Unlike in the healthy mammalian retina, macrophages in retinal degenerative states are not solely comprised of microglia but may include monocyte-derived recruits. Recent studies have applied ...transgenics, lineage-tracing, and transcriptomics to help decipher the distinct roles of these two cell types in the diseasesettings of inherited retinal degenerations and age-related macular degeneration.Literature discussed here focuses on the ectopic presence of both macrophage types in the extracellular site surrounding the outer aspect ofphotoreceptor cells (i.e.,the subretinal space), which is crucially involved in the pathobiology. From these studies we propose a working model in which perturbed photoreceptor states cause microglial dominant migration to the subretinal space as a protective response, whereas the abundant presence ofmonocyte-derived cells there instead drives and accelerates pathology. The latter, we propose, is underpinned by specific genetic and nongenetic determinants that lead to a maladaptive macrophage state.
Mononuclear phagocytes (MNPs) are central players in retinal degeneration, as seen in age-related macular degenerationand inherited retinal degeneration.A key feature of retinal degeneration is the invasion of MNPs into the subretinal space (an extracellular site critical for vision).However, it remains debated whether these cells consist of microglia and/or monocyte-derived cells, and what are their respective roles.We argue that in retinal degeneration settingsin mouse models, ‘adaptive’ MNP responses are those in which reactive microglia migrate into the subretinal space and play a role in restricting diseaseprogression.‘Maladaptive’ response, by contrast, are seen, for example, in mice with certain deficiencies in MNP-expressed genes. There, a normally subthreshold insult can result in an abundance of pathogenic monocyte-derived cells in the subretinal space.We further lay out how these adaptive/maladaptive classifications may serve as a conceptual framework to help betterunderstand the complex roles of microglia versus monocytes in the human diseasecontext.
Kupffer cells (KCs) are liver-resident macrophages that self-renew by proliferation in the adult independently from monocytes. However, how they are maintained during non-alcoholic steatohepatitis ...(NASH) remains ill defined. We found that a fraction of KCs derived from Ly-6C+ monocytes during NASH, underlying impaired KC self-renewal. Monocyte-derived KCs (MoKCs) gradually seeded the KC pool as disease progressed in a response to embryo-derived KC (EmKC) death. Those MoKCs were partly immature and exhibited a pro-inflammatory status compared to EmKCs. Yet, they engrafted the KC pool for the long term as they remained following disease regression while acquiring mature EmKC markers. While KCs as a whole favored hepatic triglyceride storage during NASH, EmKCs promoted it more efficiently than MoKCs, and the latter exacerbated liver damage, highlighting functional differences among KCs with different origins. Overall, our data reveal that KC homeostasis is impaired during NASH, altering the liver response to lipids, as well as KC ontogeny.
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•NASH alters Kupffer cell self-renewal by causing their death•Monocyte-derived KCs (MoKCs) contribute to the KC pool during NASH•MoKCs maintain on the long term after NASH regression•MoKCs are more inflammatory than EmKCs and alter the liver response during NASH
How NASH impacts on embryo-derived Kupffer cell (EmKC) homeostasis remains ill defined. Tran and colleagues demonstrate that EmKC self-renewal is impaired during NASH, leading to EmKC replacement by monocyte-derived KCs (MoKCs) that alter the liver response to lipid overload.
To establish a model of the retinal capillary circulation in pigs, which in many aspects is close to the human retina.
Using high density confocal microscopy image stacks of immunolabeled porcine ...retinal whole mounts, microvessels close to the optic nerve head were traced in three dimensions. The direction of flow of individual capillaries was deduced from their arteriolar and/or venous connections.
From major arteries, second-order arteries traversed the nerve fiber layer and resolved exclusively into the superficial vascular plexus (SVP), which dichotomized the blood flow between radial peripapillary capillaries (RPCs) on one side and the intermediate (IVP) and deep vascular plexus (DVP) on the other. Each RPC was supplied by one or several capillaries from the SVP and drained to the IVP or DVP. The DVP was a mosaic of approximately 300 to 600 μm wide anastomotic watersheds, each drained by one or two venules connected to major veins. A presumptive direction of flow could be determined for >90% of capillaries. These results suggest a model of the capillary circulation in which the three microvessel layers are serially organized with RPCs are in parallel between the SVP and IVP or DVP.
