Müller cells may have stem cell-like capability as they regenerate photoreceptor loss upon injury in some vertebrates, but not in mammals. Indeed, mammalian Müller cells undergo major cellular and molecular changes summarized as reactive gliosis. Transforming growth factor beta (TGFβ) isoforms are multifunctional cytokines that play a central role, both in wound healing and in tissue repair. Here, we studied the role of TGFβ isoforms and their signaling pathways in response to injury induction during tissue regeneration in zebrafish and scar formation in mouse. Our transcriptome analysis showed a different activation of canonical and non-canonical signaling pathways and how they shaped the injury response. In particular, TGFβ3 promotes retinal regeneration via Smad-dependent canonical pathway upon regulation of junb gene family and mycb in zebrafish Müller cells. However, in mice, TGFβ1 and TGFβ2 evoke the p38MAPK signaling pathway. The activation of this non-canonical pathway leads to retinal gliosis. Thus, the regenerative versus reparative effect of the TGFβ pathway observed may rely on the activation of different signaling cascades. This provides one explanation of the different injury response in zebrafish and mouse retina.

Background: Retinal degeneration is a disease affecting the eye, which is an immune-privileged site because of its anatomical and physiological properties. Alterations in retinal homeostasis—because of injury, disease, or aging—initiate inflammatory cascades, where peripheral leukocytes (PL) infiltrate the parenchyma, leading to retinal degeneration. So far, research on PL's role in retinal degeneration was limited to observing a few cell types at specific times or sectioning the tissue. This restricted our understanding of immune cell interactions and response duration. Methods: In vivo microscopy in preclinical mouse models can overcome these limitations enabling the spatio-temporal characterization of PL dynamics. Through in vivo imaging, we assessed structural and fluorescence changes in response to a focal injury at a defined location over time. We also utilized minimally invasive techniques, pharmacological interventions, and knockout (KO) mice to determine the role of PL in local inflammation. Furthermore, we investigated PL abundance and localization during retinal degeneration in human eyes by histological analysis to assess to which extent our preclinical study translates to human retinal degeneration. Results: We demonstrate that PL, especially T cells, play a detrimental role during retinal injury response. In mice, we observed the recruitment of helper and cytotoxic T cells in the parenchyma post-injury, and T cells also resided in the macula and peripheral retina in pathological conditions in humans. Additionally, we found that the pharmacological PL reduction and genetic depletion of T-cells reduced injured areas in murine retinas and rescued the blood–retina barrier (BRB) integrity. Both conditions promoted morphological changes of Cx3cr1⁺ cells, including microglial cells, toward an amoeboid phenotype during injury response. Interestingly, selective depletion of CD8⁺ T cells accelerated recovery of the BRB compared to broader depletions. After anti-CD8 treatment, the retinal function improved, concomitant to a beneficial immune response. Conclusions: Our data provide novel insights into the adaptive immune response to retinal injury in mice and human retinal degeneration. Such information is fundamental to understanding retinal disorders and developing therapeutics to modulate immune responses to retinal degeneration safely.

Background: Retinal degeneration results from disruptions in retinal homeostasis due to injury, disease, or aging and triggers peripheral leukocyte infiltration. Effective immune responses rely on coordinated actions of resident microglia and recruited macrophages, critical for tissue remodeling and repair. However, these phagocytes also contribute to chronic inflammation in degenerated retinas, yet the precise coordination of immune response to retinal damage remains elusive. Recent investigations have demonstrated that phagocytic cells can produce extracellular traps (ETs), which are a source of self-antigens that alter the immune response, which can potentially lead to tissue injury. Methods: Innovations in experimental systems facilitate real-time exploration of immune cell interactions and dynamic responses. We integrated in vivo imaging with ultrastructural analysis, transcriptomics, pharmacological treatments, and knockout mice to elucidate the role of phagocytes and their modulation of the local inflammatory response through extracellular traps (ETs). Deciphering these mechanisms is essential for developing novel and enhanced immunotherapeutic approaches that can redirect a specific maladaptive immune response towards favorable wound healing in the retina. Results: Our findings underscore the pivotal role of innate immune cells, especially macrophages/monocytes, in regulating retinal repair and inflammation. The absence of neutrophil and macrophage infiltration aids parenchymal integrity restoration, while their depletion, particularly macrophages/monocytes, impedes vascular recovery. We demonstrate that macrophages/monocytes, when recruited in the retina, release chromatin and granular proteins, forming ETs. Furthermore, the pharmacological inhibition of ETosis support retinal and vascular repair, surpassing the effects of blocking innate immune cell recruitment. Simultaneously, the absence of ETosis reshapes the inflammatory response, causing neutrophils, helper, and cytotoxic T-cells to be restricted primarily in the superficial capillary plexus instead of reaching the damaged photoreceptor layer. Conclusions: Our data offer novel insights into innate immunity's role in responding to retinal damage and potentially help developing innovative immunotherapeutic approaches that can shift the immune response from maladaptive to beneficial for retinal regeneration.

