A ROLE FOR INTERLEUKIN-34 IN ZEBRAFISH RETINAL PIGMENT EPITHELIUM REGENERATION

Abstract

Purpose : In zebrafish, immune responses drive intrinsic regeneration in many tissues, including the retinal pigment epithelium (RPE). Indeed, using RNA-seq, we previously identified myriad immune-related differentially expressed genes (DEGs) upregulated in RPE during regeneration, including interleukin-34 (il34), a cytokine involved in leukocyte recruitment and differentiation, among other functions. Here, we aim to validate a role for il34 during RPE regeneration.

Methods : Zebrafish harboring a mutation in il34^re03 and an RPE-specific ablation transgene (rpe65a:nfsB-eGFP) were utilized, and experiments were performed on eGFP⁺ il34^re03/re03 and il34^+/+ siblings. In some cases, macrophages/microglia were visualized using mpeg1:mCherry. Mature RPE were ablated in vivo by immersing 5 days post-fertilization (dpf) larvae in 10mM metronidazole (MTZ) for 24 hours. Larvae were processed for fixed tissue analyses at 2, 3, and 4 days post-injury (dpi). il34 expression was confirmed by in situ hybridization and pigment recovery was used as a metric to assess the extent of RPE regeneration. Pigmentation was measured using RpEGEN, an automated MATLAB-based platform, which quantifies pixel intensity values from the dorsal-most (0°) to ventral-most (180°) angular distance of the RPE. Image analysis and quantification of macrophage/microglia activity was performed in FIJI.

Results : il34 expression was specific to the central-most RPE injury site at 2dpi (n=3) when compared to 7dpf sibling controls (n=3). Analysis utilizing RpEGEN revealed significantly less pigmentation in 4dpi il34^re03/re03 larvae (n=22) when compared to il34^+/+ siblings (n=22). Angular areas of significance spanned 30°–87° and 95°–113° along the RPE, demonstrating a lack of central pigment recovery in il34 mutants. Despite the mutation, there was no significant difference in macrophage/microglia infiltration at 3dpi in il34^re03/re03 larvae (n=20) when compared to il34^+/+ siblings (n=14; p=0.5546). However, il34^re03/re03 larvae did show significant retention of eGFP⁺ cellular debris (p≤0.0001), indicating a possible phagocytic deficit in macrophages/microglia that is required for RPE regeneration.

Conclusions : Preliminary analyses support a role for il34 in macrophage/microglia function during intrinsic RPE regeneration. Broadly, these results also validate a top DEG from our initial RNA-seq screen, and other genes and pathways of interest are being examined.

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