Abstract
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)−/− retinol dehydrogenase 8 (Rdh8)−/− 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, Lcn2−/−Abca4−/−Rdh8−/− triple-knockout mice were generated. Exacerbated inflammation following light exposure was observed in Lcn2−/−Abca4−/−Rdh8−/− mice as compared with Abca4−/−Rdh8−/− mice, with upregulation of proinflammatory genes and microglial activation. RNA array analyses revealed an increase in immune response molecules such as 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-κB 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 suggest that LCN2 is involved in prosurvival responses during cell stress and plays an important role in regulating inflammation during retinal degeneration.
Introduction
Several blinding diseases of the retina are characterized by the death of retinal pigmented epithelium (RPE) and photoreceptor cells. In retinitis pigmentosa (RP), an inherited retinal disease characterized by a progressive loss of photoreceptor cells, an activation of prosurvival signaling cascades, involving upregulation of several growth factors, cytokines, and antioxidants, has been observed (1). In age-related macular degeneration (AMD), a major cause of visual impairment in elderly people, several immune responses are activated in the form of inflammatory cytokines, chemokines, Abs, and T cells in both animal models and patients (2). Accumulating evidence suggests that activation of immune responses plays an important role in progression of these blinding diseases. We previously reported an increase in acute phase protein lipocalin 2 (LCN2) coinciding with retinal degeneration in ATP-binding cassette subfamily A member 4 (Abca4)−/− retinol dehydrogenase 8 (Rdh8)−/− mice (3). LCN2 is also known as 24p3 or neutrophil gelatinase-associated lipocalin and is a member of the lipocalin superfamily known for its role in cellular transport of lipophilic molecules as fatty acids, iron, retinoids, and steroids. LCN2 is a multifunctional innate immunity protein and can augment cellular tolerance to oxidative stress (4) and, indeed, roles of LCN2 have been suggested under stress conditions and degenerative diseases. In the CNS, LCN2 deficiency has been associated with tissue inflammation (5). Lcn2-deficient mice were found to be highly sensitive to bacterial sepsis (6). Several studies have demonstrated that LCN2 protects against cellular stress, inflammation, and cell death (7–10).
Although a few studies have implicated the involvement of LCN2 in eye disease, the possible roles of LCN2 in retinal degeneration have not been fully elucidated. LCN2 was found upregulated among other acute phase response and inflammatory proteins in the retina of rodent models for diabetes and retinal ischemia/reperfusion injury (11). Increased levels of LCN2 have been reported in a mouse model of Bardet–Biedl syndrome (12). A pivotal role of LCN2 in the development of demyelinating optic neuritis in a mouse model of autoimmune optic neuritis has been demonstrated (13). Valapala et al. (14) identified LCN2 as a contributory factor in inducing chronic inflammatory response in Cryba1 conditional knockout (KO) mice, a mouse model with AMD-like pathology. Lastly, Sinha and colleagues (15) generated genetically engineered mice in which lysosome-mediated clearance in RPE cells is compromised, causing the development of features of early AMD. They further proposed the involvement of an AKT2–NF-κB–LCN2 signaling axis in activating the inflammatory responses in these mice, suggesting this pathway as a potential target for AMD treatment. In humans, Ghosh et al. (15) observed an increased infiltration of LCN2+ neutrophils in the choroid and retina of early AMD patients as compared with age-matched controls. These studies, including our observation of increased expression of LCN2 in mouse retinal degeneration (3), reinforce the possible significance of LCN2 in retinal inflammation and retinal degenerative diseases.
In the present study, Lcn2−/−Abca4−/−Rdh8−/− triple-KO mice were generated and RPE cells differentiated from human-induced pluripotent stem cells (hiPS-RPE) were employed to investigate the role of LCN2 in retinal inflammation and degeneration. Our results provide evidence that LCN2 could regulate prosurvival responses and retinal inflammation in mice and humans.
