lower oligomeric contents, whichwas then further diluted to 0.3 μM (final concentration)for the in vitro stimulation studies (Fig. 2) [22, 25–27].In vitro complement activation assay on ARPE-19 cellsAn in vitro assay was used to assess whether the fibrillarAβ1–40 induces MAC formation and deposition on ARPE-19 cells. Briefly, cells were seeded and cultured in 8-chambered glass slides. Normal human serum (NHS)(25 %) (CompTech, Tyler, TX) was added to the cell cul-tures with 0.3 μM fibrillar Aβ1–40 for 24 h. Positive controlsincluded zymosan stimulation (10 μg/ml, Sigma Aldrich,St. Louis MO) with 25 % NHS. Negative controls included25 % heat-inactivated NHS (HI-NHS) with fibrillar Aβ1–40Quantification of ATAC and CH50 assayImmunohistochemistry (MAC, IL-18, Aβ, and NF-κB)Paraffin-embedded eye tissues were prepared and sec-tioned following established protocols [28]. Sectionsfrom the paired groups (7.5 m ATAC and No ATAC;11.5 m ATAC and No ATAC) and the three age groups(2.5, 7.5, and 11.5 m) were processed simultaneously inorder to make intensity comparisons. Primary antibodiestargeting MAC, IL-18, and Aβ are described in Table 1.For the negative control sections, the primary antibodywas replaced with a matched non-specific isotype IgG(Sigma Aldrich). For visualization, the slides were de-veloped using either the Vector® VIP substrate kit orVector® AEC substrate kit.MAC, Aβ, and IL-18 immunoreactivity was scored in amasked fashion and semi-quantitatively based on a 0–3point scale (see Fig. 1 legend). Analysis and micrographswere taken using a × 60 objective lens and × 10 eyepieces(N = 3). The immunoreactivity scores of MAC, Aβ, andion(IH017–24 (Aβ17–24) anti-Aβ17–24 (clone 4G8)(I1:1000(IH(W(W00Zhao et al. Journal of Neuroinflammation  (2015) 12:121 Page 3 of 14Phosphorylated NF-κB p65(Ser 276)Rabbit polyclonal 1:751:500Phosphorylated NF-κB p50(Ser 337)Rabbit polyclonal 1:500Caspase-1 Mouse monoclonal 1:100Amyloid-beta amino acid Mouse monoclonal 1:200ATAC synthesis and its serum concentration analysisfollowed published methods [19, 20]. The final ATACconcentration was expressed in μg per 500 μL of blood(N = 6). A CH50 hemolysis assay was used to assess com-plement activation in rat serum before and after ATACadministration, following published procedures [19].Table 1 List of primary antibodiesAntigen Antibody DilutMembrane attack complex(MAC)Mouse monoclonal(clone aE11)1:501:100Membrane attack complex(MAC)Rabbit polyclonal 1:500Interleukin-18 (IL-18) Rabbit polyclonal 1:100Amyloid-beta amino acid Mouse monoclonal 1:400or zymosan, and serum-free medium with 25 % NHS orHI-NHS. MAC formation was detected by immunocyto-chemistry (Table 1) and quantified as the percentage ofRPE cells positive for MAC compared to the total numberof cells per × 40 field. The experiments were done in hexa-plicates for each stimulation condition (N = 6).1–16 (Aβ1–16) anti-Aβ1–16 (clone 6E10)GAPDH Mouse monoclonal 1:10,000IL-18 were averaged and normalized to the 7.5-month-oldgroup or the untreated control group (No ATAC).To detect the active NF-κB, an antibody against theNF-κB p65 subunit was used (Table 1). Immunoreac-tivity was scored quantitatively, in a masked fashion,using a × 60 objective lens and × 10 eyepieces (N = 3).Positive RPE nuclei were identified as containing boththe red (AEC) chromogen and blue hematoxylin coun-terstain, thus resulting in a purple appearance distinctfrom the unlabeled RPE nuclei that were blue in colorfrom only the hematoxylin counterstain. The numberof NF-κB positive nuclei was converted to percentageof all RPE nuclei in the sample area, and normalized tothe untreated control group (No ATAC) or the 7.5-month-old group.Source ApplicationsC)(WB)Dako, Burlington,ON, CanadaImmunocytochemistryWestern blotBIoss, Woburn, MA ImmunohistochemistrySanta Cruz Biotechnology,Dallas, TXImmunohistochemistryHC)(WB)BioLegend, Dedham, MA ImmunohistochemistryWestern blotC)B)Santa Cruz Biotechnology,Dallas, TXImmunohistochemistryWestern blotB) Santa Cruz Biotechnology,Dallas, TXWestern blotR&D Systems, Minneapolis,MNWestern blotBioLegend, Dedham, MA Western blotEMD Millipore, Billerica, MA Western blotFig. 