Interleukin-17A Induces IL-1β Secretion From RPE Cells Via the NLRP3 Inflammasome

Abstract

Purpose: Inflammasome activation and IL-1β production have been proposed to have an important role in age-related macular degeneration (AMD). Growing evidence is emerging for involvement of interleukin-17A (IL-17A) in AMD pathogenesis. We investigated the effects of IL-17A on the activation of inflammasome and production of IL-1β in primary human RPE cells.

Methods: Primary human RPE cells were isolated and cultured for the following experiments. Expression patterns of IL-17 receptor A (IL-17RA), IL-17 receptor C (IL-17RC), and ACT1 were analyzed by RT-PCR, flow cytometry, and immunofluorescence. IL-17A was added to the cell cultures, and cytokine expression, signaling pathways, and inflammasome machinery were investigated using real-time RT-PCR, ELISA, Western blot, flow cytometry, and small interfering RNA.

Results: Retinal pigment epithelial cells constitutively expressed IL-17RA, IL-17RC, and ACT1. IL-17A upregulated the mRNA levels of pro-IL-1β, IL-8, CCL2, and CCL20, as well as the protein level of IL-1β. IL-17A induced the phosphorylation of Akt, Erk1/2, p38 MAPK, and NF-κB p65 in RPE cells. Blocking NF-κB attenuated IL-17A–induced expression of pro-IL-1β mRNA. IL-17A enhanced pro-caspase-1 and NLRP3 mRNA expression. Inhibiting caspase-1 activity and silencing NLRP3 decreased IL-1β secretion, confirming NLRP3 as the IL-17A–responsive inflammasome on the posttranscriptional level. The mechanism of IL-17A–triggered NLRP3 activation and subsequent IL-1β secretion was found to involve the generation of reactive oxygen species.

Conclusions: Our results suggest that IL-17A triggers a key inflammatory mediator, IL-1β, from RPE cells, via NLRP3 inflammasome activation, holding therapeutic potential for AMD.

Age-related macular degeneration (AMD) is a neurodegenerative disease with a multifactorial etiology, primarily attributable to morphologic and functional abnormalities in the RPE cells.¹ Mounting evidence has highlighted the essential role of immune processes in the development, progression, and treatment of AMD. Innate and adaptive immune systems have been shown to contribute to AMD pathogenesis.^2,3 

Inflammasome activation is a key component of innate immunity.⁴ Recent efforts have drawn attention to the inflammasome machinery, particularly its product IL-1β, as an inciting factor in AMD.^5–8 It has been well documented that RPE cells can be triggered to secrete a number of inflammatory cytokines and chemokines.⁹ Among these, IL-1β is one of the initial cytokines produced following RPE activation.^10,11 Transcription of the pro-IL-1β gene and production of cytosolic pro-IL-1β are dependent on the activation of NF-κB. At the posttranscriptional level, generation of mature IL-1β is regulated by inflammasomes, which is formed by NACHT, LRR, and PYD domains-containing (NLRP) proteins, the most studied member of which is NLRP3.¹² More recently, the role of the NLRP3 inflammasome in AMD pathogenesis has been investigated extensively using AMD-related stimuli, such as A2E,⁸ Alu RNA,¹³ lysosomal activation,¹⁴ and oxidative stress.¹⁵ However, it still is not clear whether this component of innate immunity could be activated or regulated by the local inflammatory cytokine milieu generated during adaptive immune responses. 

Interleukin-17A (IL-17A) is the signature cytokine of Th17 cells and has a pivotal role in inducing expression of proinflammatory cytokines and chemokines in the pathogenesis of autoimmune and inflammatory diseases.¹⁶ Recent studies have demonstrated involvement of IL-17A in the pathogenesis of AMD. Aberrant levels of IL-17A were observed in AMD macular lesions and sera.¹⁷ Complement component C5a, which accumulated in AMD patient serum and in drusen, induced IL-17A production from CD4⁺ T cells.¹⁸ A recent epigenetic study demonstrated that hypomethylation of the IL-17 receptor C (IL-17RC) promoter was associated with AMD.¹⁹ Since previous reports showed the presence of IL-17A receptors on RPE cells,^20,21 possibility exists that IL-17A may have a role in AMD by direct modulating the immunologic functions of RPE cells. 

