Luteolin Attenuates IL-1β-Induced THP-1 Adhesion to ARPE-19 Cells via Suppression of NF-κB and MAPK Pathways

Cytokine-induced endothelial dysfunction leads to inflammation and vascular adhesion molecule production in retinal pigment epithelium (RPE) cells. Inflammation is a critical mediator in retinal degeneration (RD) diseases, including age-related macular degeneration (AMD), and RD progression may be prevented through anti-inflammatory activity in RPE cells. The flavonoid polyphenol luteolin (LU) has anti-inflammatory and antidiabetes activities, but its effects regarding retinal protection remain unknown. Here, we examined the ability of luteolin to alleviate markers of inflammation related to RD in cytokine-primed APPE-19 cells. We found that luteolin decreased the levels of interleukin- (IL-) 6, IL-8, soluble intercellular adhesion molecule-1 (sICAM-1), and monocyte chemoattractant protein-1 (MCP-1) and attenuated adherence of the human monocytic leukemia cell line THP-1 to IL-1β-stimulated ARPE-19 cells. Luteolin also increased anti-inflammatory protein heme oxygenase-1 (HO-1) levels. Interestingly, luteolin induced protein kinase B (AKT) phosphorylation, thus inhibiting nuclear factor- (NF-) κB transfer from cytoplasm into the nucleus and suppressing mitogen-activated protein kinase (MAPK) inflammatory pathways. Furthermore, cotreatment with MAPK inhibitors and luteolin decreased inflammatory cytokine and chemokine levels, and further suppressed THP-1 adhesion. Overall, these results provide evidence that luteolin protects ARPE-19 cells from IL-1β-stimulated increases of IL-6, IL-8, sICAM-1, and MCP-1 production by blocking the activation of MAPK and NF-κB signaling pathways, thus ameliorating the inflammatory response.


Introduction
The retinal pigment epithelium (RPE) is a single layer of pigment cells, which is in close contact with photoreceptors and maintains visual function [1]. RPE exposure to ultraviolet/ visible light leads to oxidative stress and chronic inflammation in the retinal tissue. Inflammation clearly plays a role in the development of age-related macular degeneration (AMD), which is a cause of severe irreversible visual impairment in elderly persons and in diabetic retinopathy [2,3]. A variety of factors promote retinal tissue degeneration and AMD progression, including genetic and environmental factors, aging, and oxidative stress [4]. AMD involves reduced photoreceptor cells and retinal pigment epithelium dysfunction in the macula and can be classified as "dry AMD" or "wet/exudative/neovascular AMD." Compared to neovascular AMD, dry AMD has a higher incidence, but involves less vision degradation and is thus less frequently a cause of "blindness" [5,6]. Neovascular AMD is characterized by excessive choroidal neovascularization (CNV) under the retina, leading to retinal edema and even detachment, thereby causing vision loss. Notably, dry AMD can potentially evolve into neovascular AMD, leading to irreversible vision loss [7,8]. Therefore, the best means of avoiding vision deterioration in elderly persons and cases of diabetic retinopathy is to prevent macular degeneration.
Available data suggest that a number of inflammatory cytokines and chemokines are elevated in the serum or ocular tissue of AMD patients-including interleukin-6 (IL-6), IL-8, monocyte chemotactic protein (MCP-1), and intercellular adhesion molecule (ICAM-1) [9][10][11]. Notably, IL-6 levels are increased in the intraocular fluids of patients with neovascular AMD compared to controls, and findings suggest that IL-6 and IL-8 are proangiogenic in AMD progression [12,13]. Intraocular MCP-1 is a chemokine for monocyte recruitment, and ICAM-1 is an adhesion molecule that facilitates leukocyte transmigration. Studies have reported that elevated MCP-1 levels contribute to subfoveal neovascular membrane development and the degree of macular edema in eyes with exudative AMD and that elevated soluble ICAM-1 (sICAM-1) levels are correlated with choroidal neovascularization [13,14]. Inflammatory mediators, including chemokines and cytokines, can upregulate ICAM-1 expression. IL-6 could directly or indirectly activate the leukocytes to induce retinal inflammation. It has been found that MCP-1 recruits leukocytes to sites of injury and activates ICAM-1; thus, ICAM-1 induced leukocyte-endothelial interactions and promoted leukocyte migration into extravascular locations involved in the inflammatory response [14,15]. More studies found that IL-8 and MCP-1 could attract neutrophils and monocytes to migrate into inflammatory tissues. IL-1β is a proinflammatory cytokine and promotes the upregulation of chemokines in RPE as model of focal retinal degeneration [15]. In the present study, we evaluated the ability of the luteolin to modulate inflammation in ARPE-19 cells-THP-1 monocytes interactions. The levels of the inflammatory cytokines IL-6, IL-8, MCP-1, and ICAM-1 in ocular tissue are significantly associated with exudative AMD occurrence and progression [4]. Moreover, IL-1β activates inflammatory-related pathways, including nuclear factor-(NF-) κB and mitogen-activated protein kinase (MAPK) signaling, thus enhancing the production of the proinflammatory mediators MCP-1, IL-6, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). Proinflammatory mediators are closely associated with the development and progression of retinal degenerative diseases [15,16].
Based on the available data, we speculate that IL-6, IL-8, MCP-1, and sICAM-1 could be target molecules for AMD therapy or prevention. In the present study, we aimed to assess the mechanisms underlying the luteolin-induced anti-inflammatory effects in ARPE-19 cells stimulated by the proinflammatory cytokine IL-1β ( Figure 1).