In the peripapillary retina of pigs, microvascular layers have a serial arrangement, with RPCs emerging from the SVP and draining to the IVP or DVP; hence, connected in parallel of this scheme. The bulk of flow, therefore, traverses the SVP and DVP successively. This organization contributes to the higher oxygen saturation in the SVP and RPCs than in the DVP. Physiopathologic implications of this model regarding retinal diseases are discussed.
The retinal pigment epithelium (RPE) forms the outer blood⁻retina barrier and facilitates the transepithelial transport of glucose into the outer retina via GLUT1. Glucose is metabolized in ...photoreceptors via the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) but also by aerobic glycolysis to generate glycerol for the synthesis of phospholipids for the renewal of their outer segments. Aerobic glycolysis in the photoreceptors also leads to a high rate of production of lactate which is transported out of the subretinal space to the choroidal circulation by the RPE. Lactate taken up by the RPE is converted to pyruvate and metabolized via OXPHOS. Excess lactate in the RPE is transported across the basolateral membrane to the choroid. The uptake of glucose by cone photoreceptor cells is enhanced by rod-derived cone viability factor (RdCVF) secreted by rods and by insulin signaling. Together, the three cells act as symbiotes: the RPE supplies the glucose from the choroidal circulation to the photoreceptors, the rods help the cones, and both produce lactate to feed the RPE. In age-related macular degeneration this delicate ménage à trois is disturbed by the chronic infiltration of inflammatory macrophages. These immune cells also rely on aerobic glycolysis and compete for glucose and produce lactate. We here review the glucose metabolism in the homeostasis of the outer retina and in macrophages and hypothesize what happens when the metabolism of photoreceptors and the RPE is disturbed by chronic inflammation.
Remodeling senescent blood vessels
The retina is a thin layer of nervous tissue at the back of the eye that transforms light into neuronal signals. The retina is essential for vision and is supported ...by networks of blood vessels. In diabetic retinopathy, a common cause of vision loss, these microvessels degenerate and regrow in an aberrant manner. Such degeneration and regrowth can compromise the functioning of retinal nerve cells. Binet
et al.
observed that, after rapid proliferation, vascular endothelial cells in diseased blood vessels engaged molecular pathways linked to cellular senescence (see the Perspective by Podrez and Byzova). Senescent vascular units summoned an inflammatory response in which neutrophils extruded neutrophil extracellular traps onto diseased vessels to remodel them. This endogenous repair mechanism promoted the elimination of senescent blood vessels and could lead to beneficial vascular remodeling.
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Remodeling of senescent vascular endothelial cells in the retina is mediated through neutrophil extracellular traps.
INTRODUCTION
Vision provides a critical survival advantage but requires a tight coupling between neuronal energy demands and their vascular supply. The degeneration and consequent aberrant regrowth of retinal vasculature is the hallmark of diseases such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration, which collectively are the most common causes of loss of sight in industrialized countries. Although considerable effort has been devoted to understanding how diseased blood vessels form, relatively little is known of the processes at play during late stages of pathological angiogenesis when blood vessels remodel and subsets of diseased vasculature regress.
RATIONALE
The retina is part of the central nervous system and thus has limited regenerative capacity. A relative exception to this rule are retinal blood vessels, which have a greater propensity to remodel depending on metabolic demand. We investigated the cellular mechanisms activated during the remodeling and regression of pathological blood vessels in retinopathy. We focused on a mouse model of oxygen-induced retinopathy, which has distinct and timed phases of vascular degeneration, neovascularization, and vascular regression. Our findings were verified in human patients with proliferative diabetic retinopathy. Understanding how diseased blood vessels remodel and yield functional networks has the potential to lead to strategies that enhance vascular normalization and helps to explain why retinas in certain patients have the propensity to repair themselves more readily than others.
RESULTS
We found that vascular remodeling in retinopathy is associated with bouts of sterile inflammation and tardy recruitment of neutrophils, an immune population typically associated with a first wave of invading leukocytes. We observed that, after rapid proliferation, vascular endothelial cells in diseased blood vessels engaged molecular pathways shared with aging and cellular damage that lead to cellular senescence. Senescent vascular units then released a secretome of cytokines and factors that attracted neutrophils and triggered the production of neutrophil extracellular traps (NETs). Through extrusion of NETs, neutrophils eliminated diseased senescent vasculature by promoting its apoptosis. By crippling the ability of neutrophils to produce NETs by genetically removing the peptidyl arginine deiminase type IV (PAD4) enzyme, clearance of senescent cells was impaired and regression of pathological angiogenesis compromised. Similar effects were observed with the neutrophil-depleting antibody anti-Ly6G or by pharmacological inhibition of the neutrophil receptor CXCR2.
