Bjo 2023 323884 - Proof - Hi 2

British Journal of Ophthalmology
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Exogenous CFH Modulates Levels of Pro-Inflammatory
Mediators to Prevent Oxidative Damage of Retinal Pigment
Epithelial Cells with the At-Risk CFH Y402H Variant
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Journal: British Journal of Ophthalmology

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Manuscript ID bjo-2023-323884
Article Type: Laboratory science

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Date Submitted by the
04-May-2023
Author:
Complete List of Authors: Velazquez-Soto, Henry; Institute of Ophthalmology Foundation Conde de

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Valenciana, Department of Immunology and Research Unit

Groman-Lupa, Sergio; Institute of Ophthalmology Foundation Conde de
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Cruz-Aguilar, Marisa; Institute of Ophthalmology Foundation Conde de

Salazar, Alberto L.; Institute of Ophthalmology Foundation Conde de
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Zenteno, Juan C.; National Autonomous University of Mexico,

Department of Biochemistry
JIMENEZ-MARTINEZ, MARIA C; National Autonomous University of
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Mexico Faculty of Medicine, ; Institute of Ophthalmology Foundation

Conde de Valenciana,
Keywords: Oxidative Stress, Immunology, Experimental – laboratory

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Page 1 of 35 British Journal of Ophthalmology
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British Journal of Ophthalmology Page 2 of 35
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3 1 Exogenous CFH Modulates Levels of Pro-Inflammatory Mediators to Prevent
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5 2 Oxidative Damage of Retinal Pigment Epithelial Cells with the At-Risk CFH
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7 3 Y402H Variant
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10 5 Henry Velazquez-Soto1, Sergio Groman-Lupa1, Marisa Cruz-Aguilar1, Alberto L.
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12 6 Salazar1, Juan C. Zenteno2,3, Maria C Jimenez Martinez1,3*
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19 10 1Department of Immunology and Research Unit, Institute of Ophthalmology “Conde
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11 de Valenciana Foundation”, Mexico City 06800, Mexico
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22 12 2Department of Genetics, Institute of Ophthalmology "Conde de Valenciana

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24 13 Foundation”, Mexico City, Mexico
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26 14 3Department of Biochemistry, Faculty of Medicine, National Autonomous University
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15 of Mexico, Mexico City 04510, Mexico

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31 17 *Correspondence: mcjimenezm@facmed.unam.mx
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3 20 Synopsis
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5 21 Addition of exogenous complement factor H prevents oxidative stress induced cell
6 22 death and modulates proinflammatory cytokine production in retinal pigment
7 23 epithelium cells with the at-risk CFH Y402H variant
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6 26 Abstract
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8 27 Background: Age-related macular degeneration (AMD) is a complex, progressive
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10 28 degenerative retinal disease. Retinal pigment epithelial (RPE) cells play an
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29 important role in the immune defense of the eye, and their dysfunction leads to the
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13 30 progressive irreversible degeneration of photoreceptors. Genetic factors, chronic
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15 31 inflammation, and oxidative stress have been implicated in AMD pathogenesis.
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32 Oxidative stress causes RPE injury, resulting in a chronic inflammatory response
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18 33 and cell death. The Y402H polymorphism in the complement factor H (CFH)
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20 34 protein is an important risk factor for AMD. However, the functional significance of
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22 35 CFH Y402H polymorphism remains unclear.

23 36 Methods: In the present study, we investigated the role of CFH in the
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25 37 proinflammatory response using an in vitro model of oxidative stress in the RPE

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27 38 with the at-risk CFH Y402H variant.
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39 Results: ARPE-19 cells with the at-risk CFH Y402H variant were highly
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30 40 susceptible to damage caused by oxidative stress, with increased levels of

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32 41 inflammatory mediators and pro-apoptotic factors that lead to cell death.
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34 42 Pretreatment of ARPE-19 cell cultures with exogenous CFH prior to the induction
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43 of oxidative stress prevented damage and cell death. This protective affect may be
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37 44 related to the negative regulation of pro-inflammatory cytokines.

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39 45 Conclusion: CFH contributes to cell homeostasis and is required to modulate the
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41 46 pro-inflammatory cytokine response under oxidative stress.

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44 48 Keywords: RPE, oxidative stress, CFH, AMD.

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59 3
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3 49 Key Messages
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5 50
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7 51 What is already known on this topic- Complement factor H has been pointed out as
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52 a critical participant in AMD pathogenesis, nevertheless, no functional studies have
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10 53 approached the role of this molecule in regulating the production of
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12 54 proinflammatory cytokine by retinal pigmented epithelium.
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14 55 What this study adds- In this study we report that addition of exogenous CFH
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15 56 decreases significatively the concentration of proinflammatory cytokines produced
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17 57 by the RPE with the at-risk CFH Y402H variant under oxidative stress.
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19 58 How this study might affect research, practice, or policy -These findings suggest a
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59 new role for CFH and helps to better understand its participation in the
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22 60 pathophysiology of AMD.
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3 63 1. Introduction
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5 64 Age-related macular degeneration (AMD) is a leading cause of legal blindness
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7 65 worldwide [1]. Several factors, such as genetic predisposition, chronic local
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66 inflammation, and oxidative stress, contribute to the etiology of AMD; however, the
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10 67 mechanisms leading to AMD development are not fully understood [2–5].
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12 68 Retinal pigment epithelium (RPE) cells are crucial for the maintenance of retinal
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14 69 homeostasis because they participate in several key functions, such as: the
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15 70 transport of nutrients, preservation of the retinal structure, phagocytosis of the
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17 71 outer segments of photoreceptors, and protection of the retina from oxidative
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19 72 damage [6,7]. The RPE is a highly oxidized microenvironment generated by a
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73 combination of visible light exposure, elevated metabolic activity, and accumulation
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22 74 of oxidized lipoproteins [8,9]. Physiological functions and aging processes, in

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24 75 combination with environmental stressors, including smoking or obesity related
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26 76 conditions force RPE cells to support excessive levels of oxidative stress,
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77 disturbing cell homeostasis [10] and causing chronic inflammation [11].

