Foxp3⁺ Tregs are recruited to the retina to repair pathological angiogenesis

Abstract

Purpose : Neovascular retinopathies are major causes of vision loss; yet current treatments to prevent the condition are inadequate. Although microglia activation has a causal role, whether Foxp3⁺ T regulatory cells (Tregs) are recruited to the retina and influence neovascular retinopathy is unknown. We hypothesized that Tregs are trafficked to the retina and their augmentation de-activates microglia and reduces vascular pathology.

Methods : Oxygen-induced retinopathy (OIR) was induced in C57BL/6J and Foxp3⁺ reporter mice by exposure to hyperoxia (75% O₂) between postnatal days (P) 7-12 and room air until P18. OIR mice were treated with an IL-2/anti-IL-2 mAb complex or the adoptive transfer of Tregs prior to or during hyperoxia. Comparisons were to room air controls. Retinal vasculopathy (confocal microscopy and ELISA’s for leakage and angiogenic factors) and Treg numbers in lymphoid organs and blood (flow cytometry) and retina (confocal microscopy) were measured (N=32-38 mice/group). Microglial activation was assessed in retina (flow cytometry, confocal microscopy) and in co-culture with Tregs and a CTLA-4 blocking antibody that inhibits suppressive Tregs (2 independent experiments each with 3 replicates). A one-way ANOVA followed by a Mann Whitney U test or a Student’s test was used for statistical analysis.

Results : In OIR, Treg numbers transiently increased in lymphoid organs and retina in the period of acute retinal ischemia at P13, but declined when neovascularization was established. Both treatments boosted Treg numbers in lymphoid organs and blood (up to 10.1-fold at P12 and 2.4-fold at P18) and retina (up to 4-fold at P18) and reduced retinal vaso-obliteration, neovascularization, leakage and pro-angiogenic mediators. Furthermore, Tregs contacted microglia, which were de-activated as demonstrated by a decrease in MHCII⁺CD45⁺CD11b⁺ cells and preservation of cell process length (p<0.01). These findings were corroborated in co-cultures, which exhibited reduced co-stimulatory molecules, CD40, CD80 and CD86 and the secretion of TNFα and IL-6, events that were reduced by CTLA-4 blockade.

Conclusions : We demonstrated for the first time that Tregs are recruited to the retina and play a critical role in repair of the vasculature. These findings indicate that manipulation of Treg numbers is a previously unrecognized, effective and promising novel avenue for therapies aimed at preventing blinding neovascular retinopathies.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.