A Proinflammatory Function of Toll-Like Receptor 2 in the Retinal Pigment Epithelium as a Novel Target for Reducing Choroidal Neovascularization in Age-Related Macular Degeneration

Abstract

Current treatments for choroidal neovascularization, a major cause of blindness for patients with age-related macular degeneration, treat symptoms but not the underlying causes of the disease. Inflammation has been strongly implicated in the pathogenesis of choroidal neovascularization. We examined the inflammatory role of Toll-like receptor 2 (TLR2) in age-related macular degeneration. TLR2 was robustly expressed by the retinal pigment epithelium in mouse and human eyes, both normal and with macular degeneration/choroidal neovascularization. Nuclear localization of NF-κB, a major downstream target of TLR2 signaling, was detected in the retinal pigment epithelium of human eyes, particularly in eyes with advanced stages of age-related macular degeneration. TLR2 antagonism effectively suppressed initiation and growth of spontaneous choroidal neovascularization in a mouse model, and the combination of anti-TLR2 and antivascular endothelial growth factor receptor 2 yielded an additive therapeutic effect on both area and number of spontaneous choroidal neovascularization lesions. Finally, in primary human fetal retinal pigment epithelium cells, ligand binding to TLR2 induced robust expression of proinflammatory cytokines, and end products of lipid oxidation had a synergistic effect on TLR2 activation. Our data illustrate a functional role for TLR2 in the pathogenesis of choroidal neovascularization, likely by promoting inflammation of the retinal pigment epithelium, and validate TLR2 as a novel therapeutic target for reducing choroidal neovascularization.

Figure 1

Expression of Toll-like receptor 2 (TLR2; red) in the retinal pigment epithelium (RPE)/choroid area of aged eyes (≥77 years) without age-related macular degeneration (AMD) and with different stages of AMD. Aged eye (77 years) without AMD (A and B), aged eye with early AMD [Age-Related Eye Disease Study (AREDS) 1] (C and D), aged eye with intermediate stage AMD (AREDS 3) (E and F), aged eye with advanced AMD (G–J), showing regions without choroidal neovascularization (CNV) (G and H) and with CNV (I and J) (asterisks). Note the absence of the RPE layer beneath regions with CNV. Arrows indicate RPE; black arrowheads, TLR2-positive staining; green arrowheads, small drusen. Brm, Bruch's membrane; IgG Control, isotype match IgG; V, vessel in CNV. Scale bar = 10 μm (A–J).

Figure 2

Activation of NF-κB in the retinal pigment epithelium (RPE) during age-related macular degeneration (AMD) pathogenesis. A, B, D, and E: In aged eyes without any signs of AMD and with early AMD (≥77 years), only a few RPE cells display nuclear localization of NF-κB (white arrows). Cells with nuclear NF-κB staining in the choroid of the eyes with no AMD and with early AMD may be vascular endothelial cells and inflammatory cells (blue arrows). C and F–P: In aged eyes with intermediate AMD and advanced AMD (≥77 years), nuclear localization of NF-κB is detected in many RPE cells (white arrows). In the CNV membrane (asterisks), staining for nuclear NF-κB is unremarkable and limited to a few cells in the retina (green arrows) and choroid (blue arrows) (G and H). RPE cells with activated NF-κB are detected near the transition zone with geographic atrophy (dashed ovals) (I and J). NF-κB–positive cells in the retina (green arrows) are likely to be inflammatory cells because of their rounded nuclear morphologic structure. More nuclear NF-κB staining in choroidal cells is detected in eyes with intermediate AMD and advanced AMD (blue arrows). Scale bars: 20 μm (A–H and K–P); 50 μm (I and J). Brm, Bruch's membrane; CNV, choroidal neovascularization.

Figure 3

Expression of Toll-like receptor 2 (TLR2) in the retinal pigment epithelium (RPE) of wild-type C57BL/6J and JR5558 spontaneous choroidal neovascularization (CNV) mice. A–F:En face view of whole-mount eyecups (with the retinae removed). Staining shows robust TLR2 expression (red) on the RPE both around and at a distance from the CNV vessels [isolectin B4 (IB4); green], whereas little TLR2 staining of CNV vessels (IB4; green) is detected in the JR5558 mice (A–C). In the whole-mount eyecups from wild-type C57BL/6J mice, strong TLR2 expression is detected on the RPE (D–F ). G–J: In eye sections from JR5558 mice, expression of TLR2 is detected readily on the apical side of the RPE layer, whereas the CNV vessels (dotted oval) are largely negative for TLR2 expression (H). G and H are from a region with CNV, whereas I and J are from a CNV-free region. H and J are higher magnification images of the boxed areas in G and I, respectively. K–M: Double immunostaining of eye sections from JR5558 mice reveals colocalization of ezrin (green), a marker for microvilli of RPE, and TLR2 (red) to the apical surface of the RPE layer. N: Semiquantitative real-time PCR for TLR2 mRNA levels in both wild-type C57BL/6J and JR5558 mice, showing significantly higher levels of TLR2 mRNA in the RPE/choroid complex than in the retina. Data are expressed as means ± SEM. ^∗∗P < 0.01, ^∗∗∗P < 0.001 (one-way analysis of variance). Scale bars: 50 μm (A–C, H, and J–M); 20 μm (D–F); 100 μm (G and I). ONL, outer nuclear layer of the photoreceptors; WT, wild-type.