Both IL-17A signaling and inflammasome machinery have been linked to the pathogenesis of AMD; however, to our knowledge no studies to date have adequately addressed the interactions between them. Interleukin-1β functions in synergy with IL-23 to promote the production of IL-17A and related cytokines from Th17 cells.^22,23 Therefore, we hypothesized that local IL-17A may serve as a trigger for inflammasome signaling and IL-1β production in RPE cells, forming a positive feedback loop. In the present study, we first aimed to examine the expression pattern of IL-17 receptor A (IL-17RA), IL-17RC, and ACT1 in primary human RPE cells. We next characterized the putative role of IL-17A in the production of IL-1β and the activation of NLRP3 inflammasome machinery in RPE cells. 

Materials and Methods

Reagents

Bay 11-7082 was purchased from Cell Signaling Technology (Danvers, MA, USA). Caspase-1 inhibitor (Z-YVAD-fmk) was obtained from Biovision (Milpitas, CA, USA). N-acetyl-L-cysteine (NAC) was obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). For flow cytometry, anti-human IL-17RA, anti-human IL-17RC, and appropriate IgG1 isotype antibody were purchased from R&D Systems, Inc. (Minneapolis, MN, USA); anti-human ACT1 was obtained from eBioscience (San Diego, CA, USA). For Western blot, antibodies against phospho-Akt, total-Akt, phospho-Erk1/2, total-Erk1/2, phospho-p38 MAPK, total-p38 MAPK, phospho-NF-κB p65, β-actin, and caspase-1 were purchased from Cell Signaling Technology. Anti–total-NF-κB p65 and anti-human NLRP3 were obtained from Abcam (Cambridge, MA, USA). Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit, and anti-mouse IgG (Cell Signaling Technology). 

RPE Cells Isolation and Culture

The study was approved by the ethics committee of the EYE & ENT Hospital of Fudan University, and was carried out in accordance with the ethical principles in the Declaration of Helsinki. Informed consents concerning the use of the posterior tissues of the donated eyes for research purposes were obtained by the Eye Bank of the EYE & ENT Hospital of Fudan University. Primary RPE cells were isolated within 24 hours of death from healthy adult human globes provided by the Eye Bank of the EYE & ENT Hospital of Fudan University.²⁴ On receipt, intact globes were rinsed in PBS. After the anterior portion of the eye was removed, the posterior poles were incubated in 2% dispase solution (Gibco, Grand Island, NY, USA) for 30 minutes in 37°C, 5% CO₂. After dispase treatment, the posterior poles were dissected into quadrants, and the retina was removed gently with forceps. Single-cell RPE layers were peeled off in small sheets, and then RPE cells were dissociated from the sheets in a solution containing 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (Life Technologies, Grand Island, NY, USA) at 37°C for 30 minutes. Afterward, the enzyme-treated tissues were dissociated into single RPE cells by gentle pipetting and centrifugation. 

The RPE cells were cultured in DMEM:F12 growth medium containing 5.5 mM glucose (Gibco), supplemented with 10% fetal bovine serum (Gibco). The cell cultures were maintained in a humidified incubator at 37°C in an atmosphere containing 95% air and 5% CO₂. The cells were passed every 3 days by trypsinization. For the following experiments, RPE cells were seeded into 6-well plates (Corning, Lowell, MA, USA). The 50% confluent RPE cell cultures were used in RPE65 immunofluorescence and the 80% to 100% confluent RPE cell cultures at passages 3 to 5 were used in other experiments. 

After treatment with IL-17A (up to 200 ng/mL), Bay 11-7082 (up to 10 μM), Z-YVAD-fmk (up to 10 μg/mL), small interfering RNA (siRNA), or NAC (5 mM), RPE cells were tested for viability using LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA) and flow cytometry. The cell viability was above 90% and did not decrease following these treatments. 

Immunofluorescence of RPE Cells

The 50% confluent cultured RPE cells were fixed in PBS containing 3.7% paraformaldehyde. They then were blocked with the appropriate normal serum in PBS for 1 hour at room temperature. They then were incubated at 4°C overnight with anti-RPE65 (1:200; Abcam). After being washed in PBS, the sections were incubated further with FITC-conjugated anti-mouse IgG (eBioscience) for 45 minutes at 37°C. Control sections were treated in the same way, but with no primary mouse anti-RPE65 monoclonal antibody. Cell nuclei were counterstained with Hoechst 33342 (Life Technologies). 