1 (See legend on next page.)Zhao et al. Journal of Neuroinflammation  (2015) 12:121 Page 4 of 14n bt aolds on r7.5ed. ganBaszemi cntrtyNFZhao et al. Journal of Neuroinflammation  (2015) 12:121 Page 5 of 14Western blotRPE/Bruch’s membrane (BM)/choroid tissues were iso-lated and pooled for animals in each of the four treat-ment groups (groups 1–4, N = 6) and in each age groupof the naive animals (2.5, 7.5, and 11.5 months; N ≥ 3).To detect the MAC deposits, the tissue samples werehomogenized in 200 μL of ice-cold MAC extraction buf-fer (50 mM Tris-HCl (pH 6.8), 150 mM NaCl, 0.1 %SDS) containing protease inhibitor cocktail (Roche Diag-nostics, Indianapolis IN) [20]. To preserve the MACprotein complex, 40 μg of total protein was mixed withequal volume of 2× non-reducing loading buffer, devoid ofboiling, and directly subjected to 5–10 % SDS-PAGE. Pro-teins were transferred to a PVDF membrane and incu-bated with a series of blocking buffers, primary antibodyagainst MAC (Table 1), and HRP conjugated secondary(See figure on previous page.)Fig. 1 Age-dependent increases of ocular MAC, Aβ and NF-κB. a Westerage from 2.5 to 11.5 months old (Kruskal-Wallis, p≤ 0.05). b Western blo(MW > 95 kDa) in the rat RPE/choroid with age from 2.5 to 11.5 monthsincrease in the soluble Aβ levels in the vitreous fluids of the 11.5 month(Mann-Whitney, p≤ 0.05). d, e Analysis of MAC deposition (d) or Aβ (e) iwith increasing age, with data normalized to the younger age group ofwith age. The percentage of RPE cells with nuclear labeling of translocatgroup compared to the 7.5 months old group (Mann-Whitney, p≤ 0.05)(7.5 and 11.5 months) showed MAC deposition on the basal side of RPEpurple color (blue arrows) and nuclei counterstained with Methyl Green.++ are given for the following examples: 7.5 months old (+), 11.5 monthmelanocytes in the 11.5 months picture appeared surrounded by red haimmunoreactivity at both ages (7.5 and 11.5 months) showed positive imwas processed with AEC, resulting in a red color (blue arrows) and nucleas follows: 7.5 months old (++), 11.5 months old (+++), and negative co11.5 months old group showed more robust NF-κB p65 immunoreactivipurple in RPE nuclei and indicated by blue arrows. RPE nuclei devoid ofretinal pigment epithelium, Ch choroidantibody (R&D Systems, Minneapolis, MN). The en-hanced chemiluminescence (ECL) method was used to de-tect the MAC protein bands from animal groups 1–4 andthe naive animals at three different ages. The glyceralde-hyde 3-phosphate dehydrogenase (GAPDH)-loading con-trol blot was done similarly using a mouse GAPDHantibody (Table 1). All protein bands were subsequentlyquantified using Image J (NIH, Bethesda MD), and theratio of MAC-to-GAPDH was calculated. The finalrelative intensity of MAC was normalized either to theyoungest age group of 2.5 months or to the No ATACcontrol group.To detect caspase-1 cleavage and NF-κB activation, theRPE/BM/choroid tissues were homogenized in 200 μL ofice-cold RIPA buffer (Thermo Scientific, Waltham, MA)containing protease inhibitor cocktail (Roche Diagnostics).Blotting procedures followed our established protocol[28]. For GAPDH, the same membrane was incubated instripping buffer and then re-probed with the GAPDHantibody (Table 1). The protein band intensity of cleavedcaspase-1 (20kD), phosphorylated NF-κB p65 subunit (65KDa), phosphorylated NF-κB p50 subunit (50 KDa), andGAPDH (36kD) was individually measured using Image Jand converted into ratios relative to GAPDH. The finalrelative intensity of cleaved caspase-1 p20 and phosphory-lated NF-κB p65 was normalized to the No ATAC controlgroup.For western blot detection of Aβ, both fibrillar Aβ1–40preparation and the RIPA buffer-extracted RPE/BM/choroid tissue lysates were mixed with 2× non-reducingloading buffer, devoid of boiling, and directly subjectedto 5–12 % SDS-free PAGE separation. Electrophoresiswas run using MES buffer (Invitrogen, pH 7.3 ~ 7.7), andproteins were transferred onto a 0.2-μm PVDF mem-brane. The anti-Aβ 6E10 antibody was used to detectthe fibrillar Aβ1–40 preparation, whereas the anti-Aβlot analysis revealed increasing MAC levels in the rat RPE/choroid withnalysis showed enhanced accumulation of high-molecular weight Aβ(Kruskal-Wallis, p≤ 0.05). c ELISA measurements showed a dramaticld (599.1 pg/mL) compared to the 6 months old group (7.5 pg/mL)at RPE/choroid