Lipocalin 2 Plays an Important Role in Regulating Inflammation in Retinal Degeneration

This information is current as Tanu Parmar, Vipul M. Parmar, Lindsay Perusek, Anouk of September 25, 2021. Georges, Masayo Takahashi, John W. Crabb and Akiko Maeda J Immunol 2018; 200:3128-3141; Prepublished online 30 March 2018;

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Supplementary http://www.jimmunol.org/content/suppl/2018/03/30/jimmunol.170157 Material 3.DCSupplemental http://www.jimmunol.org/ References This article cites 75 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/200/9/3128.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Lipocalin 2 Plays an Important Role in Regulating Inﬂammation in Retinal Degeneration

Tanu Parmar,* Vipul M. Parmar,* Lindsay Perusek,* Anouk Georges,† Masayo Takahashi,† John W. Crabb,‡ and Akiko Maeda*,†,x

It has become increasingly important to understand how retinal inflammation is regulated because inflammation plays a role in retinal degenerative diseases. Lipocalin 2 (LCN2), an acute stress response protein with multiple innate immune functions, is increased in ATP-binding cassette subfamily A member 4 (Abca4)2/2 retinol dehydrogenase 8 (Rdh8)2/2 double-knockout mice, an animal model for Stargardt disease and age-related macular degeneration (AMD). To examine roles of LCN2 in retinal inflammation and degeneration, Lcn22/2Abca42/2Rdh82/2 triple-knockout mice were generated. Exacerbated inflammation following light exposure was observed in Lcn22/2Abca42/2Rdh82/2 mice as compared with Abca42/2Rdh82/2 mice, with upregulation of proinflammatory genes and microglial activation. RNA array analyses revealed an increase in immune response molecules such as Downloaded from Ccl8, Ccl2, and Cxcl10. To further probe a possible regulatory role for LCN2 in retinal inflammation, we examined the in vitro effects of LCN2 on NF-kB signaling in human retinal pigmented epithelial (RPE) cells differentiated from induced pluripotent stem cells derived from healthy donors. We found that LCN2 induced expression of antioxidant enzymes heme oxygenase 1 and superoxide dismutase 2 in these RPE cells and could inhibit the cytotoxic effects of H2O2 and LPS. ELISA revealed increased LCN2 levels in plasma of patients with Stargardt disease, retinitis pigmentosa, and age-related macular degeneration as compared with healthy controls. Finally, overexpression of LCN2 in RPE cells displayed protection from cell death. Overall these results http://www.jimmunol.org/ suggest that LCN2 is involved in prosurvival responses during cell stress and plays an important role in regulating inflammation during retinal degeneration. The Journal of Immunology, 2018, 200: 3128–3141.

everal blinding diseases of the retina are characterized by that activation of immune responses plays an important role in the death of retinal pigmented epithelium (RPE) and progression of these blinding diseases. We previously reported an S photoreceptor cells. In retinitis pigmentosa (RP), an increase in acute phase protein lipocalin 2 (LCN2) coinciding with inherited retinal disease characterized by a progressive loss of retinal degeneration in ATP-binding cassette subfamily A member 2/2 2/2 photoreceptor cells, an activation of prosurvival signaling cascades, 4(Abca4) retinol dehydrogenase 8 (Rdh8) mice (3). LCN2 by guest on September 25, 2021 involving upregulation of several growth factors, cytokines, and is also known as 24p3 or neutrophil gelatinase-associated lipocalin antioxidants, has been observed (1). In age-related macular de- and is a member of the lipocalin superfamily known for its role in generation (AMD), a major cause of visual impairment in elderly cellular transport of lipophilic molecules as fatty acids, iron, ret- people, several immune responses are activated in the form of inoids, and steroids. LCN2 is a multifunctional innate immunity inflammatory cytokines, chemokines, Abs, and T cells in both protein and can augment cellular tolerance to oxidative stress (4) animal models and patients (2). Accumulating evidence suggests and, indeed, roles of LCN2 have been suggested under stress conditions and degenerative diseases. In the CNS, LCN2 defi- *Department of Ophthalmology and Visual Sciences, Case Western Reserve Univer- ciency has been associated with tissue inflammation (5). † sity, Cleveland, OH 44106; Laboratory for Retinal Regeneration, RIKEN Center for Lcn2-deficient mice were found to be highly sensitive to bacterial Developmental Biology, Kobe, Hyogo 650-0047, Japan; ‡Cole Eye Institute, Cleve- land Clinic, OH 44195; and xDepartment of Pharmacology, Case Western Reserve sepsis (6). Several studies have demonstrated that LCN2 protects University, Cleveland, OH 44106 against cellular stress, inflammation, and cell death (7–10). Received for publication November 15, 2017. Accepted for publication March 5, Although a few studies have implicated the involvement of 2018. LCN2 in eye disease, the possible roles of LCN2 in retinal de- This work was supported by funding from National Institutes of Health Grants generation have not been fully elucidated. LCN2 was found up- EY022658 and EY11373, the Research to Prevent Blindness Foundation, and the Ohio Lions Eye Research Foundation. regulated among other acute phase response and inflammatory Address correspondence and reprint requests to Dr. Akiko Maeda, Department of proteins in the retina of rodent models for diabetes and retinal Ophthalmology and Visual Science, Case Western Reserve University, 2085 Adel- ischemia/reperfusion injury (11). Increased levels of LCN2 have bert, IP115, 10900 Euclid Avenue, Cleveland, OH 44106. E-mail address: been reported in a mouse model of Bardet–Biedl syndrome (12). [email protected] A pivotal role of LCN2 in the development of demyelinating optic The online version of this article contains supplemental material. neuritis in a mouse model of autoimmune optic neuritis has been Abbreviations used in this article: ABCA4, ATP-binding cassette subfamily A mem- demonstrated (13). Valapala et al. (14) identified LCN2 as a ber 4; AMD, age-related macular degeneration; GFAP, glial fibrillary acidic protein; hiPS-RPE, RPE cell differentiated from human-induced pluripotent stem cell; contributory factor in inducing chronic inflammatory response in HMOX1, heme oxygenase 1; KO, knockout; LCN2, lipocalin 2; miRNA, Cryba1 conditional knockout (KO) mice, a mouse model with microRNA; POS, photoreceptor outer segment; qRT-PCR, quantitative RT-PCR; RDH8, retinol dehydrogenase 8; ROS, reactive oxygen species; RP, retinitis AMD-like pathology. Lastly, Sinha and colleagues (15) generated pigmentosa; RPE, retinal pigmented epithelium; SD-OCT, spectral domain–optic genetically engineered mice in which lysosome-mediated clear- coherence tomography; SLC22A17, solute carrier family 22 member 17; SLO, ance in RPE cells is compromised, causing the development of scanning laser ophthalmoscopy; SOD2, superoxide dismutase 2; WT, wild-type. features of early AMD. They further proposed the involve- Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 ment of an AKT2–NF-kB–LCN2 signaling axis in activating the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701573 The Journal of Immunology 3129