Glia antigen‐presenting cells (APCs) are pivotal regulators of immune surveillance within the retina, maintaining tissue homeostasis and promptly responding to insults. However, the intricate mechanisms underlying their local coordination and activation remain unclear. Our study integrates an animal model of retinal injury, retrospective analysis of human retinas, and in vitro experiments to gain insights into the crucial role of antigen presentation in neuroimmunology during retinal degeneration (RD), uncovering the involvement of various glial cells, notably Müller glia and microglia. Glial cells act as sentinels, detecting antigens released during degeneration and interacting with T‐cells via MHC molecules, which are essential for immune responses. Microglia function as APCs via the MHC Class II pathway, upregulating key molecules such as Csf1r and cytokines. In contrast, Müller cells act through the MHC Class I pathway, exhibiting upregulated antigen processing genes and promoting a CD8⁺ T‐cell response. Distinct cytokine signaling pathways, including TNF‐α and IFN Type I, contribute to the immune balance. Human retinal specimens corroborate these findings, demonstrating glial activation and MHC expression correlating with degenerative changes. In vitro assays also confirmed differential T‐cell migration responses to activated microglia and Müller cells, highlighting their role in shaping the immune milieu within the retina. In summary, our study emphasizes the involvement of retinal glial cells in modulating the immune response after insults to the retinal parenchyma. Unraveling the intricacies of glia‐mediated antigen presentation in RD is essential for developing precise therapeutic interventions for retinal pathologies.

Reactive gliosis is a hallmark of chronic degenerative diseases of the retina. As gliosis involves macroglia, we investigated their gliotic response to determine the role of S100β and intermediate filaments (IFs) GFAP, vimentin, and nestin during tissue repair in a laser-induced model of retinal degeneration. We validated the results with human retinal donor samples. Experiments were performed in zebrafish and mice using an argon laser (532 nm) to induce focal lesions in the outer retina. At different time points following injury induction, the kinetics of retinal degeneration and regeneration were assessed using hematoxylin and eosin staining (H&E). Immunofluorescence was performed to evaluate Müller cell (GS) and astrocyte (GFAP) injury response and to distinguish between both cell types. Additionally, staining was performed in human retinal sections containing drusen. Focal laser treatment elevated the expression of gliotic markers in the area of the damage, which was associated with increased expression of S100β, GFAP, vimentin, and nestin in mice and humans. In zebrafish, we detected S100β at the first time point, but not GFAP or nestin. Double-positive cells with the selected glia markers were detected in all models. However, in zebrafish, no double-positive GFAP/GS cells were found on days 10 and 17, nor were S100β/GS double-positive cells found on day 12. Macroglia cells showed a different pattern in the expression of IFs in degenerative and regenerative models. In particular, S100β may prove to be a target for suppressing chronic gliosis in retinal degeneration.

Wound repair in the retina is a complex mechanism, and a deeper understanding of it is necessary for the development of effective treatments to slow down or even prevent degenerative processes leading to photoreceptor loss. In this study, we harnessed a laser-induced retinal degeneration model (532-nm laser photocoagulation with 300 μm spot size, 60 ms duration and 60 mV pulse), enabling a profound molecular elucidation and a comprehensive, prolonged observation of the wound healing sequence in a murine laser-induced degeneration model (C57BL/6J mice, 6–12 weeks) until day 49 post-laser. Our observations included the expression of specific extracellular matrix proteins and myofibroblast activity, along with an analysis of gene expression related to extracellular matrix and adhesion molecules through RNA measurements. Furthermore, the administration of pirfenidone (10 mg/kg via drinking water), an anti-inflammatory and anti-fibrotic compound, was used to modulate scar formation after laser treatment. Our data revealed upregulated collagen expression in late regenerative phases and sustained inflammation in the damaged tissue. Notably, treatment with pirfenidone was found to mitigate scar tissue formation, effectively downregulating collagen production and diminishing the presence of inflammatory markers. However, it did not lead to the regeneration of the photoreceptor layer.