demonstrated a significant increase in immunoreactivitymonths (Mann-Whitney, p≤ 0.05). f NF-κB activation in RPE increasedNF-κB p65 subunit was higher in the retina of the 11.5 months oldRepresentative micrographs of MAC immunoreactivity at both agesd in choroid. Immunoreactivity was processed with VIP, resulting in ackground immunoreactivity (0) and semi-quantitative scoring of + andold (++), and negative control (0). Note the dark brown choroidaldue to bright field illumination. h Representative micrographs of Aβunolabeling on the basal side of RPE and in choroid. Immunoreactivityounterstained as blue. Examples of semi-quantitative scores are givenol (0). i Representative micrographs of the RPE nuclei from thethan the 7.5 months old group. Positive NF-κB p65 immunolabeling is-κB p65 are blue and indicated by black arrows. Scale bars; 10 μm. RPE4G8 antibody was used for tissue lysates (Table 1). Aβbands were developed by the ECL method. For themembrane containing tissue lysate proteins, it wasstripped and re-processed for GAPDH detection. The in-tensity of high-molecular species Aβ (MW > 95 kDa)and GAPDH (36kD) was independently measured usingImage J and converted into ratios of Aβ-to-GAPDH.The final relative intensity ratio of Aβ-to-GAPDH wasnormalized to the youngest age group of 2.5 months.ELISA for AβA chemiluminescent ELISA assay specific for the detec-tion of Aβ x-40 isoform was used to quantify Aβ in thevitreous of rat eyes (BioLegend, Dedham, MA). 6-month-old (N = 4) and 11.5-month-old (N = 6) rats weresacrificed for vitreous collection. Vitreous samples werediluted with the HRP detection antibody at a ratio of1:1. After 18 h of detection antibody incubation at 4 °C,the ELISA plate was then incubated with chemilumines-cent substrates for 15 s and imaged with a microplatereader (Synergy H1, BioTek, Winooski, VT). A non-linear 4-parameter regression model was used to gener-ate the standard curve to calculate Aβ concentrations ofall vitreous samples (Gen5 version 2.04.11, BioTek).Suspension array for IL-1β and IL-18An ELISA-based cytokine assay for the mature, secretedproducts of the inflammasome, IL-1β and IL-18, wascarried out (Bio-Plex 200 System, Bio-Rad Laboratories,Hercules CA). Vitreous from rat eyes in groups 3–4 werepooled. Experiments were carried out following methodsin our earlier publication [28].Statistical analysesNon-parametric tests were used throughout the study. Forthe two group comparisons (Figs. 1c–f, 2c, 3a, c, e, 4a–c,and 5a, b, e–g), a Mann-Whitney U test (one-tailed) wasused. For the three group comparisons, a Kruskal-Wallisand post hoc Dunn’s multiple comparisons test was usedto determine differences among age groups (Fig. 1a, b) oramong stimulation regimens (Fig. 2c). All analyses wereconducted with GraphPad Prism version 6.00 for Win-dows (GraphPad Software, La Jolla, CA). Statistical signifi-cance was set at p ≤ 0.05.ResultsAge-dependent increases of MAC, Aβ, and NF-κB in theRPE/choroidIn this study, we first asked whether MAC deposits in-crease in normal aging and, if so, is it related to inflamma-some activation in the RPE/choroid. A significant increasein MAC (MW> 580 kDa) was evident in the RPE/choroidrillitaincRef fiencgrel o.05F2Zhao et al. Journal of Neuroinflammation  (2015) 12:121 Page 6 of 14Fig. 2 Aβ induces MAC deposition on RPE in vitro. a Western blot of fibof high-molecular weight, fibrillar Aβ1–40 (MW > 170 kDa), and a concomwith increasing incubation time. Among all the time points, the 1-weekpared to monomeric and dimeric species than the other time points. bMAC deposits (Cy3, red) on ARPE-19 cells by combinatorial stimulation oreplacement abolished MAC deposition on RPE cells regardless the preslar Aβ1–40 or Zym (positive control) in the presence of NHS resulted in aNHS (Mann-Whitney, p≤ 0.05). The control bars indicate background levand this resulted in higher labeling than in HI-NHS (Mann-Whitney, p≤ 0and control, in the presence of NHS, demonstrated significance between finot between fibrillar Aβ1–40 and zymosan (Kruskal-Wallis, p≤ 0.05)ar Aβ1–40 preparation from six sampling time points. A general increasent decrease of low-molecular weight Aβ1–40 (MW < 17 kDa) are seenubation yielded a relatively higher percentage of fibrillar Aβ1–40 com-presentative confocal microscopic images showed significantly morebrillar Aβ1–40/NHS or zymosan (Zym)/NHS than by NHS alone. HI-NHSe of fibrillar Aβ1–40 or Zym. Scale bars; 20 μm. c Stimulation with fibril-eater percentage of MAC-labeled RPE cells than in the presence of HI-f MAC labeling as demonstrated by incubating ARPE19 cells with NHS,). Comparisons between the three groups, fibrillar Aβ1–40, zymosan,brillar Aβ1–40 and control, as well as between zymosan and control butFig. 