Immunofluorescence of Human Retina

Surgically isolated pieces of human retina-RPE-choroid were fixed in 4% paraformaldehyde at 4°C overnight and then washed in cold PBS for 20 minutes. Tissues were cryoprotected before freezing by overnight incubation in 20% sucrose solutions in PBS and then in 30% sucrose in PBS overnight at 4°C. Tissues were embedded in optimal cutting temperature embedding medium (Sakura Finetek, Torrance, CA, USA) and frozen in liquid nitrogen. Cryosections (1–15 μm) were cut and collected on glass slides, dried at room temperature, and stored at −80°C until use. The retina cryosections were washed with PBS and incubated with blocking solution (10% goat serum in PBS) at room temperature for 2 hours and then with goat anti–IL-17RA (1:200 dilution, sc-23124; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or rabbit anti–IL-17RC (1:200 dilution, sc-367206; Santa Cruz Biotechnology, Inc.) at 4°C overnight. After they were washed with PBS three times, sections were incubated at room temperature for 1 hour with secondary antibodies coupled to Alexa Fluor 488 (1:1000 dilution; Invitrogen) and Alexa Fluor 555 (1:1000 dilution; Invitrogen), respectively. Nuclei were counterstained with Hoechst 33342. Sliders were analyzed on a scanning laser confocal microscope. 

RT-PCR and Real-Time RT-PCR

Total RNA was extracted from RPE cells using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer's instructions. A total of 1 μg total RNA isolated was reverse transcribed with the reverse transcriptase kit (Takara Bio, Inc., Shiga, Japan) according to the manufacturer's protocol. IL-17RA, IL-17RC, ACT1, NLRP3, and GAPDH were amplified using ExTaq DNA polymerase (Takara Bio, Inc.). Polymerase chain reaction assay cycles were as follows: 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds. The PCR products were visualized on 2% agarose gels and ethidium bromide staining. To check the mRNA levels of pro-IL-1β, IL-8, CCL2, CCL20, pro-caspase-1, and NLRP3 in RPE cells, real-time RT-PCR was performed in triplicate with a real-time RT-PCR system (ABI PRISM 7500; Applied Biosystems, Foster City, CA, USA) using a SYBR detection kit (Takara Bio, Inc.) according to the standard protocol. Specific primers for NLRP3 and GAPDH were identical to those used for RT-PCR. The mRNA levels of each target gene were normalized to the levels of GAPDH and were represented as fold induction. The primer sequences of for RT-PCR and real-time RT-PCR are shown in the Table. 

Table

 

The Sequence of Primers Used for RT-PCR and Real-Time RT-PCR

 

Western Blot

After treatment, RPE cells were harvested and lysed with RIPA buffer (Shenggong, Shanghai, China). Cytosol proteins from RPE cells (40 μg/lane) were separated by electrophoresis in 8% to 12% SDS-polyacrylamide gel and then transferred onto a polyvinylidine fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Subsequently, the membrane was blocked in 2% BSA for 1 hour at room temperature and incubated with the specific primary antibodies (diluted 1:100–1:1000) overnight at 4°C. After washing with phosphate buffer solution containing Tween-20 (PBST) five times, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies (diluted 1:5000 by 2% BSA). The level of β-actin also was measured at the same time as an internal control. The results were quantified by analysis for gray scale using Gel-Pro Analyzer software (Media Cybernetics, Inc., Rockville, MD, USA). 

Flow Cytometry

To detect IL-17RA and IL-17RC expression, RPE cells were stained with anti–IL-17RA or anti–IL-17RC, and then were stained with FITC-conjugated anti-mouse IgG. To analyze the expression of ACT1, RPE cells were fixed and permeabilized in Intracellular Fixation & Permeabilization Buffer Set (eBioscience). Cells then were stained with anti-ACT1 and FITC-conjugated anti-mouse IgG. For the detection of phospho-NF-κB p65, the RPE cells were stimulated with 100 ng/mL IL-17A for 5, 15, or 30 minutes. The cells then were cooled quickly, fixed and permeabilized with BD Cytofix/Cytoperm solution (BD Biosciences, Franklin Lakes, NJ, USA), then sequentially stained with PE-labeled phospho-NF-κB p65 antibody (BD Biosciences). An isotype control mouse IgG was used for gating of phospho-protein positive cells. For ROS measurement, treated RPE cells were incubated with 10 mM CM-H2DCFDA (Invitrogen) for 30 minutes at 37°C, harvested, and analyzed. FACS data analysis was performed using the Diva (BD Biosciences) and FlowJo (TreeStar, Ashland, OR, USA) software. 

