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Neovascular age-related macular degeneration (nAMD) commonly causes vision loss from aberrant angiogenesis, termed choroidal neovascularization (CNV). Interleukin-6 (IL6) is a pro-inflammatory and pro-angiogenic cytokine that is correlated with AMD progression and nAMD activity. We hypothesize that anti-IL6 therapy is a potential nAMD therapeutic. We found that IL6 levels were increased after laser injury and expressed by macrophages. Il6 -deficiency decreased laser-induced CNV area and exogenous IL6 addition increased choroidal sprouting angiogenesis. Il6 -null mice demonstrated equally increased macrophage numbers as wildtype mice. At steady state, IL6R expression was detected on peripheral blood and ocular monocytes. After laser injury, the number of IL6R + Ly6C + monocytes in blood and IL6R + macrophages in the eye were increased. In human choroid, macrophages expressed IL6 , IL6R , and IL6ST . Furthermore, IL6R + macrophages displayed a transcriptional profile consistent with STAT3 (signal transducer and activator of transcription 3) activation and angiogenesis. Our data show that IL6 is both necessary and sufficient for choroidal angiogenesis. Macrophage-derived IL6 may stimulate choroidal angiogenesis via classical activation of IL6R + macrophages, which then stimulate angiogenesis. Targeting IL6 or the IL6R could be an effective adjunctive therapy for treatment-resistant nAMD patients.

Background Diabetic retinopathy and retinal vein occlusion are vision threatening retinal vascular diseases. Current first-line therapy targets the vascular component, but many patients are treatment-resistant due to unchecked inflammation. Non-invasive inflammatory imaging biomarkers are a significant unmet clinical need for patients. Imaging of macrophage-like cells on the surface of the retina using clinical optical coherence tomography (OCT) is an emerging field. These cells are increased in patients with retinal vascular disease, and could be a potential inflammatory biomarker. However, since OCT is limited by an axial resolution of 5–10 microns, the exact location and identity of these retinal cells is currently unknown. Methods We performed OCT followed by confocal immunofluorescence in wild-type mice to identify macrophages within 5–10 microns of the vitreoretinal interface. Next, we used Cx3cr1 CreER/ + ; Rosa26 zsGreen/ + mice to fate map retinal surface macrophages. Using confocal immunofluorescence of retinal sections and flatmounts, we quantified IBA1 + Tmem119 + CD169 neg microglia, IBA1 + Tmem119 neg CD169 neg perivascular macrophages, and IBA1 + Tmem119 neg CD169 + vitreal hyalocytes. Finally, we modeled neuroinflammation with CCL2 treatment and characterized retinal surface macrophages using flow cytometry, OCT, and confocal immunofluorescence. Results We were able to detect IBA1 + macrophages within 5–10 microns of the vitreoretinal interface in wild-type mice using OCT followed by confirmatory confocal immunofluorescence. Retinal surface macrophages were 83.5% GFP + at Week 1 and 82.4% GFP + at Week 4 using fate mapping mice. At steady state, these macrophages included 82% IBA1 + Tmem119 + CD169 neg microglia, 9% IBA1 + Tmem119 neg CD169 + vitreal hyalocytes, and 9% IBA1 + Tmem119 neg CD169 neg perivascular macrophages. After CCL2-driven neuroinflammation, many Ly6C + cells were detectable on the retinal surface using OCT followed by confocal immunofluorescence. Conclusions Macrophages within close proximity to the vitreoretinal interface are self-renewing cells, and predominantly microglia with minor populations of perivascular macrophages and vitreal hyalocytes at steady state. In the context of neuroinflammation, monocytes and monocyte-derived macrophages are a significant component of retinal surface macrophages. Human OCT-based imaging of retinal surface macrophages is a potential biomarker for inflammation during retinal vascular disease.

