Jeremy A. Lavine

Background: Age-related macular degeneration (AMD) is characterized by disruption of the choriocapillaris (CC) and retinal pigment epithelium (RPE) dysfunction, leading to drusen accumulation. The CC and RPE form a tightly interdependent unit that maintains homeostasis where the CC supplies oxygen and nutrients to the RPE, while the RPE produces vascular endothelial growth factor (VEGF) to maintain the CC. Genetic studies link alternative complement pathway variants to AMD, and complement deposition on the CC increases during both aging and AMD. Macrophages express complement protein, receptors, and inhibitors, suggesting that they may be a missing link in understanding the role of complement in AMD. In support, previous groups have shown that macrophage depletion disrupts RPE-CC homeostasis, leading to AMD-like pathology, but the mechanism remains unclear. Methods and results: To investigate the role of macrophages in CC–RPE homeostasis, we generated Cx3cr1 ^CreER Csf1r ^i−DTR and Ms4a3 ^Cre Rosa26 ^DTR mice. In Cx3cr1 ^CreER Csf1r ^i−DTR mice, tamoxifen administration induced diphtheria toxin receptor (DTR) expression, allowing ablation of all macrophages (many tamoxifen injections) or long-lived, tissue-resident macrophages (tamoxifen followed by a 3–4 week wash out period). Ms4a3 ^Cre Rosa26 ^DTR mice were used to deplete monocyte-derived macrophages. Ablation of all macrophages caused decreased CC density, increased CC apoptosis, RPE disorganization, and membrane attack complex (MAC) accumulation. Tissue-resident macrophage ablation phenocopied this result while monocyte-derived macrophage ablation had no phenotype. Additionally, long-term depletion of tissue-resident macrophages led to formation of drusen-like sub-RPE deposits and retinal thinning, mimicking AMD pathology. Finally, pharmacologic depletion of all macrophages similarly reduced CC density to genetic ablation, but C3 ^−/− mice showed an attenuated phenotype. Conclusions: These data demonstrate that long-lived, tissue-resident macrophages are essential for maintaining CC–RPE homeostasis while monocyte-derived macrophages are dispensable in this context. Further, ocular macrophage ablation led to MAC accumulation, while C3 ^−/− mice were resistant to CC regression. Together, these findings suggest that tissue-resident choroidal macrophages maintain CC-RPE homeostasis partially by complement dependent mechanisms. Further, the loss of tissue-resident choroidal macrophages over time is a potential mechanism of AMD pathogenesis.

Perivascular macrophages (pvMacs) exist on venules at the blood-retina barrier (BRB) and are important for leukocyte transendothelial migration during retinal inflammation. The venular basement membrane contains low expression regions (LERs), which act as permissive sites for extravasation in non-ocular tissues. We sought to identify LERs in retinal venules and determine their relationship with pvMacs at steady state and during inflammation. Retinal flatmounts from Pf4Cre:Rosa26zsGreen/+ mice were analyzed by confocal microscopy followed by three-dimensional reconstruction to visualize pvMacs, collagen IV, laminin, and pericytes. Qualitative and quantitative analysis of LERs was performed at steady state and after induction of the endotoxin-induced uveitis (EIU) model. The role of pvMacs and macrophages in EIU were investigated using diphtheria toxin (DT) in Pf4Cre:Rosa26DTR/+ mice or a colony-stimulating factor 1 receptor (CSF1R) antagonist, respectively. Retinal venules contained discrete LERs characterized by reduced expression of both collagen IV and laminin with concurrent absence of pericyte coverage. pvMacs resided within LERs and adjacent to LERs. During EIU, neutrophils extravasated through pvMac-adjacent LERs, resulting in increased LER area and irregular morphology. Extravascular neutrophils co-localized with collagen IV fragments, suggesting basement-membrane degradation. Depletion of pvMacs or all macrophages did not alter neutrophil or monocyte infiltration, indicating that EIU-driven inflammation is macrophage independent. LERs are features of retinal venules that serve as permissive sites for immune cell extravasation and are closely associated with pvMacs. Although macrophages are dispensable for EIU, pvMac-LER complexes likely represent regulated portals for leukocyte trafficking and are potential loci of chronic BRB breakdown during retinal inflammation.