FIGURE 1. Increased expression of Lcn2 in the retina and brain after light exposure. Abca42/2 Rdh82/2 mice (1 mo old) were exposed to light at 10,000 lx for 30 min. (A) Various tissues, including the eye, liver, heart, brain, spleen, lung, and kidney, were collected 24 h after light exposure. RNA levels are presented as fold change over non–light-exposed control tissues. (B) Immunoblot of LCN2 protein in light-exposed 1-mo-old Abca42/2Rdh82/2 mice 1, 3, and7dafterlightexposure.(C) Quantitative analy- Downloaded from ses of the immunoblot after normalization to b-actin are presented. (D) Serum was collected from 1-mo- old Abca42/2Rdh82/2 mice at different time points after light exposure. (E) Immunohistochemistry of LCN2 protein in the retinal sections of 1-mo-old Abca42/2Rdh82/2 mice 24 h after light exposure. http://www.jimmunol.org/ Scale bars, 50 mm. Error bars indicate SD of the means (n =3).*p , 0.05. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. by guest on September 25, 2021

inflammatory responses in these mice, suggesting this pathway as using the primers with LCN2-KO and common for KO allele and LCN2- a potential target for AMD treatment. In humans, Ghosh et al. (15) WT and common for WT allele: LCN2-KO, 59-CCTTCTAT GCC- 9 9 9 observed an increased infiltration of LCN2+ neutrophils in the TTCTTGACG-3 ; LCN2-common, 5 -TAGGGGATGCCACATCTCA-3 ; LCN2-WT, 59-TGGAGGTGACATTGTAGCTATTG-39. Genotyping for choroid and retina of early AMD patients as compared with age- Abca42/2Rdh82/2 mice was performed as described previously (16). All matched controls. These studies, including our observation of animal procedures and experiments were approved by the Case Western increased expression of LCN2 in mouse retinal degeneration (3), Reserve University Animal Care Committees and conformed to recom- reinforce the possible significance of LCN2 in retinal inflamma- mendations of both the American Veterinary Medical Association Panel on Euthanasia and the Association of Research for Vision and Ophthalmology. tion and retinal degenerative diseases. In the present study, Lcn22/2Abca42/2Rdh82/2 triple-KO mice Light exposure were generated and RPE cells differentiated from human-induced Mice were dark adapted for 48 h before being exposed to light. Light- pluripotent stem cells (hiPS-RPE) were employed to investigate induced degeneration was induced by exposing mice to 10,000 lx of dif- the role of LCN2 in retinal inflammation and degeneration. Our fuse white fluorescent light (150 W spiral lamp; Commercial Electric, results provide evidence that LCN2 could regulate prosurvival Cleveland, OH) for 30 min. Before such light exposure, pupils of mice were responses and retinal inflammation in mice and humans. dilated with mixture of 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Midorin-P; Santen Pharmaceutical, Osaka, Japan). After exposure, animals were kept in the dark until further evaluation. Materials and Methods Animals Histological analysis Lcn22/2 mice were purchased from The Jackson Laboratory (Bar Harbor, All procedures to make sections for retinal histology and immunohisto- ME). All mice were housed in the animal facility at the School of Medi- chemistry followed established methods (17, 18). For H&E staining eye cine, Case Western Reserve University, where they were maintained either cups were fixed in 10% formalin for 72 h followed by paraffin embedding. under complete darkness or in a 12-h light (∼10 lx)/12-h dark cycle en- Five-micrometer-thick sections were cut and stained with H&E for anal- vironment in a pathogen-free environment. Both male and female mice at ysis by light microscopy. For immunohistochemistry, eye cups were em- 1 mo of age were used in the study. Lcn22/2 mice were crossed with bedded in 4% paraformaldehyde/1% glutaraldehyde overnight followed by Abca42/2Rdh82/2 mice to generate Lcn22/2Abca42/2Rdh82/2 mice. cryosectioning. The following Abs were used for immunohistochemistry; 129SV or littermates of mutant mice were used as controls. Only the mice rabbit anti–Iba-1 Ab (1:400; Wako, Osaka, Japan), rabbit anti–glial with RPE65 leucine variant and free of rd8 mutation were employed in the fibrillary acidic protein (GFAP) Ab (1:400; Dako, Carpinteria, CA), rabbit study. Genotyping for Lcn22/2 or wild-type (WT) mice was performed NF-kB p65 Ab (1:1000; eBioscience, San Diego, CA). Secondary Abs 3130 ROLES OF LCN2 IN RETINAL DEGENERATION

20 mM HEPES (pH 7), and 10% FBS. To enrich microglial cells, neural retinas were homogenized and cultured in DMEM containing minimal essential medium nonessential amino acids (Invitrogen), penicillin/streptomycin (Invitrogen), 20 mM HEPES (pH 7.0), and 10% FBS for 7 d at 37˚C. Adherent cells to the plastic surface were treated with 0.05% trypsin (Invitrogen), and less adhesive cells were collected as microglial cells. Human primary RPE cells were purchased from Lonza (Walkersville, MD). Stimulation with photoreceptor outer segment and LPS Photoreceptor outer segment (POS) membranes were prepared from 1-mo-old Abca42/2Rdh82/2 and WT mice using a published method (19). LPS was purchased from InvivoGen (San Diego, CA). hiPS-RPE cell culture Establishment of hiPS cell lines and differentiated to RPE monolayers has been described in detail (21–25). All procedures were approved by the Institutional Review Boards at the Case Western Reserve University (Cleveland, OH) and adhered to the Declaration of Helsinki. All cell culture procedures were approved by Case Western Reserve University Institutional Biosafety Committee. All samples were obtained after donors had given informed consent.

FIGURE 2. Lcn2 increases in the RPE more than in the microglia. RPE Downloaded from cells and microglia were isolated from 1-mo-old Abca42/2Rdh82/2 mice. Apoptosis assay Cells were incubated with photoreceptor outer segments (POS, 6 mg/ml) hiPS-RPE cells (30,000–50,000) were seeded in 96-well plates. Cells were for 24 h. RNA levels of Lcn2 were measured in both RPE cells and treated with 100 mMH2O2 and recombinant LCN2 protein (R&D Systems, microglia with and without POS. Error bars indicate SD of the means. Minneapolis, MN) in doses of 1, 10, and 100 ng/ml for 24 h. On the next *p , 0.05 versus no POS treatment (n = 6). day, 1 drop/ml of CellEvent caspase-3/7 green detection reagent was added to each well for 60 min at 37˚C. Cells were then washed and ﬁxed in 4%

