Peptide Inhibitor of MyD88 Increases Neuroprotective Microglia and Photoreceptor Survival

Abstract

Purpose : Myeloid differentiation factor 88 (MyD88) is an adaptor protein for Toll-like receptors and interleukin 1 receptors that mediates inflammatory responses to cellular injury through cytokine release and migration of microglia and macrophages to injury sites. MyD88 also regulates microglia polarization into neurotoxic or neuroprotective phenotypes. In this study, we investigated the consequences of inhibiting MyD88 signaling early in retinal degeneration.

Methods : Rd10 mice (Jackson Labs) were randomly divided into therapeutic and experimental groups and injected IP with 1, 2, or 3 mg/Kg body weight of MyD88 inhibitor (MI) or control peptides (Ctrl) (Novus Biologicals). Retina function was analyzed by ERGs, microglia/macrophage were quantified by immunostaining for IBA1 and Arg1 and flow cytometry, cytokine levels were quantified by cytoplex, and iTRAQ labeling for mass spectrometry based proteomic quantification used a Q Exactive instrument.

Results : 2 mg/Kg MI injected mice showed 3.7 fold (p=0.018) and 3 fold (p=0.024) decrease in apoptotic retinal cells compared to Ctrl and untreated rd10 mice, respectively. ERGs showed significantly higher scotopic responses in 2 mg/Kg MI injected mice compared to Ctrl or untreated mice (p<0.05). We demonstrated that the number of CD45^highCD11b⁺ macrophages and CD45^lowCD11b⁺ microglia in the retina did not differ between MI and Ctrl injected mice. However, IBA1⁺ cells in the ONL were reduced by half in MI injected mice compared with Ctrl (p=0.0254), suggesting reduced macrophage/microglia migration contributes to an anti-inflammatory environment. Furthermore, there were 2-fold more IBA1⁺ cells that were positive for the Arg1 marker of neuroprotective microglia (p=0.0058) in the MI injected mice. Cytokine analysis demonstrated that the anti-inflammatory cytokine IL-27 was significantly higher in MI injected mice compared with Ctrl (p=0.03), and intraocular injection of IL-27 into rd10 mice significantly increased photopic and scotopic ERG responses (p<0.05). Finally, proteomics analysis demonstrated upregulation of multiple anti-apoptotic chaperone proteins in MI injected mouse retinas.

Conclusions : Inhibiting MyD88 led to increased photoreceptor survival, which was associated with increased polarization into Arg+ neuroprotective microglia. Potential neuroprotective mechanisms involved increased IL-27 and chaperones.

This is a 2020 ARVO Annual Meeting abstract.