Role of Chronic Inflammation in Myopia Progression: Clinical Evidence and Experimental Validation

Abstract

Prevention and treatment of myopia is an important public problem worldwide. We found a higher incidence of myopia among patients with inflammatory diseases such as type 1 diabetes mellitus (7.9%), uveitis (3.7%), or systemic lupus erythematosus (3.5%) compared to those without inflammatory diseases (p<0.001) using data from children (<18years old) in the National Health Insurance Research database. We then examined the inhibition of myopia by atropine in Syrian hamsters with monocular form deprivation (MFD), an experimental myopia model. We found atropine downregulated inflammation in MFD eyes. The expression levels of c-Fos, nuclear factor κB (NFκB), interleukin (IL)-6, and tumor necrosis factor (TNF)-α were upregulated in myopic eyes and downregulated upon treatment with atropine. The relationship between the inflammatory response and myopia was investigated by treating MFD hamsters with the immunosuppressive agent cyclosporine A (CSA) or the inflammatory stimulators lipopolysaccharide (LPS) or peptidoglycan (PGN). Myopia progression was slowed by CSA application but was enhanced by LPS and PGN administration. The levels of c-Fos, NF-κB, IL-6, and TNF-α were upregulated in LPS- and PGN-treated eyes and downregulated by CSA treatment. These findings provide clinical and experimental evidence that inflammation plays a crucial role in the development of myopia.

Keywords: Inflammation; Interleukin 6; Myopia; Nuclear factor κB; Tumor necrosis factor alpha; c-Fos.

Supplementary Fig. 1

Inflammatory molecules increased in occluded eyes and down-regulated by atropine (1%) treatment. MFD was induced in Syrian hamster and the myopic eyes treated with PBS or atropine (1%) for 21 days. Immunofluorescence staining of NF-κB, c-FOS, TNF-α, IL-6 and IL-10 in occluded (myopic) eyes.

Supplementary Fig. 2

Relative expression levels of NF-κB, c-FOS, TNF-α, IL-6 and IL-10 showed in Supplementary Fig. 1. The expression levels of each proteins in atropine treated occluded eyes were compared to PBS treated occluded eyes. The fluorescence intensities were determined by Image J software.

Supplementary Fig. 3

Expression levels of inflammation-related transcription factors following treatment with LPS or PGN. Immunohistochemical analysis of c-Fos and NF-κB expression in control (L) and MFD eyes (R).

Supplementary Fig. 4

Expression of inflammatory and anti-inflammatory cytokines upon treatment with LPS and PGN. Immunohistochemical analysis of TNF-α and IL-10 expression in control (L) and MFD eyes (R).

Supplementary Fig. 5

MFD was induced in guinea pigs by covering the right eye with a cloth attached to the skin at a distance of at least 1 cm from the eye.

Supplementary Fig. 6

Immunohistochemical analysis of MMP2, COL1, and TGF-β expression in control eyes (L) and MFD eyes (R) treated with 1% atropine (MFD/atropine) or PBS (MFD) or left untreated.

Supplementary Fig. 7

Immunohistochemical analysis of c-Fos and IL-10 expression in control eyes (L) and MFD eyes (R) treated with 1% atropine (MFD/atropine) or PBS (MFD) or left untreated.

Fig. 1

Association between systemic inflammatory diseases and myopia incidence. Cumulative incidence of myopia is shown for control subjects and patients with (a) type I diabetes mellitus, (b) uveitis, or (c) systemic lupus erythromatosus.

Fig. 2

Effect of atropine on the expression of genes and signaling pathways involved in tissue remodeling. (a) Effect of atropine on LPS-induced activation of NF-κB and AP1 via inhibition of the PI3K–AKT and MAPK pathways. ARPE-19 and primary retinal pigmented epithelial cells were treated with PBS (control), 100 ng/ml LPS, or LPS + 100 μM atropine for 30 min and then harvested for western blot analysis to determine the phosphorylation status of ERK (Thr202/Tyr204), AKT (Ser473), PI3K (p85(Tyr458)/p55(Tyr199)), NF-κB (p65, Ser536), and c-Fos (Ser32). Fold changes of phosphorylated signaling molecules were normalized to levels of non-phosphorylated proteins. The intensities of each band were determined by ImageJ software. (b) Primary scleral fibroblast cells were treated with or without 100 μM atropine for 24 h. Immunofluorescence analysis of MMP2 and COL1 expression in primary scleral fibroblasts; nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). (c, d) Gelatin zymograph of pro- and active MMP2 in ARPE-19 retinal pigment epithelial cells (c) or scleral fibroblasts (d) treated with 1 μg/ml LPS with or without atropine. The MMP2 inhibitor diacerein was used as the control.