CONCLUSION
We conclude that neutrophils, through the release of NETs, targeted pathological senescent vasculature for clearance and thus prepare the ischemic retina for reparative vascular regeneration. These findings imply that elimination of senescent blood vessels leads to beneficial vascular remodeling. Although cellular senescence is not necessarily synonymous with aging, our study may provide insight into a general mechanism in which senescent endothelial cells trigger NETosis and predispose to thrombotic events such as myocardial infarction, atherosclerosis, and stroke, which are typically seen in older populations.
Senescent blood vessels trigger neutrophil extracellular traps in retinopathy.
(
A
) Human samples and a mouse model were used to elucidate mechanisms of vascular remodeling in retinopathy. (
B
) Upon rapid proliferation, vascular cells in pathological tufts triggered pathways of cellular senescence, leading to cytokine secretion and the recruitment of neutrophils. (
C
) Factors secreted by senescent cells triggered NETosis. (
D
) NETs promoted the removal of senescent endothelial cells, ultimately leading to regression of pathological angiogenesis and promoting the regeneration of functional vessels.
In developed countries, the leading causes of blindness such as diabetic retinopathy are characterized by disorganized vasculature that can become fibrotic. Although many such pathological vessels often naturally regress and spare sight-threatening complications, the underlying mechanisms remain unknown. Here, we used orthogonal approaches in human patients with proliferative diabetic retinopathy and a mouse model of ischemic retinopathies to identify an unconventional role for neutrophils in vascular remodeling during late-stage sterile inflammation. Senescent vasculature released a secretome that attracted neutrophils and triggered the production of neutrophil extracellular traps (NETs). NETs ultimately cleared diseased endothelial cells and remodeled unhealthy vessels. Genetic or pharmacological inhibition of NETosis prevented the regression of senescent vessels and prolonged disease. Thus, clearance of senescent retinal blood vessels leads to reparative vascular remodeling.
Adaptive optics (AO)-enhanced en face retinal imaging, termed here AO ophthalmoscopy (AOO) has reached a level of robustness which fuels its increasing use in research and clinical centers. Here we ...will review the contribution of clinical AOO to the understanding and monitoring of 1) age-related macular degeneration and 2) vascular diseases. The main contributions of AOO to the phenotyping of AMD are a better identification of drusen, a better delineation of the limits of atrophy, and the identification of novel features such as punctate hyperreflectivity and mobile melanin-containing clumps. Characterization of progression of atrophy is facilitated by time-lapse imaging. In vessels, AOO enables the observation and measurement of parietal structures and the observation of microscopic pathological features such as small hemorrhages and inflammatory cell accumulations.
The innate immune system is activated in a number of degenerative and inflammatory retinal disorders such as age-related macular degeneration (AMD). Retinal microglia, choroidal macrophages, and ...recruited monocytes, collectively termed 'retinal mononuclear phagocytes', are critical determinants of ocular disease outcome. Many publications have described the presence of these cells in mouse models for retinal disease; however, only limited aspects of their behavior have been uncovered, and these have only been uncovered using a single detection method. The workflow presented here describes a comprehensive analysis strategy that allows characterization of retinal mononuclear phagocytes in vivo and in situ. We present standardized working steps for scanning laser ophthalmoscopy of microglia from MacGreen reporter mice (mice expressing the macrophage colony-stimulating factor receptor GFP transgene throughout the mononuclear phagocyte system), quantitative analysis of Iba1-stained retinal sections and flat mounts, CD11b-based retinal flow cytometry, and qRT-PCR analysis of key microglia markers. The protocol can be completed within 3 d, and we present data from retinas treated with laser-induced choroidal neovascularization (CNV), bright white-light exposure, and Fam161a-associated inherited retinal degeneration. The assays can be applied to any of the existing mouse models for retinal disorders and may be valuable for documenting immune responses in studies for immunomodulatory therapies.
Age‐related macular degeneration in its neovascular form (NV AMD) is the leading cause of vision loss among adults above the age of 60. Epidemiological data suggest that in men, overall abdominal ...obesity is the second most important environmental risk factor after smoking for progression to late‐stage NV AMD. To date, the mechanisms that underscore this observation remain ill‐defined. Given the impact of high‐fat diets on gut microbiota, we investigated whether commensal microbes influence the evolution of AMD. Using mouse models of NV AMD, microbiotal transplants, and other paradigms that modify the gut microbiome, we uncoupled weight gain from confounding factors and demonstrate that high‐fat diets exacerbate choroidal neovascularization (CNV) by altering gut microbiota. Gut dysbiosis leads to heightened intestinal permeability and chronic low‐grade inflammation characteristic of inflammaging with elevated production of IL‐6, IL‐1β, TNF‐α, and VEGF‐A that ultimately aggravate pathological angiogenesis.