Age-related macular degeneration (AMD) is genetically associated with complement. Dendritic cells (DCs) play key roles during innate and adaptive immunity, and express complement components and their receptors. We investigated ocular DC heterogeneity and the role of DCs in the laser-induced choroidal neovascularization (CNV) model. In order to determine the function of DCs, we used two models of DC deficiency: the Flt3 −/− and Flt3l −/− mouse. We identified three types of ocular DCs: plasmacytoid DC, classical DC-1, and classical DC-2. At steady-state, classical DCs were found in the iris and choroid but were not detectable in the retina. Plasmacytoid DCs existed at very low levels in iris, choroid, and retina. After laser injury, the number of each DC subset was up-regulated in the choroid and retina. In Flt3 −/− mice, we found reduced numbers of classical DCs at steady-state, but each DC subset equally increased after laser injury between wildtype and Flt3 −/− mice. In Flt3l −/− mice, each DC subsets was severely reduced after laser injury. Neither Flt3 −/− or Flt3l −/− mice demonstrated reduced CNV area compared to wildtype mice. DCs do not play any significant role during the laser-induced CNV model of neovascular AMD.

The blood-retina barrier (BRB), which is disrupted in diabetic retinopathy (DR) and uveitis, is an important anatomical characteristic of the retina, regulating nutrient, waste, water, protein, and immune cell flux. The BRB is composed of endothelial cell tight junctions, pericytes, astrocyte end feet, a collagen basement membrane, and perivascular macrophages. Despite the importance of the BRB, retinal perivascular macrophage function remains unknown. We found that retinal perivascular macrophages resided on postcapillary venules in the superficial vascular plexus and expressed MHC class II. Using single-cell RNA-Seq, we found that perivascular macrophages expressed a prochemotactic transcriptome and identified platelet factor 4 (Pf4, also known as CXCL4) as a perivascular macrophage marker. We used Pf4Cre mice to specifically deplete perivascular macrophages. To model retinal inflammation, we performed intraocular CCL2 injections. Ly6C+ monocytes crossed the BRB proximal to perivascular macrophages. Depletion of perivascular macrophages severely hampered Ly6C+ monocyte infiltration. These data suggest that retinal perivascular macrophages orchestrate immune cell migration across the BRB, with implications for inflammatory ocular diseases including DR and uveitis.

Patients with neovascular AMD (nAMD) suffer vision loss from destructive angiogenesis, termed choroidal neovascularization (CNV). Macrophages are found in CNV lesions from patients with nAMD. Additionally, Ccr2-/- mice, which lack classical monocyte-derived macrophages, show reduced CNV size. However, macrophages are highly diverse cells that can perform multiple functions. We performed single-cell RNA-Seq on immune cells from WT and Ccr2-/- eyes to uncover macrophage heterogeneity during the laser-induced CNV mouse model of nAMD. We identified 12 macrophage clusters, including Spp1+ macrophages. Spp1+ macrophages were enriched from WT lasered eyes and expressed a proangiogenic transcriptome via multiple pathways, including vascular endothelial growth factor signaling, endothelial cell sprouting, cytokine signaling, and fibrosis. Additionally, Spp1+ macrophages expressed the marker CD11c, and CD11c+ macrophages were increased by laser and present in CNV lesions. Finally, CD11c+ macrophage depletion reduced CNV size by 40%. These findings broaden our understanding of ocular macrophage heterogeneity and implicate CD11c+ macrophages as potential therapeutic targets for treatment-resistant patients with nAMD.