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29 78 Genetic predispositions play an essential role in AMD pathogenesis. Different
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31 79 variants of complement system proteins are associated with an increased risk of
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33 80 AMD development and progression [12]. A single nucleotide polymorphism
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34 81 predicting a Y402H replacement in the gene coding complement factor H (CFH) is

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36 82 an important risk factor for AMD [13]. CFH is a primary inhibitor of complement
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38 83 alternative pathways. By acting as a cofactor for factor I it prevents the formation of
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84 C3 convertase [14]. CFH is present in human and mouse ocular tissues, such as
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41 85 the RPE and choroid. Moreover, CFH and other complement proteins have been
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43 86 found in the drusen of patients with AMD suggesting that inflammation is an
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45 87 important component of this disease [15]. However, the mechanism through which
46 88 the CFH Y402H variant confers an increased risk of AMD remains unclear.
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48 89 In vitro studies have shown that the at-risk CFH Y402H variant leads to
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50 90 dysregulation of the complement pathway, with increased levels of the C3 protein
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91 [16]. Interestingly, the Y402H polymorphism is located in a short consensus repeat
53 92 (SCR) 7 of the protein, which is important for mediating CFH binding to polyanions
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55 93 such as heparan sulfate chains, C-reactive proteins, malondialdehyde, and
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59 5
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3 94 glycosaminoglycans [17,18]. This risk variant is associated with high levels of
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5 95 complement activation products and proinflammatory cytokines in the blood of
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7 96 patients with AMD [19]. Moreover, inefficient binding of the at-risk CFH Y402H
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97 variant to malondialdehyde induces macrophages and monocytes to secrete many
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10 98 pro-inflammatory cytokines, suggesting that systemic cytokines may also be
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12 99 associated with genetic factors and thus contribute to AMD [20].
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14 100 Similarly, studies in RPE in vitro showed that the poorly binding affinity of CFH
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15 101 Y402H to C-reactive protein resulted in elevated complement activation and altered
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17 102 secretion of the pro-inflammatory cytokines including tumor necrosis factor alpha
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19 103 (TNF-α) [17,18] and an upregulation of oxidative stress markers [21].
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21 104 Lipid metabolism in AMD is altered and associated with CFH Y402H. It shows
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22 105 impaired binding to malondialdehyde, a product of lipid oxidation. Transgenic aged

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24 106 mice expressing homozygous at-risk CFH variants present an AMD-like phenotype
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26 107 with changes in plasma and eyecup lipoproteins as a consequence of the Y402H
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108 polymorphism [22].
29 109 Several animal and in vitro models have been used to investigate the association
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31 110 between CFH and AMD. Mouse models provide a conduit for dissecting the
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33 111 molecular mechanisms linking complement responses to AMD pathobiology [23].
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112 Given the complexity of the disease, none of the available models fully recapitulate
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36 113 human AMD; however, they have provided important insights into the roles of the
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38 114 complement system and CFH in AMD. The in vitro generation of RPE monolayers
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40 115 from primary donor eye cultures has made it possible to study cell dynamics under
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41 116 experimental conditions, which has increased our knowledge of the cellular and
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43 117 molecular mechanisms of AMD. However, the limited life span and absence of the
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45 118 at-risk CFH variant in normal human RPE cells represents an obstacle for
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119 molecular analysis.
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48 120 Spontaneously generated human RPE cell line (ARPE-19) cells are a valuable
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50 121 source of human RPE cells. As the ARPE-19 cell line has been shown to maintain
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52 122 many of the properties of native cells of the RPE, it has frequently been used as an
53 123 in vitro model for the study of retinal disorders, such as AMD. Previous assays
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55 124 using ARPE-19 cells showed that an oxidized environment increases the
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3 125 expression of complement receptors and accumulated C3 as well as its regulators
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5 126 complement CFH, properdin and NLRP3, as a mechanism to maintain cell
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7 127 homeostasis following oxidative stress [24].
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128 With all these backgrounds raised we explored the role of CFH in regulating cell
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10 129 death and inflammation induced by oxidative stress condition in RPE cells culture
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12 130 with the at risk CFH Y402H variant.
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14 131
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15 132 2. Materials and Methods
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17 133 2.1. Cell culture and induction of oxidative stress
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19 134 The ARPE-19 was purchased from the American Type Culture Collection (ATCC
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135 CRL-2302) and cultured in 25 ml flasks containing Dulbecco's modified Eagle's
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22 136 nutrient F12 medium (DMEM / F12, STEMCELL Technologies) supplemented with
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24 137 10% fetal bovine serum (GIBCO, BRL), 100 U/ml penicillin, 100 mg/ml
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26 138 streptomycin (GIBCO, BRL), 1mM glutamate (Stem cell, Sartorius) and sodium
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139 pyruvate (Stem cell, Sartorius). The cell culture was kept under a humid
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29 140 atmosphere of 37 °C and 5% carbon dioxide (CO2). Cells were harvested and
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31 141 adjusted to 2.5 x 105 cells then seeded in 24 wells plates until they reached 80%
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33 142 confluence. To induce an oxidative stress condition, the cells were treated with 400
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34 143 μM or 800μM hydrogen peroxide (H2O2) for 24 h, harvested, washed, and

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36 144 resuspended in 1X Phosphate-buffered saline (PBS) for treatment and subsequent
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38 145 analysis.
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41 147 2.2. Genomic DNA extraction (gDNA)