Enzyme-Linked Immunosorbent Assay (ELISA)

Retinal pigment epithelial cells were seeded in 12-well plates and grown to 80% confluence. After treatment, supernatants were collected and subsequently centrifuged to pellet cell debris. Supernatants were analyzed using human ELISA development kits for IL-1β (R&D Systems) according to the manufacturer's instructions. 

KnockDown of NLRP3 Expression

To reduce endogenous NLRP3 expression, RPE cells were transfected with 80 pmol small interfering RNA (siRNA) oligonucleotides (Santa Cruz Biotechnology) following the manufacturer's instructions. The cells were transfected with either a siRNA oligonucleotide against NLRP3 or a nontargeted control siRNA oligonucleotide. After incubation, the RPE cells were stimulated with IL-17A (100 ng/mL) for 24 hours, the cell-culture medium was collected for IL-1β ELISA analysis, and the cells were harvested for the detection of caspase-1 and NLRP3 expression using RT-PCR and Western blot. 

Statistical Analysis

Results were expressed as the mean ± SEM. A 1-way ANOVA was used to compare variances within and among groups. Data were evaluated statistically using post hoc 2-tailed Student's t-tests. Statistical significance was set at P < 0.05. 

Results

IL-17A Receptors are Constitutively Expressed on RPE Cells

IL-17A binds to a heterodimeric receptor consisting of IL-17RA and IL-17RC, and the downstream signaling pathway requires an essential signaling adaptor, ACT1 (also known as TNFR-associated factor [TRAF]3IP2 or CIKS).²⁵ To determine whether human RPE cells might be responsive to IL-17A, we isolated and cultured primary human RPE cells (Fig. 1A), and then performed RT-PCR to detect mRNA transcripts of IL-17RA, IL-17RC, and ACT1. As shown in Figure 1B, RPE cells constitutively expressed IL-17RA, IL-17RC, and ACT1 mRNA. Furthermore, analyzing their protein expression by flow cytometry confirmed that RPE cells constitutively expressed IL-17RA, IL-17RC, and ACT1 (Fig. 1C). To rule out the possibility that the expression of these receptors in RPE cells was due to culture conditions, we performed immunofluorescence using healthy retinal tissue. A positive staining of IL-17RA and IL-17RC was observed in RPE layer (Fig. 1D). These results suggest that RPE cells may be directly influenced by IL-17A via signaling through heterodimeric IL-17RA and IL-17RC. 

Figure 1

 

Expression of IL-17RA, IL-17RC, and ACT1 in RPE cells. (A) Cultured RPE cells morphologic analysis. Left: On the seventh day of the primary culture, RPE cells contained some pigment. Right: Positive RPE65 staining, indicated by the green color, was detected in RPE cells. (B) The mRNA expression of IL-17RA, IL-17RC, and ACT1 in cultured RPE cells were analyzed by RT-PCR. One experiment representative of three experiments. (C) The protein expression of IL-17RA, IL-17RC, and ACT1 in cultured RPE cells were detected by flow cytometry. One experiment representative of three experiments. (D) Immunofluorescence analysis of IL-17RA and IL-17RC in human retinal tissue. Retinal sections from healthy human donor eyes were stained with either goat polyclonal anti–IL-17RA or rabbit monoclonal anti–IL-17RC as indicated. Negative controls were generated by omission of the primary antibody. Secondary antibodies were coupled to Alexa Fluor 488 (green) and Alexa Fluor 555 (red) respectively. Nuclei were stained with Hoechst 33342. Sections were analyzed under a confocal microscope. BrM, Bruch's membrane; INL, inner nuclear layer; ONL, outer nuclear layer; Ch, choroid.