Materials and Methods
Animals
Lcn2−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in the animal facility at the School of Medicine, Case Western Reserve University, where they were maintained either under complete darkness or in a 12-h light (∼10 lx)/12-h dark cycle environment in a pathogen-free environment. Both male and female mice at 1 mo of age were used in the study. Lcn2−/− mice were crossed with Abca4−/−Rdh8−/− mice to generate Lcn2−/−Abca4−/−Rdh8−/− mice. 129SV or littermates of mutant mice were used as controls. Only the mice with RPE65 leucine variant and free of rd8 mutation were employed in the study. Genotyping for Lcn2−/− or wild-type (WT) mice was performed using the primers with LCN2-KO and common for KO allele and LCN2-WT and common for WT allele: LCN2-KO, 5′-CCTTCTAT GCCTTCTTGACG-3′; LCN2-common, 5′-TAGGGGATGCCACATCTCA-3′; LCN2-WT, 5′-TGGAGGTGACATTGTAGCTATTG-3′. Genotyping for Abca4−/−Rdh8−/− mice was performed as described previously (16). All animal procedures and experiments were approved by the Case Western Reserve University Animal Care Committees and conformed to recommendations of both the American Veterinary Medical Association Panel on Euthanasia and the Association of Research for Vision and Ophthalmology.
Light exposure
Mice were dark adapted for 48 h before being exposed to light. Light-induced degeneration was induced by exposing mice to 10,000 lx of diffuse white fluorescent light (150 W spiral lamp; Commercial Electric, Cleveland, OH) for 30 min. Before such light exposure, pupils of mice were 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.
Histological analysis
All procedures to make sections for retinal histology and immunohistochemistry followed established methods (17, 18). For H&E staining eye cups were fixed in 10% formalin for 72 h followed by paraffin embedding. Five-micrometer-thick sections were cut and stained with H&E for analysis by light microscopy. For immunohistochemistry, eye cups were embedded in 4% paraformaldehyde/1% glutaraldehyde overnight followed by cryosectioning. The following Abs were used for immunohistochemistry; rabbit anti–Iba-1 Ab (1:400; Wako, Osaka, Japan), rabbit anti–glial fibrillary acidic protein (GFAP) Ab (1:400; Dako, Carpinteria, CA), rabbit NF-κB p65 Ab (1:1000; eBioscience, San Diego, CA). Secondary Abs used were anti-mouse, anti-rabbit Alexa Fluor 555 (Invitrogen, Carlsbad, CA). Images were captured by a confocal microscope (LSM; Carl Zeiss, Thornwood, NY).
Scanning laser ophthalmoscopy and spectral domain–optic coherence tomography imaging
HRAII (Heidelberg Engineering, Heidelberg, Germany) for scanning laser ophthalmoscopy (SLO) and ultra-high resolution spectral domain–optic coherence tomography (SD-OCT; Bioptigen SD-OCT Envisu C2200; Bioptigen, Research Triangle Park, NC) were employed for in vivo imaging of mouse retinas. Mice were anesthetized by i.p. injection of a mixture (20 μl/g body weight) containing ketamine (6 mg/ml) and xylazine (0.44 mg/ml) in 10 mM sodium phosphate (pH 7.2) with 100 mM NaCl. Pupils were dilated with a mixture of 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Midorin-P; Santen Pharmaceutical). Numbers of autofluorescent particles in SLO images were counted per image.
Quantitative RT-PCR
Total RNA was extracted with the RNeasy mini kit (Qiagen, Germantown, MD). Primers used in the study are listed in Supplemental Table I. All procedures for quantitative RT-PCR (qRT-PCR) were carried out as described previously (19).
Immunoblot
Enucleated eyes were harvested from the animals and lysed in ice-cold lysis buffer (150 mM NaCl, 1 mM EDTA, 0.2% Nonidet P-40, and 20 mM Tris-HCl [pH 7.5]) containing protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO). Tissue lysate was spun at 10,000 rpm for 10 min at 4°C. Proteins from each sample were transferred onto Immobilon-P membranes (Millipore, Bedford, MA) after SDS-PAGE gel electrophoresis. Membranes were incubated in 1% BSA solution containing a 1:1000 dilution of either anti-LCN2 rabbit polyclonal Ab (sc-50350; Santa Cruz Biotechnology, Santa Cruz, CA) or anti–β-actin Ab (Santa Cruz Biotechnology). Signals were visualized with alkaline phosphatase (Promega, Madison, WI) at a dilution of 1:10,000. The intensities of the bands were normalized to the actin band using ImageJ software (National Institutes of Health, Bethesda, MD).
Isolation of primary RPE and microglia cells
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 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 (ILLUMIN-i; Endure Medical, Cumming, GA). The RPE layer was peeled from eye cups and cultured in DMEM containing minimal essential medium nonessential amino acids (Invitrogen), penicillin/streptomycin (Invitrogen), 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 Abca4−/−Rdh8−/− 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.