Synopsis
Obesity is an important risk factor for neovascular AMD. We show that high‐fat diets induce gut microbial dysbiosis, resulting in heightened intestinal permeability, which leads to chronic low‐grade inflammation and ultimately increased choroidal neovascularization (CNV).
A high‐fat diet (HFD) induces a shift in commensal gut microbiota and increases permeability of the gut barrier.
Intestinal dysbiosis increases circulating pathogen‐associated molecular patterns (PAMPs), leading to low‐grade endotoxemia that triggers an inflammatory response through pattern recognition receptors (PRRs).
Increased concentrations of circulating and local inflammatory cytokines such as IL‐6, IL‐1β, TNF‐α, and VEGF‐A exacerbate CNV.
Microbiotal transplants confirm that HFD aggravates CNV through gut microbiota.
Obesity is an important risk factor for neovascular AMD. We show that high‐fat diets induce gut microbial dysbiosis, resulting in heightened intestinal permeability, which leads to chronic low‐grade inflammation and ultimately increased choroidal neovascularization (CNV).
The role of microglia, present in the retina early in development before vascularization, remains ill defined. The authors investigated whether microglia are implicated in retinal blood vessel ...formation.
The microglia and vasculature of developing human fetal and rodent retinas were examined by labeling the endothelial cells with lectin and the microglia with CD18 antibody or green fluorescent protein driven by the promoter of the chemokine receptor CX(3)CR1. Rodent ischemic proliferative retinopathy induced by hyperoxia or hypercapnia, which model retinopathy of prematurity, and ex vivo retinal explants were used to assess microglial involvement in vascular pathology. Microglial participation in developmental retinal vessel formation was further studied in neonatal rats after pharmacologic macrophage depletion with the use of clodronate liposomes and subsequent intravitreal injection of microglia.
Microglia intimately appose developing vessels of human and murine retinas. Ischemic retinopathy models exhibit decreased microglia concomitant with the characteristic reductions in vasculature observed in these retinopathies. Retinal explants exposed to conditions resulting in ischemic retinopathies (in vivo) reveal that antioxidants protect against microglial loss. Depletion of resident retinal microglia, but not systemic macrophages, reduced developmental vessel growth and density, which were restored by intravitreal microglial injection.
These observations suggest that proper retinal blood vessel formation requires an adequate resident microglial population because diminished microglia are associated with decreased vascularity in models of ischemic retinopathy and retinal vascular development. In light of these findings, the traditional definition of microglia as merely immunocompetent cells should be reconsidered to encompass this new function related to blood vessel formation.
The cone function is essential to mediate high visual acuity, color vision, and daylight vision. Inherited cone dystrophies and age-related macular degeneration affect a substantial percentage of the ...world population. To identify and isolate the most competent cells for transplantation and integration into the retina, cone tracing during development would be an important added value. To that aim, the Chrnb4-EGFP mouse line was characterized throughout retinogenesis. It revealed a sub-population of early retinal progenitors expressing the reporter gene that is progressively restricted to mature cones during retina development. The presence of the native CHRNB4 protein was confirmed in EGFP-positive cells, and it presents a similar pattern in the human retina. Sub-retinal transplantations of distinct subpopulations of Chrnb4-EGFP-expressing cells revealed the embryonic day 15.5 high-EGFP population the most efficient cells to interact with host retinas to provoke the appearance of EGFP-positive cones in the photoreceptor layer. Importantly, transplantations into the DsRed retinas revealed material exchanges between donor and host retinas, as >80% of transplanted EGFP-positive cones also were DsRed positive. Whether this cell material fusion is of significant therapeutic advantage requires further thorough investigations. The Chrnb4-EGFP mouse line definitely opens new research perspectives in cone genesis and retina repair.
In adult mouse and human retina, the beta-4 subunit of the Acetylcholine receptor (CHRNB4) is expressed only in cones. The Chrnb4-EGFP mouse served for cone lineage-tracing and transplantation studies. Transplantation of Chrnb4-EGFP cone precursors into DsRed retina revealed frequent bidirectional cellular material exchanges between donor and host cones.
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