IL-17A Triggers IL-1β Secretion From RPE Cells

We postulated that IL-17A might be an inducer of inflammatory mediators in human RPE cells. To verify this assumption, RPE cells were incubated with IL-17A (100 ng/mL) for 24 hours, and the mRNA levels of pro-IL-1β, IL-8, CCL2, and CCL20 were determined by real-time RT-PCR. IL-17A significantly upregulated the mRNA expression of pro-IL-1β, IL-8, CCL2, and CCL20 (Fig. 2A). IL-1β has been shown to be one of the key cytokines activating Th17 cells. In addition, the production of mature IL-1β from RPE cells stimulated by IL-17A was assayed by ELISA. As shown in Figure 2B, IL-17A had a stimulatory effect on the production of mature IL-1β in a dose-dependent manner. 

Figure 2

 

IL-17A induces the transcription of pro-IL-1β and secretion of IL-1β in RPE cells. (A) Retinal pigment epithelial cells were stimulated by 100 ng/mL IL-17A for 24 hours. Pro-IL-1β, IL-8, CCL2, and CCL20 gene expressions then were assessed by real-time RT-PCR and normalized against the amount of GAPDH mRNA. Gene expression is graphed as mean fold induction over medium control. The data represent the mean ± SEM of three experiments with similar results (*P < 0.05, **P < 0.01). (B) Retinal pigment epithelial cells were stimulated by different concentrations of IL-17A for 24 hours. Mature IL-1β secretion into culture supernatant was assessed using ELISA. The data represent the mean ± SEM of three experiments with similar results (*P < 0.05, **P < 0.01).

IL-17A Upregulates Pro-IL-1β mRNA in RPE Cells Via NF-κB

To further explore the responsiveness of RPE cells to IL-17A, we analyzed the downstream signaling molecules activated using Western blot. As shown in Figure 3A, significant phosphorylation of Akt, Erk1/2, p38 MAPK, and NF-κB p65 was detected in RPE cells following treatment of IL-17A. The production of IL-1β involves the induction of pro-IL-1β and the processing of pro-IL-1β into mature IL-1β. In most cases, the induction of pro-IL-1β requires the activation of NF-κB signaling cascade. Next, we sought to determine whether NF-κB could be responsible for IL-17A–induced pro-IL-1β upregulation in RPE cells. Using flow cytometry, we confirmed that NF-κB p65 phosphorylation was enhanced in IL-17A–treated RPE cells (Fig. 3B). Furthermore, using Bay 11-7082, an NF-κB inhibitor, we demonstrated that IL-17A–induced pro-IL-1β mRNA was significantly suppressed (Fig. 3C). These results suggest that activation of NF-κB signaling is involved in the IL-17A–mediated of pro-IL-1β induction. 

Figure 3

 

The role of NF-κB signaling in the upregulation of pro-IL-1β mRNA in RPE cells. (A) Kinetic of Akt, Erk1/2, p38 MAPK, and NF-κB p65 phosphorylations was determined by Western blot in RPE cells treated by 100 ng/mL IL-17A. One experiment representative of three experiments. (B) Retinal pigment epithelial cells were exposed to 100 ng/mL IL-17A for indicated time. NF-κB p65 phosphorylation was determined by flow cytometry. Values denote phospho-p65⁺ cells (%) from three independent experiments. (C) Retinal pigment epithelial cells were treated with IL-17A (100 ng/mL), in the presence NF-κB inhibitor Bay 11-7082 in indicated concentrations (5 and 10 μM). After 24 hours, the cells were analyzed for pro-IL-1β mRNA levels by real-time RT-PCR. Gene expression is graphed as mean fold induction over medium control. The data represent the mean ± SEM of three experiments with similar results (*P < 0.05, **P < 0.01).