paraformaldehyde for 15 min, followed by DAPI staining. Cells were vi- http://www.jimmunol.org/ used were anti-mouse, anti-rabbit Alexa Fluor 555 (Invitrogen, Carlsbad, sualized and imaged under inverted fluorescence microscope. CA). Images were captured by a confocal microscope (LSM; Carl Zeiss, Thornwood, NY). Cell viability assay Scanning laser ophthalmoscopy and spectral domain–optic Assays were performed on 96-well plates, with 1 3 104 cells seeded in coherence tomography imaging each well. Cells were incubated with and without recombinant LCN2 (R&D Systems) for 24 h. After 2 h incubation, 10 ml of the WST-1 HRAII (Heidelberg Engineering, Heidelberg, Germany) for scanning laser solution (Roche, Mannheim, Germany) was added to the culture me- ophthalmoscopy (SLO) and ultra-high resolution spectral domain–optic co- dium and incubated for 2 h at 37˚C. Absorbance was measured using a herence tomography (SD-OCT; Bioptigen SD-OCT Envisu C2200; Bio- microplate ELISA reader (Multiskan FC microplate reader; Fisher ptigen, Research Triangle Park, NC) were employed for in vivo imaging of Scientific, Pittsburgh, PA). All experiments were conducted in triplicate by guest on September 25, 2021 mouse retinas. Mice were anesthetized by i.p. injection of a mixture (20 ml/g and replicated at least three times. Viable cells number was calculated body weight) containing ketamine (6 mg/ml) and xylazine (0.44 mg/ml) in by comparing the absorbance values of the samples after background 10 mM sodium phosphate (pH 7.2) with 100 mM NaCl. Pupils were dilated subtraction. with a mixture of 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Midorin-P; Santen Pharmaceutical). Numbers of autofluorescent particles in ELISA SLO images were counted per image. Amounts of CCL2, TNF-a, and LCN2 in serum, plasma, and tissue ho- Quantitative RT-PCR mogenates were quantified by ELISA kits (R&D Systems) according to the manufacturer’s instructions. Eyes were homogenized in 500 ml of Nonidet Total RNA was extracted with the RNeasy mini kit (Qiagen, Germantown, P-40 lysis buffer containing 20 mM Tris (pH 8), 137 mM NaCl, and 1% MD). Primers used in the study are listed in Supplemental Table I. All Nonidet P-40. Protein concentrations were measured using a NanoDrop procedures for quantitative RT-PCR (qRT-PCR) were carried out as de- (Thermo Fisher Scientific, Waltham, MA). scribed previously (19). Immunoblot RNA array analysis Enucleated eyes were harvested from the animals and lysed in ice-cold lysis Detection and quantification of gene expression was performed using a buffer (150 mM NaCl, 1 mM EDTA, 0.2% Nonidet P-40, and 20 mM Tris- mouse inflammatory cytokine and receptors array (PAMM-011ZF; HCl [pH 7.5]) containing protease inhibitor mixture (Sigma-Aldrich, St. SABiosciences/Qiagen, Frederick, MD) according to the manufac- Louis, MO). Tissue lysate was spun at 10,000 rpm for 10 min at 4˚C. turer’s instructions. This PCR-based array was selected, as it in- Proteins from each sample were transferred onto Immobilon-P membranes cludes 84 diverse genes important in the immune response, including (Millipore, Bedford, MA) after SDS-PAGE gel electrophoresis. Mem- genes encoding CC chemokines, CXC chemokines, IL cytokines, branes were incubated in 1% BSA solution containing a 1:1000 dilution of other cytokines, chemokine receptors, and cytokine receptors, as well either anti-LCN2 rabbit polyclonal Ab (sc-50350; Santa Cruz Biotech- as other genes involved in the inflammatory response. MicroRNA nology, Santa Cruz, CA) or anti–b-actin Ab (Santa Cruz Biotechnology). (miRNA) array was also performed using an inflammatory response Signals were visualized with alkaline phosphatase (Promega, Madison, miRNA PCR array (MIMM-105Z; SABiosciences/Qiagen) according to the manufacturer’s instructions. Real-time PCR amplification was WI) at a dilution of 1:10,000. The intensities of the bands were normalized 2 to the actin band using ImageJ software (National Institutes of Health, performed using RT SYBR Green PCR master mix (Qiagen). Bethesda, MD). Primers were designed using Web tool Primer3 and synthesized by Eurofins MWG Operon (Huntsville, AL). Data analysis was performed DD Isolation of primary RPE and microglia cells using the CT method according to the manufacturer’s protocol (SABiosciences). Primary mouse RPE cells and retinal microglial cells were prepared from 2-wk- old mice based on previously published methods (19, 20). Enucleated eyes were Immunocytochemistry for NF-kB localization incubated with 2% dispase (Invitrogen) in DMEM (Invitrogen) for 1 h at 37˚C, and neural retinas and eyecups were separated under a surgical microscope hiPS-RPE cells (30,000–50,000) were seeded in 96-well plates. Cells (ILLUMIN-i; Endure Medical, Cumming, GA). The RPE layer was peeled were coincubated with 1 mg/ml LPS and 1 ng/ml LCN2 for 24 h. On the from eye cups and cultured in DMEM containing minimal essential medium next day, the culture medium was removed and cells were fixed by nonessential amino acids (Invitrogen), penicillin/streptomycin (Invitrogen), adding 100 ml of 4% formalin to the wells for 15 min, followed by The Journal of Immunology 3131 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Deficiency of Lcn2 resulted in severe light-induced retinal degeneration in Abca42/2Rdh82/2 mice. (A) Representative retinal histology (upper panel) and SLO (lower panel) of 1-mo-old Lcn22/2Abca42/2Rdh82/2 and Abca42/2Rdh82/2 mice 7 d after light exposure at 10,000 lx for 30 min are shown. Scale bars, 20 mm. (B) Outer nuclear layer (ONL) thickness measurements of 1-mo-old Lcn22/2Abca42/2Rdh82/2 and Abca42/2Rdh82/2 mice 7 d after light exposure is presented. (C) Numbers of autofluorescent (AF) spots detected by SLO in 1-mo-old Lcn22/2Abca42/2Rdh82/2 and Abca42/2 Rdh82/2 mice 7 d after light exposure were counted. Error bars indicate SD of the means (n = 3). *p , 0.05. washing in PBS twice for 5 min. Fixed cells were permeabilized with 100 ml Next, PBS was removed and 100 ml of Nonidet P-40 lysis buffer was added of 0.2% Triton X-100 in PBS for 1 min at room temperature. The cells were and incubated on the plate on ice for 5 min. Cells were scraped, collected, then incubated with rabbit anti-mouse p65 (1:100) (eBioscience) in PBS and centrifuged for 10 min (14,000 rpm) at 4˚C. Supernatant was collected containing 10% goat serum overnight at 4˚C. Cells were then washed twice and total protein was measured using a Nanodrop (Thermo Fisher Scientific). with PBS and incubated with Alexa Fluor 488–labeled goat anti-rabbit IgG Total endogenous levels of total NF-kB p65 protein was then measured using Ab (1:200) (Molecular Probes) in PBS at room temperature for 2 h. The cells the PathScan total NF-kB p65 sandwich ELISA kit (no. 7174; Cell Signaling were washed twice for 5 min with PBS. DAPI (1:500; Vector Laboratories, Technology, Danvers, MA) according to the manufacturer’s instructions. Burlingame, CA) was added to the cells to visualize the nuclei. Cells were Endogenous levels of phospho–NF-kB p65 protein were measured using the then washed twice with PBS and imaged under fluorescent microscope. Five PathScan phospho–NF-kB p65 sandwich ELISA kit (no. 7173; Cell Sig- high-power fields (3100 magnification) were randomly selected in each naling Technology) according to the manufacturer’s instructions. ELISA sample for analysis. Positive nuclear staining in LPS-treated cells was used results were normalized to the total protein content per well. as positive control for NF-kB staining. The percentage of nuclear staining for NF-kB p65 was scored by counting the positive-stained cells and the total Human plasma analysis number of cells quantified in random microscopic fields using All participants in the study underwent a complete ophthalmic exami- the Metamorph image analysis software (Molecular Devices, San Jose, CA). nation and visual function tests by ophthalmologists. Informed consent Measurement of NF-kB activity by ELISA was obtained from each person for the present study. Procedures followed the Declaration of Helsinki guidelines and were approved by the Insti- hiPS-RPE cells (30,000–50,000) were seeded in 96-well plates. Cells were tutional Review Boards of Case Western Reserve University, RIKEN, coincubated with 1 mg/ml LPS and 1 ng/ml LCN2 for 24 h. On the next Institute of Biomedical Research and Innovation Hospital, and Cleveland day, media were removed and cells were rinsed once with ice-cold PBS. Clinic. Nonfasting blood samples were collected at the Institute of 3132 ROLES OF LCN2 IN RETINAL DEGENERATION

