Loss of neurofibromin enhances Inflammation in the Oxygen-induced Retinopathy Model

Abstract

Purpose : Persons with neurofibromatosis type 1 (NF1) experience chronic inflammation and oxidative stress and develop a characteristic retinal neovascularization (NV) that mimics features of Retinopathy of prematurity (ROP). We propose that neurofibromin, the NF1 gene product, controls macrophage polarization and loss of neurofibromin in myeloid cells enhances NV in the OIR model.

Methods : We used a mouse model deficient in Nf1 in myeloid cells (Nf1^-/- MΦ) selectively. Bone marrow Nf1^-/- and WT MΦs were assessed for functional changes in oxidative (O₂^-) and nitrative species (NO) production (Chemiluminescence). Metabolic profiles of polarized Nf1^-/- and WT MΦs were assessed by Seahorse XF analyzer. Next, Nf1^-/- and WT mice were subjected to OIR and P17 OIR retinas from each genotype were assessed for NV area, vessel dropout (Image J), and inflammatory/M1 vs reparative/M2 MΦs quantification (IHC and flow cytometry). Finally, blood samples from preterm infants with type 1 ROP and matched controls were analyzed for inflammatory monocyte frequency (flow cytometry). Two-tailed Student’s t-test and One-way ANOVA were used for statistical analysis.

Results : Compared to the WT MΦ, Nf1^-/-MΦ produced excessive quantities of O₂^- and NO and over-expressed iNOS after exposure to LPS and IFN-γ (*P<0.05), which is characteristic of inflammatory/M1 MΦ. M1MΦs are highly reliant on glycolysis, and Nf1^-/- murine M1MΦs exhibited enhanced metabolic potential and glycolysis compared to WT M1MΦs (**P<0.001). In the OIR model, Nf1^-/- MΦ pups demonstrated enhanced NV, increased vessel dropout, and an abundance of M1MΦs (F4/80+CD86+, P=0.001) near neovascular tufts compared to WT pups. Mechanistically, Nf1^-/- M1MΦs amplify Akt phosphorylation and suppress ERK kinase activity compared to WT M1MΦs. Both Akt and Erk inhibitors suppressed glycolysis in WT M1MΦs, but only Akt inhibition suppressed glycolysis in Nf1^-/- M1MΦs (*P<0.01). Finally, neonates with Type I ROP had increased numbers of circulating intermediate monocytes (CD14++CD16+) (*P<0.01) when compared to matched controls without ROP, mirroring results in NF1 persons.

Conclusions : We conclude that loss of neurofibromin promotes M1MΦ polarization, function, and glucose utilization via a PI3K-Akt dependent mechanism and that Nf1^-/-M1MΦs participate in NV (Figure 1). Further, our human data suggest that inflammatory monocyte/macrophages participate in human ROP.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Proposed scheme