Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 Feb 28.
Published in final edited form as: Discov Med. 2014 Jul-Aug;18(97):7–14.

Cholesterol Crystals Induce Inflammatory Cytokines Expression in nARPE-19 Cells by Activating the NF-κB Pathway

Yijun Hu 1, Haijiang Lin 2, Bernard Dib 3, Alp Atik 4, Peggy Bouzika 5, Christopher Lin 6, Yueran Yan 7, Shibo Tang 8, Joan W Miller 9, Demetrios G Vavvas 10
PMCID: PMC5330255  NIHMSID: NIHMS718003  PMID: 25091484

Abstract

Purpose

To investigate the expression of inflammatory cytokines in ARPE-19 cells after stimulation with cholesterol crystals.

Methods

APRE-19 cells were cultured, primed with IL-1α, and treated with cholesterol crystals under different concentrations. Inflammatory cytokines (mature-IL-1β, IL-6, and IL-8) in supernatant and inflammatory cytokines (pro-IL-1β, IL-18) in cell lysate were detected by western blot. The NF-κB pathway inhibitor BAY 11-7082 was used to determine the pathway of cytokine expression.

Results

Cholesterol crystals did not induce the nucleotide-binding domain leucine-rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome, but did increase pro-IL-1β expression in ARPE-19 cells. Cholesterol crystals increased pro-IL-1β expression by activating the NF-κB pathway. Cholesterol crystal activation of the NF-κB pathway also leads to increased IL-6 and IL-8 expression.

Conclusion

Cholesterol crystals can induce inflammatory cytokine expression in ARPE-19 cells by activating the NF-κB pathway.

Introduction

Age-related macular degeneration (AMD) is one of the leading causes of blindness in the world in individuals aged 55 or older (Klein et al., 2004). There are at least 1.75 million people suffering from AMD in the U.S. and this number is estimated to increase by 50% by the year 2020 (Friedman et al., 2004). Early AMD is characterized by macular drusen, which represents accumulation of extracellular deposits between the retinal pigment epithelium (RPE) and the inner layer of Bruch’s membrane. Advanced AMD can be further divided into the dry and the wet forms. Geographic atrophy is a fundamental characteristic of dry AMD while the wet form is characterized by choroidal neovascularization (CNV) at the macula. RPE dysfunction is the initial pathogenesis of AMD (Ambati et al., 2003). Its damage can lead to subsequent disruption of photoreceptors and the choroidal vasculature, which in turn can cause dry or wet AMD (McLeod et al., 2009; Vogt et al., 2011).

AMD is associated with multiple genes and environmental factors. High serum cholesterol is one of the risk factors for AMD (Feehan et al., 2011; Klein et al., 2010; 2003). Serum cholesterol exists mainly in two forms, a soluble esterified form and an insoluble unesterified form (crystal). Studies have shown that esterified cholesterol can induce the NLRP3 inflammasome in macrophages (Duewell et al., 2010; Rajamaki et al., 2010) and its derivatives can induce proinflammatory cytokines such as interleukin-6 (IL-6) and interleukin-8 (IL-8) (Larrayoz et al., 2010), which are shown to be associated with AMD (Miao et al., 2012; Roh et al., 2009). The NLRP3 inflammasome has also been shown to contribute to the pathogenesis of AMD (Marneros, 2013; Tarallo et al., 2012). Activation of the NLRP3 inflammasome leads to the cleavage of pro-interleukin-1 beta (pro-IL-1β) and pro-interleukin-18 (pro-IL-18) into their biologically active forms, which are then secreted by the cells (Franchi et al., 2009). Since the RPE plays an important role in the pathogenesis of AMD, it would be meaningful to find out whether cholesterol can induce inflammasome and proinflammatory cytokine expression in RPE cells. Unesterified cholesterol is one of the major components of drusen (Curcio et al., 2005; Wang et al., 2010). Whether unesterified cholesterol deposition can induce the NLRP3 inflammasome and proinflammatory cytokines in RPE cells was not investigated. The present study has shown that though cholesterol crystals cannot induce inflammasome activation, they can induce proinflammatory cytokine expression in ARPE-19 cells by activating the NF-κB pathway.