Cell Viability Assay.
We assessed the inhibitory effect of luteolin on cell viability using CCK-8 kits (Sigma-Aldrich) as described previously [35]. Cells were seeded at 10 5 cells/well into 96-well plates and treated with luteolin at concentrations of 1-100 μM for 24 h. After treatment, the CCK-8 solution was added, and the plates were incubated at 37°C for 2 h. Finally, cell viability was measured at 450 nm using a microplate reader (Multiskan FC; Thermo, Waltham, MA, USA). The CCK-8 assay with each concentration was carried out in triplicate, and cell viability was reported as a percentage relative to the cells without luteolin treatment. 2.6. Western Blot Analysis. Protein samples were separated on 10% SDS polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Next, the PVDF membranes were incubated overnight at 4°C with specific primary antibodies against β-actin (Sigma), COX-2, iNOS, HO-1, AKT, pAKT (Santa Cruz, CA, USA), JNK, pJNK, p38, and pp38 (Cell Signaling Technology, Danvers, MA, USA). Then, the membranes were washed three times using tris-buffered saline with Tween 20 (TBST) buffer and incubated with secondary antibodies at room temperature for 1 h. Proteins were detected using Luminol/Enhancer solution (Millipore), and signals were detected using the BioSpectrum 600 system (UVP, Upland, CA, USA) to quantitate protein bands.  Mediators of Inflammation cells. Finally, the extent of adhesion of THP-1 cells to ARPE19 cells was observed under fluorescence microscope (4 per view; magnification, ×200; Olympus Corporation, Tokyo, Japan). The control groups were treated with IL-1β alone, and all experiments were repeated three times.
2.8. Immunofluorescence Staining. ARPE19 cells were seeded into 6-well plates until reaching 50-60% confluence and pretreated with or without luteolin (1, 3, 10, and 30 μM) for 1 h, followed by addition of IL-1β for 15 min. Then, the medium was suctioned out, and the cells were washed with PBS. The cells were fixed with 4% (w/v) paraformaldehyde and incubated with anti-NF-κB p65 antibody overnight at 4°C. The next day, the medium was removed, and the cells were washed with PBS and then incubated with secondary antibodies at room temperature for 1 h. Then, the cells were washed 2-3 times with PBS, followed by the addition of fluorescent dye (BODIPY493/503 or BODIPY581/591). The cells were again washed with PBS to remove the dye, and 4',6-diamidino-2-phenylindole (DAPI; Sigma) was added to stain the nucleus. Finally, images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan).
2.9. Statistical Analysis. Image Lab software (Bio-Rad) was used to quantify the intensity of western blot bands, and ImageJ software (W. Rasband, NIH, USA) to determine the numbers of THP-1 cells in the adhesion assay. Data were presented as the mean ± standard deviation (SD) from at least three independent experiments. Statistical analyses included one-way analysis of variance (ANOVA) followed by the Tukey's post hoc test. A p value of < 0.05 was considered significant.