IL-17A Activates the NLRP3 Inflammasome in RPE Cells

Inflammasome activation results in the processing of pro-IL-1β into the mature IL-1β on the posttranscriptional level. Next, we aimed to study NLRP3 inflammasome complex involved in IL-17A–triggered IL-1β production. Retinal pigment epithelial cells upregulated the expression of pro-caspase-1 and NLRP3 mRNA in response to IL-17A (Fig. 4A). Interleukin-17A also induced higher amounts of active caspase-1 subunit (p20) and NLRP3 at protein levels, suggesting NLRP3 inflammasome activation (Fig. 4B). Interleukin-1β secretion following IL-17A treatment was inhibited in a concentration-dependent manner by Z-YVAD-fmk, the specific inhibitor of caspase-1, confirming the role of caspase-1 in IL-17A–induced IL-1β secretion (Fig. 4C). To further examine the role of NLRP3 in IL-17A–triggered IL-1β release from RPE cells, we adopted siRNA. Transfection with siRNAs efficiently downregulated NLRP3 mRNA and protein (Fig. 4D) expression in IL-17A–treated RPE cells. Knockdown of NLRP3 significantly inhibited IL-17A-mediated IL-1β release, indicating a crucial role of the NLRP3 inflammasome in the response of RPE cells to IL-17A (Fig. 4E). 

Figure 4

 

The involvement of NLRP3 inflammasome in the production of IL-1β by RPE cells stimulated with IL-17A. (A) Retinal pigment epithelial cells were stimulated with 100 ng/mL IL-17A for 24 hours, and the mRNA expression of pro-caspase-1 and NLRP3 was determined using real-time RT-PCR and normalized against the amount of GAPDH mRNA. Gene expression is graphed as mean fold induction over medium control. The data represent the mean ± SEM of three independent experiments (**P < 0.01). (B) Retinal pigment epithelial cells were treated with 100 ng/mL IL-17A for 24 hours. The cells were lysed and the whole-cell extracts were assessed by Western blot using antibodies against NLRP3 and the active form of caspase-1 (p20). Representative blots of three independent experiments are shown. (C) Retinal pigment epithelial cells were treated with caspase-1 inhibitor Z-YVAD-fmk in indicated concentrations (1 and 10 μg/mL) for 30 minutes and then stimulated with 100 ng/mL IL-17A. Culture supernatants were collected after 24 hours and assayed with IL-1β ELISA. The data represent the mean ± SEM of three experiments with similar results (*P < 0.05, **P < 0.01). (D) Retinal pigment epithelial cells were transfected with siRNA oligos specific for NLRP3 (siNLRP3) or a nonspecific siRNA oligo (siControl), and subsequently stimulated with 100 ng/mL IL-17A for 24 hours, and the levels of NLRP3 mRNA (above) and protein (below) were analyzed using RT-PCR and Western blot. One experiment representative of three experiments. (E) Small interfering RNA–transfected RPE cells then were stimulated with 100 ng/mL IL-17A, and then culture supernatants were collected after 24 hours and assayed with IL-1β ELISA. The data represent the mean ± SEM of three experiments with similar results (*P < 0.05).

ROS Generation is Required for IL-17A-Induced IL-1β Secretion From PRE Cells

Reactive oxygen species (ROS) have been proposed to perform an important role in the activation of NLRP3 inflammasome, and IL-17A has been reported to be an inducer of ROS.^26,27 Therefore, we hypothesized that ROS formation in RPE cells might be involved in IL-17A–mediated inflammasome activation and IL-1β secretion. Indeed, as determined by CM-H2DCFDA labeling and flow cytometry, ROS formation in RPE cells increased significantly following stimulation with IL-17A (Fig. 5A). Preconditioning with the antioxidant NAC significantly modulated the expression of caspase-1 and NLRP3 at mRNA and protein levels (Figs. 5B, 5C). Furthermore, IL-17A–induced IL-1β production was significantly inhibited by NAC. These data indicated that activation of the NLRP3 inflammasome by IL-17A is dependent on ROS generation in RPE cells. 

Figure 5

 

IL-17A activation of inflammasome and IL-1β secretion in RPE cells is regulated by ROS. (A) Retinal pigment epithelial cells were incubated with 100 ng/mL IL-17A for 6 and 24 hours. The cells then were collected and labeled with CM-H2DCFDA, and ROS production was measured by flow cytometry. Representative histograms and mean fluorescence intensities (mean ± SEM) were obtained from five independent experiments (*P < 0.05, **P < 0.01). (B) Retinal pigment epithelial cells were treated with 100 ng/mL IL-17A in combination of NAC (5 mM) for 24 hours, and the mRNA levels of pro-caspase-1 and NLRP3 were determined using real-time RT-PCR and normalized against the amount of GAPDH mRNA. Gene expression is graphed as mean fold induction over medium control. The data represent the mean ± SEM of three experiments with similar results (*P < 0.05). (C) Retinal pigment epithelial cells were treated with 100 ng/mL IL-17A for 24 hours, in the presence of NAC (5 mM). NLRP3 and the active form of caspase-1 (p20) then were analyzed by Western blot. Representative blots of three independent experiments are shown. (D) Retinal pigment epithelial cells were treated with 100 ng/mL IL-17A in the presence of 5 mM NAC, and then culture supernatants were collected after 24 hours and assayed with IL-1β ELISA. The data represent the mean ± SEM of three experiments with similar results (*P < 0.05).