The retina is a central nervous system tissue with immune-privileged status, protected by the blood-retina barrier and maintained by specialized resident tissue macrophages: microglia, hyalocytes, and perivascular macrophages. These cells exhibit specific ontogeny, spatial localization, and immunologic functions. Each population contributes to homeostasis, phagocytosis, and immunoregulation in their local microenvironment. Microglia are the most abundant immune cells in the retina, and their key functions include vascular and synaptic regulation. Hyalocytes are abundant in the vitreous and at the vitreoretinal interface with key functions including vasoregression and maintenance of a clear visual axis. Perivascular macrophages are present along major venules in the superficial retina and may regulate immune cell transendothelial migration. This review summarizes key vitreoretinal myeloid cell functions, compares/contrasts the relative abundances of antigen presentation machinery and T cell costimulatory molecules, and places these expression levels into the context of immune surveillance in the experimental autoimmune uveitis model.

Levodopa (L-DOPA), a precursor for melanin and dopamine, has been linked to reduced intravitreal injection burden and delayed onset of neovascular age-related macular degeneration (AMD). Further, L-DOPA and dopamine receptor D2 (DRD2) agonists inhibit laser-induced choroidal neovascularization (CNV). However, the contributions of endogenous versus exogenous L-DOPA signaling, their effects in alternative CNV models, and the contributions of DRD2 versus GPR143, the receptor for L-DOPA, signaling remain unresolved. Choroidal sprouting assays (CSA) were performed using wild-type (WT) and tyrosinase-mutant (Tyr-/-) mice with and without dopamine pathway agonists and antagonists. CNV number and area were measured in pigmented Vldlr-/- and albino Vldlr-/-Tyr-/- mice with and without L-DOPA or the DRD2 agonist quinpirole. Endogenous L-DOPA deficiency (Tyr-/-) did not affect CSA sprouting or CNV in Vldlr-/- mice. Exogenous L-DOPA suppressed angiogenesis ex vivo in both WT and Tyr-/- choroidal explants in a dose-dependent manner. In vivo, L-DOPA reduced CNV lesion number, lesion area, and macrophage infiltration in albino but not pigmented Vldlr-/- mice. Dopamine and quinpirole produced modest anti-angiogenic effects, and eticlopride partially reversed L-DOPA inhibition in choroidal explants. Quinpirole suppressed CNV lesion number, lesion area, and macrophage infiltration in pigmented Vldlr-/- mice. Our findings show that L-DOPA's anti-angiogenic effects are exogenous, more effective in tyrosinase-mutant mice, and mediated by both the DRD2 and non-DRD2 pathways, potentially GPR143. The saturation of GPR143 signaling in pigmented eyes provides a mechanistic basis for reduced responsiveness, highlighting the importance of pigmentation biology in the development of L-DOPA-based therapeutics.

Effective therapies for treatment resistant neovascular age-related macular degeneration (nAMD) remain an unmet need. Beta-adrenergic receptor (AR) blockers can decrease laser-induced choroidal neovascularization (CNV) size in mice. We have shown that monocyte-derived macrophages (MDMs) and interleukin-6 (IL-6) are necessary for beta-AR blockers to inhibit CNV. However, the specific beta-AR and the mechanism of this pathway are not fully elucidated. We hypothesized that beta2-AR (Adrb2) signaling on MDMs increases IL-6 production and stimulates CNV. Previously published single-cell RNA-sequencing data was reanalyzed to determine which mononuclear phagocytes express beta-ARs. Adrb2flox/flox: Cx3cr1CreER/+ mice (Adrb2ΔMacs) or Adrb2flox/flox (Adrb2flox) controls were given tamoxifen injections at either four weeks before or at the time of laser-induced CNV to knockout Adrb2 in tissue resident or all macrophages, respectively. Mice underwent laser induced-CNV, and eyes were collected for choroidal wholemount immunofluorescence imaging to measure CNV area, multiparameter flow cytometry to analyze macrophage heterogeneity, and ELISAs to quantitate IL-6 levels. Adrb2 was the predominantly expressed beta-AR and was found on microglia, macrophages, and monocytes. Adrb2 deletion in tissue resident macrophages had no effect upon CNV area. Adrb2 deletion in all macrophages decreased CNV area by 1.4-fold. Adrb2ΔMacs posterior eye cups demonstrated similar levels of pro-angiogenic CD11c+ macrophages compared to Adrb2flox controls, but Ly6CnegCD11cneg macrophages were significantly increased. IL-6 levels increased with laser in Adrb2flox controls, but IL-6 levels in Adrb2ΔMacs posterior eye cups were unchanged. Beta2-AR deletion in ocular-infiltrating macrophages decreases laser-induced CNV area. Beta2-AR expression regulates IL-6 expression in monocyte-derived macrophages.