Methods and Materials

Materials

The human RPE cell line ARPE-19 was purchased from ATCC (Manassas, VA, US). DMEM/F-12, HEPES medium (#11330-057) was purchased from Life Technologies (Grand Island, NY, US). FBS (#SCRR-30-2020) was obtained from ATCC. Penicillin-Streptomycin (#15140122) was obtained from Life Technologies. Recombinant human IL-1α (200-LA-002) was obtained from R&D Systems (Minneapolis, MN, US). Cholesterol crystals (#C8667) were purchased from Sigma-Aldrich (St. Louis, MO, US). Bay 11-7082 inhibitor (#196870) was obtained from EMD Millipore. Anti-IL-18 antibody (#ab137664), anti-IL-6 antibody (#ab32530), and anti-β-actin antibody (ab8227) were obtained from Abcam (Cambridge, MA, US). Anti-IL-1β antibody (#MAB201) and anti-IL-8 antibody (#MAB208) were purchased from R&D Systems. HRP-linked secondary antibodies (7074S, 7076S) were obtained from Cell Signaling Technology (Danvers, MA, US).

ARPE-19 cell culture

ARPE-19 cells were cultured in 150mm dishes using DMEM/F-12, HEPES medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated with humidified 5% CO2 at 37°C and passaged when they were 80% confluent.

Preparation of cholesterol crystals solution

Cholesterol crystals were ground with a grinder and subsequently sterilized with UV light for 30 minutes. ARPE-19 culture medium was added to the cholesterol crystals to make a 6 mg/mL stock solution. Sonication was applied to the stock solution until the cholesterol crystals were evenly suspended in the culture medium.

Priming and treatment of ARPE-19 cells and sample collection

ARPE-19 cells were seeded in 6-well plates at a density of 1×106 cells/well. The cells were primed with 50 ng/mL IL-1α for 24 hours (Tseng et al., 2013). After priming, the cells were treated with cholesterol crystals under different concentrations along with 50 ng/mL IL-1α for 6 hours. After treatment, the culture medium was collected and centrifuged at 13.3 rpm for 15 minutes at 4°C. The supernatant was collected and stored at −80°C. Lysis buffer with proteinase inhibitors was added to the cells. The cells were scraped off the plates and transferred to 1.5 mL tubes. After sonication, the cell lysates were centrifuged at 13.3 rpm for 15 minutes at 4°C and the supernatant was collected and stored at −80°C.

Western blot

Total protein concentration was determined by DC™ Protein Assay (Bio-Rad, Philadelphia, PA, US). Equal amounts of total protein (for pro-IL-1β and pro-IL-18) or culture medium (for mature-IL-1β, IL-6, and IL-8) were loaded on each lane and the samples were run with electrophoresis. The proteins were transferred to a PVDF membrane and the membrane was blocked with non-fat milk and incubated with primary antibodies against IL-1β, IL-18, IL-6, IL-8, and β-actin. The membrane was then washed and incubated with secondary antibodies. The membrane was developed with enhanced chemiluminescence. The intensity of protein bands was measured using the software Image Lab 4.1 (Bio-Rad, Hercules, CA, US). Relative intensity of pro-IL-1β and pro-IL-18 was calculated by dividing the pro-IL-1β or pro-IL-18 band intensity by each corresponding β-actin. Relative intensity of IL-6 and IL-8 was calculated by dividing the band intensity of each treatment group by that of the IL-1α group. The difference in relative intensity between any two treatment groups was analyzed by unpaired t test.