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43 148 gDNA was obtained from 5 × 106 cells using a Blood and Tissue extraction kit
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45 149 (QIAGEN). The cells cultured in 25 ml culture flasks were dissociated with
46 150 trypsin/EDTA as previously described, and the cell button was resuspended in 200
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48 151 μl of 1X PBS to continue with the manufacturer´s protocol. The concentration and
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50 152 purity of the extracted gDNA was determined by spectrophotometry (Nanodrop,
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153 Thermofisher) and its integrity was verified by electrophoresis in 1.5% agarose gel,
53 154 stained with SYBR® Gold Nucleic Acid Gel Stain (Thermofisher Scientific) for
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55 155 visualization under UV light in a photo documenter.
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3 156
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5 157 2.3. ARPE-19 genotyping
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7 158 The region of the CFH gene that contains the single nucleotide polymorphism
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159 (SNP) rs1061170 (c.1204T>C; p.Tyr402His) was amplified by Polymerase Chain
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10 160 Reaction (PCR) using the oligonucleotides CFH_Fw:5'-AGT TCG TCT TCA GTT
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12 161 ATA C-3' and CFH_Rv:5'-TGG TCT GCG CTT TTG GAA GAG c-3. ’ The
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14 162 amplification reaction consisted of a mixture of 60 ng of gDNA, 5 l of Buffer A2
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163 KAPA2G 5X, 0.5l of KAPA dNTP Mix 10mM, 1.25l of each 10μM
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oligonucleotide, 0.1l of KAPA2G Fast DNA Polymerase 5 U/μL and sufficient
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19 165 nuclease-free water to achieve 25 l of reaction. The amplification conditions
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21 166 consisted of an initial denaturation cycle at 95 °C for 3 min, followed by 38 cycles
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23 167 consisting of denaturation at 95 °C for 1 min, hybridization at 59.5 °C for 30 s,
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168 extension at 72 °C for 1 min, and a final extension cycle at 72 °C for 3 min. Five 
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26 169 microliters of the PCR product were analyzed on a 1.5 % agarose gel and
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28 170 electrophoresed. The remaining samples were purified using a DNA Clean and
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171 Concentrator Kit (ZYMO Research). Genotyping of the CFH variant was carried out
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172 by automated direct sequencing with the BigDye Terminator Cycle Sequencing kit
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33 173 (Applied Biosystems), following the manufacturer's protocol and using a
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35 174 temperature program consisting of 25 denaturation cycles at 97 °C for 30 s,
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175 alignment at 50 °C for 30 s and an extension at 60 °C for 1 min. The samples were
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38 176 analyzed using an ABI Prism 3130 Genetic Analyzer sequencer (Applied
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40 177 Biosystems) and the obtained sequences were compared with a reference
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42 178 published in ENSEMBL (NM_000186.4).
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45 180 2.4. RNA extraction and cDNA synthesis
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47 181 Total RNA was extracted from 1 × 105 ARPE-19 cells using Trizol assay [25].
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49 182 These samples were treated with RNase-free DNase I (QIAGEN) according to the
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183 manufacturer's protocol. cDNA was synthesized from 2 g of total RNA in a final
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52 184 volume of 20 l using the Superscript III First Strand Synthesis System (Thermo
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54 185 Fisher Scientific) with oligo (dT). PCR was carried out with 125 ng of cDNA as a
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56 186 template in a 15 l reaction mix containing Kapa Taq Readymix PCR kit (Sigma-
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59 8
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3 187 Aldrich) and the specific primers for CRALBP (CRALBP_Fw:5´-
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5 188 TGGTGTCCTCTCTAGTCGGG-3,’ CRALBP_Rv:5'-
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7 189 GTTCAGCTGGCAGGAGATGT-3') and RPE65 (RPE65_Fw:5'-
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190 ACAACTTGGCCCTGACTTCC-3,’ RPE65_Rv:5'-TGAAGAGCATGACCACTCGG-
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10 191 3´). The transcripts were analyzed using the GeneAmp PCR System 9700
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12 192 thermocycler (Applied Biosytem) and PCR conditions of 35 cycles formed by:
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14 193 denaturation at 95 °C for 30 s, hybridization at 60 °C for 15 s, and extension at 72
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15 194 °C for 1 min, were done. Furthermore, the amplified products were analyzed by
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17 195 electrophoresis.
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197 2.5. Flow cytometry
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22 198 The cultured ARPE-19 cells were analyzed using flow cytometry. The cells were
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24 199 harvested by treatment with 0.25% trypsin- 0.5% EDTA (Gibco, Carlsbad, CA,
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26 200 USA), washed with 1X PBS and the cell pellet was resuspended in 2% fetal bovine
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201 serum in 1X PBS for 1 h at room temperature.

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29 202 The identification of surface molecules was carried out by incubating the cells with
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31 203 human monoclonal APC-conjugated anti-Toll like receptor 4 (TLR4), FITC-
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33 204 conjugated CD14 and PE- conjugated CD86 antibodies (all from Biolegend, San
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34 205 Diego, CA, USA) for 20 min at 4 °C, according to the manufacturer's
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36 206 recommendations. Cells were washed with 1X PBS and fixed with 4%
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38 207 paraformaldehyde (Sigma-Aldrich, Inc.) for 20 min at room temperature, washed,
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208 and acquired with a BD FACSVerse Flow Cytometer (BD Biosciences, San Jose,
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41 209 CA, USA). The results were analyzed using Flow Jo ver. 10 from Beckton
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43 210 Dickinson).
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46 212 2.6. Cell viability assay
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48 213 The effect of oxidative stress on ARPE-19 cell viability was evaluated by staining
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50 214 them with 7-Amino-Actinomicin (7-AAD; Molecular Probes, Invitrogen,
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215 Carlsbad,CA,USA). ARPE-19 cells were cultured with or without the addition of
53 216 exogenous CFH (5 µg/ml; Sigma Aldrich) and in the presence or absence of
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55 217 400μM H2O2 as an inducer of oxidative stress. In some experiments, CFH was
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3 218 added to the cell culture 1 h before or simultaneously with H2O2. Cells with or
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5 219 without stimuli were harvested and washed twice in 1X PBS containing 2% BSA
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7 220 and subsequently resuspended in 100 μL of 1X PBS. The exclusion of viable cells
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221 was carried out by staining with 5 μL of 7-AAD for 5 min according to the
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10 222 manufacturer's instructions and subsequently analyzed by flow cytometry.
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14 224 2.7. Intracellular for staining cytochrome c and NF-κB
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15 225 Nuclear factor kappa b (NF-κB) and release of cytochrome C from the
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17 226 intermembrane space of the mitochondria into the cytosol was measured using a
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19 227 monoclonal fluorescein isothiocyanate (FITC)-conjugated anti NF-κB or FITC-
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228 conjugated anti- cytochrome C antibody (Biolegend, San Diego, CA, US). ARPE-
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22 229 19 cells treated with H2O2 for 3, 6, 12, or 24 h were permeabilized and
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24 230 subsequently fixed with 1% PFA in 1X PBS. The mean fluorescence intensity (MFI)
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26 231 for both molecules was measured by flow cytometry and analyzed using FlowJo
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232 software, ver.10 (Beckton Dickinson).