Discussion

In this study, we determined that primary human RPE cells constitutively expressed IL-17RA, IL-17RC, and ACT1. Treatment with IL-17A caused an increase in IL-1β production via ROS production leading to NLRP3 activation in RPE cells. Recent efforts have drawn attention to the Th17 cells and Th17 cytokines, particularly IL-17A, as a pathogenic factor in AMD. Serum levels of IL-17A were reported to be significantly higher in AMD patients compared to normal controls, while serum levels of IL-22, another Th17 family cytokine, also were significantly higher in AMD patients. The elevation of IL-17A expression was found not only in the peripheral blood but also in macular tissues with AMD lesions.^17–19 Since AMD patients usually present without systemic inflammation, we hypothesized that local IL-17A signaling was more relevant in AMD pathogenesis. 

Although Th17 cells, a subtype of activated CD4⁺ T cells, often are deemed the major producers of IL-17A, the source of IL-17A in the outer retina is not known. Other immune competent cells, such as γδT cells, neutrophils, and even macrophages, under specific conditions may produce IL-17A.^28–30 In a recent study, using Nrf2^−/− mice as a model with pathologic features similar to human AMD, Zhao et al.³¹ showed that lymphocytes infiltrated in the sub-RPE space were mainly γδT cells, which produced IL-17A as a proinflammatory signal. Histologic evidence has shown the presence of macrophages near many AMD lesions. Therefore, it is worthwhile to determine further the exact cellular sources of local IL-17A in the progression of AMD.³² 

The elevated expression of IL-17RC recently was observed in chorioretinal tissues from AMD patients.¹⁷ Therefore, the RPE cells, being resident at the chorioretinal interface, would appear a potential target of IL-17A immunologic effect. A recent report showed that IL-17A promoted ARPE-19 cells to secrete inflammatory mediators and compromised the ARPE-19 monolayer barrier function.²⁰ In this report, Chen el al.²⁰ found ARPE-19 cells express IL-17RC but no IL-17RA, while in our study, we demonstrated that RPE cells express IL-17RA and IL-17RC, which are essential to IL-17A signaling. This discrepancy could be explained by the origin of the RPE cells adopted in the studies. ACT1 has been implicated in the control of immune and inflammatory responses. The binding of IL-17A to the IL-17RA/RC heterodimer leads to the ACT1 recruitment through a SEFIR-SEFIR-dependent interaction. This results in incorporation of TRAF6 into the signaling complex and the subsequent downstream activation of the NF-κB and MAPK pathways, leading to the induction of target genes.²⁴ The expression of ACT1 in RPE cells has never been characterized before to our knowledge, and our results provided support for the RPE cells to be targets of IL-17A. 

The most notable effects of IL-17A on RPE cells might be the increased production of inflammatory cytokines and chemokines. In current study, IL-17A was able to significantly stimulate the expression of IL-8, CCL2, and CCL20 by RPE cells. Our results were consistent with previous studies showing the enhanced production of these cytokines by IL-17A.²⁰ These cytokines and chemokines have been implicated previously in the development of AMD. A strong correlation has been obtained between elevated levels of IL-8 concentrations in aqueous humor and exudative AMD patients.³³ Murine models have shown that CCL2 is involved in drusen formation and RPE changes seen in the early stages of AMD.³⁴ CCL20 is a chemokine involved in the attraction of Th17 cells. These cytokines and chemokines might promote immune cells trafficking into the inflammatory sites within macular tissues of AMD patients and eventually causing the pathology of AMD. 