Apoptosis assay
hiPS-RPE cells (30,000–50,000) were seeded in 96-well plates. Cells were treated with 100 μM H2O2
Cell viability assay
Assays were performed on 96-well plates, with 1 × 104
ELISA
RNA array analysis
Detection and quantification of gene expression was performed using a mouse inflammatory cytokine and receptors array (PAMM-011ZF; SABiosciences/Qiagen, Frederick, MD) according to the manufacturer’s instructions. This PCR-based array was selected, as it includes 84 diverse genes important in the immune response, including genes encoding CC chemokines, CXC chemokines, IL cytokines, other cytokines, chemokine receptors, and cytokine receptors, as well as other genes involved in the inflammatory response. MicroRNA (miRNA) array was also performed using an inflammatory response miRNA PCR array (MIMM-105Z; SABiosciences/Qiagen) according to the manufacturer’s instructions. Real-time PCR amplification was performed using RT2 SYBR Green PCR master mix (Qiagen). Primers were designed using Web tool Primer3 and synthesized by Eurofins MWG Operon (Huntsville, AL). Data analysis was performed using the ΔΔCT method according to the manufacturer’s protocol (SABiosciences).
Immunocytochemistry for NF-κB localization
hiPS-RPE cells (30,000–50,000) were seeded in 96-well plates. Cells were coincubated with 1 μg/ml LPS and 1 ng/ml LCN2 for 24 h. On the next day, the culture medium was removed and cells were fixed by adding 100 μl of 4% formalin to the wells for 15 min, followed by washing in PBS twice for 5 min. Fixed cells were permeabilized with 100 μl of 0.2% Triton X-100 in PBS for 1 min at room temperature. The cells were then incubated with rabbit anti-mouse p65 (1:100) (eBioscience) in PBS containing 10% goat serum overnight at 4°C. Cells were then washed twice with PBS and incubated with Alexa Fluor 488–labeled goat anti-rabbit IgG Ab (1:200) (Molecular Probes) in PBS at room temperature for 2 h. The cells were washed twice for 5 min with PBS. DAPI (1:500; Vector Laboratories, Burlingame, CA) was added to the cells to visualize the nuclei. Cells were then washed twice with PBS and imaged under fluorescent microscope. Five high-power fields (×100 magnification) were randomly selected in each sample for analysis. Positive nuclear staining in LPS-treated cells was used as positive control for NF-κB staining. The percentage of nuclear staining for NF-κB p65 was scored by counting the positive-stained cells and the total number of cells quantified in random microscopic fields using the Metamorph image analysis software (Molecular Devices, San Jose, CA).
Measurement of NF-κB activity by ELISA
Human plasma analysis
All participants in the study underwent a complete ophthalmic examination and visual function tests by ophthalmologists. Informed consent was obtained from each person for the present study. Procedures followed the Declaration of Helsinki guidelines and were approved by the Institutional Review Boards of Case Western Reserve University, RIKEN, Institute of Biomedical Research and Innovation Hospital, and Cleveland Clinic. Nonfasting blood samples were collected at the Institute of Biomedical Research and Innovation Hospital or at the Cole Eye Institute from patients diagnosed with Stargardt disease (n = 11), RP (n = 117), and the wet form of AMD (n = 57). Blood was also collected from 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 underwent cataract surgery. Plasma was prepared as previously described and stored under argon at −80°C until analysis (26).
Overexpression of LCN2 in ARPE19 cells
LCN2 mRNA was then examined by RT-PCR 72 h after transfection using primers shown in Supplemental Table I.
Statistical analysis
Statistical analyses were performed using the t test for comparing two groups, and one-way ANOVA was used to detect differences among three or more groups. Mann–Whitney U statistics were employed to analyze human plasma samples. Results were presented as mean ± SD. The results were considered statistically significant at p < 0.05.
Results
Increased expression of Lcn2 in the retina and brain after light exposure
To examine expressional changes of acute stress protein LCN2 in various tissues under stress conditions that can induce retinal degeneration, 1-mo-old Abca4−/−Rdh8−/− mice were exposed to light at 10,000 lx for 30 min and qRT-PCR was performed with isolated tissues, including the eye, liver, heart, brain, spleen, lung, and kidney. A 2-fold or higher increase of Lcn2 was observed in the eye (15.38 ± 3.86) and brain (4.97 ± 0.55) when compared with dark-adapted controls (Fig. 1A). Lcn2 expression was not obvious in these tissues prior to light exposure except spleen, where low levels of Lcn2 expression were detected. To determine the kinetics of LCN2 levels in the eye after light exposure, LCN2 expression was examined by immunoblot 1, 3, and 7 d after illumination at 10,000 lx for 30 min. LCN2 protein in the eye was found most upregulated 1 d after light exposure (Fig. 1B, 1C). Greatest LCN2 increase in serum was observed 3 d after light exposure in mice (Fig. 1D). Immunohistochemistry indicated that secreted LCN2 protein was detected in RPE and microglial cells in the inner nuclear layer of the retina (Fig. 1E). Because incubation with POS can induce production of inflammatory cytokines and chemokines from RPE and microglial cells (19), RPE and microglia cells were isolated from 2-wk-old Abca4−/−Rdh8−/− mice. When these cells were incubated with POS for 24 h, a 14-fold or 5-fold increase in Lcn2 was observed in RPE or microglial cells, respectively (Fig. 2).
