Abstract
Purpose :
Determine if NETs form in eyes with experimental uveitis, and measure the impact of inhibiting NET formation on ocular inflammation in Primed Mycobacterial Uveitis (PMU) and Experimental Autoimmune Uveitis (EAU).
Methods :
For chemical inhibition of NETs, uveitis was induced with either the PMU or IRBP immunization EAU protocol in C57BL6 mice. Mice were divided into groups treated with either PBS, dexamethasone (Dex), or chloride amidine (Cl-Am) a PAD4 inhibitor that significantly reduced NET formation in a mouse model of ANCA vasculitis. For genetic inhibition of NETs, PMU or EAU was induced in C57BL6 and PAD4 deficient animals. Ocular inflammation was scored on the day of peak inflammation in PMU using optical coherence tomography (OCT) or by fundus score in EAU. To detect intraocular NETs, immunohistochemistry (IHC) was performed on ocular sections with antibodies to detect myeloperoxidase (MPO), citrullinated H3, and extracellular DNA. Additionally, intraocular NET concentration was determined using an ELISA that detects extracellular DNA complexed with MPO. Comparisons between treatment groups were made using Kruskal Wallis or unpaired t-test. Differences in NET concentration between right and left eyes in PMU animals were compared using paired t-test. Significant differences were determined as p<0.05.
Results :
NETs were detected in inflamed eyes by IHC (A) and ELISA (B). NETs concentrations were significantly elevated in inflamed (right eyes) of PMU animals compared to uninflamed fellow (left eyes) for all treatments. In PMU, no treatment significantly decreased NET concentration compared to PBS. In EAU, treatment with Dex and Cl-Am decreased aqueous NET concentration compared to PBS, but not significantly. There was a mild, non-significant decrease in inflammation score in PMU animals treated with Cl-Am (C). In EAU, Cl- Am treatment also non-significantly decreased inflammation score compared to PBS (D). Only Dex significantly decreased fundus scores in EAU animals. PAD4 deficiency did not impact NET concentration or clinical score in PMU or EAU.
Conclusions :
NETs are present in eyes with uveitis, but their formation is not PAD4 dependent. Treatment with Cl-Am has a mild anti-inflammatory impact on uveitis, but this effect is likely mediated by inhibition of other protein deiminases and not PAD4
This is a 2021 ARVO Annual Meeting abstract.