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Lei Wang, Yara Gorashi, Marisol Cano, Katayoon B Ebrahimi, James T Handa; PTX3 Suppresses 4-HNE Induced Production of IL-1b, but not IL-18, by Retinal Pigment Epithelial (RPE) Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5808.
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© ARVO (1962-2015); The Authors (2016-present)
PTX3, a prognostic biomarker for diseases involving chronic, low-grade inflammation, plays an active anti-inflammatory role in certain medical conditions. Our earlier studies have shown that PTX3 is locally expressed by RPE cells, and its dysfunction is linked to the development of age-related macular degeneration (AMD). Here, we want to further explore how PTX3 modulates the specific production of IL-1b, a pro-inflammatory cytokine associated with degenerative diseases, from stressed RPE cells and the implications for AMD.
Two month old C57Bl6J mice, or PTX3 deficient (PTX3-/-) mice were given 5 ug/ml 4-HNE by intravitreal injection in one eye and an equal volume of vehicle in the other. After 24 h, eyes were fixed, cryopreserved, and sectioned for immunostaining. Total RNA and protein were extracted from RPE/choroids for quantitative PCR and western blot analysis. ARPE-19 cells were transfected with siRNA to PTX3, or scrambled control, and treated with 50 uM 4-HNE for 24 h. Protein was collected for western blot and ELISA.
After an injection of sub-lethal dose of 4-HNE to the wt mice, increased expression of IL-1b, but not IL-18 (p>0.05), were observed at both mRNA and protein levels (1.4-, 1.6- fold, p<0.05). This increase was accompanied by a 2.8 fold (p<0.05) elevation in Casp1, a key component of inflammasome that cleaves IL-1b to its mature active form, suggesting that IL-1b production by RPE cells is modulated at both the transcriptional and post-translational level. In PTX3-/- mice injected with 4-HNE, IL-1b production and inflammasome activation were both exacerbated, but pre-injection with recombinant PTX3 could rescue the profile. Importantly, PTX3-/- mice showed increased F4/80 macrophages in the choroid relative to wt mice, reflecting the influence of enhanced IL-1b production. Observations in mice were then confirmed using human ARPE-19 cells. Noticeably, PTX3 knockdown exacerbated the 4-HNE induced secretion of IL-1b into the culture medium, but SB290157, a selective C3a receptor antagonist, abrogated the effect of PTX3 knockdown, indicating that C3a plays a key role in the PTX3 modulation of IL-1b production in RPE cells.
PTX3, via a complement-dependent mechanism, selectively suppresses the oxidative stress induced production of IL1b in RPE cells. Further characterization of PTX3’s role may lead to a therapeutic solution for AMD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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