Rohan Bir Singh1, Tomas Blanco1, Sharad K Mittal1, Yukako Taketani1, Sunil K Chauhan1, Yihe Chen1, Reza Dana2. 1. Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA. 2. Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA. Electronic address: Reza_Dana@meei.harvard.edu.
Abstract
PURPOSE: In this study, we quantify Pigment Epithelium-derived Factor (PEDF) secreted by corneal epithelial cells and evaluate its immunomodulatory functions in a murine model of dry eye disease (DED). METHODS: We induced DED in female C57BL/6 mice using a controlled environment chamber for 14 days. We quantified mRNA expression of Serpinf1 gene and PEDF protein synthesis by corneal epithelial cells (CEpCs) using RT-PCR and ELISA. CEpCs from normal or DED mice were cultured with IFNγ-stimulated-dendritic cells (DCs) for 24 h, and expression of MHC-II and CD86 by DCs was determined using flow cytometry. Next, we either added recombinant PEDF (rPEDF) or anti-PEDF antibody to co-culture, and DC expression of the above maturation markers was quantified. Lastly, we treated DED mice with either topical rPEDF, anti-PEDF Ab or murine serum albumin (MSA), and DC maturation, expression of pro-inflammatory cytokines, and DED severity were investigated. RESULTS: Serpinf1 mRNA expression and PEDF protein production levels by CEpCs were upregulated in DED. CEpCs from DED mice exhibited an enhanced suppressive effect on the expression of MHC-II and CD86 by DCs, compared to normal mice. This effect was abolished by blocking endogenous PEDF with anti-PEDF Ab or enhanced by supplementing with rPEDF. Treatment with anti-PEDF antibody blocked the effect of endogenous-PEDF and increased DC maturation, expression of pro-inflammatory cytokines in conjunctivae, and exacerbated disease severity in DED mice. Conversely, topical rPEDF enhanced the suppressive effect of endogenous PEDF on DC maturation, decreased expression of pro-inflammatory cytokines in conjunctivae, and reduced disease severity. CONCLUSIONS: The results from our study elucidate the role of PEDF in impeding DC maturation, and suppression of ocular surface inflammation, explicating a promising therapeutic potential of PEDF in limiting the corneal epitheliopathy as a consequence of DED.
PURPOSE: In this study, we quantify Pigment Epithelium-derived Factor (PEDF) secreted by corneal epithelial cells and evaluate its immunomodulatory functions in a murine model of dry eye disease (DED). METHODS: We induced DED in female C57BL/6 mice using a controlled environment chamber for 14 days. We quantified mRNA expression of Serpinf1 gene and PEDF protein synthesis by corneal epithelial cells (CEpCs) using RT-PCR and ELISA. CEpCs from normal or DED mice were cultured with IFNγ-stimulated-dendritic cells (DCs) for 24 h, and expression of MHC-II and CD86 by DCs was determined using flow cytometry. Next, we either added recombinant PEDF (rPEDF) or anti-PEDF antibody to co-culture, and DC expression of the above maturation markers was quantified. Lastly, we treated DED mice with either topical rPEDF, anti-PEDF Ab or murineserum albumin (MSA), and DC maturation, expression of pro-inflammatory cytokines, and DED severity were investigated. RESULTS:Serpinf1 mRNA expression and PEDF protein production levels by CEpCs were upregulated in DED. CEpCs from DED mice exhibited an enhanced suppressive effect on the expression of MHC-II and CD86 by DCs, compared to normal mice. This effect was abolished by blocking endogenous PEDF with anti-PEDF Ab or enhanced by supplementing with rPEDF. Treatment with anti-PEDF antibody blocked the effect of endogenous-PEDF and increased DC maturation, expression of pro-inflammatory cytokines in conjunctivae, and exacerbated disease severity in DED mice. Conversely, topical rPEDF enhanced the suppressive effect of endogenous PEDF on DC maturation, decreased expression of pro-inflammatory cytokines in conjunctivae, and reduced disease severity. CONCLUSIONS: The results from our study elucidate the role of PEDF in impeding DC maturation, and suppression of ocular surface inflammation, explicating a promising therapeutic potential of PEDF in limiting the corneal epitheliopathy as a consequence of DED.
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Authors: Rohan B Singh; Tomas Blanco; Sharad K Mittal; Hamid Alemi; Sunil K Chauhan; Yihe Chen; Reza Dana Journal: Am J Pathol Date: 2021-01-14 Impact factor: 4.307