Interleukin‐2 induces extracellular matrix synthesis and TGF‐β2 expression in retinal pigment epithelial cells

Abstract Macular fibrosis is a vital obstacle of vision acuity improvement of age‐related macular degeneration patients. This study was to investigate the effects of interleukin 2 (IL‐2) on epithelial‐mesenchymal transition (EMT), extracellular matrix (ECM) synthesis and transforming growth factor β2 (TGF‐β2) expression in retinal pigment epithelial (RPE) cells. 10 μg/L IL‐2 was used to induce fibrosis in RPE cells for various times. Western blot was used to detect the EMT marker α‐smooth muscle actin (α‐SMA), ECM markers fibronectin (Fn) and type 1 collagen (COL‐1), TGF‐β2, and the activation of the JAK/STAT3 and NF‐κB signaling pathway. Furthermore, JAK/STAT3 and NF‐κB signaling pathways were specifically blocked by WP1066 or BAY11‐7082, respectively, and the expression of α‐SMA, COL‐1, Fn and TGF‐β2 protein were detected. Wound healing and Transwell assays were used to measure cell migration ability of IL‐2 with or without WP1066 or BAY11‐7082. After induction of IL‐2, the expressions of Fn, COL‐1, TGF‐β2 protein were significantly increased, and this effect was correlated with IL‐2 treatment duration, while α‐SMA protein expression did not change significantly. Both WP1066 and BAY11‐7082 could effectively downregulate the expression of Fn, COL‐1 and TGF‐β2 induced by IL‐2. What's more, both NF‐κB and JAK/STAT3 inhibitors could suppress the activation of the other signaling pathway. Additionally, JAK/STAT3 inhibitor WP1066 and NF‐κB inhibitor BAY 11‐7082 could obviously decrease RPE cells migration capability induced by IL‐2. IL‐2 promotes cell migration, ECM synthesis and TGF‐β2 expression in RPE cells via JAK/STAT3 and NF‐κB signaling pathways, which may play an important role in proliferative vitreoretinopathy.


| INTRODUC TI ON
As a major cause of severe loss of vision among the old, age-related macular degeneration (AMD) is a complex multifactorial disease clinically caused by the degeneration of the photoreceptors and retinal pigment epithelium (RPE) cells in the old (Yonekawa & Kim, 2014). AMD is broadly classified into the wet form (neovascular or exudative) and dry form. Both two types of AMD pathology start with the formation of insoluble aggregates and drusen, which forms in the extracellular matrix between Brush's membrane and RPE (Mitchell, Liew, Gopinath, & Wong, 2018). Nowadays the main therapeutic medication of neovascular age-related macular degeneration (nAMD)-anti-vascular endothelial growth factor (VEGF)includes bevacizumab and ranibizumab (Solomon, Lindsley, Vedula, Krzystolik, & Hawkins, 2019). Delightedly, the treatment could prevent the decrease or even improve vision acuity. However, after frequently, costly and invasive intravitreal injections, up to one-third of patients could not benefit from the therapy due to the development of macular fibrosis or atrophy (Little, Ma, Yang, Chen, & Xu, 2018).
Considering TGF-β is a dominant factor in the development of AMD and macular fibrosis or atrophy , the mechanism of enhanced TGF-β activity after anti-VEGF by intravitreal injection may provide a new way to prevent the development of nAMD and macular fibrosis or atrophy. While inflammation is an inevitable response after intravitreal injection and it plays important roles in nAMD (Kauppinen, Paterno, Blasiak, Salminen, & Kaarniranta, 2016;Rezar-Dreindl et al., 2016;Roh et al., 2009). IL-2, a characteristic inflammatory factor, plays major roles in many eye diseases and is involved in fibrosis in many tissues (Jung, Woo, & Park, 2019;Wang, Wang, Zhu, Geng, & Yang, 2015). The expression level of IL-2 has been found to be highly expressed in the aqueous humour of polypoidal choroidal vasculopathy (PCV) and proliferative vitreoretinopathy (PVR) patients (Ricker et al., 2011;Roybal et al., 2018;Sakurada et al., 2015). In addition, the inflammation pathways associated to IL-2 were activated in AMD (Makarev et al., 2014;Newman et al., 2012). Various studies have confirmed that IL-2 could interact with TGF-β to enhance their activities (Battaglia et al., 2013;Chambers et al., 2014;Freudenberg et al., 2018;Tischner, Wiegers, Fiegl, Drach, & Villunger, 2012). It is speculated that persistent activation of IL-2 may be an important contributor to the increase of TGF-β effects. However, whether IL-2 could directly induce the occurrence of EMT and synthesis of ECM and whether there was a connection between TGF-β activity and IL-2 in RPE cells remain unknown.
According to these effects of IL-2 and fibrosis in AMD, we hypothesized that IL-2 may serve as an inducer function by interacting with TGF-β and activating certain pathways. To validate, we simulated the inflammatory microenvironment of the AMD with IL-2 in this study, then used Western blot method to detect the EMT marker, the ECM markers, and the activation of the JAK/STAT3 and NF-κB signaling pathway. In addition, Wound healing and Transwell assays were enrolled to detect cell migration capability. We found that IL-2 could promote RPE cells migration, ECM markers fibronectin (Fn) and type 1 collagen (COL-1), TGF-β2 but not EMT marker α-smooth muscle actin (α-SMA) depending on the activation of JAK/ STAT3 and NF-κB signaling pathways.