3 (See legend on next page.)Zhao et al. Journal of Neuroinflammation  (2015) 12:121 Page 7 of 14E.(3ATACHatioloF-κRP. Um,ubupsZhao et al. Journal of Neuroinflammation  (2015) 12:121 Page 8 of 14homogenates of rats ranging in age from 2.5 to11.5 months using western blot. The normalized MAClevels were 1.88-fold higher at 7.5 months and 2.75-foldhigher at 11.5 months when compared to the samplesobtained from 2.5-month-old rats (Fig. 1a).Aβ is a known pathological activator of complementcascade in Alzheimer’s disease (AD) [29]. Its ocularpresence has been reported in drusen of postmortemeyes [30] and in rodent eyes [31]. To correlate Aβ accu-mulation with MAC formation, we semi-quantitativelycompared the levels of high-molecular weight Aβ spe-cies (MW > 95 kDa) among the RPE/choroid homoge-nates from different ages. We found an age-dependentincrease of high-molecular weight Aβ from 2.5 to11.5 months (Fig. 1b). Based on the knowledge thatbiosynthesized Aβ is present in both retina and the vit-reous compartment of the eye [32], we quantified theAβ in rat vitreous samples at two ages. With increasingage, the vitreal Aβ concentration increased, by almost80-fold, from 7.49 ± 5.16 pg/mL at 6 months to 599.10 ±159.25 pg/mL at 11.5 months of age (Fig. 1c).To support these results, we also assessed MAC for-mation and Aβ accumulation in retinal cross sections byimmunohistochemistry. We demonstrated that there are(See figure on previous page.)Fig. 3 Systemic ATAC administration did not alter NF-κB activation in RPadministration in 7.5-month-old (1.98 μg/500 μL) and in 11.5-month-oldhigher ATAC blood concentrations than age-matched controls withoutinhibition on total complement activation by ATAC was measured by aATAC were performed to calculate the half maximal inhibitory concentrreflect higher levels of complement activity. Note that the IC50 levels areto untreated controls at both ages. c ATAC administration did not affect Np > 0.05). d Representative micrographs of NF-κB p65 immunoreactivity innuclear translocalization have purple nuclei and are marked by blue arrowsare marked by black arrows. Scale bar; 10 μm. RPE retinal pigment epitheliuATAC administration contained the same amount of phosphorylated p65 sThe level of phosphorylated p50, however, was extremely low in both groincreasingly higher levels of immunoreactivity of bothMAC and Aβ in the 11.5 month animals compared tothe 7.5-month-old animals, particularly in the choroidand the basal side of RPE (Fig. 1d, e, g, h).NF-κB is a major transcription factor that responds toa variety of pro-inflammatory signals by nuclear translo-calization to upregulate the expression of target genes.Previously, we demonstrated that Aβ activates NF-κB,which can be specifically inhibited by NF-κB antagonists(e.g., vinpocetine or BAY 11–7082) [28]. In the presentstudy, by using an antibody targeting the phosphory-lated p65 subunit of the translocated NF-κB, we ob-served an increase in the percentage of RPE nucleiharboring the phosphorylated p65 subunit, mirroringthe age-related increase we observed with both MACand Aβ (Fig. 1f, i).Aβ promotes MAC formation in cultured ARPE-19 cellsThe AD literature suggests that Aβ binds to C1q andthereby promotes the classic complement cascade [29].To further understand the relationship between MAC andAβ in the RPE/choroid, we undertook stimulation studiesto assess MAC formation on RPE in vitro. Cells werestimulated with 0.3-μM fibrillar Aβ1–40 (MW> 170 kDa),which was prepared from synthetic Aβ1–40 peptide andtested by western blot. In the presence of 25 % NHS, weobserved MAC deposition on cells as ascertained byimmunocytochemistry with an anti-MAC antibody andimaged by confocal microscopy. MAC formation was pre-vented in control experiments in which cells were