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31 234 2.8. Cytokine measurement
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33 235 The cytokines produced after the induction of oxidative stress in ARPE-19 cells,
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34 236 with or without the addition of exogenous human plasma Complement Factor H
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36 237 (CFH; Sigma Aldrich, Saint Louis, MO, USA) at different concentrations (1, 2.5,
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38 238 and 5 µg/mL), were determined using the cytometric bead array (CBA) Human
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239 Inflammatory Cytokines Kit (BD Biosciences) according to the manufacturer's
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41 240 specifications. The assay allowed the simultaneous and quantitative measurement
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43 241 of cytokines such as interleukin (IL)-8, IL-1β, IL-6, IL-10, TNF-α and IL-12p70 in the
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45 242 cell culture supernatant. The induced culture supernatants were added to a mixture
46 243 of capture antibodies, bead reagents, and PE-conjugated detection antibodies. The
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48 244 mixture was then incubated at room temperature, in the dark, and washed. The
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50 245 cytokine panel was analyzed using FCAP Array Software (BD Biosciences).
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246 Detection limits for this method were as follows: 3.6 pg/mL of IL-8, 7.2 pg/mL of IL-
53 247 1β, 2.5 pg/mL of IL-6, 3.3 pg/mL of IL-10, 3.7 pg/ml of TNF-α, and 1.9 pg/mL of IL-
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55 248 12p70.
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5 250 2.9. Statistics
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7 251 All experiments were performed independently at least three times under each
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252 condition. All analyses were performed using GraphPad Prism 5.0 (GraphPad
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10 253 Software Inc., La Jolla, CA, USA). A normality test was performed on all the data
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12 254 obtained from the previous ANOVA and post-hoc statistical analyses. Statistical
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14 255 significance was set at P-value of less than 0.05.
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15 256
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17 257 3. Results
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19 258 3.1. ARPE-19 cell genotyping
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259 Aging, genetic factors, such as the complement factor H (CFH)Y402H
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22 260 polymorphism, and damage to the RPE induced by oxidative stress have been
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24 261 suggested as the main contributors to the development of AMD. As the ARPE-19
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26 262 cell line has been shown to maintain many of the properties of native cells of the
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263 RPE, it has frequently been used as an in vitro model for the study of retinal
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29 264 disorders.
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31 265 In this study, we used a cell line with an at-risk CFH Y402H variant to investigate
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33 266 the production of pro- and anti-inflammatory cytokines under oxidative stress.
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34 267 Thus, we initially determined the presence of the genetically at-risk CFH Y402H
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36 268 variant in ARPE-19 cells by direct sequencing. The gDNA of ARPE-19 cells was
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38 269 used as a template to amplify exon 9 of CFH by PCR. After sequencing the PCR
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270 product, we identified a heterozygous missense variant consisting of a c.1204 T to
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41 271 C transition, which was predicted to change tyrosine (TAT) to histidine (CAT) at
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43 272 residue 402 (Figure 1).
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46 274 As it is common for differentiated cells to alter some of their functional properties
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48 275 after several culture passages, we characterized the ARPE-19 cell line with respect
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50 276 to the expression of differentiation markers such as retinaldehyde-binding protein
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277 (CRALBP) and protein-specific RTE (RPE65) by real time-polymerase chain
53 278 reaction (RT-PCR). After amplification of the transcripts for differentiation markers,
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55 279 they were separated by electrophoresis into a single transcript of 593 bp for
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3 280 CRALBP and 598 bp for RPE65 (supplementary figure 1). As a negative control (-),
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5 281 the expression of CRALBP was also investigated in peripheral blood monocytes.
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7 282 The presence of a genetic variant that confers the risk of developing AMD and the
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283 expression of differentiation markers validated the idea that the ARPE-19 cell line
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10 284 could be suitable for establishing an in vitro model of oxidative stress and studying
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12 285 the participation of the Y402H genetic variant in the generation of an inflammatory
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14 286 environment.
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15 287
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17 288 3.2. Identification of molecules of immunological importance in ARPE-19
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19 289 It has been shown that the cells of the RTE express Toll-like receptors and co-
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290 stimulatory molecules which are considered key factors in ocular inflammation due
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22 291 to their ability to secrete or potentiate various inflammatory mediators [26,27].

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24 292 The expression of molecules of potential immunological importance on the surface
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26 293 of ARPE-19 cells was determined by detection with specific antibodies conjugated
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294 to different fluorochromes and analyzed by flow cytometry.

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29 295 Under stable conditions, and in the absence of external stimuli, ARPE-19 cells
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31 296 constitutively express basal levels of receptors, such as TLR4 and its co-receptor
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33 297 CD14. The co-stimulatory molecule, CD86, was also identified. While CD86
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34 298 showed low basal expression levels, TLR4 was the most highly expressed
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36 299 molecule, followed by CD14. Representative histograms for the detection of the
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38 300 molecules analyzed in this study are shown in supplementary figure 2.
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301
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41 302
42
43 303
On
44
45 304 3.3. H2O2-induced cell death.
46 305 To establish an in vitro model of cell stress induction, ARPE-19 cells were treated
47
ly
48 306 with 400μM or 800μM of H2O2 for 24 h. The effect of exposure to oxidative stress
49
50 307 was evaluated using a 7-amino-actinomycin-D (7-AAD) dye exclusion test, which
51
52
308 allows the evaluation of cell viability by flow cytometry. Living cells keep their
53 309 membranes intact, which prevents the passage of dyes, such as 7-AAD. In non-
54
55
56
57
58
59 12
1
2
3 310 viable cells, the membranes are damaged and permeable; therefore, the dye easily
4
5 311 penetrates and binds to double-stranded DNA.
6
7 312 ARPE-19 cells without H2O2 treatment were used as controls, representing more
8
313 than 80% viable cells. In contrast, treatment of cells with H2O2 significantly
9
10 314 decreased the membrane permeability of more than 70% of the cells (Figure 2a
11
Co
12 315 and 2c).
13
14 316 The representative dot plots in figure 4(a) show the distribution of ARPE-19 cells
nf
15 317 before and after the induction of oxidative damage and subsequent treatment with
16
17 318 7-AAD. All samples were analyzed using the same gates. Within stressed cells,
ide
18
19 319 living cells (30%) and apoptotic cells (70%) were identified. Our results indicate
20
21
320 that at the H202 concentration used in this assay, induced an oxidative stress state
nt
22 321 in ARPE-19 cells, which significantly affected membrane permeability and

23
24 322 compromised cell viability.
ial
25
26 323
27
:F
324 3.4. Release of apoptotic and inflammatory markers induced by oxidative stress in
28
29 325 ARPE-19 cells
or
30
31 326 Alterations in the integrity of the plasma and mitochondrial membranes due to
32
33 327 oxidative stress leads to the release of apoptogenic factors normally confined to
Re
34 328 the intermembranous space, such as cytochrome C. This induces an irreversible

35
36 329 commitment towards cell death. Therefore, in response to the synthesis of pro-
vie
37
38 330 inflammatory cytokines, the canonical NF-κβ pathway also triggers events that lead
39
40
331 cells to apoptosis. Hence, we considered it important to evaluate apoptosis
w
41 332 markers such as cytochrome C and NF-κβ. Intracellular molecules were identified
42
43 333 using specific antibodies and analyzed using flow cytometry. The mean
On
44
45 334 fluorescence intensity (MFI) was determined at different times of oxidative stress
46 335 induction (supplementary figure 3).
47
ly
48 336
49
50 337 Figure supplementary 3 (a) and (b) show an increase in the level of cytochrome C
51
52
338 released by ARPE-19 cells after exposure to H2O2. The release of cytochrome C
53 339 into the cytosol increases in a time-dependent manner. At the early stages of
54
55 340 oxidative stress induction, the cytosolic cytochrome C content was low and
56
57
58
59 13
1
2
3 341 increased over time, reaching a maximum at 24 h. The cytosolic content of
4
5 342 cytochrome C at 24 h post-oxidative stress was four times higher post-treatment.
6
7 343 Furthermore, the induction of NF-κB correlated with levels of cytoplasmic
8
344 cytochrome C. Maximum synthesis of these components was observed 24 h after
9
10 345 subjecting ARPE-19 cells to oxidative stress.
11
Co
12 346 Taken together, these results suggest that ARPE-19 cells harboring the at-risk
13
14 347 CFH Y402H variant are highly susceptible to oxidative stress-induced damage. In
nf
15 348 response to oxidative damage, proinflammatory and pro-apoptotic factors, such as
16
17 349 cytochrome C and NF-κB, can be detected.
ide
18
19 350
20
21
351 3.5. Exogenous CFH protein protects ARPE -19 cells from damage caused by
nt
22 352 oxidative stress

23
24 353 As we observed that oxidative stress affected the ARPE-19 cells viability, we
ial
25
26 354 considered it important to evaluate whether the presence of exogenous CFH could
27
:F
355 rescue cells from oxidative damage and prevent apoptosis.