Figure 4

Intravitreal anti–Toll-like receptor 2 (TLR2) neutralizing antibody significantly suppresses spontaneous choroidal neovascularization (CNV) initiation and growth. A: Representative fundus fluorescein angiography (FFA) images showing the CNV lesions (hyperfluorescent spots; asterisks) in different treatment groups. Note the substantial reduction in the number of CNV lesions in the anti–vascular endothelial growth factor receptor 2 (VEGFR2) and anti-TLR2 combination treatment groups. B: Quantification of the FFA data from A, showing a significant reduction in the average number of CNV lesions per eye and the average CNV area per eye in the anti-VEGFR2 groups and the anti-TLR2 groups, both as individual treatment and as combination treatment. IgGs = rat IgG2A at 4.8 μg and mouse IgG1 at 3.2 μg per injection. Data are expressed as means ± SEM. n = 12 to 33 eyes. ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus IgG control-treated group (one-way analysis of variance); ^†P < 0.05, ^††P < 0.01 (one-way analysis of variance).

Figure 5

Toll-like receptor 2 (TLR2) antagonism in the eye reduces macrophage association with choroidal neovascularization (CNV) in the JR5558 mouse. Representative images of macrophage staining using whole-mount eyecups without retinae from all treatment groups. Although both anti–vascular endothelial growth factor receptor 2 (VEGFR2) and anti-TLR2 treatments effectively suppress growth of CNV vessels, only anti-TLR2 treatment reduces the number of CNV-associated macrophages (F4/80; red) in the eye. Macrophages and their nuclei in the vehicle group are highlighted with arrows, whereas some of the RPE cells and their nuclei are highlighted with arrowheads. Scale bar = 200 μm. IB4, isolectin B4 vascular staining (green).

Figure 6

Toll-like receptor 2 (TLR2) mediates expression of proinflammatory genes in primary human retinal pigment epithelium (RPE) cells. A: Pam2CSK4 (Pam2), a synthetic diacylated lipoprotein and TLR2-selective ligand, induces robust expression of IL6, MCP1, CXCL8, IL1B, and TLR2 by primary human RPE cells, an induction that is suppressed by 4 μg/mL TLR2-specific neutralizing antibody. B: Induction of IL1B, IL6, and TLR2 expression in human primary RPE cells by Pam2CSK4 is not affected by neutralizing antibodies against TLR1 (4 μg/mL) or TLR6 (4 μg/mL). Note that at least two different donor eyes were used for primary human RPE isolation and culture for this experiment. Data are expressed as means ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 (one-way analysis of variance).

Figure 7

Carboxyethylpyrrole (CEP)-dipeptide has a significant synergistic effect with the Toll-like receptor 2 (TLR2)-selective ligand Pam2CSK4 (Pam2) on inducing expression of proinflammatory genes in human primary retinal pigment epithelium (RPE) cells. A–C, E, and F: CEP alone does not induce the expression of IL6, MCP1, CXCL8, IL1B, or TLR2, whereas Pam2CSK4 significantly induces expression of these genes in human fetal RPE (hfRPE). Combination treatment with CEP and Pam2SCK4 results in a synergistic induction of these proinflammatory genes, and anti-TLR2 neutralizing antibody (4 μg/mL) significantly suppresses the effects of the CEP and Pam2CSK4 combination treatment. D: CEP, Pam2CSK4, and the combination fails to induce VEGFA expression compared with the control in hfRPE cells. Pam2CSK4 at 100 ng/mL (78.6 nmol/L) (A–D); Pam2CSK4 at 50 ng/mL (39.3 nmol/L) (E and F); CEP at 10 μg/mL (26.2 μmol/L) (A–F). Data are expressed as means ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 (one-way analysis of variance).

Figure 8

Synergistic effects of carboxyethylpyrrole (CEP)-dipeptide with gamma irradiation-inactivated Chlamydia pneumoniae (Cpn) in inducing Toll-like receptor 2 (TLR2)-mediated expression of proinflammatory genes in retinal pigment epithelium (RPE) cells. Primary human fetal RPE (hfRPE) cells were treated with 100 μg/mL Cpn alone or Cpn plus 26.2 μmol/L CEP for either 6 (A–E) or 48 (F–J) hours. A synergistic effect for IL6 expression is detected at 48 hours, whereas synergistic effects for the expression of MCP1, CXCL8, and IL1B are detected at both 6 and 48 hours. The induction of expression of proinflammatory genes by treatment with Cpn and CEP is significantly suppressed in the presence of an anti-TLR2 neutralizing antibody (4 μg/mL) (B–D and F–I). Treatment with Cpn either alone or in combination with CEP does not induce VEGFA expression in hfRPE at 6 or 48 hours compared with control. Anti-TLR2 antibody significantly suppresses background vascular endothelial growth factor (VEGF) expression in the control group at 6 hours and in the group treatment with Cpn plus CEP at 48 hours (E and J). Data are expressed as means ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 (one-way analysis of variance).