ONL

INL Iba-1/DAPI

ONL

INL Downloaded from

GFAP/DAPI http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Lcn22/2Abca42/2Rdh82/2 mice showed stronger gliosis than did Abca42/2Rdh82/2 mice after light exposure. Lcn22/2Abca42/2Rdh82/2 and Abca42/2Rdh82/2 miceat1moofagewereexposedtolightat10,000lxfor30min.(A) Immunohistochemistry using anti–Iba-1 (in red), a marker of microglia/ macrophages, in non–light exposed tissues and 24 h after light exposure is presented (upper panel). Immunohistochemistry using anti-GFAP (in green), a marker of Mu¨ller cells, in non–light exposed tissues and 24 h after light exposure is presented (lower panel). Nuclei were stained with DAPI (in blue). Scale bars, 50 mm. (B) RNA samples were collected from 1-mo-old Lcn22/2Abca42/2Rdh82/2, Abca42/2Rdh82/2, and WT mice. Expression levels of Gfap are normalized by Gapdh expression and shown by fold change. Error bars indicate SD of the means (n =6).*p , 0.05. INL, inner nuclear layer; ONL, outer nuclear layer.

Biomedical Research and Innovation Hospital or at the Cole Eye Institute or more groups. Mann–Whitney U statistics were employed to analyze from patients diagnosed with Stargardt disease (n = 11), RP (n =117), human plasma samples. Results were presented as mean 6 SD. The results and the wet form of AMD (n = 57). Blood was also collected from were considered statistically signiﬁcant at p , 0.05. healthy controls (n = 77) who had no retinal degeneration as determined by ophthalmologic examination. Healthy controls include family members of patients with inherited retinal disorder and individuals who Results underwent cataract surgery. Plasma was prepared as previously described Increased expression of Lcn2 in the retina and brain after light and stored under argon at 280˚C until analysis (26). exposure Overexpression of LCN2 in ARPE19 cells To examine expressional changes of acute stress protein LCN2 in various tissues under stress conditions that can induce retinal pcDNA3.1-LCN2 (OHu27037D) was purchased from GenScript 2/2 2/2 (Piscataway, NJ). ARPE19 cells (American Type Culture Collection, degeneration, 1-mo-old Abca4 Rdh8 mice were exposed to Manassas, VA) were transiently transfected with 1 mgofpcDNA3.1- light at 10,000 lx for 30 min and qRT-PCR was performed with LCN2 or pcDNA3.1 plasmids using Lipofectamine 3000 (Thermo isolated tissues, including the eye, liver, heart, brain, spleen, lung, Fisher Scientiﬁc) according to the manufacturer’s protocol. Expression and kidney. A 2-fold or higher increase of Lcn2 was observed in of LCN2 mRNA was then examined by RT-PCR 72 h after transfection 6 6 using primers shown in Supplemental Table I. the eye (15.38 3.86) and brain (4.97 0.55) when compared with dark-adapted controls (Fig. 1A). Lcn2 expression was not Statistical analysis obvious in these tissues prior to light exposure except spleen, Statistical analyses were performed using the t test for comparing two where low levels of Lcn2 expression were detected. To determine groups, and one-way ANOVA was used to detect differences among three the kinetics of LCN2 levels in the eye after light exposure, LCN2 The Journal of Immunology 3133

FIGURE 5. Increased expression of inﬂam- matory response in Lcn22/2Abca42/2Rdh82/2 mice. (A) Scatter plot comparing the expression of genes involved in the inﬂammatory response in Lcn22/2Abca42/2Rdh82/2 and Abca42/2 Rdh82/2 mice 24 h after light exposure at 10,000

lx for 30 min is presented. Each circle represents Downloaded from an individual gene with upregulated genes (in red) and downregulated genes (in green). Genes with no change in regulation (,2-fold in either direction) are within the boundary lines (in black). Genes outside the boundary lines have $2-fold altered expression. (B) RNA samples from whole eyes were collected from 1-mo-old http://www.jimmunol.org/ Lcn22/2Abca42/2Rdh82/2 and Abca42/2 Rdh82/2 mice 24 h after light exposure. Ex- pression levels of Ccl8, Ccl2, Cxcl10, Ccr5, Tnf, and Il2a are normalized by Gapdh expression and shown by fold change. (C and D) CCL2 and TNF protein levels were measured in 1-mo-old Lcn22/2Abca42/2Rdh82/2 and Abca42/2 Rdh82/2 mice 24 h after light exposure. Error bars indicate SD of the means (n = 6). *p , 0.05. by guest on September 25, 2021

expressionwasexaminedbyimmunoblot1,3,and7dafterillu- exposed to light at 10,000 lx for 30 min. WT littermates were mination at 10,000 lx for 30 min. LCN2 protein in the eye was found subjected to the same light exposure. Histology sections of the most upregulated 1 d after light exposure (Fig. 1B, 1C). Greatest retina demonstrated a decrease in outer nuclear layer thickness in LCN2increaseinserumwasobserved3dafterlightexposurein Lcn22/2Abca42/2Rdh82/2 mice as compared with Abca42/2 mice (Fig. 1D). Immunohistochemistry indicated that secreted LCN2 Rdh82/2 mice 7 d after light exposure (Fig. 3A, upper panels, 3B). protein was detected in RPE and microglial cells in the inner nuclear In vivo imaging of the retina by SLO was performed 7 d after light layer of the retina (Fig. 1E). Because incubation with POS can in- exposure (Fig. 3A, lower panels). Increased number of auto- duce production of inflammatory cytokines and chemokines from fluorescent spots, which are activated microglial cells and mac- RPE and microglial cells (19), RPE and microglia cells were isolated rophages (19), were counted in Lcn22/2Abca42/2Rdh82/2 mice from 2-wk-old Abca42/2Rdh82/2 mice. When these cells were in- as compared with Abca42/2Rdh82/2 mice (Fig. 3C). Lcn22/2 mice cubated with POS for 24 h, a 14-fold or 5-fold increase in Lcn2 was did not develop light-induced retinal degeneration (see “Deficiency observed in RPE or microglial cells, respectively (Fig. 2). of Lcn2 is associated with inflammatory changes”below). Deficiency of Lcn2 results in severe light-induced retinal Lcn22/2Abca42/2Rdh82/2 mice show stronger gliosis than do degeneration in Abca42/2Rdh82/2 mice Abca42/2Rdh82/2 mice after light exposure To examine effects of Lcn2 deficiency in retinal degeneration, 1-mo-old To investigate effects of Lcn2 deficiency to retinal glial cells after Lcn22/2Abca42/2Rdh82/2 and Abca42/2Rdh82/2 mice were light exposure, we examined the expression of Iba-1 for microglial 3134 ROLES OF LCN2 IN RETINAL DEGENERATION

Table I. Fold changes in differentially expressed genes in Lcn22/2Abca42/2Rdh82/2 versus Abca42/2Rdh82/2 mice 24 h after light exposure