incu-bated with 25 % HI-NHS. Levels of MAC resulting fromfibrillar Aβ1–40-stimulation were compared to MAC de-position on cells treated with zymosan (positive control)or serum-free medium (negative control) in the presenceof NHS or HI-NHS. NHS alone resulted in 3.73 % of RPEcells positive for MAC. However, when combined with fi-brillar Aβ1–40 or zymosan, MAC deposition increased to14.91 % and 17.52 % of total RPE cells counted, respect-ively (Fig. 2).ATAC did not affect local NF-κB activation in RPEa The amount of ATAC in blood was measured after 40 days of drug.5 μg/500 μL) animals. Animals treated with ATAC showed significantlyC treatment at both ages (Mann-Whitney, p≤ 0.05). b The degree of50 hemolysis assay. Dilutions of the sera containing different levels ofn (IC50) for total complement activity. Higher IC50 values proportionallywer (i.e., curves shifted to the left) for ATAC-treated animals comparedB activation in RPE at both ages of 7.5 and 11.5 months (Mann-Whitney,E/choroid from each group in c. RPE cells that demonstrate NF-κB p65nlabeled RPE nuclei are counterstained with hematoxylin (blue only) andCh choroid. e, f RPE/choroid tissue lysates from 11.5-month-old rats withunit as in rats without ATAC in drinking-water (Mann-Whitney, p > 0.05).While ATAC inhibits complement cascade, it is unclearwhether systemic administration of ATAC would reducelocal inflammation in the eye. To test this, animals weretreated with oral administration of ATAC in drinking-water (60 mg/100 mL) for 40 days. Animals readily ac-cepted drinking-water laced with ATAC ad libitum, andno overt signs of side effects or toxicity were noted, con-sistent with our earlier studies in which ATAC was givenin food [19].After 40 days, the average ATAC level, as measured byfluorescence spectroscopy, was 1.98 and 3.5 μg/500 μLin the 7.5 and 11.5-month-old ATAC-treated rats, re-spectively. As expected, no appreciable amount of ATACwas evident in control rats at either age group (Fig. 3a).A CH50 assay, which measures the amount of hemolysisin blood due to complement activation, has been usedZhao et al. Journal of Neuroinflammation  (2015) 12:121 Page 9 of 14before as a surrogate marker for complement activityboth clinically and in experimental studies [19, 33]. Ourresults showed that sera from the ATAC-treated animalsdisplayed a significant three- to fourfold decrease inhemolysis relative to the sera of the control animals in bothage groups studied, thus confirming that orally adminis-tered ATAC was present and effective at inhibiting comple-ment activation in the sera of treated rats (Fig. 3b). Next,we assessed the nuclear translocation of the p65 subunit inRPE after ATAC treatment. Intriguingly, systemic adminis-tration of ATAC did not change NF-κB p65 nuclear trans-location when compared to control animals at 7.5 orFig. 4 ATAC treatment suppresses MAC deposition in the RPE/choroid. a, banalysis showed that ATAC significantly reduced MAC deposits in rat RPE/chothe rat RPE/choroid was significantly lower in the ATAC-treated group compap ≤ 0.05). d Representative micrographs from each group in c showed MBlue arrows identify positive MAC deposits labeled with VIP chromogen (retinal pigment epithelium, Ch choroid11.5 months old (Fig. 3c, d). Further western blot analysesof phosphorylated NF-κB p65 and p50 subunits in 11.5-month-old rats supported this, and no significant differ-ence in normalized band intensity was found betweenanimals receiving ATAC or drinking-water (Fig. 3e, f).ATAC reduced MAC deposition in the RPE/choroidOur earlier studies showed that ATAC was effective atdecreasing MAC in the central nervous system [19, 20],and our next question was whether the observed sys-temic level of ATAC was sufficient to inhibit MAC for-mation locally in the eye. To answer this, ATAC-treatedAt both ages of 7.5 months (a) and 11.5 months (b), western blotroid (Mann-Whitney, p≤ 0.05). c At both ages, MAC immunoreactivity inred to the age-matched, No ATAC control group (Mann-Whitney,AC immunoreactivity on the basal side of RPE cells and in choroid.purple). Nuclei were counterstained as green. Scale bar; 10 μm. RPEZhao et al. Journal of Neuroinflammation  (2015) 12:121 Page 10 of 14animals (7.5 and 11.5 months old) were sacrificed, and tis-sue lysate of RPE, choroid, and BM underwent westernblot analysis. At both ages, animals treated with ATACdemonstrated less MAC deposits (MW> 580 kDa) in theFig. 