28
29 356 Thus, to evaluate the effect of CFH on cell viability after the induction of oxidative
or
30
31 357 stress, ARPE-19 cells were cultured in the presence of any of the following
32
33 358 treatments: a simultaneous combination of exogenous CFH and H2O2 for 24 h, or
Re
34 359 treatment for 1 h with exogenous CFH followed by H2O2 induced-oxidative stress
35
36 360 for 24 h. Cell viability was evaluated by flow cytometry to determine the percentage
vie
37
38 361 of cells that were negative for 7-AAD staining. The data was then compared with
39
40
362 that obtained from cells cultured in the presence of exogenous CFH or H2O2 as the
w
41 363 only stimuli. ARPE-19 cells without any treatment were used as positive controls.
42
43 364 Figure 3 shows the percentage of viable cells after 12 h of treatment. ARPE-19
On
44
45 365 cells treated with exogenous CFH did not show deleterious changes and
46 366 maintained a viability similar to that of unstimulated cells (80%). In contrast, the
47
ly
48 367 induction of oxidative stress by H2O2 significantly affected cell survival (20% of
49
50 368 viable cells), as shown in previous studies.
51
52
369 The addition of exogenous CFH to the cell culture increased the percentage of
53 370 viable cells when added simultaneously or before the induction of oxidative stress.
54
55 371 The simultaneous addition of exogenous CFH and H2O2 to the cell culture
56
57
58
59 14
1
2
3 372 increased the percentage viability (55%) almost two-fold with respect to the
4
5 373 condition where only oxidative stress was induced. Similarly, when the cultures
6
7 374 were treated with exogenous CFH and subsequently subjected to oxidative stress,
8
375 the percentage of viable cells (74%) increased 2.3 times with respect to cells
9
10 376 treated only with H2O2 or 1.3 times with respect to cells treated simultaneously
11
Co
12 377 with exogenous CFH and H2O2.
13
14 378 Therefore, CFH synthesized by ARPE-19 cells possessing the at-risk CFH Y402H
nf
15 379 variant may not be completely efficient in modulating the oxidative effect caused by
16
17 380 the addition of H2O2. However, when exogenous CFH was incorporated into the
ide
18
19 381 culture medium, ARPE-19 cells could modulate the effect of oxidative stress. This
20
21
382 cellular protection was more effective when CFH was present in the cell culture
nt
22 383 before stimulation with oxidative stress. Furthermore, although exogenous CFH
23
24 384 contributed to the protection of cells against oxidative damage, the percentage of
ial
25
26 385 viable cells did not reach the level observed in untreated cells.
27
:F
386
28
29 387 3.6 Effect of exogenous CFH on the synthesis of pro-inflammatory cytokines
or
30
31 388 induced by oxidative stress
32
33 389 Oxidative stress and the inflammatory process are known to be associated with
Re
34 390 AMD. When a cell damage signal is generated, caspase activation mechanisms
35
36 391 are triggered, which subsequently activate the synthesis of pro-inflammatory
vie
37
38 392 cytokines. Since our previous results showed that the addition of exogenous CFH
39
40
393 to the cell culture prevented apoptosis induced by exposure to oxidative stress, we
w
41 394 evaluated whether this protection was driven by the modulation of pro-inflammatory
42
43 395 cytokines.
On
44
45 396 ARPE-19 cells were treated for 1 h with exogenous CFH (5 µg / ml) prior to the
46 397 addition of H2O2, or simultaneous to the addition of H2O2, maintaining the stress
47
ly
48 398 condition for 12 h. Additionally, we evaluated the cytokine profile depending on the
49
50 399 concentration of exogenous CFH added simultaneously with H2O2 to the cell
51
52
400 culture. Cytokines in the supernatants of the ARPE-19 cell cultures were quantified
53 401 using the CBA method. It is important to note that under some conditions, the
54
55 402 evaluated cytokines showed concentrations below the estimated detection limit.
56
57
58
59 15
1
2
3 403 Oxidative stress did not induce changes in the synthesis of IL-1β. IL-1β was not
4
5 404 detected in the supernatant of ARPE-19 cells treated simultaneously with 5 µg/ml
6
7 405 of exogenous CFH and H2O2. Even though the synthesis of IL-1β showed an
8
406 increased expression trend with respect to unstimulated cells, it was not
9
10 407 statistically significant.
11
Co
12 408
13
14 409 The induction of oxidative stress triggered the significant overexpression of IL-8,
nf
15 410 which increased 30 times with respect to non-stimulated cells or those stimulated
16
17 411 with exogenous CFH. This shows that the synthesis of IL-8 is inhibited by CFH,
ide
18
19 412 regardless of the concentration. Similarly, exogenous CFH regulates IL-8 synthesis
20
21
413 when present in the cell culture before or simultaneously with H2O2. Furthermore,
nt
22 414 IL-6 also significantly increased after the induction of oxidative stress compared
23
24 415 with when 5μg exogenous CFH was added previously or simultaneously with H2O2.
ial
25
26 416 The behavior of TNF-α, was similar to that observed for IL-6. The synthesis of
27
:F
417 TNF-α was increased 2.3 times in ARPE-19 cells under oxidative stress. Treatment
28
29 418 of cells with exogenous CFH at the highest concentration tested, added before or
or
30
31 419 simultaneously with H2O2, significantly decreased (approximately 3 times) the
32
33 420 synthesis of TNF-α with respect to treatment of cells with only H2O2.
Re
34 421 Finally, IL-10, an anti-inflammatory cytokine, was synthesized in response to

35
36 422 oxidative stress similar to levels in untreated cells. Lower IL-10 concentrations
vie
37
38 423 were observed when exogenous CFH was added in combination with H2O2. The
39
40
424 addition of exogenous CFH to cell cultures inhibited IL-10 synthesis induced by
w
41 425 oxidative damage.