Gene Symbols Gene Names Log2 Fold Change 24 h after Light p Value Upregulated Chemokines (C-C) CCL8 Chemokine (C-C motif) ligand 8 27.78 1.28 3 10221 CCL20 Chemokine (C-C motif) ligand 20 6.45 1.5 3 10221 CCL5 Chemokine (C-C motif) ligand 5 4.55 4.06 3 10221 CCL4 Chemokine (C-C motif) ligand 4 2.96 4.1 3 10221 CCL6 Chemokine (C-C motif) ligand 6 2.47 1.11 3 10220 CCL2 Chemokine (C-C motif) ligand 2 2.45 1.17 3 10220 CCL17 Chemokine (C-C motif) ligand 17 2.07 2.41 3 10220 Chemokines (C-X-C) CXCL5 Chemokine (C-C motif) ligand 5 17.21 2.96 3 10220 CXCL13 Chemokine (C-C motif) ligand 13 10.60 4.84 3 10220 CXCL10 Chemokine (C-C motif) ligand 10 3.02 5.26 3 10220 CXCL1 Chemokine (C-C motif) ligand 1 2.52 6.54 3 10220 Chemokine receptors CCR1 C-C motif chemokine receptor 1 10.60 7.96 3 10220 CCR5 C-C motif chemokine receptor 5 4.39 8.96 3 10220 CCR10 C-C motif chemokine receptor 10 2.48 8.6 3 10219 ILs/receptors Downloaded from SPP1 Secreted phosphoprotein 1 6.99 5.87 3 10219 IL1A Interleukin 1a 4.77 1.66 3 10218 IL10 Interleukin 10 4.04 3.83 3 10218 MIF Macrophage inhibitory factor 3.49 1.11 3 10217 TNF Tumor necrosis factor 2.76 1.42 3 10217 IL1 Interleukin 1 2.52 5.42 3 10217 IL15 Interleukin 15 2.50 2.42 3 10217 http://www.jimmunol.org/ IL2 Interleukin 2 2.38 3.42 3 10216 Downregulated CX3CL1 Chemokine (C-X3-C motif) ligand 1 24.06 1.42 3 10217 VEGFA Vascular endothelial growth factor A 23.84 3.42 3 10217 CXCL15 Chemokine (C-C motif) ligand 15 22.22 5.42 3 10219 Signiﬁcantly expressed genes (p , 0.05, fold changes $2.0) are listed. Minus signs indicate reduced expression.

2 2 2 2 cells and GFAP for Mu¨ller cells. Activation of glial cells is asso- Abca4 / Rdh8 / mice. Table I lists the fold changes of the 2/2 2/2 2/2 2/2 2/2 ciated with retinal inflammation (27). Lcn2 Abca4 Rdh8 genes compared in the two groups of mice. Lcn2 Abca4 by guest on September 25, 2021 and Abca42/2Rdh82/2 mice at 1 mo of age were exposed to light at Rdh82/2 mice displayed upregulation of 22 inflammatory medi- 10,000 lx for 30 min, and eyes were collected 24 h thereafter. ators, including chemokines, chemokine receptors, and ILs, as Immunohistochemistry with anti–Iba-1 Ab showed increased pro- compared with Abca42/2Rdh82/2 mice. Conversely, Cx3cl1, tein expression and more Iba-1+ microglial cell numbers in Lcn22/2 Vegfa, and Cxcl15 were downregulated in Lcn22/2Abca42/2 Abca42/2Rdh82/2 mice as compared with Abca42/2Rdh82/2 mice Rdh82/2 mice. Validation by qRT-PCR for Ccl8, Ccl2, Cxcl10, (Fig. 4A, upper panels). Immunohistochemical staining with anti- Ccr5, Tnf, and Il2a was carried out using a separate set of mice GFAP Ab revealed stronger gliosis with an increased expression of (Fig. 5B). Production of CCL2 and TNF was quantified with eyes 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 GFAP in Mu¨ller cells of Lcn2 / Abca4 / Rdh8 / mice (Fig. 4A, of 1-mo-old Lcn2 / Abca4 / Rdh8 / and Abca4 / Rdh8 / lower panels). qRT-PCR of Gfap displayed a similar pattern of mice 24 h after light exposure at 10,000 lx for 30 min. Increased increase as observed in immunohistochemistry (Fig. 4B). These levels of CCL2 and TNF in Lcn22/2Abca42/2Rdh82/2 mice results indicate that Lcn2 deficiency contributes to stronger response (CCL2, 34.75 6 1.5 pg/ml; TNF, 6.02 6 0.04 pg/ml) compared of retinal glial cells that are associated with retinal inflammation. with Abca42/2Rdh82/2 mice (CCL2, 5.75 6 0.7 pg/ml; TNF, 2 2 1.26 6 0.02 pg/ml) were measured (Fig. 5C, 5D). Increased expression of inflammatory response in Lcn2 / Abca42/2Rdh82/2 mice Deficiency of Lcn2 is associated with inflammatory changes To further dissect LCN2-mediated effects on inflammatory genes caused by light exposure and immune pathways, we employed the RT2 real-time PCR array Because Lcn22/2Abca42/2Rdh82/2 mice displayed more severe kit for 84 key immune genes involved in mediating inflammation. light-induced retinal degeneration and inflammation as compared Lcn22/2Abca42/2Rdh82/2 and Abca42/2Rdh82/2 mice (1 mo with Abca42/2Rdh82/2 mice (Figs. 3, 4, 5), roles of LCN2 in old) were exposed to light at 10,000 lx for 30 min. Dark-adapted retinal inflammation after light exposure were examined using mice were used for controls. The PCR array was performed Lcn22/2 mice. When 6-wk-old Lcn22/2 mice were exposed to 1 d (24 h) after light exposure. Total RNA was isolated from all light at 10,000 lx for 30 min, increased levels of inflammatory groups of mice. Equal amounts were converted to cDNA and then changes were observed 24 h after illumination as compared with subjected to pathway-focused gene expression profiling using real- WT mice, including increased expression of Iba-1/Aif-1 and Gfap time PCR. Scatter plots comparing the differential expression of (Fig. 6A). Although increased immune reactions were observed in genes in light-exposed Lcn22/2Abca42/2Rdh82/2 and Abca42/2 Lcn22/2 mice, this light condition did not cause obvious retinal Rdh82/2 mice were generated (Fig. 5A). Each symbol represents degenerative changes in Lcn22/2 and WT mice; in contrast, an individual gene. The boundary lines indicate a 2-fold differ- Abca42/2Rdh82/2 mice showed retinal structural changes 7 d ence. Genes outside the boundary lines have $2-fold altered ex- after illumination (Fig. 6B). No retinal degeneration was observed pression in Lcn22/2Abca42/2Rdh82/2 mice compared with in 6-wk-old and 4-mo-old Lcn22/2 mice when they were kept The Journal of Immunology 3135