| Culture and treatment of retinal pigment epithelial cells
The ARPE19 cell line was purchased from ATCC. A total of 1 × 10 6 cells within 12 passages were seeded into a culture flask with DMEM/F12 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The medium was renewed by serum-free DMEM/ F12 when the cells get to 70% confluence, and then cells were treated with IL-2 for different durations (0, 12, 24, 36 and 48 hr) or at different concentrations (0, 0.5, 1, 2, 4, 6 and 8 μg/L). Cells in the control group were treated with an equal volume of medium.

| Western blot
Western blot was performed as described in our previous study . Briefly, RPE cell lysis was loaded at a concentration of 20 μg per well to separate in SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked using 1% BSA (Beijing Biodee Biotechnology Co., Ltd) in tris-buffered saline containing 0.1% Tween-20 for 1 hr at room temperature and probed using antibodies shown in Table 1 overnight at 4°C. After incubation with appropriate secondary antibodies, protein signals were detected using enhanced chemiluminescence western blotting detection kit (Bio-Rad).

| Wound healing and Transwell ® migration assay
Wound healing and Transwell ® migration assay were performed as described in our previous study . The cells were in serum-free medium containing 10 μg/L IL-2 with or without WP1066 (S2796; Selleck) or BAY 11-7082 (S2913; Selleck).

| Cell viability detection using CCK8 kit
Approximately 10,000 cells were seeded in a 96 well plate. The medium was replaced with serum-free medium containing IL-2 and renewed 24 hr later. Cells were treated with different time (0, 24 and 48 hr) and concentration (0, 1, 5, 10, 15 and 20 μg/L) grades. After induction, 100 μl/ml CCK8 solution in medium was added into the microplate. 4 hr later, absorbance at 450 nm and relevance at 650 were measured using a microplate reader.

| Statistical analysis
GraphPad Prism 6.0 (GraphPad Software, Inc.) was employed to perform all of the statistical analyses. All of the data are reported as the mean ± SEM with at least three independent experiments. Statistical comparisons of the Western blot data were analyzed using one-way ANOVA, whereas differences between groups were compared using Tukey's honest significant difference (HSD) test. Differences of p < .05 were considered statistically significant.

| IL-2 stimulates RPE cells migrate
Varies cytokines were verified to be involved in fibrosis disease among which IL-2 was also discovered to function in prolapsed lumbar intervertebral disc and hypertension after glaucoma surgery (Jung et al., 2019;Wang et al., 2015). Whether IL-2 had the analogous effect was unknown on RPE cells. We first investigated the impact of IL-2 on the migration ability of RPE cells using Transwell and wound healing assays. The results showed that the relative wound area was much less in the IL-2 treated group than the control group (p < .05, Figure 1a,b). Similar results were also observed in the Transwell assay (p < .01, Figure 1c,d). The results indicated that IL-2 promoted the migration capability of RPE cells in vitro.

| IL-2 promotes ECM synthesis and TGF-β2 expression in RPE cells
Previous studies have shown that IL-2 could promote cell migration and ECM synthesis in nucleus pulposus cells (Wang et al., 2015). However, the ability of IL-2 to promote EMT and ECM synthesis in RPE cells was  Figure 2b). Therefore, the results indicate that IL-2 could promote ECM synthesis but not EMT in RPE cells.

| Role of the JAK/STAT3 and NF-κB signaling pathway in ECM synthesis in RPE cells treated with IL-2
Previous studies have shown that IL-2 can activate the JAK/STAT3 and NF-κB signaling pathway in some cells (Fujii, 2007;Marzec et al., 2008). However, whether JAK/STAT3 and NF-κB signaling pathway play important roles in RPE cells was not clear. Our results confirmed that IL-2 could activate the JAK/STAT3 and NF-κB signaling pathways in RPE cells (Figure 3a-c). Then we wondered whether the JAK/STAT3 and NF-κB signaling pathways were involved in the IL-2 induced expression of ECM proteins in RPE cells. RPE cells were treated with the JAK/STAT3 inhibitor WP1066 (4 μM) to block the JAK/STAT3 signaling pathway prior to IL-2 for 2 hr. Consistent with prior observations, after treatment with 10 μg/L IL-2, the protein expression levels of Fn, COL-1 and TGF-β2 were significantly increased compared with those in the control group. Nonetheless, the induction of Fn, COL-1 and TGF-β2 protein expression by IL-2 was significantly inhibited by blocking the JAK/STAT3 signaling pathway with WP1066 or BAY (Figure 3d-i).
Similarly, when BAY (10 μM) was used to block the NF-κB signaling pathway, the induction of Fn, COL-1 and TGF-β2 protein expression by IL-2 was significantly inhibited (Figure 3j-o). Intriguingly, either WP1066 or BAY were applied to block the specific pathway, the other pathway was blocked significantly (Figure 3i,n). These results might indicate that STAT3 and NK-κB signaling pathways might interact with each other and played important roles in fibrosis in RPE cells together.