Results

Cholesterol crystals do not induce the NLRP3 inflammasome, but do increase pro-IL-1β expression in ARPE-19 cells

Since the NLRP3 inflammasome has been shown to be associated with the pathogenesis of AMD (Marneros, 2013; Tarallo et al., 2012), we were interested in knowing whether cholesterol crystals can induce the NLRP3 inflammasome in ARPE-19 cells. Western blot showed that cholesterol crystals did not induce detectable mature-IL-1β after priming (Figure 1). However, it did induce pro-IL-1β production in ARPE-19 cells. Cholesterol concentrations of 1 mg/mL and 2 mg/mL induced about 1- and 2.5-times more pro-IL-1β, respectively, compared to priming alone (Figure 2A, 2B). Treatment with cholesterol alone did not induce detectable pro-IL-1β. Our results suggest that by increasing pro-IL-1β expression, cholesterol can stimulate the RPE cells to a status where, if the inflammasome is activated, massive amounts of mature-IL-1β may be released from the cells and cause severe inflammation.

Figure 1.

Figure 1

Open in a new tab

Western blot analysis of mature-IL-1β in cell culture medium. Cholesterol crystals did not induce detectable mature-IL-1β after priming in ARPE-19 cells.

Figure 2.

Figure 2

Open in a new tab

Cholesterol crystals increased pro-IL-1β expression, but not pro-IL-18, in ARPE-19 cells. (A) Western blot for pro-IL-1β and β-actin; (C) Western blot for pro-IL-18 and β-actin; (B, D) Relative intensity was calculated by dividing the pro-IL-1β/pro-IL-18 band intensity by each corresponding β-actin. Data expressed as mean±SE. *p<0.05.

The other cytokine processed after inflammasome activation is pro-IL-18 (Franchi et al., 2009). Our study showed that ARPE-19 cells constitutively produced pro-IL-18 during the ‘inactive’ state. However, priming and cholesterol treatment did not change pro-IL-18 levels (Figure 2C, 2D).

Cholesterol crystals increase pro-IL-1β expression by activating the NF-κB pathway

NF-κB pathway is the major pathway to activate pro-IL-1β expression (Broz and Monack, 2011). To determine whether cholesterol crystals increased pro-IL-1β expression by activating this pathway, we used the NF-κB pathway inhibitor BAY 11-7082 (dissolved in DMSO) along with cholesterol treatment. Our results showed that 20 mM BAY 11-7082 significantly inhibited pro-IL-1β expression induced by 1 mg/ml and 2 mg/ml cholesterol, suggesting that cholesterol crystals induce pro-IL-1β expression by activating the NF-κB pathway (Figure 3).

Figure 3.

Figure 3

Open in a new tab

Western blot for pro-IL-1β and β-actin. NF-κB pathway inhibitor BAY 11-7082 inhibited pro-IL-1β expression induced by cholesterol crystals.

Activation of the NF-κB pathway also leads to IL-6 and IL-8 expression

The NF-κB pathway is a common route leading to expression of many cytokines including IL-6 and IL-8 in RPE cells (Chen et al., 2011; Liu et al., 2012). Since the NF-κB pathway is activated by cholesterol crystals, we were interested to find out whether the expression of inflammatory cytokines such as IL-6 and IL-8 is also increased. Western blot showed that cholesterol could increase IL-6 expression after priming and that the effect was dose-dependent (Figure 4A, 4B). Priming alone was able to induce detectable IL-6 expression, which was further increased with cholesterol treatment. Specifically, 1 mg/mL of cholesterol induced 53% more IL-6 expression compared to priming alone, while a cholesterol dose of 2 mg/mL induced an IL-6 level that was 323% higher than after priming alone. Cholesterol treatment alone did not induce detectable IL-6 expression in ARPE-19 cells.

Figure 4.