5 ATAC treatment inhibited inflammasome activation in the RPE/choroblot analysis showed that ATAC significantly inhibited pro-caspase-1 (MW 4(Mann-Whitney, p≤ 0.05). c Images of western blot demonstrates a concomcaspase-1 band after ATAC treatment in both age groups. d Representative miactivation, on the basal side of RPE cells and in choroid from treated and untreIL-18 labeling (VIP, purple). Scale bar; 10 μm. RPE retinal pigment epithelium, Chdownregulated by ATAC treatment. Labeling was normalized to 100 % for themeasurements of vitreous samples taken from treated and untreated control aIL-18 (f) and IL-1β (g) concentrations after ATAC treatment (Mann-Whitney, p≤RPE/choroid compared to non-ATAC-treated animals.Furthermore, ATAC treatment was more effective at sup-pressing MAC in the younger group (7.5 months old). Thisis intriguing, as the younger group demonstrated a lowerid. a–c At both ages of 7.5 months (a) and 11.5 months (b), western5 kDa) cleavage into active caspase-1 (MW 20 kDa) in rat RPE/choroiditant increase in the pro-caspase-1 band and decrease in the activecrographs illustrate immunoreactivity for IL-18, a product of inflammasomeated animals at 7.5 and 11.5 months of age. Blue arrows indicate positivechoroid. e IL-18 immunoreactivity in the rat RPE/choroid was significantlyuntreated animals in each age group (Mann-Whitney, p≤ 0.05). f, g ELISAnimals at 11.5 months of age. Note the dramatic reduction in secreted0.05)Zhao et al. Journal of Neuroinflammation  (2015) 12:121 Page 11 of 14ATAC concentration in sera (1.98 μg/500 μL), but with aproportionally greater inhibition of MAC (50 % inhibition)compared to the older group with 3.5 μg/500 μL ATAC insera, yet only 25 % inhibition of MAC (Figs. 3a and 4a, b).To better understand the distribution of MAC in the ocu-lar compartments after systemic ATAC, we assessed MACin retinal cross sections by immunohistochemistry. MACimmunoreactivity was robust in the choroid and BM ofnon-ATAC-treated rats at both ages, and it was reduced byATAC treatment at both ages (Fig. 4c, d).ATAC suppressed inflammasome activation in the RPE/choroidEarlier studies in non-ocular systems suggest that MACformation is a potential trigger for inflammasome activa-tion [15, 16]. Here we used an inhibitor of MAC, ATAC, tosuppress inflammasome activation in the RPE/choroid. Totest whether MAC promotes inflammasome activation, wefirst examined the level of pro-caspase-1 (MW 45 kDa)cleavage in RPE/choroid homogenates using western blot.At both ages tested (7.5 and 11.5 months), ATAC success-fully lowered cleaved caspase-1 (MW 20 kDa) by ~90 %(7.5 months old) and by ~50 % (11.5 months old), whencompared to untreated controls (Fig. 5a–c). Next, weassessed the levels of two mature products of inflamma-some activation, IL-1β and IL-18. Compared to the ATACgroup, we found that IL-18 immunoreactivity was higherin the untreated rat RPE/choroid. On closer visual examin-ation, it was evident that the majority of the IL-18 immu-nolabeling was located to the basal side of RPE and at theRPE/choroid interface (Fig. 5d, e). We next tested secretedlevels of IL-1β and IL-18 in the vitreous by custom-madeELISA assays. Secreted levels of IL-1β and IL-18 were 3-and 2.5-fold lower in the vitreous of animals treated withATAC, respectively, compared to the untreated age-matched controls (Fig. 5f, g).DiscussionWith normal aging, the RPE/choroid complex undergoesmany changes including drusen deposition, thickening ofBM, and thinning of the choroid. The RPE also undergoesa number of age-associated changes, including loss ofmelanin granules, accumulation of lipofuscin, changesin pro-inflammatory cytokine secretion, and even celldeath [34, 35]. However, the cellular mechanisms under-lying these changes in the RPE/choroid remain largely un-known. Here, we provide a new perspective by investigatingthe relationship among aging, MAC formation, and inflam-masome activation.Aβ facilitates MAC formation and MAC-induced inflamma-some activation in the RPE/choroidIn AD, Aβ is a known activator of the classic comple-ment pathway [36, 