Fig. 3

Levels of tissue remodeling proteins and inflammatory molecules increase over time in occluded eyes. MFD was induced in Syrian hamsters, and the occluded eyes were collected at days 0, 2, 7, 14 and 21. (a) Immunohistochemical analysis of TGF-β, MMP2, and collagen I expression in occluded eyes. (b) Immunohistochemical analysis of NF-κB, c-FOS, TNF-α, IL-6, and IL-10 levels in occluded eyes.

Fig. 4

Effect of atropine on myopia progression. (a) The RE was determined as the difference in diopter measurements taken after and before MFD. The ANOVA test was used to determine significant differences (p < 0.0001), and Dunnett's multiple comparisons test was used for paired comparisons between PBS- and atropine-treated eyes. p < 0.05 was considered statistically significant. (b) The axial length was determined as the difference in diopter measurements taken after and before MFD. ANOVA was used to determine significant differences (p = 0.0042) and Dunnett's multiple comparisons test was used for paired comparisons between PBS- and atropine-treated eyes. p < 0.05 was considered statistically significant. (c–g) Immunohistochemical analysis of mAChR1 and 3 (c), MMP2 and COL1 (d), TGF-β (e), c-FOS and NF-κB (f), and IL-6, TNF-α, and IL-10 (g) in control eyes (control [L]), occluded eyes (control [R]), 1% atropine-treated control eyes (atropine [L]), and 1% atropine-treated occluded eyes (atropine [R]).

Fig. 5

Effect of suppressing or increasing inflammation in the eye on myopia progression. (a) The RE was determined as the difference in diopter measurements taken after and before MFD in hamsters treated for 21 days with 3% CSA. (b) Immunohistochemical analysis of TGF-β and MMP2 expression in control eyes (control [L]), occluded eyes (control [R]), 3% CSA-treated control eyes (CSA [L]), and 3% CSA-treated occluded eyes (CSA [R]). (c) The refractive error was determined as the difference in diopter measurements taken after and before MFD in hamsters treated for 21 days with LPS or PGN. *Indicates statistically significant compared with the untreated control. (d) Immunohistochemical analysis of TGF-β and MMP2 expression in control eyes ([L]) and form-deprived occluded eyes ([R]) with or without LPS or PGN. (e) The refractive error was determined as the difference in diopter measurements in hamsters treated for 21 days with LPS or PGN. *Indicates statistically significant compared with PBS-treated control. (f) Immunohistochemical analysis of TGF-β, MMP2, and collagen I expression in eyes treated with or without LPS or PGN.

Fig. 6

Expression levels of inflammation-related transcription factors and cytokines in cyclosporine A-treated myopic eyes. (a) Immunohistochemical analysis of c-Fos and NF-κB expression in control eyes (control [L]), occluded eyes (control [R]), 3% CSA-treated control eyes (CSA [L]), and 1% atropine-treated occluded eyes (CSA [R]). (b) Immunohistochemical analysis of IL-6, TNF-α, and IL-10 expression in control eyes (control [L]), occluded eyes (control [R]), 3% CSA-treated control eyes (CSA [L]), and 3% CSA-treated occluded eyes (CSA [R]).

Fig. 7

Model of the association between inflammation and myopia progression. Activated mAChR3 (M3R) activates phosphoinositide 3-kinase (PI3K)–AKT and mitogen-associated protein kinase (MAPK) signaling pathways, in turn activating NF-κB and AP1 (i.e., the Jun.-Fos heterodimer) and stimulating the expression of the target genes NF-κB, MMP2, TGFβ, IL-1β and -6, and TNF-α. MMP2 and TGF-β promote tissue remodeling and TNF-α may act in a paracrine feedback loop in the retina or sclera to activate NF-κB during myopia progression.