Figure 4

Open in a new tab

Cholesterol crystals induced IL-6 and IL-8 expression in ARPE-19 cells. (A) Western blot shows IL-6 induced by cholesterol crystals. (B) The experiment was repeated three times independently. Data expressed as mean±SE. *p<0.05. (C) Western blot shows IL-8 induced by cholesterol crystals. (D) The experiment was repeated three times independently. Data expressed as mean±SE. *p<0.05.

Cholesterol treatment after priming also increased IL-8 expression in a dose-dependent manner. Priming alone induced detectable IL-8 expression in ARPE-19 cells. However, treatment with 1 mg/mL cholesterol after priming induced 24% more IL-8 expression compared to priming alone. This expression was increased with a cholesterol concentration of 2 mg/mL, which was 253% higher than after priming alone. No detectable IL-8 expression was induced by cholesterol treatment alone (Figure 4C, 4D).

To further confirm that the increased IL-6 and IL-8 expression was due to activation of the NF-κB pathway, we used BAY 11-7082 along with cholesterol. Our results showed that BAY 11-7082 significantly inhibited cholesterol induced expression of IL-6 and IL-8, which confirmed that activation of the NF-κB pathway was the mechanism of increased expression of these two cytokines (Figure 5).

Figure 5.

Figure 5

Open in a new tab

NF-κB pathway inhibitor BAY 11-7082 inhibited IL-6 and IL-8 expression induced by cholesterol, suggesting that cholesterol crystals induced IL-6 and IL-8 expression by activating the NF-κB pathway. (A) Western blot for IL-6; (B) Western blot for IL-8.

Discussion

Inflammation has been attributed as a key contributor to the pathogenesis of AMD (Ambati et al., 2013; Patel and Chan, 2008). The NLRP3 inflammasome has been shown to contribute to the pathogenesis of both dry and wet AMD in recent studies (Marneros, 2013; Tarallo et al., 2012). Activation of the NLRP3 inflammasome leads to cleavage of pro-IL-1β into its biologically active form: mature-IL-1β (Franchi et al., 2009). Mature-IL-1β released from cells can induce RPE cell death and angiogenesis. Different methods have been used to induce the NLRP3 inflammasome in ARPE-19 cells (Anderson et al., 2013; Tseng et al., 2013). Our study has shown that treatment with cholesterol crystals after priming does not induce detectable mature-IL-1β in the culture medium, suggesting that this inflammasome is not activated by cholesterol, or western blot is not sensitive enough to detect mature-IL-1β released by ARPE-19 cells. However, cholesterol crystals do increase pro-IL-1β expression in ARPE-19 cells after priming. A critical step before activation of the NLRP3 inflammasome is to produce abundant pro-IL-1β (Franchi et al., 2009; Hornung and Latz, 2010). Our study suggests that by increasing pro-IL-1β expression, cholesterol can stimulate the RPE cells to a status where, if the inflammasome is activated, large amounts of mature IL-1β may be released from the cells and cause severe inflammation.

Proinflammatory cytokines such as IL-6 and IL-8 have been shown to be associated with the development of CNV (Izumi-Nagai et al., 2008; 2007b; Miao et al., 2012; Roh et al., 2009). IL-6 is a pleiotropic proinflammatory cytokine which can exaggerate local immune and inflammatory responses. IL-6 has been shown to be associated with progression of AMD (Seddon et al., 2005), and its level is associated with macular thickness or CNV size in exudative AMD (Miao et al., 2012; Roh et al., 2009). In vivo studies have shown that IL-6 levels are increased in CNV and that IL-6 signaling is critical for CNV development (Izumi-Nagai et al., 2008; 2007b). Moreover, IL-6 can activate macrophages (Zhang et al., 2013) and induce VEGF expression (Cohen et al., 1996), both of which are believed to contribute to the development of CNV (Marneros, 2013; Patel and Chan, 2008). Our results have shown that cholesterol crystals after priming can induce IL-6 expression, while cholesterol crystals alone do not have the same effect. Although IL-1α is mainly released by necrotic cells, its expression is also increased in aged cells, activated macrophages, and monocytes (Hirsiger et al., 2012). Our results suggest that in an aging retina, cholesterol may stimulate IL-6 expression in RPE cells on the basis of increased IL-1α levels from aging cells and activated macrophages. The IL-6 induced by cholesterol may further contribute to the development of CNV.