Background Neovascular age-related macular degeneration causes vision loss from destructive angiogenesis, termed choroidal neovascularization (CNV). Cx3cr1 −/− mice display alterations in non-classical monocytes and microglia with increased CNV size, suggesting that non-classical monocytes may inhibit CNV formation. NR4A1 is a transcription factor that is necessary for maturation of non-classical monocytes from classical monocytes. While Nr4a1 −/− mice are deficient in non-classical monocytes, results are confounded by macrophage hyper-activation. Nr4a1 se2/se2 mice lack a transcriptional activator, resulting in non-classical monocyte loss without macrophage hyper-activation. Main body We subjected Nr4a1 −/− and Nr4a1 se2/se2 mice to the laser-induced CNV model and performed multi-parameter flow cytometry. We found that both models lack non-classical monocytes, but only Nr4a1 −/− mice displayed increased CNV area. Additionally, CD11c + macrophages were increased in Nr4a1 −/− mice. Single-cell transcriptomic analysis uncovered that CD11c + macrophages were enriched from Nr4a1 −/− mice and expressed a pro-angiogenic transcriptomic profile that was disparate from prior reports of macrophage hyper-activation. Conclusions These results suggest that non-classical monocytes are dispensable during CNV, and NR4A1 deficiency results in increased recruitment of pro-angiogenic macrophages.

Background Myeloid cells are heterogeneous cells that are critical for spontaneous choroidal neovascularization (CNV) in the Vldlr −/− mouse model. However, the specific myeloid cell subtype necessary for CNV remains unknown. Methods and results To investigate the role of monocytes, we bred Ccr2 −/− and Nr4a1 −/− mice into the Vldlr −/− background. We found that Ccr2 and Nr4a1 deficiency had no effect upon macrophage counts, CNV lesion number, or total CNV area. Next, we investigated the role of microglia by generating Vldlr −/− Tmem119 CreER/+ Rosa26 DTR/+ mice. Diphtheria toxin (DT) treatment reduced macrophage counts at CNV lesions and CNV lesion number, but did not affect total CNV lesion area. To target microglia via a second strategy, we generated Vldlr −/− Cx3cr1 CreER Csf1r iDTR mice and treated them with a single low dose of tamoxifen to target microglia without affecting choroidal macrophages. DT treatment in Vldlr −/− Cx3cr1 CreER Csf1r iDTR mice decreased macrophage counts at CNV lesions and CNV lesion number but again had no effect upon total CNV lesion area. To target choroidal macrophages and microglia, we treated Vldlr −/− Cx3cr1 CreER Csf1r iDTR mice with 9 tamoxifen treatments. DT-treated mice showed dramatic reductions in macrophage counts, CNV number, and total lesion area. Conclusions These data suggest that monocytes and monocyte-derived macrophages are dispensable, microglia are likely initiators for CNV development, and choroidal macrophages are potential key contributors to CNV growth and/or maintenance in the Vldlr −/− model.

Background Neovascular age-related macular degeneration (nAMD) commonly causes vision loss from aberrant angiogenesis, termed choroidal neovascularization (CNV). Macrophages are heterogeneous cells that are necessary for experimental CNV, present in human CNV samples, and can display diverse functions, which are dependent upon both their origin and tissue microenvironment. Despite these associations, choroidal macrophage heterogeneity remains unexplored. Methods We performed multi-parameter flow cytometry on wildtype (WT) and Ccr2 −/− mice after laser injury to identify macrophage subtypes, and determine which subsets originate from classical monocytes. To fate map tissue resident macrophages at steady state and after laser injury, we used the Cx3cr1 CreER/+ ; Rosa26 zsGFP/+ mouse model. We reanalyzed previously published single-cell RNA-seq of human choroid samples from healthy and nAMD patients to investigate human macrophage heterogeneity, disease association, and function. Results We identified 4 macrophage subsets in mice: microglia, MHCII + CD11c − , MHCII + CD11c + , and MHCII − . Microglia are tissue resident macrophages at steady state and unaffected by laser injury. At steady state, MHCII − macrophages are long lived, tissue resident macrophages, while MHCII + CD11c − and MHCII + CD11c + macrophages are partially replenished from blood monocytes. After laser injury, MHCII + CD11c − macrophages are entirely derived from classical monocytes, MHCII − macrophages originate from classical monocytes (90%) and an expansion of tissue resident macrophages (10%), and MHCII + CD11c + macrophages are derived from classical monocytes (70%), non-classical monocytes (10%), and an expansion of tissue resident macrophages (20%). Single-cell RNA-seq analysis of human choroid found 5 macrophage subsets: two MHCII + CD11C − and three MHCII + CD11C + populations. One MHCII + CD11C + subset was 78% derived from a patient with nAMD. Differential expression analysis identified up-regulation of pro-angiogenic gene expression in one MHCII + CD11C − and two MHCII + CD11C + subsets, including the disease-associated cluster. The upregulated MHCII + CD11C − pro-angiogenic genes were unique compared to the increased MHCII + CD11C + angiogenesis genes. Conclusions Macrophage origin impacts heterogeneity at steady state and after laser injury in mice. Both mice and human patients demonstrate similar macrophage subtypes. Two discrete pro-angiogenic macrophage populations exist in the human choroid. Targeting specific, pro-angiogenic macrophage subsets is a potential novel therapeutic for nAMD.

