Activation of the arginase 1/ornithine pathway suppresses ischemia/reperfusion‐induced neuronal injury by suppressing HDAC3

Abstract

PurposeWe have discovered that treatment with PEGylated arginase 1 (PEG‐A1) protects against inflammation and neurovascular injury in retina models of ischemia/reperfusion (IR) injury whereas deletion of myeloid A1 exacerbates the damage. We also found that PEG‐A1 treatment limits LPS‐induced inflammation in macrophages (MΦ) by reducing inducible nitric oxide synthase (iNOS)‐derived nitrative and oxidative stress and promoting a reparative MΦ phenotype. This project aimed to elucidate the mechanisms underlying the protective effects of A1. Arginase hydrolyzes L‐arginine to form L‐ornithine and urea. Ornithine decarboxylase (ODC) converts Lornithine to putrescine and polyamines which can inhibit histone deacetylase 3 (HDAC3). Thus, we examined the involvement of ODC and HDAC3 in retinal IR injury.MethodsPeritoneal MΦ were isolated from myeloid A1 KO or floxed control mice and treated with LPS (100 ng/mL) ± PEG‐A1 (1 μg/mL), the ODC inhibitor DFMO (5 mM), or the specific HDAC3 inhibitor RGFP966 (2 μM), for 24 h. Expression of HDACs was examined by qPCR and western blotting. Levels of inflammatory markers, iNOS, NLRP3, pro‐interleukin (IL)‐1β, and tumor necrosis factor α (TNF‐α) were measured using western blotting on cell lysates and nitric oxide (NO) production was measured in media. A cohort of WT mice were subjected to retinal IR injury and treated with RGFP966 (10 mg/Kg, i.p., 1h after IR and then every 48 h). Retinal thickness was examined in live mice using optical coherence tomography (OCT) at 7 days followed by sacrifice and assessment of neurodegeneration using NeuN labeling on retina flat mounts.ResultsA1 deletion or ODC inhibition increased HDAC3 expression and exacerbated the inflammatory response (iNOS, NLRP3, pro‐IL‐1β) in LPS‐treated MΦ. PEG‐A1 treatment dampened the upregulation of HDAC3 and reduced the inflammatory response. HDAC3 inhibition ameliorated the inflammatory response in A1 KO MΦ by reducing NO production and TNF‐α expression. In vivo, HDAC3 co‐localized with Iba1+ microglia/MΦ at 48 h after IR in WT retina sections and was further increased in A1‐deficient, IR‐injured retinas as measured by western blotting. HDAC3 inhibition improved neuronal cell survival and prevented retinal thinning. The number of surviving neurons with HDAC3 inhibitor treatment was 74±15% of sham vs 63±12% with vehicle (mean±S.D.) and retinal thickness was 99±3% of sham vs 92±4% with vehicle at 7 days after IR.ConclusionA1 mediates its anti‐inflammatory effects via ODC‐mediated suppression of HDAC3, which is a central player in the MΦ inflammatory response. Targeting HDAC3 offers a novel strategy for limiting neurovascular injury and promoting repair in the early stages of ischemic retinopathy.Support or Funding InformationThis work was supported by: NIH grant R01‐EY11766 to R.B.C., VA grant BX001233 to R.B.C., the Culver Vision Discovery Institute at Augusta University, and AHA postdoctoral fellowship 18POST34060036 to A.Y.F.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.