CNV development is also associated with inflammatory cells such as macrophages and neutrophils (Patel and Chan, 2008; Zhou et al., 2005). IL-8 is a member of the CXC chemokine family and a potent chemoattractant for neutrophils and monocytes. Moreover, IL-8 is angiogenic (Heidemann et al., 2003) and can induce VEGF expression (Martin et al., 2009). Studies have shown that IL-8 levels correlate with macular volume or CNV size in exudative AMD (Miao et al., 2012; Roh et al., 2009). In the present study, IL-8 levels were increased by cholesterol crystals after priming. This finding suggests that in aging eyes, cholesterol can induce IL-8 expression by RPE cells. The increased expression of IL-8 may promote CNV development by inducing angiogenesis and recruiting inflammatory cells such as macrophages and neutrophils.

The NF-κB pathway is a common mechanism that regulates expression of many cytokines including IL-1β, IL-6, and IL-8 in RPE cells (Chen et al., 2011; Liu et al., 2012). Moreover, it is also critical for priming the NLRP3 inflammasome in RPE cells (Kerur et al., 2013). It has been shown that the NF-κB pathway may also be involved in the development of CNV (Izumi-Nagai et al., 2008; 2007a) and that cholesterol derivatives can induce inflammatory cytokine expression in ARPE-19 cells by activating the NF-κB pathway (Larrayoz et al., 2010). Our results show that cholesterol crystals can also activate the NF-κB pathway in ARPE-19 cells. Since activation of the NF-κB pathway leads to expression of various inflammatory cytokines and chemokines that may contribute to the pathogenesis of AMD (Chen et al., 2011; Liu et al., 2012), cholesterol therefore can create an inflammatory micro-environment for the development and progression of AMD. This is probably one of the reasons why high serum cholesterol level is one of the risk factors of AMD (Feehan et al., 2011; Klein et al., 2010; 2003).

There are several limitations to the present study. First of all, only an in vitro study was performed. The effects of cholesterol crystals on cytokine expression and the significance of cholesterol on the pathogenesis of AMD need to be validated by in vivo studies. Secondly, only a limited number of inflammatory cytokines were evaluated. The effects of cholesterol on expression of other cytokines associated with AMD should be further investigated. Thirdly, the possibility that cholesterol may induce cytokine expression by activating other pathways is also worth exploring.

Conclusion

In conclusion, our study has shown that cholesterol crystals can induce inflammatory cytokines expression in ARPE-19 cells by activating the NF-κB pathway. In vivo studies are required to verify this finding and the significance of cholesterol in the pathogenesis of AMD.

Acknowledgments

This study is supported by National Institutes of Health Grants EY12850 and R01EY019688 and by Research to Prevent Blindness, Inc., and was supported by NEIR21EY023079-01/A1, the Yeatts Family Foundation, a 2013 Macula Society Research Grant award, an RPB Physician Scientist Award, and the Lions unrestricted fund to MEEI.

Footnotes

Disclosure

The authors report no conflicts of interest.

Contributor Information

Yijun Hu, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, postal code, China and Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA

Haijiang Lin, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA

Bernard Dib, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA.

Alp Atik, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA.

Peggy Bouzika, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA.

Christopher Lin, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA.

Yueran Yan, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA.

Shibo Tang, Aier School of Ophthalmology, Central South University, Changsha, Hunan, postal code, China.