Increased expression of Lcn2 in the retina and brain after light exposure. Abca4−/−Rdh8−/− 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 Abca4−/−Rdh8−/− mice 1, 3, and 7 d after light exposure. (C) Quantitative analyses of the immunoblot after normalization to β-actin are presented. (D) Serum was collected from 1-mo-old Abca4−/−Rdh8−/− mice at different time points after light exposure. (E) Immunohistochemistry of LCN2 protein in the retinal sections of 1-mo-old Abca4−/−Rdh8−/− mice 24 h after light exposure. Scale bars, 50 μm. Error bars indicate SD of the means (n = 3). *p < 0.05. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.
Lcn2 increases in the RPE more than in the microglia. RPE cells and microglia were isolated from 1-mo-old Abca4−/−Rdh8−/− mice. Cells were incubated with photoreceptor outer segments (POS, 6 mg/ml) for 24 h. RNA levels of Lcn2 were measured in both RPE cells and microglia with and without POS. Error bars indicate SD of the means. *p < 0.05 versus no POS treatment (n = 6).
Deficiency of Lcn2 results in severe light-induced retinal degeneration in Abca4−/−Rdh8−/− mice
To examine effects of Lcn2 deficiency in retinal degeneration, 1-mo-old Lcn2−/−Abca4−/−Rdh8−/− and Abca4−/−Rdh8−/− mice were exposed to light at 10,000 lx for 30 min. WT littermates were subjected to the same light exposure. Histology sections of the retina demonstrated a decrease in outer nuclear layer thickness in Lcn2−/−Abca4−/−Rdh8−/− mice as compared with Abca4−/−Rdh8−/− mice 7 d after light exposure (Fig. 3A, upper panels, 3B). In vivo imaging of the retina by SLO was performed 7 d after light exposure (Fig. 3A, lower panels). Increased number of autofluorescent spots, which are activated microglial cells and macrophages (19), were counted in Lcn2−/−Abca4−/−Rdh8−/− mice as compared with Abca4−/−Rdh8−/− mice (Fig. 3C). Lcn2−/− mice did not develop light-induced retinal degeneration (see “Deficiency of Lcn2 is associated with inflammatory changes” below).
Deficiency of Lcn2 resulted in severe light-induced retinal degeneration in Abca4−/−Rdh8−/− mice. (A) Representative retinal histology (upper panel) and SLO (lower panel) of 1-mo-old Lcn2−/−Abca4−/−Rdh8−/− and Abca4−/−Rdh8−/− mice 7 d after light exposure at 10,000 lx for 30 min are shown. Scale bars, 20 μm. (B) Outer nuclear layer (ONL) thickness measurements of 1-mo-old Lcn2−/−Abca4−/−Rdh8−/− and Abca4−/−Rdh8−/− mice 7 d after light exposure is presented. (C) Numbers of autofluorescent (AF) spots detected by SLO in 1-mo-old Lcn2−/−Abca4−/−Rdh8−/− and Abca4−/−Rdh8−/− mice 7 d after light exposure were counted. Error bars indicate SD of the means (n = 3). *p < 0.05.