FIGURE 6. Lcn22/2 mice displayed more preserved retinas with milder inflammation. (A) Retinas were isolated from 1-mo-old Lcn22/2 and WT mice 1 d after light exposure at 10,000 lx for 30 min, and expression of Iba-1/Aif-1 and Gfap was examined by qRT-PCR. Error bars indicate SD of the means (n = 3). *p , 0.05. (B) Downloaded from Mice were exposed to light and in vivo retinal images were obtained using SD-OCT 7 d later. Representative images are presented (n = 3). Scale bars, 50 mm. ONL, outer nuclear layer. http://www.jimmunol.org/ by guest on September 25, 2021 under regular lighting conditions. Because inflammatory changes (Fig. 7C). These observations suggest that LCN2 contributes to in light-exposed Lcn22/2 mice suggest regulatory roles of LCN2 inhibition of NF-kB activation in human RPE cells. hiPS-RPE cells in inflammation, expression of miRNA, which can modulate can express LCN2 as well as murine RPE cells when these cells transcriptional expression of inflammatory molecules, was ex- were incubated with POS (Supplemental Fig. 1). amined by miRNA array. Changes in miRNA expression obtained LCN2 protects against oxidative stress by increasing the from Lcn22/2 and WT mice are presented in Table II. These re- expression of antioxidant enzymes heme oxygenase 1 and sults indicate that loss of Lcn2 is prone to accelerating inflam- superoxide dismutase 2 in hiPS-RPE cells mation. Several in vitro studies have demonstrated that LCN2 protects LCN2 attenuates LPS-stimulated NF-kB activation in RPE against cellular stress and exposure to H2O2 (33–37). Other studies cells have reported that oxidative stress plays a major role in degen- The LCN2 gene promoter region contains binding sites for several erative retinal diseases such as RP and AMD (38–41). To inves- transcription factors, including NF-kB (28), which prompted us to tigate the effects of LCN2 on H2O2-induced oxidative stress in examine the effects of LCN2 on NF-kB signaling in RPE cells. RPE cells, 1 ng/ml LCN2 was incubated with hiPS-RPE cells Notably, the NF-kB pathway is a prototypical proinflammatory under oxidative stress conditions. Incubation with 100 mMH2O2 signaling pathway involved in the expression of multiple proin- for 24 h in the absence of LCN2 resulted in reduced cell viability flammatory genes such as cytokines, chemokines, and adhesion to 49.51 6 11.59%, indicating that about half of the cell pop- molecules (29–32). hiPS-RPE cells were incubated with 1 mg/ml ulation died (Fig. 8A). When 1 ng/ml LCN2 was added to these LPS for 24 h to investigate whether LCN2 can inhibit the nuclear incubation conditions, protective effects of LCN2 against H2O2- translocation of NF-kB p65. Immunocytochemistry with anti-p65 induced cell death were observed. LCN2 supplementation main- Ab revealed that LPS-stimulated hiPS-RPE cells showed p65 tained cell viability nearly to the control level at 87.62 6 12.74%. staining in the nuclei, whereas unstimulated cells showed stronger With increasing doses of recombinant LCN2, increased expression staining in the cytoplasm. Supplementation of LCN2 in the culture of antioxidant enzymes heme oxygenase 1 (HMOX1) and super- medium decreased the numbers of cells with LPS-induced nuclear oxide dismutase 2 (SOD2) in hiPS-RPE cells was observed translocation of p65 (Fig. 7A, 7B). Furthermore, phosphorylation of (Fig. 8B, 8C), suggesting that an upregulation of HMOX1and NF-kB p65, which indicates activation of NF-kB, was examined SOD2 is an adaptive mechanism to protect cells from oxidative using ELISA. In contrast to the finding that levels of total NF-kB damage. HMOX1 and SOD2 are known to act as the first-line p65 remained unchanged, a decrease in phosphorylated NF-kBp65 antioxidant enzyme defense system against reactive oxygen spe- was observed when LCN2 was supplemented to hiPS-RPE cells cies (ROS) and particularly superoxide anion radicals (40). 3136 ROLES OF LCN2 IN RETINAL DEGENERATION

Table II. Fold changes in differentially expressed miRNA genes in Lcn22/2 mice versus WT mice 24 h after light exposure

Gene Symbols Functional Grouping of Genes Log2 Fold Change 24 h after Light p Value miR-429-3p Gpr68, Hmgb3, Il13, Ntf3, Prkca, Ripk2, 9.06 5.63 3 10257 Vegfa let-7a-5p Casp3, Ccr7, Fgf11, Fgf5, Gdf6, Il13, 7.01 2.67 3 10255 Masp1 let-7b-5p Casp3, Ccr7, Fgf11, Fgf5, Gdf6, Il13 7.01 2.00 3 10253 let-7e-5p Casp3, Ccr7, Fgf11, Fgf5, Gdf6, Il13 7.01 5.24 3 10247 let-7f-5p Casp3, Ccr7, Fgf11, Fgf5, Gdf6, Il13, Masp 7.01 6.42 3 10247 miR-1192 Atrn, Bcl11a, Clcf1, Cyp26b1, Fgf7, Ptpra 7.01 2.68 3 10245 miR-135a-5p Fgf11, Nampt, Sdcbp, Sp3, Tnfsf4, Txndc5 7.01 2.84 3 10245 miR-140-5p Bmp2, Fgf9, Hdac4, Hdac7, Rac1, Spred1 7.01 2.92 3 10245 miR-144-3p Cxcl12, Eda, Gdf10, Lifr, Ptgs2, Tnfsf11, Ttn 7.01 3.87 3 10245 miR-155-5p Cebpb, Cyp26b1, Fgf7, Gdf6, Ms4a1, Sdcbp 7.01 5.34 3 10244 miR-15a-5p Cd28, Eda, Fgf7, Ghr, Ifnk, Il10ra, Pik3r1 7.01 9.51 3 10261 miR-186-5p Cast, Cntnap2, Cxcl13, Gdf6, Il13ra1, Pdgfc 7.01 9.02 3 10259 miR-19a-3p Cast, Cbfb, Chst1, Cntfr, Cxcl12, F3, 7.01 1.01 3 10256 Impdh1 miR-19b-3p Cast, Cbfb, Chst1, Cntfr, Cxcl12, F3, 7.01 3.54 3 10250 Impdh1 2 miR-20a-5p F3, Mgll, Mink1, Osm, Pdcd1lg2, Ptger3, 7.01 5.42 3 10 50 Downloaded from Stat3 miR-20b-5p F3, Mgll, Mink1, Osm, Pdcd1lg2, Ptger3, 7.01 2.72 3 10248 Stat3 miR-26b-5p Cmtm4, Inhbb, Pawr, Ppp3cb, Prkcd, Prkcq 7.01 3.36 3 10248 miR-291a-3p Bcl11a, Bcl6, Cdkn1a, Cyp26b1, Dock2, F3 7.01 3.94 3 10248 miR-294-3p Bcl11a, Bcl6, Cdkn1a, Cyp26b1, Dock2, F3 7.01 5.88 3 10248 miR-295-3p Bcl11a, Bcl6, Cdkn1a, Cyp26b1, Dock2, F3 7.01 9.03 3 10247 http://www.jimmunol.org/ miR-29b-3p Atrn, Bcl11a, Hdac4, Il1rap, Lif, Pdgfc, 7.01 2.84 3 10246 Tnfrsf1a miR-29c-3p Atrn, Bcl11a, Hdac4, Il1rap, Lif, Pdgfc, 7.01 5.88 3 10247 Tnfrsf1a miR-301a-3p Cast, Cbfb, Chst1, Eda, Erbb2ip, Hprt1, 7.01 5.63 3 10252 Impdh1 miR-301b-3p Cast, Cbfb, Chst1, Eda, Erbb2ip, Hprt1, 7.01 2.67 3 10255 Impdh1 miR-302b-3p Bcl11a, Bcl6, Cdkn1a, Cyp26b1, Dock2, F3 7.01 2.00 3 10253 miR-302d-3p Cd28, Eda, Fgf7, Ghr, Ifnk, Il10ra, Pik3r1 7.01 5.24 3 10249 247 miR-322-5p Cd28, Eda, Fgf7, Ghr, Ifnk, Il10ra, Pik3r1 7.01 6.42 3 10 by guest on September 25, 2021 miR-325-3p Bcl11a, Bmi1, Cast, Cmtm6, Cntnap2, 7.01 2.68 3 10245 Cyp26b1 miR-340-5p Bcl11a, Bmi1, Cast, Cmtm6, Cntnap2, 7.01 5.88 3 10248 Cyp26b1 miR-106b-5p Atrn, Bmp3, Bmp4, Cd40lg, Chst2, Gfra2 6.36 1.01 3 10256 miR-466d-3p Cast, Cbfb, Chst1, Eda, Erbb2ip, Hprt1, 4.79 2.72 3 10248 Impdh1 miR-669k-3p Cast, Cbfb, Chst1, Eda, Erbb2ip, Hprt1, Tnf 4.03 5.63 3 10257 miR-130b-3p Cast, Cbfb, Chst1, Eda, Erbb2ip, Hprt1, 3.63 2.67 3 10255 Impdh1 miR-16-5p Cd28, Eda, Fgf7, Ghr, Ifnk, Il10ra, Pik3r1, 3.18 2.00 3 10253 Spr miR-23a-3p Btla, Ccl7, Cxcl12, Erbb2ip, Fas, Grem1 2.95 5.24 3 10247 let-7g-5p Casp3, Ccr7, Fgf11, Fgf5, Gdf6, Il13 2.81 6.42 3 10247 miR-350-3p Cntnap2, Cyp26b1, Ghr, II1rap, II1m 2.57 2.68 3 10245 miR-23b-3p Btla, Ccl7, Cxcl12, Erbb2ip, Fas, Grem1 2.43 1.01 3 10258 miR-181a-5p Cd4, Il1a, Il7, Lif, Phf20l1, Prkcd, Tnf 2.25 5.42 3 10251 miR-126a-5p Ap3b1, Cast, Cntnap2, Fgf7, Gfra2, Hdac4 2.2 2.72 3 10249 miR-181c-5p Cd4, Il1a, Il7, Lif, Phf20l1, Prkcd, Tnf 2.2 3.36 3 10248 miR-9-5p Ap3b1, Cmtm6, Cxcl11, Hdac5, Inhbb, 2.19 3.94 3 10249 Pdgfc miR-30e-5p Cbfb, Chst1, Chst2, Hdac9, Hipk2 2.1 9.03 3 10240 miR-200c-3p Gpr68, Hmgb3, Il13, Ntf3, Prkca, Ripk2, 2.06 3.36 3 10248 Vegfa Signiﬁcantly expressed genes (p , 0.05, fold changes $2.0) are listed.

