Targeting neutrophils as a novel approach for AMD therapy

Abstract

Purpose : Microglia have been established as immunomodulatory cells in age-related macular degeneration (AMD). However, while other pro-inflammatory cell types, such as neutrophils, have been shown to be important in various inflammatory diseases, a role for neutrophils in AMD remains uncertain. This study was undertaken to determine whether neutrophils are involved in AMD progression.

Methods : Human AMD and age-matched control sections from the posterior pole were stained with anti-neutrophil elastase and co-immunostained with antibodies to Lipocalin-2 (LCN-2). Retina from 5 and 10 month old Cryba1^fl/fl (WT) or Cryba1 KO (lacks the Cryba1 gene, which encodes for bA3/A1-crystallin) mice were used for RNAseq analysis. One year old Cryba1^fl/fl and Cryba1 cKO (lacks the Cryba1 gene in RPE cells) mice were injected with DMSO (vehicle control) or Triciribine (a potent inhibitor of AKT2) intravitreally and western blots were performed on extracts from the treated and control retinas for AKT2, NF-kB–p65 and LCN-2.

Results : Our data show significant neutrophil infiltration in the sub-macular choroid and retina of human AMD samples compared to age-matched controls. Interestingly, the neutrophils in the control retina do not express LCN-2, but the infiltrating neutrophils in the AMD patient samples show elevated expression of LCN-2. Our previous data indicated that LCN-2 plays a pivotal role in activating the inflammatory response in AMD. RNAseq analysis from retinal samples obtained from Cryba1 KO mice suggested that there is significant increase in the expression of pro-inflammatory genes which are known to trigger neutrophil infiltration into inflammatory foci. These inflammatory changes were also associated with increased microglial activation in the retina of the Cryba1 KO mice. Moreover, retinal samples from Cryba1 cKO and human AMD patient samples showed increased expression of LCN-2, NF-kB-p65 and AKT2 with respect to control. Intravitreal Triciribine treatment to Cryba1 cKO mice significantly ameliorated LCN-2 expression by inhibiting the AKT2-dependent pathway in the retina.

Conclusions : Our studies suggest a critical role for neutrophils during the inflammatory response in AMD. Association of increased microglial activation with neutrophil infiltration during AMD could be of immense importance in understanding the para to chronic inflammatory transition in the disease and thereby provide novel therapeutic strategies to counter AMD.

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