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.
Vascular endothelial growth factor-A (VEGF) is the angiogenic factor promoting the pathological neovascularization in age-related macular degeneration (AMD) or diabetic macular edema (DME). Evidences ...have suggested a neurotrophic and neuroprotective role of VEGF, albeit in retina, cellular mechanisms underlying the VEGF neuroprotection remain elusive. Using purified adult retinal ganglion cells (RGCs) in culture, we demonstrated here that VEGF is released by RGCs themselves to promote their own survival, while VEGF neutralization by specific antibodies or traps drastically reduced the RGC survival. These results indicate an autocrine VEGF neuroprotection on RGCs. In parallel, VEGF produced by mixed retinal cells or by mesenchymal stem cells exerted a paracrine neuroprotection on RGCs. Such neuroprotective effect was obtained using the recombinant VEGF-B, suggesting the involvement of VEGF-R1 pathway in VEGF-elicited RGC survival. Finally, glaucomatous patients injected with VEGF traps (ranibizumab or aflibercept) due to either AMD or DME comorbidity, showed a significant reduction of RGC axon fiber layer thickness, consistent with the plausible reduction of the VEGF autocrine stimulation of RGCs. Our results provide evidence of the autocrine neuroprotective function of VEGF on RGCs is crucially involved to preserve injured RGCs such as in glaucomatous patients.
Abstract
Changes in cell function occur by specific patterns of intracellular Ca
2+
, activating Ca
2+
-sensitive proteins. The anoctamin (TMEM16) protein family has Ca
2+
-dependent ion channel ...activity, which provides transmembrane ion transport, and/or Ca
2+
-dependent phosphatidyl-scramblase activity. Using amino acid sequence analysis combined with measurements of ion channel function, we clarified the so far unknown Ano4 function as Ca
2+
-dependent, non-selective monovalent cation channel; heterologous Ano4 expression in HEK293 cells elicits Ca
2+
activated conductance with weak selectivity of K
+
> Na
+
> Li
+
. Endogenously expressed Ca
2+
-dependent cation channels in the retinal pigment epithelium were identified as Ano4 by KO mouse-derived primary RPE cells and siRNA against Ano4. Exchanging a negatively charged amino acid in the putative pore region (AA702–855) into a positive one (E775K) turns Ano4-elicited currents into Cl
−
currents evidencing its importance for ion selectivity. The molecular identification of Ano4 as a Ca
2+
-activated cation channel advances the understanding of its role in Ca
2+
signaling.
A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here, we examined the mechanisms underlying this susceptibility. We found that ...monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1, and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1), which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD, OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD.
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•10q26 AMD-risk haplotype carrying monocytes overexpress HTRA1 and OPN•HTRA1 locates to mononuclear phagocytes in eyes of patients with AMD•HTRA1 proteolysis of TSP-1 curbs CD47-dependent OPN repression•HTRA1 induced OPN promotes pathogenic subretinal MP accumulation
A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Beguier et al. provide a mechanistic understanding of this susceptibility by linking this risk haplotype to overexpression of the peptidase HTRA1 and thereby to the accumulation of macrophages in the subretinal space and pathogenic inflammation.
Chloride channels (Cl channels) play an essential role for the retinal pigment epithelium (RPE). They provide a plasma membrane conductance for Cl− important for transepithelial transport and volume ...regulation. Ca2+-dependent chloride channels (CaCC) in the RPE were found to adapt Cl− transport to specific needs by increasing intracellular free Ca2+. Although a variety of Cl channels have been identified in the RPE, the molecular identity of the CaCC remains controversial. Sagittal sections of mouse retina were stained against anoctamin2 (Ano2) and analyzed by confocal microscopy. Membrane currents from ARPE-19 cells and primary murine RPE cells were recorded in the whole-cell configuration of the patch-clamp technique. Expression of Ano2 was assessed via immunocytochemistry, PCR and western-blot and down-regulated via siRNA approaches. In the mouse retina, Ano2 was found in the basolateral membrane of the RPE. In primary mouse RPE cells, Ano2 was localized predominantly in the cell membrane. Ano2 mRNA and protein were also detected in rat and primate RPE as well as ARPE-19 cells. Whole-cell currents were elicited by increasing intracellular free Ca2+ via ATP application. These currents were identified as Cl− currents by their reversal potential and blocker sensitivity. Knock-down of Ano2 by siRNA decreased both the Ca2+ dependent chloride conductance and protein expression of Ano2. The biophysical and pharmacological properties of CaCC in ARPE-19 and primary mouse RPE cells resemble those described in previous publications using RPE cells from different species. The siRNA knock-down suggests that Ano2 contributes to Ca2+-dependent chloride conductance in the RPE.