42
43 426 These results indicate that oxidative stress induced in ARPE-19 cells results in the
On
44
45 427 expression of pro-inflammatory cytokines, which is regulated by exogenous CFH,
46 428 suggesting that CFH is important for maintaining cell homeostatic function. (Figure
47
ly
48 429 5)
49
50 430
51
52
431
53 432
54
55 433
56
57
58
59 16
1
2
3 434 4. Discussion
4
5 435 The RPE performs different complex functions that are necessary to maintain
6
7 436 photoreceptor homeostasis. Alterations in the RPE cell layer lead to deleterious
8
437 effects and photoreceptor death, triggering diseases, such as AMD.
9
10 438 The risk of developing AMD is mainly due to advanced age, environmental stress,
11
Co
12 439 and genetic factors [5,13,16,28]. Among genetic factors, the at-risk CFH Y402H
13
14 440 variant is strongly associated with AMD [29–31]. However, little is known about its
nf
15 441 impact on disease pathophysiology.
16
17 442 The in vitro generation of RPE monolayers has enabled the study of cell dynamics
ide
18
19 443 under experimental conditions, which has increased our knowledge of the cellular
20
21
444 and molecular mechanisms of AMD. The ARPE-19 cell line used in this study
nt
22 445 maintained many of the differentiation characteristics of RPE, such as the

23
24 446 expression of RPE65 and CRALBP, which are involved in the vitamin A cycle and
ial
25
26 447 regeneration of visual pigment, respectively. Molecules of immunological
27
:F
448 importance, such as TLR4, its CD14 co-receptor, and co-stimulatory molecules
28
29 449 such as CD86 on the surface of ARPE-19 cells, were also identified as part of their
or
30
31 450 cellular characterization. It is well known that the Toll-like receptors and co-
32
33 451 stimulatory molecules expressed in RPE cells are important for inducing ocular
Re
34 452 inflammation mechanisms because of their ability to secrete or potentiate various

35
36 453 inflammatory mediators [26,32,33]. Additionally, direct sequencing of CFH
vie
37
38 454 confirmed the heterozygous presence of the Y402H variant in ARPE-19 cells, one
39
40
455 of the main genetic variants associated with AMD development. These
w
41 456 characteristics of ARPE-19 cells allowed us to consider them an ideal cell model
42
43 457 for studying the effect of the at-risk CFH variant on the inflammatory response to
On
44
45 458 oxidative stress.
46 459 It is known that one of the main generators of oxidative stress in the RPE cells is
47
ly
48 460 H2O2. In this study, we used H2O2 to mimic the conditions of physiological oxidative
49
50 461 stress in vitro and evaluate the role of the at-risk CFH Y402H variant in ARPE-19
51
52
462 cells in generating the inflammatory cytokine profile under these conditions. The
53 463 RPE is susceptible to oxidative stress but is protected by the production of
54
55 464 antioxidant agents and its ability to regulate apoptosis under certain conditions [8].
56
57
58
59 17
1
2
3 465 However, the inadequate neutralization of oxidative stress affects mitochondrial
4
5 466 function and can lead to cellular damage. The main event after damage to the
6
7 467 mitochondria is increased permeability and loss of membrane potential, which
8
468 allows the release of pro-apoptotic molecules such as cytochrome C into the
9
10 469 cytoplasm [34–36]. Furthermore, the activation of caspases 9 and 3 has been
11
Co
12 470 demonstrated in RPE cells treated with H2O2 [35].
13
14 471 In our oxidative stress model using H2O2, ARPE-19 cells showed different degrees
nf
15 472 of alteration in the permeability of their plasma membrane, which correlated with
16
17 473 the amount of the internalized fluorescent marker (7AAD), and allowed the
ide
18
19 474 identification of living cells with intact plasma membranes and apoptotic cells. The
20
21
475 increased release of cytochrome C in stressed cells confirmed apoptosis. This
nt
22 476 release increased in a time-dependent manner, which correlated with the

23
24 477 permeability of the plasma membrane.
ial
25
26 478 Our assays showed that release of cytochrome C was accompanied by the
27
:F
479 activation of the NF-kB transcription factor. These results suggest that oxidative
28
29 480 stress triggers a signal transduction cascade that induces the expression of genes
or
30
31 481 encoding NF-kB-dependent proinflammatory cytokines. However, further studies
32
33 482 are needed to investigate the possible signaling pathways that lead to apoptosis.
Re
34 483 Intervention with caspases and the proteolytic pathway of the Bcl-2 family could be
35
36 484 involved in cytochrome C release. This could be feasible considering that in the
vie
37
38 485 mitochondria, Bcl-2 proteins modulate the apoptotic signal that releases apoptotic
39
40
486 factors such as cytochrome C and activates effectors such as caspases 9 and 3
w
41 487 that promote cell death [37,38]. Additionally, signal transduction through Fas or
42
43 488 TNF-α receptors with caspase 8 activation also stimulates cytochrome C release
On
44
45 489 through the cleavage pathway of Bcl-2 family products [39].
46 490 The synthesis of pro-inflammatory cytokines is dependent on the activation of
47
ly
48 491 transcription factors, or the action of other cytokines leading to the establishment of
49
50 492 a state of inflammation that contributes to the elimination of agents that cause cell
51
52
493 damage; however, under disease conditions, exacerbation of the inflammatory
53 494 response leads to cell and tissue damage. In RPE cells, the expression of IL-6 and
54
55 495 IL-8 for example, is induced in vitro by TNF-α, a potent initiator of the immune
56
57
58
59 18
1
2
3 496 response. The synthesis of these cytokines by RPE cells plays an important role in
4
5 497 the immune processes of the eye to achieve homeostasis in stressful situations;
6
7 498 however, its exacerbated or poorly controlled action induces severe tissue
8
499 damage, leading to the development of diseases such as AMD [40].
9
10 500 Our results showed that ARPE-19 cells were highly susceptible to damage caused
11
Co
12 501 by oxidative stress, with increased levels of inflammatory mediators and pro-
13
14 502 apoptotic factors that lead to cell death. However, it is unknown whether the
nf
15 503 presence of Y402H polymorphism is related to the inability of cells to achieve
16
17 504 homeostasis under oxidative stress. Therefore, we evaluated whether the
ide
18
19 505 presence of exogenous wild type CFH could restore the ability of cells to modulate
20
21
506 inflammatory responses and survive under prolonged conditions of oxidative
nt
22 507 stress.
23
24 508 Exogenous CFH incorporated into the cell culture prior to the induction of oxidative
ial
25
26 509 stress prevented damage and cell death. This protective effect was related to the
27
:F
510 negative regulation of pro-inflammatory cytokines induced by exogenous CFH. In

28
29 511 the absence of exogenous CFH, the synthesis of pro- and anti-inflammatory
or
30
31 512 cytokines significantly increased, causing deleterious effects on cell viability.
32
33 513 Similar alterations in cytokine profiles were observed when ARPE-19 cells were
Re
34 514 grown simultaneously in the presence of both stimuli. However, it is important to