Luteolin Inhibited Inflammatory Mediator Expression
and Increased Anti-Inflammatory Protein HO-1 Expression in IL-1β-Stimulated ARPE19 Cells. First, we performed a CCK-8 assay to assess luteolin cytotoxicity in ARPE-19 cells. Luteolin concentrations of ≤50 μM showed no significant cytotoxicity in ARPE-19 cells, while cell numbers were significantly reduced at concentrations of ≥100 μM (Figure 2(a)). Therefore, all subsequent experiments were performed using 1-30 μM luteolin. ARPE-19 cells were seeded in 6-well plates, treated with luteolin, and then stimulated with 1 ng/mL IL-1β. Compared with IL-1β alone, additional treatment with luteolin at ≥1 μM significantly inhibited expression of the inflammatory mediator iNOS (Figures 2(b) and 2(c)) but did not significantly inhibit COX-2 expression (data not shown). Notably, compared with IL-1β alone, treatment with 1 and 30 μM luteolin significantly increased expression of the anti-inflammatory protein HO-1 (Figures 2(d) and 2(e)). Previous studies have indicated that AKT activation may contribute to inhibiting the NF-κB inflammatory pathway in inflammation-related diseases [36]. Our results showed that luteolin at ≥1 μM enhanced the expression of phosphorylated AKT proteins in IL-1β-stimulated ARPE19 cells compared with in cells treated with IL-1β alone (Figures 2(f) and 2(g)).

Discussion
In many retinal degenerative (RD) diseases, the pathogenesis is inflammation-induced, involving the recruitment and activation of microglia and macrophages, the expression of inflammatory mediators (COX-2 and iNOS), and photoreceptor cell death. The proinflammatory cytokine IL-1β triggers inflammatory responses and attracts inflammatory cells to migrate into the retina, promoting retinal impairment and degeneration in RD diseases [38,39]. Numerous studies reported that IL-1β activates the expression of other proinflammatory cytokines and modulates chemokine expression. Proinflammatory cytokines can target and induce retinal inflammation in RD pathogenesis [36,37] and the phytochemical luteolin has excellent anti-inflammatory properties [32,33]. Therefore, here we performed a detailed exploration of the anti-inflammatory effects of luteolin in IL-1β-stimulated ARPE19 cells.
Prior studies have indicated that the cytokines IL-6 and IL-8 are proangiogenic, while the chemokine MCP-1 and cell adhesion molecule ICAM-1 facilitate leukocyte transmigration into ocular tissue, in AMD development and progression [4,[12][13][14]. Elevated IL-1β levels in the vitreous or retina lead to photoreceptor cell death in retinal detachment patients and in a mouse model, while reduced IL-1β levels inhibit photoreceptor cell death [40]. In the present study, we demonstrated that luteolin significant inhibited cytokine and chemokine release in IL-1β-stimulated ARPE-19 cells (Figures 3(a)-3(d)). Prior studies have also shown that ICAM-1 is upregulated in response to inflammatory mediators and mediates leukocyte adhesion and transmigration on the RPE, while decreased ICAM-1 levels can suppress  Mediators of Inflammation monocyte adhesion in RPE cells [41,42]. Here, we demonstrated that luteolin inhibited sICAM-1 levels and attenuated THP-1 cell adhesion to IL-1β-stimulated ARPE-19 cells (Figure 3(e)). We also found that luteolin significantly inhibited iNOS protein expression and increased HO-1 protein expression (Figures 2(b) and 2(d)). These findings support that luteolin is indeed an anti-inflammatory phytochemical that can attenuate proinflammatory cytokine-induced inflammation in ARPE-19 cells. IL-1β activation of NF-κB signaling is closely associated with RD diseases [15,16] and pAKT is related to inhibition of the NF-κB inflammatory pathway [36]. Previous studies   Mediators of Inflammation show that IL-1β-activates NF-κB, resulting in its translocation from the cytoplasm into the nucleus, followed by induction of cytokine and chemokine expressions in ARPE-19 cells [43]. Our present results showed that luteolin promoted AKT phosphorylation (Figures 2(f) and 2(g)), inhibited NF-κB p65 activation, and suppressed THP-1 cell adhesion ( Figure 4). These findings suggest that luteolin promotes AKT phosphorylation to block NF-κB p65 activation and thereby suppresses THP-1 cell adherence to IL-1β-stimulated ARPE-19 cells. Many studies have indicated that MAPK signaling plays an important role in AMD [44]. We observed that luteolin decreased the expression levels of IL-6, IL-8, sICAM-1, and MCP-1 in IL-1β-stimulated ARPE-19 cells. Thus, we further investigated whether luteolin attenuated   Mediators of Inflammation inflammation by suppressing MAPK pathways. Our results demonstrated that luteolin significantly inhibited the phosphorylation of the MAPKs JNK 1/2, ERK 1/2, and p38, supporting that luteolin may block MAPK pathways to decrease production of IL-6, IL-8I, sICAM-1, and MCP-1 ( Figures 5(a), 5(b), 6(a), 6(b), 7(a), and 7(b)).