The immune response is a critical regulator of zebrafish retinal pigment epithelium regeneration

Abstract

Loss of the retinal pigment epithelium (RPE) due to dysfunction or disease can lead to blindness in humans. Harnessing the intrinsic ability of the RPE to self-repair is an attractive therapeutic strategy; however, mammalian RPE is limited in its regenerative capacity. Zebrafish possess tremendous intrinsic regenerative potential in ocular tissues, including the RPE, but little is known about the mechanisms that drive RPE regeneration. Here, utilizing zebrafish, we identified elements of the immune response as critical mediators of intrinsic RPE regeneration. Macrophages/microglia are responsive to RPE damage and are required for the timely progression of the regenerative response and our data highlight that inflammation post-RPE injury is also critical for normal RPE regeneration. To our knowledge, these data are the first to identify the molecular and cellular underpinnings of RPE regeneration in any system and hold significant potential for translational approaches aimed towards promoting a pro-regenerative environment in mammalian RPE.

Retinal ganglion cell survival after severe optic nerve injury is modulated by crosstalk between JAK/STAT signaling and innate immune responses in the zebrafish retina

“…2) and therefore we investigated how components of the innate immune system respond to ONT in zebrafish and whether they contribute to RGC vulnerability. We focused on macrophages/microglia, leukocytes that accumulate in the zebrafish retina after a variety of injury types and facilitate repair and regeneration (Leach et al, 2020; White et al, 2017). In zebrafish, the 4C4 antibody recognizes an unidentified protein expressed by macrophages/microglia (Craig et al, 2008).…”

“…CSF1R activity is required for macrophage/microglia differentiation (Lin et al, 2008; Sherr et al, 1985) and PLX3397 has been utilized effectively in zebrafish (e.g. Conedera et al, 2019; Leach et al, 2020; Van Dyck et al, 2021). Animals were immersed in 500nM PLX3397 one day prior to ONT and retinae were collected at 7dpi.…”

“…Library preparation, quality control analysis, and next generation sequencing were performed by the Health Sciences Sequencing Core at Children’s Hospital of Pittsburgh as previously described (Leach et al, 2020). cDNA sequencing libraries were prepared using a SmartSeq HT kit (Takara Bio) and Illumina Nextera XT kit (Illumina Inc.).…”

mTOR activity is essential for retinal pigment epithelium regeneration in zebrafish

“…To test this hypothesis, we utilized mpeg1:mCherry;rpe65:nfsB-eGFP transgenic fish, in which macrophages and microglia are labeled by mCherry (52). MTZ + and MTZcontrol mpeg1:mCherry;rpe65:nfsB-eGFP larvae were treated with rapamycin or DMSO from 4dpf until 3dpi (Fig 6A ), the time at which macrophage/microglia infiltration peaks after RPE ablation (14). Immunostaining for mCherry revealed no visible accumulation of macrophages/microglia in the RPE layer of DMSO-treated MTZlarvae (Fig 6B ), while, as expected, significant macrophage/microglia recruitment to the RPE layer in DMSO-treated MTZ+ larvae was observed (Fig 6D ,G).…”

“…Indeed, inflammation is necessary for RPE and retinal regeneration in zebrafish (14,32,64). With this in mind, we utilized Dexamethasone (Dex) to dampen inflammation systemically (14,64) to determine whether inflammation was responsible for mTOR activity in the RPE layer at later stages post-injury. As expected, DMSO-treated MTZ + larvae showed significantly increased p-S6 levels in the RPE layer at 2dpi when compared to MTZcontrols (Fig 7K,M,O).…”