Joan W. Miller, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA

Demetrios G. Vavvas, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA

References

  1. Ambati J, Ambati BK, Yoo SH, Ianchulev S, Adamis AP. Age-related macular degeneration: etiology, pathogenesis, and therapeutic strategies. Surv Ophthalmol. 2003;48(3):257–293. doi: 10.1016/s0039-6257(03)00030-4. [DOI] [PubMed] [Google Scholar]
  2. Ambati J, Atkinson JP, Gelfand BD. Immunology of age-related macular degeneration. Nat Rev Immunol. 2013;13(6):438–451. doi: 10.1038/nri3459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson OA, Finkelstein A, Shima DT. A2E induces IL-1ss production in retinal pigment epithelial cells via the NLRP3 inflammasome. PLoS One. 2013;8(6):e67263. doi: 10.1371/journal.pone.0067263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Broz P, Monack DM. Molecular mechanisms of inflammasome activation during microbial infections. Immunol Rev. 2011;243(1):174–190. doi: 10.1111/j.1600-065X.2011.01041.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen Y, Kijlstra A, Yang P. IL-17A stimulates the production of inflammatory mediators via Erk1/2, p38 MAPK, PI3K/Akt, and NF-kappaB pathways in ARPE-19 cells. Mol Vis. 2011;17:3072–3077. [PMC free article] [PubMed] [Google Scholar]
  6. Cohen T, Nahari D, Cerem LW, Neufeld G, Levi BZ. Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem. 1996;271(2):736–741. doi: 10.1074/jbc.271.2.736. [DOI] [PubMed] [Google Scholar]
  7. Curcio CA, Presley JB, Malek G, Medeiros NE, Avery DV, Kruth HS. Esterified and unesterified cholesterol in drusen and basal deposits of eyes with age-related maculopathy. Exp Eye Res. 2005;81(6):731–741. doi: 10.1016/j.exer.2005.04.012. [DOI] [PubMed] [Google Scholar]
  8. Duewell P, Kono H, Rayner KJ, Sirois CM, Vladimer G, Bauernfeind FG, Abela GS, Franchi L, Nunez G, Schnurr M, Espevik T, Lien E, Fitzgerald KA, Rock KL, Moore KJ, Wright SD, Hornung V, Latz E. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature. 2010;464(7293):1357–1361. doi: 10.1038/nature08938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Feehan M, Hartman J, Durante R, Morrison MA, Miller JW, Kim IK, Deangelis MM. Identifying subtypes of patients with neovascu-ar age-related macular degeneration by genotypic and cardiovascular risk characteristics. BMC Med Genet. 2011;12:83. doi: 10.1186/1471-2350-12-83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Franchi L, Eigenbrod T, Munoz-Planillo R, Nunez G. The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis. Nat Immunol. 2009;10(3):241–247. doi: 10.1038/ni.1703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Friedman DS, O’colmain BJ, Munoz B, Tomany SC, Mccarty C, De Jong PT, Nemesure B, Mitchell P, Kempen J. Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol. 2004;122(4):564–572. doi: 10.1001/archopht.122.4.564. [DOI] [PubMed] [Google Scholar]
  12. Heidemann J, Ogawa H, Dwinell MB, Rafiee P, Maaser C, Gockel HR, Otterson MF, Ota DM, Lugering N, Domschke W, Binion DG. Angiogenic effects of interleukin 8 (CXCL8) in human intestinal microvascular endothelial cells are mediated by CXCR2. J Biol Chem. 2003;278(10):8508–8515. doi: 10.1074/jbc.M208231200. [DOI] [PubMed] [Google Scholar]