LCN2 has antiapoptotic effects in the RPE apoptosis. Increased numbers of apoptotic cells were detected Retinal inﬂammation has detrimental effects on cell viability (27, in LPS-treated cells in the absence of LCN2. When hiPS-RPE 42–44). To examine whether LCN2 could protect RPE cells from cells were pretreated with 10 or 100 ng/ml LCN2 for 24 h inﬂammation-associated cell death, hiPS-RPE cells were incu- before incubation with 1 mg/ml LPS, a reduction in the number bated with 1 mg/ml LPS for 24 h in the absence and presence of of apoptotic cells was observed as compared with the LPS LCN2. Caspase-3/7 expression was measured to assess cell only–treated group (Fig. 9). Anti-immune reactions of LCN2 The Journal of Immunology 3137

the possible involvement of a receptor-mediated effect, expression of LCN2 receptor in RPE cells was examined. Expression of SLC22A17 was detected in human RPE cells, including hiPS- RPE, ARPE19 (a human RPE derived cell line), and primary human RPE cells (Fig. 10A). When Abca42/2Rdh82/2 mice were exposed to light exposure at 10,000 lx for 30 min, qRT- PCR revealed that 24p3r expression increased in the RPE cells after light exposure (Fig. 10B), suggesting the possible involvement of RPE–LCN2 interaction during retinal degeneration pathogenesis. Patients with Stargardt disease, RP, and AMD have higher LCN2 plasma levels To investigate roles of LCN2 in human retinal degenerative diseases, LCN2 plasma levels in patients with Stargardt disease, RP, and AMD were measured and compared with age-matched control individuals without any retinal diseases (Supplemental Fig. 3). Determined LCN2 plasma concentrations were as follows: 45.13

6 5.37 ng/ml in Stargardt disease (n = 11), 44.78 6 31.73 ng/ml Downloaded from in RP (n = 117), 48.41 6 28.52 ng/ml in AMD (n = 57), and 17.04 6 11.9 ng/ml in controls (n = 77). Patients with these retinal degenerative diseases exhibited higher plasma levels of LCN2 as compared with control individuals, suggesting roles of LCN2 in human diseases.

Overexpression of LCN2 prevented LPS-induced cell death in http://www.jimmunol.org/ RPE cells Lastly, protective effects of LCN2 from RPE cell death were examined. ARPE19 cells were transfected with plasmids for LCN2 expression, and then these cells were cultured with 1 mg/ml LPS for 24 h to induce inﬂammation-associated cell death. LCN2- transfected cells demonstrated better cell viability as compared with control vector–transfected cells (Fig. 11A). Transfection of

LCN2 successfully increased LCN2 levels in ARPE19 cells by guest on September 25, 2021 (Fig. 11B).

Discussion Millions of people around the world suffer from retinal degenerative diseases such as inherited retinal dystrophies and AMD, with major debilitating impact on daily life. Apoptosis and inflammation play important roles in the pathogenesis of these diseases. Previous studies have demonstrated that retinal inflammation contributes to retinal degeneration (19) and that in- FIGURE 7. LCN2 attenuated the LPS-stimulated NF-kB activation in A m creased expression of an acute stress protein LCN2 is observed RPE cells. ( ) hiPS-RPE cells were incubated with 1 g/ml LPS and 1 ng/ml 2/2 2/2 recombinant LCN2 for 24 h. Fluorescence images show anti–NF-kBp65 in Abca4 Rdh8 mice after light exposure (3). In the pre- staining (in red) and nucleus stained by DAPI (in blue). Scale bars, 20 mm. sent study, Lcn2 expression was increased in the brain as well as 2/2 2/2 (B) Quantitative analyses of the ratio of p65–red nuclear fluorescence versus in the eye 1 d after light exposure in Abca4 Rdh8 mice cytoplasmic fluorescence are presented. Data are presented as mean 6 SD with LCN2 production observed in RPE and retinal microglial determined for each experiment independently. *p , 0.05 versus no LCN2 cells. We also compared Lcn22/2Abca42/2Rdh82/2 mice with treatment (n =15).(C) hiPS-RPE cells were incubated with 1 mg/ml LPS and Abca42/2Rdh82/2 mice after exposure to light and found that 1 ng/ml recombinant LCN2 for 24 h, and then levels of phosphorylated deficiency of Lcn2 resulted in more severe retinal damage and k k NF- B p65 and total NF- B p65 were quantified using ELISA. Data are increased expression of inflammatory cytokines, including Ccl8, presented as mean 6 SD. *p , 0.05 versus no LCN2 treatment (n =6). Ccl2,andCxcl10. Lcn2 loss also resulted in increased expression of one of the receptors of CCL3, namely Ccr5. Our previous were observed in human RPE-derived cells when these cells work has shown that CCL2 and CCL3 have distinct roles in the 2/2 2/2 were cultured with LCN2 (Supplemental Fig. 2). These results pathogenesis of retinal degeneration in Abca4 Rdh8 mice suggest that LCN2 provides protection from inflammation- (27). In other published studies, mice lacking Lcn2 exhibited associated cell death. impaired migration of astrocytes to injury sites with decreased Cxcl10 expression (48). This observation suggests that LCN2 Expression of LCN2 receptor in RPE cells protein, secreted under inflammatory conditions, could amplify LCN2 is known to modulate cell homeostasis by its interaction with neuroinflammation by inducing neural immune cells to secrete specific cell-surface receptors, namely murine 24p3r (45–47) and chemokines such as CXCL10 for recruiting additional inflam- the solute carrier family 22 member 17 (SLC22A17), which be- matory cells. Unexpectedly, we observed in the present study 2 2 longs to the major cation transporter family in humans. To explore more activated microglial cells in light-exposed Lcn2 / 3138 ROLES OF LCN2 IN RETINAL DEGENERATION