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•Ano2 is expressed in the basolateral membrane of the RPE.•ATP application elicits Ca2+-dependent Cl− conductance in RPE cells that is reduced by siAno2.•Ano2 contributes to Ca2+-dependent Cl− conductance in both ARPE-19 and primary mouse RPE cells.
To decipher the role of monocyte-derived macrophages (Mφs) in vascular remodeling of the occluded vein following experimental branch retinal vein occlusion (BRVO).
The inflammation induced by ...laser-induced BRVO on mice retina was evaluated at different time points by RT-PCR looking at inflammatory markers mRNA level expression, Icam-1, Cd11b, F4/80, Ccl2, and Ccr2 and by quantification of Iba1-positive macrophage (Mφ) density on Iba1-stained retinal flatmount. Repeated intraperitoneal EdU injection combined with liposome clodronate-induced monocyte (Mo) depletion in wildtype mice was used to differentiate Mo-derived Mφs from resident Mφs. Liposome clodronate Mo-depleted wildtype mice and Ccr2-deficient mice were used to evaluate the role of all CCR2
and CCR2
Mo-derived Mφs on EC apoptosis in the occluded vein.
cd11b, ICAM-1, F4/80, Ccl2, and Ccr2 mRNA expression were increased 1, 3, and 7 days after vein occlusion. The number of parenchymal (parMφs) and perivascular (vasMφs) macrophages was increased 3 and 7 days after BRVO. The systemic depletion of all circulating Mos decreased significantly the BRVO-induced parMφs and vasMφs macrophage accumulation, while the deletion of CCR2
-inflammatory Mo only diminished the accumulation of parMφs, but not vasMφs. Finally, apoptotic ECs of the vein were more numerous in fully depleted, liposome clodronate-treated mice, than in Ccr2
mice that only lack the recruitment of CCR2
inflammatory Mos.
BRVO triggers the recruitment of blood-derived parMφs and vasMφs. Interestingly, vasMφs accumulation was independent of CCR2. The observation that the inhibition of the recruitment of all infiltrating Mφs increases the vein EC apoptosis, while CCR2 deficiency does not, demonstrates that CCR2
Mo-derived vasMφs protect the ECs against apoptosis in the occluded vein.
Hypoxia is potentially one of the essential triggers in the pathogenesis of wet age-related macular degeneration (wetAMD), characterized by choroidal neovascularization (CNV) which is driven by the ...accumulation of subretinal mononuclear phagocytes (MP) that include monocyte-derived cells. Here we show that systemic hypoxia (10% O
) increased subretinal MP infiltration and inhibited inflammation resolution after laser-induced subretinal injury in vivo. Accordingly, hypoxic (2% O
) human monocytes (Mo) resisted elimination by RPE cells in co-culture. In Mos from hypoxic mice, Thrombospondin 1 mRNA (Thbs1) was most downregulated compared to normoxic animals and hypoxia repressed Thbs-1 expression in human monocytes in vitro. Hypoxic ambient air inhibited MP clearance during the resolution phase of laser-injury in wildtype animals, but had no effect on the exaggerated subretinal MP infiltration observed in normoxic Thbs1
-mice. Recombinant Thrombospondin 1 protein (TSP-1) completely reversed the pathogenic effect of hypoxia in Thbs1
-mice, and accelerated inflammation resolution and inhibited CNV in wildtype mice. Together, our results demonstrate that systemic hypoxia disturbs TSP-1-dependent subretinal immune suppression and promotes pathogenic subretinal inflammation and can be therapeutically countered by local recombinant TSP-1.