  13. Hirsiger S, Simmen HP, Werner CM, Wanner GA, Rittirsch D. Danger signals activating the immune response after trauma. Mediators Inflamm. 2012;2012:315941. doi: 10.1155/2012/315941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hornung V, Latz E. Critical functions of priming and lysosomal damage for NLRP3 activation. Eur J Immunol. 2010;40(3):620–623. doi: 10.1002/eji.200940185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Izumi-Nagai K, Nagai N, Ohgami K, Satofuka S, Ozawa Y, Tsubota K, Ohno S, Oike Y, Ishida S. Inhibition of choroidal neovascularization with an anti-inflammatory carotenoid astaxanthin. Invest Ophthalmol Vis Sci. 2008;49(4):1679–1685. doi: 10.1167/iovs.07-1426. [DOI] [PubMed] [Google Scholar]
  16. Izumi-Nagai K, Nagai N, Ohgami K, Satofuka S, Ozawa Y, Tsubota K, Umezawa K, Ohno S, Oike Y, Ishida S. Macular pigment lutein is antiinflammatory in preventing choroidal neovascularization. Arterioscler Thromb Vasc Biol. 2007a;27(12):2555–2562. doi: 10.1161/ATVBAHA.107.151431. [DOI] [PubMed] [Google Scholar]
  17. Izumi-Nagai K, Nagai N, Ozawa Y, Mihara M, Ohsugi Y, Kurihara T, Koto T, Satofuka S, Inoue M, Tsubota K, Okano H, Oike Y, Ishida S. Interleukin-6 receptor-mediated activation of signal transducer and activator of transcription-3 (STAT3) promotes choroidal neo-vascularization. Am J Pathol. 2007b;170(6):2149–2158. doi: 10.2353/ajpath.2007.061018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kerur N, Hirano Y, Tarallo V, Fowler BJ, Bastos-Carvalho A, Yasuma T, Yasuma R, Kim Y, Hinton DR, Kirschning CJ, Gelfand BD, Ambati J. TLR-independent and P2X7-dependent signaling mediate Alu RNA-induced NLRP3 inflammasome activation in geographic atrophy. Invest Ophthalmol Vis Sci. 2013;54(12):7395–7401. doi: 10.1167/iovs.13-12500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Klein R, Cruickshanks KJ, Nash SD, Krantz EM, Nieto FJ, Huang GH, Pankow JS, Klein BE. The prevalence of age-related macular degeneration and associated risk factors. Arch Ophthalmol. 2010;128(6):750–758. doi: 10.1001/archophthalmol.2010.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klein R, Klein BE, Tomany SC, Cruickshanks KJ. The association of cardiovascular disease with the long-term incidence of age-related maculopathy: the Beaver Dam Eye Study. Ophthalmology. 2003;110(6):1273–1280. doi: 10.1016/S0161-6420(03)00599-2. [DOI] [PubMed] [Google Scholar]
  21. Klein R, Peto T, Bird A, Vannewkirk MR. The epidemiology of age-related macular degeneration. Am J Ophthalmol. 2004;137(3):486–495. doi: 10.1016/j.ajo.2003.11.069. [DOI] [PubMed] [Google Scholar]
  22. Larrayoz IM, Huang JD, Lee JW, Pascual I, Rodriguez IR. 7-ketocholesterol-induced inflammation: involvement of multiple kinase signaling pathways via NFkappaB but independently of reactive oxygen species formation. Invest Ophthalmol Vis Sci. 2010;51(10):4942–4955. doi: 10.1167/iovs.09-4854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liu XC, Liu XF, Jian CX, Li CJ, He SZ. IL-33 is induced by amyloid-beta stimulation and regulates inflammatory cytokine production in retinal pigment epithelium cells. Inflammation. 2012;35(2):776–784. doi: 10.1007/s10753-011-9379-4. [DOI] [PubMed] [Google Scholar]
  24. Marneros AG. NLRP3 Inflammasome Blockade Inhibits VEGF-A-Induced Age-Related Macular Degeneration. Cell Rep. 2013;4(5):945–958. doi: 10.1016/j.celrep.2013.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Martin D, Galisteo R, Gutkind JS. CXCL8/IL8 stimulates vascular endothelial growth factor (VEGF) expression and the autocrine activation of VEGFR2 in endothelial cells by activating NFkappaB through the CBM (Carma3/Bcl10/Malt1) complex. J Biol Chem. 2009;284(10):6038–6042. doi: 10.1074/jbc.C800207200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mcleod DS, Grebe R, Bhutto I, Merges C, Baba T, Lutty GA. Relationship between RPE and choriocapillaris in age-related macular degeneration. Invest Ophthalmol Vis Sci. 2009;50(10):4982–4991. doi: 10.1167/iovs.09-3639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Miao H, Tao Y, Li XX. Inflammatory cytokines in aqueous humor of patients with choroidal neovascularization. Mol Vis. 2012;18:574–580. [PMC free article] [PubMed] [Google Scholar]