Lcn2−/−Abca4−/−Rdh8−/− mice show stronger gliosis than do Abca4−/−Rdh8−/− mice after light exposure
To investigate effects of Lcn2 deficiency to retinal glial cells after light exposure, we examined the expression of Iba-1 for microglial cells and GFAP for Müller cells. Activation of glial cells is associated with retinal inflammation (27). Lcn2−/−Abca4−/−Rdh8−/− and Abca4−/−Rdh8−/− mice at 1 mo of age were exposed to light at 10,000 lx for 30 min, and eyes were collected 24 h thereafter. Immunohistochemistry with anti–Iba-1 Ab showed increased protein expression and more Iba-1+ microglial cell numbers in Lcn2−/−Abca4−/−Rdh8−/− mice as compared with Abca4−/−Rdh8−/− mice (Fig. 4A, upper panels). Immunohistochemical staining with anti-GFAP Ab revealed stronger gliosis with an increased expression of GFAP in Müller cells of Lcn2−/−Abca4−/−Rdh8−/− mice (Fig. 4A, lower panels). qRT-PCR of Gfap displayed a similar pattern of increase as observed in immunohistochemistry (Fig. 4B). These results indicate that Lcn2 deficiency contributes to stronger response of retinal glial cells that are associated with retinal inflammation.
Lcn2−/−Abca4−/−Rdh8−/− mice showed stronger gliosis than did Abca4−/−Rdh8−/− mice after light exposure. Lcn2−/−Abca4−/−Rdh8−/− and Abca4−/−Rdh8−/− mice at 1 mo of age were exposed to light at 10,000 lx for 30 min. (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 Müller 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 μm. (B) RNA samples were collected from 1-mo-old Lcn2−/−Abca4−/−Rdh8−/−, Abca4−/−Rdh8−/−, 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.
Increased expression of inflammatory response in Lcn2−/−Abca4−/−Rdh8−/− mice
To further dissect LCN2-mediated effects on inflammatory genes and immune pathways, we employed the RT2 real-time PCR array kit for 84 key immune genes involved in mediating inflammation. Lcn2−/−Abca4−/−Rdh8−/− and Abca4−/−Rdh8−/− mice (1 mo old) were exposed to light at 10,000 lx for 30 min. Dark-adapted mice were used for controls. The PCR array was performed 1 d (24 h) after light exposure. Total RNA was isolated from all groups of mice. Equal amounts were converted to cDNA and then subjected to pathway-focused gene expression profiling using real-time PCR. Scatter plots comparing the differential expression of genes in light-exposed Lcn2−/−Abca4−/−Rdh8−/− and Abca4−/−Rdh8−/− mice were generated (Fig. 5A). Each symbol represents an individual gene. The boundary lines indicate a 2-fold difference. Genes outside the boundary lines have ≥2-fold altered expression in Lcn2−/−Abca4−/−Rdh8−/− mice compared with Abca4−/−Rdh8−/− mice. Table I lists the fold changes of the genes compared in the two groups of mice. Lcn2−/−Abca4−/−Rdh8−/− mice displayed upregulation of 22 inflammatory mediators, including chemokines, chemokine receptors, and ILs, as compared with Abca4−/−Rdh8−/− mice. Conversely, Cx3cl1, Vegfa, and Cxcl15 were downregulated in Lcn2−/−Abca4−/−Rdh8−/− mice. Validation by qRT-PCR for Ccl8, Ccl2, Cxcl10, Ccr5, Tnf, and Il2a was carried out using a separate set of mice (Fig. 5B). Production of CCL2 and TNF was quantified with eyes of 1-mo-old Lcn2−/−Abca4−/−Rdh8−/− and Abca4−/−Rdh8−/− mice 24 h after light exposure at 10,000 lx for 30 min. Increased levels of CCL2 and TNF in Lcn2−/−Abca4−/−Rdh8−/− mice (CCL2, 34.75 ± 1.5 pg/ml; TNF, 6.02 ± 0.04 pg/ml) compared with Abca4−/−Rdh8−/− mice (CCL2, 5.75 ± 0.7 pg/ml; TNF, 1.26 ± 0.02 pg/ml) were measured (Fig. 5C, 5D).
Increased expression of inflammatory response in Lcn2−/−Abca4−/−Rdh8−/− mice. (A) Scatter plot comparing the expression of genes involved in the inflammatory response in Lcn2−/−Abca4−/−Rdh8−/− and Abca4−/−Rdh8−/− mice 24 h after light exposure at 10,000 lx for 30 min is presented. Each circle represents 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 Lcn2−/−Abca4−/−Rdh8−/− and Abca4−/−Rdh8−/− mice 24 h after light exposure. Expression levels of Ccl8, Ccl2, Cxcl10, Ccr5, Tnf, and Il2α are normalized by Gapdh expression and shown by fold change. (C and D) CCL2 and TNF protein levels were measured in 1-mo-old Lcn2−/−Abca4−/−Rdh8−/− and Abca4−/−Rdh8−/− mice 24 h after light exposure. Error bars indicate SD of the means (n = 6). *p < 0.05.