37]. In the eye, it is also abundant,co-localizes with complement factors in drusen, anddemonstrates an age-associated increase [38]. We previ-ously showed by pathway analysis (Ingenuity, GSEA)that the complement system is triggered by Aβ stimula-tion of RPE in vitro [22]. However, little has been doneto assess the potential interaction between Aβ and thecomplement terminal product, MAC. Here we reportage-associated increases in MAC and Aβ in the RPE/choroid complex and soluble Aβ in the vitreous fluids(Fig. 1). The observed age-associated MAC formation inrat RPE/choroid is consistent with our earlier findings ofMAC deposition in BM and choroid of older postmor-tem human eyes [10]. Since MAC and Aβ are located atthe interface of RPE and choroid, we assessed MAC for-mation on RPE in the presence of Aβ and NHS andshowed for the first time that fibrillar Aβ is an effectiveinducer of the complement cascade resulting in MACdeposition (Fig. 2). MAC mediated inflammasome acti-vation was shown previously in cells derived from bonemarrow and lung epithelial cells in vitro [15, 16]. Ourdata support their findings and extend it to an ocularcell type, the RPE.From our work, and those of others, it is plausible thatthe inhibition of MAC in the RPE/choroid will dampeninflammasome activation in RPE. We found that ATACadministration concomitantly prevented the full-lengthcaspase-1 from being cleaved into an enzymatically ma-ture caspase-1 p20 subunit, the signature event of inflam-masome activation in the same animals in which weobserved a reduction in MAC levels (Figs. 4 and 5). How-ever, NF-κB activation, a primer for inflammasome activa-tion, was not affected by ATAC treatment, demonstratedby statistically equivalent amounts of the phosphorylatedp65 subunit in both retinal sections and RPE/choroid ly-sates (Fig. 3). Intriguingly, the phosphorylated p50 sub-unit’s level was extremely low in both ATAC anddrinking-water-treated rats. Although the p50/p65 hetero-dimer is considered the primary form of NF-κB complexin a wide variety of cell types, there are other active NF-κB dimeric combinations that do not require either one orboth of them [39]. All of these suggest that ATAC’s inhibi-tory effects spare NF-κB activation that involves p65 orp50 and are specific for MAC. Hence, the significant re-duction in inflammasome activation products, IL-18 andIL-1β, which we observed after ATAC treatment, is likelydue to inefficient post-translational processing by maturecaspase-1, rather than due to altered pro-IL-18 and pro-IL-1β production by NF-κB pathway (Fig. 5). Whetherand how the specific inhibition of NF-κB pathway can inturn affect MAC formation remains elusive and is beyondthe scope of the current study. Although the literaturesuggests NF-κB signaling regulates multiple genes in thecomplement cascades, such as CFB, C3, and C4, our dataindicates no effects on C5a production when NF-κBactivation is blocked by vinpocetine in vivo (see Additionalfile 1), and thus, likely not affecting MAC formation aswell [28, 40–42].Age-associated differences in MAC deposition and MACinhibition by ATACAnother interesting outcome of our study is the differencein ATAC efficacy in the two age groups tested. The youn-ger rats had lower ATAC levels in blood (~2 μg/500 μL)than the older rats (~3.5 μg/500 μL) (Fig. 3), yet there wasa proportionally greater decrease in MAC, caspase-1cleavage, and secreted IL-18 in the younger rats comparedto the older group, suggesting that ATAC treatment wasmore efficacious in the younger group (Figs. 4 and 5). Theexact mechanism behind this finding is not clear. Onepossible explanation is that, with age, there is more Aβ ac-cumulation in the rat eye, leading to more robust NF-κBactivation (p65 nuclear translocalization) and more MACdeposition, which is not proportional to the increase inATAC concentration, or its activity, and thus over-whelmed ATAC’s suppressive effects leading to reduceefficacy in the older rats compared to that observed inyounger rats (Figs. 1 and 2).ConclusionsIn summary, we have shown an age-dependent increase inAβ, MAC, and NF-κB in rat RPE/choroid. We have alsodemonstrated that Aβ, a drusen component, is an effectivepriming signal for NF-κB activation in vitro and in vivo[28] and