  28. Patel M, Chan CC. Immunopathological aspects of age-related macular degeneration. Semin Immunopathol. 2008;30(2):97–110. doi: 10.1007/s00281-008-0112-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rajamaki K, Lappalainen J, Oorni K, Valimaki E, Matikainen S, Kovanen PT, Eklund KK. Cholesterol crystals activate the NLRP3 inflammasome in human macrophages: a novel link between cholesterol metabolism and inflammation. PLoS One. 2010;5(7):e11765. doi: 10.1371/journal.pone.0011765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Roh MI, Kim HS, Song JH, Lim JB, Koh HJ, Kwon OW. Concentration of cytokines in the aqueous humor of patients with naive, recurrent and regressed CNV associated with amd after bevacizumab treatment. Retina. 2009;29(4):523–529. doi: 10.1097/IAE.0b013e318195cb15. [DOI] [PubMed] [Google Scholar]
  31. Seddon JM, George S, Rosner B, Rifai N. Progression of age-related macular degeneration: prospective assessment of C-reactive protein, interleukin 6, and other cardiovascular biomarkers. Arch Ophthalmol. 2005;123(6):774–782. doi: 10.1001/archopht.123.6.774. [DOI] [PubMed] [Google Scholar]
  32. Tarallo V, Hirano Y, Gelfand BD, Dridi S, Kerur N, Kim Y, Cho WG, Kaneko H, Fowler BJ, Bogdanovich S, Albuquerque RJ, Hauswirth WW, Chiodo VA, Kugel JF, Goodrich JA, Ponicsan SL, Chaudhuri G, Murphy MP, Dunaief JL, Ambati BK, et al. DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88. Cell. 2012;149(4):847–859. doi: 10.1016/j.cell.2012.03.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tseng WA, Thein T, Kinnunen K, Lashkari K, Gregory MS, D’amore PA, Ksander BR. NLRP3 inflammasome activation in retinal pigment epithelial cells by lysosomal destabilization: implications for age-related macular degeneration. Invest Ophthalmol Vis Sci. 2013;54(1):110–120. doi: 10.1167/iovs.12-10655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vogt SD, Curcio CA, Wang L, Li CM, Mcgwin G, Jr, Medeiros NE, Philp NJ, Kimble JA, Read RW. Retinal pigment epithelial expression of complement regulator CD46 is altered early in the course of geographic atrophy. Exp Eye Res. 2011;93(4):413–423. doi: 10.1016/j.exer.2011.06.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wang L, Clark ME, Crossman DK, Kojima K, Messinger JD, Mobley JA, Curcio CA. Abundant lipid and protein components of drusen. PLoS One. 2010;5(4):e10329. doi: 10.1371/journal.pone.0010329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhang C, Li Y, Wu Y, Wang L, Wang X, Du J. Interleukin-6/signal transducer and activator of transcription 3 (STAT3) pathway is essential for macrophage infiltration and myoblast proliferation during muscle regeneration. J Biol Chem. 2013;288(3):1489–1499. doi: 10.1074/jbc.M112.419788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zhou J, Pham L, Zhang N, He S, Gamulescu MA, Spee C, Ryan SJ, Hinton DR. Neutrophils promote experimental choroidal neovascularization. Mol Vis. 2005;11:414–424. [PubMed] [Google Scholar]

RESOURCES