Deficiency of Lcn2 is associated with inflammatory changes caused by light exposure
Because Lcn2−/−Abca4−/−Rdh8−/− mice displayed more severe light-induced retinal degeneration and inflammation as compared with Abca4−/−Rdh8−/− mice (Figs. 3, 4, 5), roles of LCN2 in retinal inflammation after light exposure were examined using Lcn2−/− mice. When 6-wk-old Lcn2−/− mice were exposed to light at 10,000 lx for 30 min, increased levels of inflammatory changes were observed 24 h after illumination as compared with WT mice, including increased expression of Iba-1/Aif-1 and Gfap (Fig. 6A). Although increased immune reactions were observed in Lcn2−/− mice, this light condition did not cause obvious retinal degenerative changes in Lcn2−/− and WT mice; in contrast, Abca4−/−Rdh8−/− mice showed retinal structural changes 7 d after illumination (Fig. 6B). No retinal degeneration was observed in 6-wk-old and 4-mo-old Lcn2−/− mice when they were kept under regular lighting conditions. Because inflammatory changes in light-exposed Lcn2−/− mice suggest regulatory roles of LCN2 in inflammation, expression of miRNA, which can modulate transcriptional expression of inflammatory molecules, was examined by miRNA array. Changes in miRNA expression obtained from Lcn2−/− and WT mice are presented in Table II. These results indicate that loss of Lcn2 is prone to accelerating inflammation.
Lcn2−/− mice displayed more preserved retinas with milder inflammation. (A) Retinas were isolated from 1-mo-old Lcn2−/− 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) 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 μm. ONL, outer nuclear layer.
LCN2 attenuates LPS-stimulated NF-κB activation in RPE cells
The LCN2 gene promoter region contains binding sites for several transcription factors, including NF-κB (28), which prompted us to examine the effects of LCN2 on NF-κB signaling in RPE cells. Notably, the NF-κB pathway is a prototypical proinflammatory signaling pathway involved in the expression of multiple proinflammatory genes such as cytokines, chemokines, and adhesion molecules (29–32). hiPS-RPE cells were incubated with 1 μg/ml LPS for 24 h to investigate whether LCN2 can inhibit the nuclear translocation of NF-κB p65. Immunocytochemistry with anti-p65 Ab revealed that LPS-stimulated hiPS-RPE cells showed p65 staining in the nuclei, whereas unstimulated cells showed stronger staining in the cytoplasm. Supplementation of LCN2 in the culture medium decreased the numbers of cells with LPS-induced nuclear translocation of p65 (Fig. 7A, 7B). Furthermore, phosphorylation of NF-κB p65, which indicates activation of NF-κB, was examined using ELISA. In contrast to the finding that levels of total NF-κB p65 remained unchanged, a decrease in phosphorylated NF-κB p65 was observed when LCN2 was supplemented to hiPS-RPE cells (Fig. 7C). These observations suggest that LCN2 contributes to inhibition of NF-κB activation in human RPE cells. hiPS-RPE cells can express LCN2 as well as murine RPE cells when these cells were incubated with POS (Supplemental Fig. 1).
LCN2 attenuated the LPS-stimulated NF-κB activation in RPE cells. (A) hiPS-RPE cells were incubated with 1 μg/ml LPS and 1 ng/ml recombinant LCN2 for 24 h. Fluorescence images show anti–NF-κB p65 staining (in red) and nucleus stained by DAPI (in blue). Scale bars, 20 μm. (B) Quantitative analyses of the ratio of p65–red nuclear fluorescence versus cytoplasmic fluorescence are presented. Data are presented as mean ± SD determined for each experiment independently. *p < 0.05 versus no LCN2 treatment (n = 15). (C) hiPS-RPE cells were incubated with 1 μg/ml LPS and 1 ng/ml recombinant LCN2 for 24 h, and then levels of phosphorylated NF-κB p65 and total NF-κB p65 were quantified using ELISA. Data are presented as mean ± SD. *p < 0.05 versus no LCN2 treatment (n = 6).