FIGURE 8. LCN2 protected against oxidative stress by increasing the expression of antioxidant enzymes HMOX1 and SOD2 in hiPS-RPE cells. (A) hiPS-RPE cells from healthy volunteers were incubated with 1 mg/ml LPS and 1 ng/ml recombinant LCN2 for 24 h. Cell viability was assessed using a WST-1 assay. (B and C) RNA was extracted from the cells after 24 h and expression levels of HMOX1 and SOD2 were measured. Error bars indicate SD of the means (n = 15). *p , 0.05. Downloaded from http://www.jimmunol.org/

Abca42/2Rdh82/2 mice as compared with Abca42/2Rdh82/2 after light exposure in Abca42/2Rdh82/2 mice, implicating mice, with Lcn22/2Abca42/2Rdh82/2 mice exhibiting in- LCN2 receptor and agonist interactions in RPE cells. Although by guest on September 25, 2021 creased Cxcl10 expression. Activation of microglial cells in LCN2 signaling pathways are not fully elucidated, it is note- response to chemokines, including CCL2 produced from the worthy that the LCN2 promoter region contains the binding sites RPE, is a hallmark of inflammation in retinal degeneration of several transcription factors such as STAT1, STAT3, CREB, (27). Activated microglial cells in the subretinal space display and C/EBPb and NF-kB (28). increased production of proinflammatory and chemotactic cytokines (27). The present study demonstrated protective roles of LCN2 in retinal degeneration as other reported studies; however, there are conflicting observations (49–56), and both pro- and anti- inflammatory properties of this glycoprotein have been reported. LCN2 expression can be induced by several cytokines and growth factors, including IL-6, IL-1b, IL-10, IL-17, TNF, and TGF (57–62). Such LCN2 induction is dependent on the activation of NF-kB transcriptional activity, which is suggested as a positive regulator of LCN2 expression itself (28). Another report suggested that LCN2 plays a role as an anti-inflammatory regulator of macrophage polarization and NF-kB/STAT3 pathway activation (52). This study demonstrated that LPS stimulation elicited an increase in the activation of NF-kB, c-Jun, and STAT3 signaling pathways in Lcn22/2 bone marrow–derived macrophages. Pretreatment with recombinant LCN2 attenuated LPS- stimulated degradation of Ik-Ba and STAT3 phosphorylation as well as LPS-induced gene expression of IL-6 and inducible NO synthase in Lcn22/2 bone marrow–derived macrophages. In this study, we investigated the regulation of LCN2 in RPE cells. FIGURE 9. LCN2 displayed antiapoptotic effects in the RPE. hiPS-RPE Pretreatment of hiPS-RPE cells with LCN2 before exposing to cells from healthy volunteers were incubated with 1 mg/ml LPS, and the LPS resulted in a significant decrease in nuclear translocation of indicated concentration of recombinant LCN2 for 24 h is shown. Active NF-kB p65, suggesting a negative feedback loop involving caspase-3/7 was observed using fluorescence microscopy. Quantitative LCN2. We also found that LCN2 murine receptor 24p3r ex- analyses of the percentage of apoptotic cells were performed from three pression in RPE cells increased in a similar fashion as LCN2 independent experiments. *p , 0.05. The Journal of Immunology 3139

Our present results show that expression of LCN2 in hiPS-RPE cells suppresses H2O2-induced cell death and prolongs cell survival. We found that the transcript levels of the key oxidative stress–catalyzing enzymes HMOX1 and SOD2 increased with increasing LCN2 concentrations, supporting an antioxidant role for LCN2 in RPE cells. HMOX1 not only regulates the cellular content of the pro-oxidant heme, but it also produces catabolites with regulatory and protective functions (66). SOD mRNA levels increase following a wide range of mechanical, chemical, and biological stimuli that increase ROS, such as UVB and x- irradiation, ozone, and LPS (67). Increased gene expression of SODs and heme oxygenases are adaptive cellular defense mech- anisms against oxidative stress. In our animal study, loss of Lcn2 in mice resulted in more severe retinal degeneration after light exposure. In the retina, LCN2 may serve to protect from a broad array of ROS produced by light exposure (68–70), including ROS generated in the visual cycle from the conversion of 11-cis-retinal to all-trans-retinal (19, 68).

LCN2 concentration in biological ﬂuids under healthy Downloaded from conditions is low, but the protein can be upregulated by in- ﬂammation and becomes detectable at various stages in several diseases (71–74). We found LCN2 plasma levels elevated .2-fold in patients with Stargardt disease, RP, and AMD compared with healthy controls. In this study, LCN2

expression was observed in the RPE and in the inner retina. http://www.jimmunol.org/ An earlier report demonstrated that RPE is a main source of secreted LCN2 in the eye (14). Because breakdown of the blood retinal barrier can be observed during the process of retinal degeneration (19), such comprised blood retinal barrier might contribute to leakage of LCN2 produced in the eyeandtoincreasedlevelsofLCN2inplasmaofpatients. Activated immune cells, which produce LCN2, could mi- FIGURE 10. Expression of LCN2 receptor in RPE cells. (A)RNAwas grate into the blood vessels and produce LCN2 into the by guest on September 25, 2021 extracted from hiPS-RPE cells, ARPE19 (a human RPE derived cell line) blood. This result not only implies potentially important cells, and human primary RPE cells. Expression of SLC22A17 was examined. roles for circulating LCN2 in the pathogenesis of retinal Error bars indicate SD of the means (n =6).(B) Abca42/2Rdh82/2 mice degenerative diseases, but also the possibility that LCN2 (1 mo old) were exposed to light at 10,000 lx for 30 min. RNA was extracted could serve as a biomarker of early disease onset and pro- from RPE cells at different times points after light exposure. Expression levels gression (75). of 24p3r are presented as fold induction over non–light exposed control. Error In conclusion, this study provides evidence that LCN2 may , bars indicate SD of the means (n =3).*p 0.05. serve to protect the retina from inﬂammation-induced degeneration through regulation of cytokine and chemokine The antioxidant function of LCN2 has been well characterized production, and by preserving cell viability and attenuating (33, 35, 63) and the protein has been shown to be cytoprotective apoptosis through regulation of anti-oxidant enzymes. LCN2, against oxidative stress (37, 64, 65). LCN2 protects against cel- secreted mainly from RPE cells in the retina, could be a lular stress from exposure to H2O2, and that overexpression of critical mediator in retinal inﬂammation and degeneration LCN2 allows cells to better tolerate oxidative stress conditions (6). processes.

FIGURE 11. Overexpression of LCN2 in ARPE19 cells preserved cell viability against LPS- induced cell death. (A) ARPE19 cells were transfected with LCN2 in pcDNA3 vector. These cells were cultured with 1 mg/ml LPS for 24 h, and cell viability was examined using a WST-1 assay. Error bars indicate SD of the means (n = 3). *p , 0.05. (B) LCN2 expression in transfected ARPE19 cells was quantiﬁed by qRT-PCR. Error bars indicate SD of the means (n = 3). *p , 0.05. 3140 ROLES OF LCN2 IN RETINAL DEGENERATION

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