promotes the NLRP3 inflammasome activationin the rat eye [21]. Suppression of MAC leads to a con-comitant suppression of NLRP3 inflammasome activationmeasured by caspase-1 cleavage and secretion of matureIL-18 and IL-1β as depicted in the schematic summary(Fig. 6). An inherent limitation of this study is that rodentsdo not have a macula/fovea, and thus renders this animalmodel less useful as it does not reproduce “macular” dis-ease. However, this model is useful to understand thebasic, cellular changes in the retina that are associatedwith chronic inflammation, aging, and age-related retinaldiseases. Our work suggests that MAC-induced NLRP3inflammasome activation may be an important cause ofC in RPE/choroid. Age-associated changes in RPE/choroid include an-κBom4).Zhao et al. Journal of Neuroinflammation  (2015) 12:121 Page 12 of 14Fig. 6 Proposed inflammasome activation mechanisms with Aβ and MAincreased accumulation of Aβ that acts as a “Signal 1” to activate the NFand pro-IL-1β (2). Next, assembly and activation of the NLRP3 inflammasmembrane (3). NLRP3 activation results in pro-caspase-1 auto-cleavage (enzyme to facilitate the production and secretion of active, mature IL-18 anMAC-induced inflammasome activation on RPEpathway (1). This upregulates the transcription of NLRP3, pro-IL-18e is triggered by MAC deposition (“Signal 2”) on the RPE cellThe cleaved caspase-1 then functions as a cytokine-processingd IL-1β (5). ATAC compound works as a MAC inhibitor, suppressingZhao et al. Journal of Neuroinflammation  (2015) 12:121 Page 13 of 14the chronic pro-inflammatory environment in the outerretina of a normal aging eye.Additional fileAdditional file 1: C5a is not affected by Vinpocetine-mediatedNF-κB inhibition. Description of data: Since the intraocular Aβ1–40injection has been previously implicated in promoting NF-κB activationand NLRP3 inflammasome activation [21], we assessed the level ofactivated C5 (C5a) as a surrogate marker for complement activation andMAC formation when NF-κB activity was specifically inhibited by vinpocetine.Western blot of retina protein lysates shows equal amounts of C5a (MW 41KDa; rabbit polyclonal C5a complement antibody, cat# 250565) in thevinpocetine-treated group, when compared to vehicle controls(Mann-Whitney, p > 0.05; N = 5). For a full description of the experimentalprocedures, readers are referred to our previous publication [28].Competing interestsTZ, JG, JV, ET, AW, SC, JZC, and JAM declare that they have no competinginterests. JPG, ML, and PLM declare a licensed patent (Aurin Biotech Inc.)relevant to the work.Authors’ contributionsTZ, JG, JV, ET, AW, and SC performed the experiments, analyzed the data,and participated in manuscript writing. JZC designed the study, performedthe experiments, analyzed the data, and participated in manuscript writing.JPG and ML performed the experiments and analyzed the data. PLMdesigned the study and analyzed the data. JAM designed the study,obtained funding, analyzed the data, and critically revised the manuscript. Allauthors read and approved the final manuscript.AcknowledgementsThe authors acknowledge Matthew Wong and Elliott To for expert technicalassistance and acknowledge the support by Vancouver Hospital + UBCFoundation. This study was funded by Canadian Institutes of Health Researchgrants (CIHR MOP 97806, MOP 126195) to JAM.Grant supportCIHR (MOP 97806, MOP 126195) and VGH + UBC Hospital FoundationAuthor details1Department of Ophthalmology and Visual Sciences, Faculty of Medicine,University of British Columbia, 2550 Willow Street, Vancouver V5Z 3N9BC,Canada. 2Kinsmen Lab of Neurological Research, University of BritishColumbia, Vancouver, BC, Canada.Received: 10 February 2015 Accepted: 4 June 2015References1. Wong WL, Su X, Li X, Cheung CM, Klein R, Cheng CY, et al. Globalprevalence of age-related macular degeneration and disease burden projectionfor 2020 and 2040: a systematic review and meta-analysis. Lancet Glob Health.2014;2:e106–16.2. Ardeljan D, Chan CC. Aging is not a disease: distinguishing age-relatedmacular degeneration from aging. Prog Retin Eye Res. 2013;37:68–89.3. Rodriguez-Muela N, Koga H, Garcia-Ledo L, de la Villa P, de la Rosa EJ,Cuervo AM, et al. 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