LCN2 protects against oxidative stress by increasing the expression of antioxidant enzymes heme oxygenase 1 and superoxide dismutase 2 in hiPS-RPE cells
Several in vitro studies have demonstrated that LCN2 protects against cellular stress and exposure to H2O2 (33–37). Other studies have reported that oxidative stress plays a major role in degenerative retinal diseases such as RP and AMD (38–41). To investigate the effects of LCN2 on H2O2-induced oxidative stress in RPE cells, 1 ng/ml LCN2 was incubated with hiPS-RPE cells under oxidative stress conditions. Incubation with 100 μM H2O2 for 24 h in the absence of LCN2 resulted in reduced cell viability to 49.51 ± 11.59%, indicating that about half of the cell population died (Fig. 8A). When 1 ng/ml LCN2 was added to these incubation conditions, protective effects of LCN2 against H2O2-induced cell death were observed. LCN2 supplementation maintained cell viability nearly to the control level at 87.62 ± 12.74%. With increasing doses of recombinant LCN2, increased expression of antioxidant enzymes heme oxygenase 1 (HMOX1) and superoxide dismutase 2 (SOD2) in hiPS-RPE cells was observed (Fig. 8B, 8C), suggesting that an upregulation of HMOX1and SOD2 is an adaptive mechanism to protect cells from oxidative damage. HMOX1 and SOD2 are known to act as the first-line antioxidant enzyme defense system against reactive oxygen species (ROS) and particularly superoxide anion radicals (40).
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 μg/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.
LCN2 has antiapoptotic effects in the RPE
Retinal inflammation has detrimental effects on cell viability (27, 42–44). To examine whether LCN2 could protect RPE cells from inflammation-associated cell death, hiPS-RPE cells were incubated with 1 μg/ml LPS for 24 h in the absence and presence of LCN2. Caspase-3/7 expression was measured to assess cell apoptosis. Increased numbers of apoptotic cells were detected in LPS-treated cells in the absence of LCN2. When hiPS-RPE cells were pretreated with 10 or 100 ng/ml LCN2 for 24 h before incubation with 1 μg/ml LPS, a reduction in the number of apoptotic cells was observed as compared with the LPS only–treated group (Fig. 9). Anti-immune reactions of LCN2 were observed in human RPE-derived cells when these cells were cultured with LCN2 (Supplemental Fig. 2). These results suggest that LCN2 provides protection from inflammation-associated cell death.
LCN2 displayed antiapoptotic effects in the RPE. hiPS-RPE cells from healthy volunteers were incubated with 1 μg/ml LPS, and the indicated concentration of recombinant LCN2 for 24 h is shown. Active caspase-3/7 was observed using fluorescence microscopy. Quantitative analyses of the percentage of apoptotic cells were performed from three independent experiments. *p < 0.05.
Expression of LCN2 receptor in RPE cells
LCN2 is known to modulate cell homeostasis by its interaction with specific cell-surface receptors, namely murine 24p3r (45–47) and the solute carrier family 22 member 17 (SLC22A17), which belongs to the major cation transporter family in humans. To explore 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 Abca4−/−Rdh8−/− 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.
Expression of LCN2 receptor in RPE cells. (A) RNA was extracted from hiPS-RPE cells, ARPE19 (a human RPE derived cell line) cells, and human primary RPE cells. Expression of SLC22A17 was examined. Error bars indicate SD of the means (n = 6). (B) Abca4−/−Rdh8−/− mice (1 mo old) were exposed to light at 10,000 lx for 30 min. RNA was extracted from RPE cells at different times points after light exposure. Expression levels of 24p3r are presented as fold induction over non–light exposed control. Error bars indicate SD of the means (n = 3). *p < 0.05.
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 ± 5.37 ng/ml in Stargardt disease (n = 11), 44.78 ± 31.73 ng/ml in RP (n = 117), 48.41 ± 28.52 ng/ml in AMD (n = 57), and 17.04 ± 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 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 μg/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 (Fig. 11B).
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 μg/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.