Macrophage function is determined by microenvironment and origin. Brain and retinal microglia are both derived from yolk sac progenitors, yet their microenvironments differ. Utilizing single-cell RNA sequencing (scRNA-seq) data from mice, we tested the hypothesis that retinal and brain microglia exhibit distinct transcriptional profiles due to their unique microenvironments. Eyes and brains from 2-4 month wildtype mice were combined (20 eyes; 3 brains) to yield one biologically diverse sample per organ. Each tissue was digested into single cell suspensions, enriched for immune cells, and sorted for scRNA-seq. Analysis was performed in Seurat v3 including clustering, integration, and differential expression. Multi-parameter flow cytometry was used for validation of scRNA-seq results. Lymphocytic choriomeningitis virus (LCMV) Clone 13, which produces a systemic, chronic, and neurotropic infection, was used to validate scRNA-seq and flow cytometry results . Cluster analysis of integrated gene expression data from eye and brain identified 6 microglial clusters. Differential expression analysis revealed that eye microglia were enriched for more pro-inflammatory processes including antigen processing via MHC class I (14.0-fold, and ) and positive regulation of T-cell immunity (8.4-fold) compared to brain microglia. Multi-parameter flow cytometry confirmed that retinal microglia expressed 3.2-fold greater H2-Db and 263.3-fold more H2-Kb than brain microglia. On Day 13 and 29 after LCMV infection, CD8 T-cell density was greater in the retina than the brain. Our data demonstrate that the microenvironment of retina and brain differs, resulting in microglia-specific gene expression changes. Specifically, retinal microglia express greater MHC class I by scRNA-seq and multi-parameter flow cytometry, resulting in a possibly enhanced capability to stimulate CD8 T-cell inflammation during LCMV infection. These results may explain tissue-specific differences between retina and brain during systemic viral infections and CD8 T-cell driven autoimmune disease.

Neuropsychiatric symptoms of systemic lupus erythematosus (NP-SLE) affect over one-half of SLE patients, yet underlying mechanisms remain largely unknown. We demonstrate that SLE-prone mice (CReCOM) develop NP-SLE, including behavioral deficits prior to systemic autoimmunity, reduced brain volumes, decreased vascular integrity, and brain-infiltrating leukocytes. NP-SLE microglia exhibit numerical expansion, increased synaptic uptake, and a more metabolically active phenotype. Microglia from multiple SLE-prone models express a \"NP-SLE signature\" unrelated to type I interferon. Rather, the signature is associated with lipid metabolism, scavenger receptor activity and downregulation of inflammatory and chemotaxis processes, suggesting a more regulatory, anti-inflammatory profile. NP-SLE microglia also express genes associated with disease-associated microglia (DAM), a subset of microglia thought to be instrumental in neurodegenerative diseases. Further, expression of \"NP-SLE\" and \"DAM\" signatures correlate with the severity of behavioral deficits in young SLE-prone mice prior to overt systemic disease. Our data are the first to demonstrate the predictive value of our newly identified microglia-specific \"NP-SLE\" and \"DAM\" signatures as a surrogate for NP-SLE clinical outcomes and suggests that microglia-intrinsic defects precede contributions from systemic SLE for neuropsychiatric manifestations.