| JAK/STAT3 and NF-κB signaling pathways were involved in migration capability regulation
Cell migration is a central process in fibrosis disease (   Fibrosis is a complicated pathophysiological process including cell migration, apoptosis, inflammation, EMT, ECM synthesis and so on (Chen et al., 2019;Mack, 2018). As a pleiotropic inflammatory cytokine, IL-2 is involved in cell proliferation, differentiation, immunoregulation and autoimmune or inflammatory diseases through the activation of intracellular signal transduction pathways (Klatzmann & Abbas, 2015). Several studies showed that IL-2 could promote cell proliferation and migration (Llavero, Artaso, Lacerda, Parada, & Zugaza, 2016;Tauriainen et al., 2015). However, our results showed that IL-2 could promote RPE cells migration but not proliferation ( Figure S1A,B) in vitro. Considering cell proliferation and migration are two separate independent process and there was no proliferation pathways activated by IL-2, IL-2 may specifically promote RPE cells migration in nAMD rather than induce cell proliferation according our in vitro results. Whether IL-2 could promote RPE cells proliferate directly or indirectly in vivo needs further research.

| D ISCUSS I ON
As a pleiotropic effects inflammatory factor, IL-2 also plays an important role in promoting fibrosis (Taylor et al., 2018;Wang et al., 2016). IL-2 plays important role in human nucleus pulposus cells migration and ECM synthesis (Wang et al., 2015). Moreover, a previous study showed that IL-2 could mediate the EMT process through interacting with TGF-β1 (Qian et al., 2017). However, whether IL-2 could induce EMT and ECM synthesis in RPE cells and whether there is a relationship between this cytokine and TGF-β2 remain unknown.
According to the results, IL-2 induced RPE cells ECM synthesis.
Intriguingly, IL-2 could not induce the expression of the EMT-specific protein α-SMA, which indicated that in RPE cells, IL-2 might specifically promote the synthesis of ECM rather than induce EMT.
TGF-β was considered a key member in the development of fibrosis. Our previous and several other studies found that IL-1, IL-6 and IL-17A could enhance the effects of TGF-β on fibrosis (Fabre, Kared, Friedman, & Shoukry, 2014;Qian et al., 2017;Van Den Akker et al., 2017). A previous study showed anti-VEGF could reduce TGF-β1 expression but not cytokines in the aqueous humor of nAMD patients (Rezar-Dreindl et al., 2016). Besides, our results showed IL-2 could induce the expression of TGF-β2 in RPE cells. These results indicated that cytokines could promote TGF-β work. Inflammation might link to fibrosis in macular and nAMD relapse. However, the mechanism of interaction between IL-2 and TGF-β2 requires further study.
IL-2 is involved in cell fibrosis, immunoregulation, development and other biological behaviors via activation of the STAT3 and NK-κB signaling pathways (Jung et al., 2018;Luo et al., 2018;Mitrokhin, Gorbacheva, Mladenov, & Kamkin, 2018;Park et al., 2017;Wu et al., 2018). In our study, IL-2 could activate STAT3 and NK-κB signaling pathways. Moreover, WP1066 and BAY, the specific inhibitor of F I G U R E 4 Both inhibition of JAK/STAT3 and NF-κB signalling pathway lower migration rate. (a) RPE cells were pre-seeded in the culture insert. When cells were 70% confluence, the insert was removed and serum-free medium was added. Before 10 μg/L IL-2 were loaded, WP1066 or BAY has been added into the medium for 1 hr. (b) Transwell assay of 10 μg/L IL-2 and IL-2 with 4 μM WP1066 or 10 μM BAY preteated for 1 hr. (c) The wound area was measured and analyzed with Image J software and there was a significance in 48 hr between the IL-2 and IL-2 with WP1066 or BAY group ( , DMSO; , IL-2; , IL-2+WP1066; , IL-2+BAY ECM is known to acquire substantial changes in composition, structure, and mechanics, which together exacerbate disease etiology through promotion of angiogenesis, cell differentiation, and immune activation (Shelef et al., 2012). Fn was verified to activate FAK consequently promote HLEC cell migration .
Our results showed that with STAT3 and NF-κB pathways inhibitors, the migration rate of RPE cells induced by IL-2 dropped as well. So decreased ECM synthesis resulted from downregulated JAK/STAT3 and NF-κB signaling pathways might reduce focal adhesion and thus migration capability.
These results might indicate that STAT3 and NK-κB signaling pathways might interact with each other and played important roles in IL-2-induced fibrosis in RPE cells together. Our findings will offer new insights into our understanding of the molecular mechanisms underlying the pathogenesis of nAMD.

ACK N OWLED G M ENTS
We thank the School of Life Science and Biotechnology of Xi'an Jiaotong University for assisting in the study.

CO N FLI C T O F I NTE R E S T
All of the authors declare that there is no interest.