35
36 515 note that, in some cases, the levels of cytokines were dependent on the
vie
37
38 516 concentration of exogenous CFH such as, IL-1β, IL-12, or TNF-α; high
39
40
517 concentrations of exogenous CFH inhibited the synthesis of these cytokines.
w
41 518 In basal conditions, RPE cells express very low levels of IL-1β which can be
42
43 519 slightly induced by treatment with TNF-α or antibodies cross-linking of CD48 on the
On
44
45 520 surface of cells, suggesting a minor role in the inflammatory process of the
46 521 posterior segment of the eye [41]. However, in our study, no significant differences
47
ly
48 522 were found in the levels of IL-1β synthesis in the ARPE-19 cell supernatant after
49
50 523 any treatment. These results may indicate that the availability of CFH in the
51
52
524 environment before an oxidative insult occurs is required to modulate the pro-
53 525 inflammatory cytokine response and protect cells from oxidative damage.
54
55
56
57
58
59 19
1
2
3 526 Therefore, it is possible that the elevation of pro-inflammatory cytokines under
4
5 527 oxidative stress observed in this study was related to the inability of the at-risk
6
7 528 Y402H variant of CFH to regulate complement activation. Although our assays did
8
529 not elucidate the mechanisms by which the at-risk variant in CFH promotes the
9
10 530 dysregulated expression of pro-inflammatory cytokines, a plethora of evidence in
11
Co
12 531 the literature has been reported. Oxidative stress triggers the inflammatory
13
14 532 response and activates death signals. The activation of complements and their
nf
15 533 bioactive fragments is also a potent stimulator of cytokine secretion that leads to
16
17 534 the amplification of inflammatory responses [42].
ide
18
19 535 Earlier studies have shown that the CFH Y402H polymorphism may be associated
20
21
536 with increased activation of the complement cascade, where activation of
nt
22 537 complement proteins such as C3a and C5a may regulate the expression of
23
24 538 proinflammatory cytokines [43,44]. Studies on ARPE-19 cells have shown
ial
25
26 539 increased intracellular concentrations of complement regulators CFH, properdin,
27
:F
540 and central complement protein C3 following oxidative stress induction. Properdin
28
29 541 serves as a positive complement regulator; therefore, a higher concentration of this
or
30
31 542 protein in ARPE-19 cells results in enhanced cellular C3 cleavage [24]. It is
32
33 543 potentially feasible that properdin activity prevails over that of the negative
Re
34 544 complement regulator CFH, which can be functionally decreased by the presence
35
36 545 of the risk variant Y402H.
vie
37
38 546 Other studies have reported that the complement activation product C5a is
39
40
547 associated with the at-risk variant in the CFH gene and promotes the activation of
w
41 548 the NF-kB pathway in RPE in vitro [45]. Concurrently, the NF-kB pathway mediates
42
43 549 the expression of classic inflammatory cytokines (IL-6, IL-8, TNF-α) and those
On
44
45 550 dependent of the activation of the NLRP3 inflammasomes such as IL-1β and IL-18
46 551 [46]. The addition of H2O2 to ARPE-19 cells increases the inflammasome activation
47
ly
48 552 NLRP3 and subsequently enhances the secretion of proinflammatory cytokines

49
50 553 [24]. Thus, it has been suggested that the CFH Y402H variant contributes to AMD
51
52
554 through increased activation of complement and NF-kβ pathways that result in
53 555 exacerbated secretion of inflammatory cytokines.
54
55
56
57
58
59 20
1
2
3 556 Finally, our results show that CFH is important for modulating the synthesis of
4
5 557 proinflammatory cytokines and preventing cell damage in response to oxidative
6
7 558 stress.
8
559 Further studies are needed to verify the signaling pathway(s) induced by
9
10 560 exogenous CFH to regulate the secretion of proinflammatory cytokines, and to
11
Co
12 561 investigate the endogenous expression of CFH at the mRNA and protein level.
13
14 562
nf
15 563
16
17 564
ide
18
19 565 Funding: This work was supported by the Institute of Ophthalmology, “Fundación
20
21
566 Conde de Valenciana”, and Department of Biochemistry, Faculty of Medicine,
nt
22 567 National Autonomous University of Mexico.

23
24 568 Acknowledgments: The authors thank Veronica Romero for her technical support.
ial
25
26 569
27
:F
570 Conflicts of Interest: The authors declare no conflicts of interest.

28
29 571
or
30
31 572 Data Availability Statement: All data are included in this article and supplementary
32
33 573 files.
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34 574
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59 21
1
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Sources. Antioxidants 2019;8:548. doi:10.3390/antiox8110548
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19 658 retinal pigment epithelial cells participates in transmembrane signaling in
20 659 response to photoreceptor outer segments. J Gen Physiol 2004;124:139–49.
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662 genetic, and functional expression. Exp Eye Res 2003;76:321–31.
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40 708 42 Reis ES, Mastellos DC, Hajishengallis G, et al. New insights into the
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42 710 doi:10.1038/s41577-019-0168-x
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711 43 Mullins RF, Dewald AD, Streb LM, et al. Elevated membrane attack complex
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46 712 in human choroid with high risk complement factor H genotypes. Exp Eye Res
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49 714 44 Takabayashi T, Vannier E, Clark BD, et al. A new biologic role for C3a and
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51 716 1996;156:3455–60.
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53 717 45 Cao S, Wang JCC, Gao J, et al. CFH Y402H polymorphism and the
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718 complement activation product C5a: effects on NF-κB activation and
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59 25
1
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3 719 inflammasome gene regulation. Br J Ophthalmol 2016;100:713–8.
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720 doi:10.1136/bjophthalmol-2015-307213
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6
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721 46 Bauernfeind FG, Horvath G, Stutz A, et al. Cutting edge: NF-kappaB
8 722 activating pattern recognition and cytokine receptors license NLRP3
9 723 inflammasome activation by regulating NLRP3 expression. J Immunol
10 724 2009;183:787–91. doi:10.4049/jimmunol.0901363
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19 729 FIGURES LEGENDS
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731 Figure 1. Partial Sanger sequence of the cfh gene showing the heterozygous
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732 c.1204 T>C variant (arrow) in ARPE-19 cells.