The activation of MAPK and Akt pathways might be an important signaling event in the response of RPE cells to proinflammatory cytokines.³⁵ In the present study, activation of Erk1/2, p38 MAPK and Akt pathways was observed in IL-17A–treated RPE cells, which are indicated by elevated phosphorylated/total protein ratio. Our findings are in accordance with earlier reports using ARPE-19 cell line.³⁵ The promoter region of the human pro-IL-1β gene has been cloned and has been shown to contain consensus binding motifs for NF-κB.³⁶ Here, we showed that Bay 11-7082 reduced pro-IL-1β expression in RPE cells, suggesting a crucial role for NF-κB signaling as the initial step in the NLRP3 inflammasome activation. 

IL-1β performs a fundamental role in orchestrating inflammatory responses, majorly via the regulation of gene expression. IL-1β–responsive genes coordinate all phases of local inflammation and also attract and activate cells of the adaptive immune system. Th17 cells possess IL-1β receptors and are stimulated by IL-1β to generate IL-17A. Therefore, excess IL-1β production might result in the augmentation of Th17-dominant pathology. Myloid cells serve as major producers of this Th17-promoting cytokine. However, our data offered convincing support for RPE cells as an important source of IL-1β, especially in the early development of AMD with marginal infiltration of myloid cells in the retinal tissue. To our knowledge, no studies to date have adequately addressed the possibility of a feedback mechanism occurring via the activity of IL-17A on IL-1β secreted from RPE cells under conditions of retinal inflammation. 

Mature IL-1β is generated via the cleavage of the inactive pro-IL-1β precursor by caspase-1 (p20). The inflammasome complexes activate caspase-1 by facilitating the cleavage of its zymogen precursor, pro-caspase-1. Active caspase-1 (p20) then catalyzes the proteolytic cleavage of pro-IL-1β into IL-1β.⁴ The NLRP3 inflammasome-mediated activation of caspase-1 and processing of pro-IL-1β into IL-1β has been reported previously in RPE cells.¹⁴ In our study, Z-YVAD-fmk abolished the caspase-1 activity and reduced the IL-1β production, suggesting that the IL-17A-induced IL-1β release from RPE cells also was mediated by caspase-1. 

The induction of NLRP3 expression via activation of NF-κB is a critical checkpoint for NLRP3 inflammasome activation.⁴ Accordingly, in the present study, we observed the upregulation of NLRP3 and p65 expression following the treatment of IL-17A, highlighting an IL-17A–induced activation of NLRP3 inflammasome. The activation of NLRP3 inflammasome requires two signals, a “priming signal” and an “activation signal.” The “priming signal” majorly channels through NF-κB, upregulating the transcription of pro-IL-1β. The second signal for inflammasome activation is relayed by various mechanisms including generation of intracellular ROS. Retinal pigment epithelial cells are major targets of oxidative stress because of their high oxygen consumption, high levels of polyunsaturated lipids, and the long periods of exposure to light. Disturbances in the oxidant-antioxidant system in retina may have an important role in the pathogenesis of AMD.³⁷ Here, we demonstrated that ROS generation was necessary for NLRP3 inflammasome activation in RPE cells and subsequent IL-1β release in response to IL-17A. Therefore, we hypothesize that IL-17A activates RPE cells in a positive feedback loop producing inflammatory mediators and ROS. One of the major limitations of present study is lack of in vivo comparison of IL-17A as well as NLRP3 inflammasome components between healthy and AMD retinal tissue. More in vivo evidences will be needed to confirm the hypothesis in the context of AMD, and to decipher how the local microenvironment and RPE-derived cytokines influence IL-17A-producing cells. 

In summary, the present study revealed the presence of IL-17RA, IL-17RC, and ACT1 in cultured human RPE cells. Interleukin-17A stimulated RPE cells to produce mature IL-1β through activation of NF-κB pathway and NLRP3 inflammasome, thereby supporting a mechanism bridging between innate and adaptive immune responses in AMD. This mechanism must be addressed in future investigations, but this study may contribute to our understanding of retinal immunity and translate into better therapy for AMD. 

Acknowledgments

Supported by the National Natural Science Foundations of China (81200675, 81470624) and the Fundamental Research Funds for the Central Universities. The authors alone are responsible for the content and writing of the paper. 

Disclosure: S. Zhang, None; N. Yu, None; R. Zhang, None; S. Zhang, None; J. Wu, None