Interferon Regulatory Factors in Microglia and their role in retinal homeostasis and formation of choroidal neovascularization

Abstract

Purpose : Interferon regulatory factors (Irf1-9) are transcription factors that play an important role in differentiation, cell growth and apoptosis of myeloid cells, such as retinal microglia (MG). The aim of this study was to determine the expression pattern of Irf1-9 in retinal MG and to explore the role of the most abundantly expressed member Irf8 in tissue homeostasis, MG activation and laser-induced choroidal neovascularization (CNV).

Methods : Retinal MG cells defined as CD45⁺CD11b⁺Ly6C^-Ly6G^- of Cx3cr1^GFP/+ mice were sorted by fluorescence-activated cell sorting (FACS) and processed for RNA sequencing. To explore Irf8 dependent downstream factors, adult Irf8^-/- Cx3cr1^GFP/+ mice (Irf8 KO) and age-matched Irf8^+/+ Cx3cr1^GFP/+ mice were phenotyped by flow cytometry. Furthermore, Irf8 KO and control mice were characterized using fundus imaging, optical coherence tomography and electroretinography. MG morphology was assessed by immunohistochemistry (IHC) and 3D-IMARIS reconstruction. Finally, the role of Irf8 in the formation of laser-induced CNV was analyzed by RNA sequencing, fundus fluorescein angiography (FAG) and IHC.

Results : RNASeq data demonstrated that Irf8 is the most abundantly expressed Irf family member in retinal MG. Irf8 KO mice exhibited a severely altered MG morphology in both inner plexiform layer (IPL) and outer plexiform layer (OPL) and reduced cell numbers compared to controls. FACS analysis demonstrated a downregulation of MG markers such as CD64 (p<0.001) and CX3CR1 (p<0.05) and an upregulation of CD14 (p<0.01) and F4/80 (p<0.05) in Irf8 KO mice indicating a disturbed cell homeostasis. In vivo experiments however revealed no structural and functional retinal changes in Irf8 KO mice. In the laser-induced CNV model, Irf8 deficiency was associated with reduced MG cell number around CNV (p<0.001) and an increased CNV size (p<0.005). RNASeq data revealed a downregulation of MG markers (CD11b, Hexb and Sall1), cell migratory genes (Adora1, Adora3, Adgrg1, P2ry12) and angiogenic genes (Axl, Ctsh, Ctss) seven days following laser-CNV induction.

Conclusions : Irf8 plays an important role in retinal MG homeostasis and pathological angiogenesis but does not influence retinal structure and function. Our findings indicate that Irf8 is critical for transforming resident MG into a reactive phenotype thereby suppressing retinal inflammation and formation of CNV.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.