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28 734 Figure 2. Percentage of viable ARPE-19 cells post-treatment with different

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735 concentrations of H2O2 for 12 h to induce a state of oxidative stress. Viable cells
or
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31 736 exclude the 7-AAD dye, whereas non-viable cells allow dye penetration and
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33 737 intercalation with DNA. (a) Representative dot plots of cells without staining,
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35 738 unstimulated, stimulated with 400μM H2O2, and 800 μM H2O2. (b) Histogram
36 739 representation for 7-AAD mean fluorescence intensity in all evaluated conditions.
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38 740 (c) Mean ± standard deviation (SD) for three independent viability assays.
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40 741
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742 Figure 3. The percentage of viable ARPE-19 cells post-treatment with different
43 743 stimuli. The ARPE-19 cells were cultured for 24 h in the presence of exogenous
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45 744 CFH, H2O2 or a combination simultaneous H2O2 and CFH or sequential CFH (1h)
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47 745 followed by H2O2, and stained with 7-AAD for analysis by flow cytometry. Results
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48 746 are representative of three independent assays. (a) The dot blot represents the
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50 747 percentage of viable and dead cells post-treatment. (b) The histograms represent
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52 748 the MFI for viable and dead cells. (c) The mean ± SD for three independent viability
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749 assays.
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59 26
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3 750 Figure 4. The profile of pro- and anti-inflammatory cytokines in ARPE-19 cells
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5 751 secreted in response to oxidative stress and the addition of exogenous CFH. The
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7 752 cells were cultured in the presence of different stimuli and the cytokines in the
8
753 culture supernatants were determined. The graphs are representative of three
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10 754 independent experiments. CFH added to the culture 1 h before H2O2, and CFH and
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12 755 H2O2 added simultaneously. Simultaneous treatment of cells with different
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14 756 concentrations of CFH (1, 2.5 or 5 µg / ml) and 400μM H202. *p <0.05
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17 758 Figure 5. Model explaining the role of exogenous functional CFH in RPE under
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19 759 oxidative stress. RPE cells under oxidative stress increase NF-κB expression and
20
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760 secrete proinflammatory cytokines (IL-8, IL-6, IL-1β, TNF-α) and IL-10.
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22 761 Furthermore, cytochrome C is also released and detected in the cytoplasm of

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24 762 apoptotic cells. The addition of exogenous CFH protects cells from oxidative
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26 763 stress-related death and diminishes cytokine production. Created with
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764 BioRender.com
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5 Exogenous CFH Modulates Levels of Pro-Inflammatory Mediators to Prevent
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3,6,12, and 24 h or left unstimulated. Intracellular cytochrome C release and NF-κB
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31 expression were evaluated. (a) and (c) Representative histograms depicting MFI for
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33 cytochrome C and NF- κB respectively. Black: un-stained cells, grey: unstimulated cells,
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36 stimulated for 24h. (b) and (d) Graphic depicts the mean MFI ± SD for three independent
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Bjo 2023 323884 - Proof - Hi 2

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British Journal of Ophthalmology

Journal: British Journal of Ophthalmology

Article Type: Laboratory science

Complete List of Authors: Velazquez-Soto, Henry; Institute of Ophthalmology Foundation Conde de

Valenciana, Department of Immunology and Research Unit

Cruz-Aguilar, Marisa; Institute of Ophthalmology Foundation Conde de

Zenteno, Juan C.; National Autonomous University of Mexico,

Mexico Faculty of Medicine, ; Institute of Ophthalmology Foundation

Keywords: Oxidative Stress, Immunology, Experimental – laboratory

22 12 2Department of Genetics, Institute of Ophthalmology "Conde de Valenciana

15 of Mexico, Mexico City 04510, Mexico

22 35 CFH Y402H polymorphism remains unclear.

25 37 proinflammatory response using an in vitro model of oxidative stress in the RPE

30 40 susceptible to damage caused by oxidative stress, with increased levels of

37 44 related to the negative regulation of pro-inflammatory cytokines.

41 46 pro-inflammatory cytokine response under oxidative stress.

44 48 Keywords: RPE, oxidative stress, CFH, AMD.

22 74 of oxidized lipoproteins [8,9]. Physiological functions and aging processes, in

77 disturbing cell homeostasis [10] and causing chronic inflammation [11].

34 81 predicting a Y402H replacement in the gene coding complement factor H (CFH) is

22 105 impaired binding to malondialdehyde, a product of lipid oxidation. Transgenic aged

34 143 μM or 800μM hydrogen peroxide (H2O2) for 24 h, harvested, washed, and

41 147 2.2. Genomic DNA extraction (gDNA)

201 serum in 1X PBS for 1 h at room temperature.

232 software, ver.10 (Beckton Dickinson).

22 291 to their ability to secrete or potentiate various inflammatory mediators [26,27].

294 to different fluorochromes and analyzed by flow cytometry.

22 321 in ARPE-19 cells, which significantly affected membrane permeability and

34 328 the intermembranous space, such as cytochrome C. This induces an irreversible

22 352 oxidative stress

355 rescue cells from oxidative damage and prevent apoptosis.

34 421 Finally, IL-10, an anti-inflammatory cytokine, was synthesized in response to

41 425 oxidative damage.

22 445 maintained many of the differentiation characteristics of RPE, such as the

34 452 inflammation mechanisms because of their ability to secrete or potentiate various

22 476 release increased in a time-dependent manner, which correlated with the

510 negative regulation of pro-inflammatory cytokines induced by exogenous CFH. In

34 514 grown simultaneously in the presence of both stimuli. However, it is important to

48 552 NLRP3 and subsequently enhances the secretion of proinflammatory cytokines

22 567 National Autonomous University of Mexico.

570 Conflicts of Interest: The authors declare no conflicts of interest.

591 Reviews 2005;85:845–81. doi:10.1152/physrev.00021.2004

22 624 2022;58:658. doi:10.3390/medicina58050658

30 629 18 Weismann D, Hartvigsen K, Lauer N, et al. Complement factor H binds

37 634 macular degeneration. Am J Ophthalmol 2013;156:1176–83.

41 637 polymorphism Y402H associates with inflammation, visual acuity, and

44 639 2007;42:1116–22. doi:10.1016/j.exger.2007.08.001

48 642 Ophthalmol 2012;153:460-467.e1. doi:10.1016/j.ajo.2011.08.033

28 664 28 Velilla S, García-Medina JJ, García-Layana A, et al. Smoking and age-

30 666 2013;2013:895147. doi:10.1155/2013/895147

Immunol 2009;9:729–40. doi:10.1038/nri2620

37 670 analysis of the association between complement factor H Y402H polymorphisms

675 related macular degeneration. Proc Natl Acad Sci U S A 2005;102:7227–32.

28 700 photoreceptor degeneration in retinal degeneration slow (rds) mice. J Neurosci

41 709 immune functions of complement. Nat Rev Immunol 2019;19:503–16.

732 c.1204 T>C variant (arrow) in ARPE-19 cells.

28 734 Figure 2. Percentage of viable ARPE-19 cells post-treatment with different

22 761 Furthermore, cytochrome C is also released and detected in the cytoplasm of

37 a negative control (-).

detection with specific antibodies coupled to different fluorochromes. Histograms are

ARPE-19 cells without staining (negative control) or stained with APC-conjugated-Anti-

37 assays for cytochrome C and NF- κB, respectively.