To compare in vitro, on the human reconstituted corneal epithelial SkinEthics model, and in vivo, using an acute rabbit toxicological model, the effects of a benzalkonium chloride (BAK)-preserved ...solution of latanoprost and a preservative-free (PF) latanoprost solution.
In vitro, the three-dimensional (3D) reconstituted human corneal epithelia (HCE) were treated with PBS, BAK-latanoprost, PF-latanoprost, or 0.02% BAK for 24 hours followed or not followed by a 24 hour post incubation recovery period. Cellular viability was evaluated using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test at 24 hours and the apoptotic cells were counted using TUNEL labeling on frozen sections at 24 hours and 24 hours plus 24 hours. In vivo, rabbits received 50 μL of the same solutions, which were applied at 5 minute intervals a total of 15 times. Ocular surface toxicity was investigated using slit lamp biomicroscopy examination, conjunctival impression cytology (CIC), and corneal in vivo confocal microscopy (IVCM). Standard immunohistology also assessed inflammatory CD45-positive cells.
In vitro, BAK-latanoprost and 0.02% BAK induced significant apoptosis in the apical layers that correlated with the significant decrease of cell viability as assessed by the MTT test. PF-latanoprost slightly decreased cell viability and few apoptotic cells were found in the superficial layers, without reaching statistical significance compared with PBS. In vivo, clinical observation and IVCM images showed the lowest ocular surface toxicity with PBS and PF-latanoprost, while BAK-latanoprost and BAK induced abnormal corneoconjunctival aspects. PF-latanoprost showed the lowest CIC score, close to the PBS score and induced fewer CD45-positive cells in both the limbus and the conjunctiva compared with BAK and latanoprost, as assessed by immunohistology.
We confirm that rabbit corneoconjunctival surfaces presented better tolerance when treated with PF-latanoprost compared with the standard BAK-latanoprost preparation or the BAK solution.
Abstract
Background
Forkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of ...other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood–retina barrier of the immune privileged eye.
Methods
We investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1β and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing.
Results
RPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1β to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells.
Conclusion
Our data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.
Glaucoma is one of the leading causes of irreversible blindness in the world. The major risk factor is elevated intraocular pressure (IOP) leading to progressive retinal ganglion cell (RGC) death ...from the optic nerve (ON) to visual pathways in the brain. Glaucoma has been reported to share mechanisms with neurodegenerative disorders. We therefore hypothesize that neuroinflammatory mechanisms in central visual pathways may contribute to the spread of glaucoma disease. The aim of the present study was to analyze the neuroinflammation processes that occur from the pathological retina to the superior colliculi (SCs) in a rat model of unilateral ocular hypertension induced by episcleral vein cauterization (EVC).
Six weeks after unilateral (right eye) EVC in male Long-Evans rats, we evaluated both the neurodegenerative process and the neuroinflammatory state in visual pathway tissues. RGCs immunolabeled (Brn3a(+)) in ipsilateral whole flat-mounted retina demonstrated peripheral RGC loss associated with tissue macrophage/microglia activation (CD68(+)). Gene expression analysis of hypertensive and normotensive retinas revealed a significant increase of pro-inflammatory genes such as CCL2, IL-1β, and Nox2 mRNA expression compared to naïve eyes. Importantly, we found an upregulation of pro-inflammatory markers such as IL-1β and TNFα and astrocyte and tissue macrophage/microglia activation in hypertensive and normotensive RGC projection sites in the SCs compared to a naïve SC. To understand how neuroinflammation in the hypertensive retina is sufficient to damage both right and left SCs and the normotensive retina, we used an inflammatory model consisting in an unilateral stereotaxic injection of TNFα (25 ng/μl) in the right SC of naïve rats. Two weeks after TNFα injection, using an optomotor test, we observed that rats had visual deficiency in both eyes. Furthermore, both SCs showed an upregulation of genes and proteins for astrocytes, microglia, and pro-inflammatory cytokines, notably IL-1β. In addition, both retinas exhibited a significant increase of inflammatory markers compared to a naïve retina.
All these data evidence the complex role played by the SCs in the propagation of neuroinflammatory events induced by unilateral ocular hypertension and provide a new insight into the spread of neurodegenerative diseases such as glaucoma.
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