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 increased expression of an acute stress protein LCN2 is observed in Abca4−/−Rdh8−/− mice after light exposure (3). In the present study, Lcn2 expression was increased in the brain as well as in the eye 1 d after light exposure in Abca4−/−Rdh8−/− mice with LCN2 production observed in RPE and retinal microglial cells. We also compared Lcn2−/−Abca4−/−Rdh8−/− mice with Abca4−/−Rdh8−/− mice after exposure to light and found that deficiency of Lcn2 resulted in more severe retinal damage and increased expression of inflammatory cytokines, including Ccl8, Ccl2, and Cxcl10. Lcn2 loss also resulted in increased expression of one of the receptors of CCL3, namely Ccr5. Our previous work has shown that CCL2 and CCL3 have distinct roles in the pathogenesis of retinal degeneration in Abca4−/−Rdh8−/− mice (27). In other published studies, mice lacking Lcn2 exhibited impaired migration of astrocytes to injury sites with decreased Cxcl10 expression (48). This observation suggests that LCN2 protein, secreted under inflammatory conditions, could amplify neuroinflammation by inducing neural immune cells to secrete chemokines such as CXCL10 for recruiting additional inflammatory cells. Unexpectedly, we observed in the present study more activated microglial cells in light-exposed Lcn2−/−Abca4−/−Rdh8−/− mice as compared with Abca4−/−Rdh8−/− mice, with Lcn2−/−Abca4−/−Rdh8−/− mice exhibiting increased Cxcl10 expression. Activation of microglial cells in response to chemokines, including CCL2 produced from the RPE, is a hallmark of inflammation in retinal degeneration (27). Activated microglial cells in the subretinal space display 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-1β, IL-10, IL-17, TNF, and TGF (57–62). Such LCN2 induction is dependent on the activation of NF-κB 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-κB/STAT3 pathway activation (52). This study demonstrated that LPS stimulation elicited an increase in the activation of NF-κB, c-Jun, and STAT3 signaling pathways in Lcn2−/− 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 Lcn2−/− bone marrow–derived macrophages. In this study, we investigated the regulation of LCN2 in RPE cells. Pretreatment of hiPS-RPE cells with LCN2 before exposing to LPS resulted in a significant decrease in nuclear translocation of NF-κB p65, suggesting a negative feedback loop involving LCN2. We also found that LCN2 murine receptor 24p3r expression in RPE cells increased in a similar fashion as LCN2 after light exposure in Abca4−/−Rdh8−/− mice, implicating LCN2 receptor and agonist interactions in RPE cells. Although LCN2 signaling pathways are not fully elucidated, it is noteworthy that the LCN2 promoter region contains the binding sites of several transcription factors such as STAT1, STAT3, CREB, and C/EBPβ and NF-κB (28).
The antioxidant function of LCN2 has been well characterized (33, 35, 63) and the protein has been shown to be cytoprotective against oxidative stress (37, 64, 65). LCN2 protects against cellular stress from exposure to H2O2, and that overexpression of LCN2 allows cells to better tolerate oxidative stress conditions (6). 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 mechanisms 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 conditions is low, but the protein can be upregulated by inflammation 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. 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 eye and to increased levels of LCN2 in plasma of patients. Activated immune cells, which produce LCN2, could migrate into the blood vessels and produce LCN2 into the blood. This result not only implies potentially important roles for circulating LCN2 in the pathogenesis of retinal degenerative diseases, but also the possibility that LCN2 could serve as a biomarker of early disease onset and progression (75).
In conclusion, this study provides evidence that LCN2 may serve to protect the retina from inflammation-induced degeneration through regulation of cytokine and chemokine production, and by preserving cell viability and attenuating apoptosis through regulation of anti-oxidant enzymes. LCN2, secreted mainly from RPE cells in the retina, could be a critical mediator in retinal inflammation and degeneration processes.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank Catherine Dollar and Scott Howell (Visual Science Research Center, Case Western Reserve University), Tatiana Reidel (Department of Ophthalmology and Visual Sciences, University Hospital of Cleveland), Dr. Yuki Arai, Dr. Akiko Yoshida, and Kanako Kawai (RIKEN) for technical assistance and comments.
Footnotes
This work was supported by funding from National Institutes of Health Grants EY022658 and EY11373, the Research to Prevent Blindness Foundation, and the Ohio Lions Eye Research Foundation.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- ABCA4
- ATP-binding cassette subfamily A member 4
- AMD
- age-related macular degeneration
- GFAP
- glial fibrillary acidic protein
- hiPS-RPE
- RPE cell differentiated from human-induced pluripotent stem cell
- HMOX1
- heme oxygenase 1
- KO
- knockout
- LCN2
- lipocalin 2
- miRNA
- microRNA
- POS
- photoreceptor outer segment
- qRT-PCR
- quantitative RT-PCR
- RDH8
- retinol dehydrogenase 8
- ROS
- reactive oxygen species
- RP
- retinitis pigmentosa
- RPE
- retinal pigmented epithelium
- SD-OCT
- spectral domain–optic coherence tomography
- SLC22A17
- solute carrier family 22 member 17
- SLO
- scanning laser ophthalmoscopy
- SOD2
- superoxide dismutase 2
- WT
- wild-type.
- Received November 15, 2017.
- Accepted March 5, 2018.
- Copyright © 2018 by The American Association of Immunologists, Inc.
References
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