Loss of Mir146b with aging contributes to inflammation and mitochondrial dysfunction in thioglycollate-elicited peritoneal macrophages.

Macrophages undergo programmatic changes with age, leading to altered cytokine polarization and immune dysfunction, shifting these critical immune cells from protective sentinels to disease promoters. The molecular mechanisms underlying macrophage inflammaging are poorly understood. Using an unbiased RNA sequencing (RNA-seq) approach, we identified Mir146b as a microRNA whose expression progressively and unidirectionally declined with age in thioglycollate-elicited murine macrophages. Mir146b deficiency led to altered macrophage cytokine expression and reduced mitochondrial metabolic activity, two hallmarks of cellular aging. Single-cell RNA-seq identified patterns of altered inflammation and interferon gamma signaling in Mir146b-deficient macrophages. Identification of Mir146b as a potential regulator of macrophage aging provides novel insights into immune dysfunction associated with aging.

Ocular phenotypes in a mouse model of impaired glucocerebrosidase activity.

Mutations in the GBA1 gene encoding glucocerebrosidase (GCase) are linked to Gaucher (GD) and Parkinson's Disease (PD). Since some GD and PD patients develop ocular phenotypes, we determined whether ocular phenotypes might result from impaired GCase activity and the corresponding accumulation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph) in the Gba1D409V/D409V knock-in (Gba KI/KI; "KI") mouse. Gba KI mice developed age-dependent pupil dilation deficits to an anti-muscarinic agent; histologically, the iris covered the anterior part of the lens with adhesions between the iris and the anterior surface of the lens (posterior synechia). This may prevent pupil dilation in general, beyond an un-responsiveness of the iris to anti-muscarinics. Gba KI mice displayed atrophy and pigment dispersion of the iris, and occlusion of the iridocorneal angle by pigment-laden cells, reminiscent of secondary open angle glaucoma. Gba KI mice showed progressive thinning of the retina consistent with retinal degeneration. GluSph levels were increased in the anterior and posterior segments of the eye, suggesting that accumulation of lipids in the eye may contribute to degeneration in this compartment. We conclude that the Gba KI model provides robust and reproducible eye phenotypes which may be used to test for efficacy and establish biomarkers for GBA1-related therapies.

SARM1 depletion rescues NMNAT1-dependent photoreceptor cell death and retinal degeneration.

Leber congenital amaurosis type nine is an autosomal recessive retinopathy caused by mutations of the NAD+ synthesis enzyme NMNAT1. Despite the ubiquitous expression of NMNAT1, patients do not manifest pathologies other than retinal degeneration. Here we demonstrate that widespread NMNAT1 depletion in adult mice mirrors the human pathology, with selective loss of photoreceptors highlighting the exquisite vulnerability of these cells to NMNAT1 loss. Conditional deletion demonstrates that NMNAT1 is required within the photoreceptor. Mechanistically, loss of NMNAT1 activates the NADase SARM1, the central executioner of axon degeneration, to trigger photoreceptor death and vision loss. Hence, the essential function of NMNAT1 in photoreceptors is to inhibit SARM1, highlighting an unexpected shared mechanism between axonal degeneration and photoreceptor neurodegeneration. These results define a novel SARM1-dependent photoreceptor cell death pathway and identifies SARM1 as a therapeutic candidate for retinopathies.

Impaired monocyte cholesterol clearance initiates age-related retinal degeneration and vision loss.

Advanced age-related macular degeneration (AMD), the leading cause of blindness among people over 50 years of age, is characterized by atrophic neurodegeneration or pathologic angiogenesis. Early AMD is characterized by extracellular cholesterol-rich deposits underneath the retinal pigment epithelium (RPE) called drusen or in the subretinal space called subretinal drusenoid deposits (SDD) that drive disease progression. However, mechanisms of drusen and SDD biogenesis remain poorly understood. Although human AMD is characterized by abnormalities in cholesterol homeostasis and shares phenotypic features with atherosclerosis, it is unclear whether systemic immunity or local tissue metabolism regulates this homeostasis. Here, we demonstrate that targeted deletion of macrophage cholesterol ABC transporters A1 (ABCA1) and -G1 (ABCG1) leads to age-associated extracellular cholesterol-rich deposits underneath the neurosensory retina similar to SDD seen in early human AMD. These mice also develop impaired dark adaptation, a cardinal feature of RPE cell dysfunction seen in human AMD patients even before central vision is affected. Subretinal deposits in these mice progressively worsen with age, with concomitant accumulation of cholesterol metabolites including several oxysterols and cholesterol esters causing lipotoxicity that manifests as photoreceptor dysfunction and neurodegeneration. These findings suggest that impaired macrophage cholesterol transport initiates several key elements of early human AMD, demonstrating the importance of systemic immunity and aging in promoting disease manifestation. Polymorphisms in genes involved with cholesterol transport and homeostasis are associated with a significantly higher risk of developing AMD, thus making these studies translationally relevant by identifying potential targets for therapy.

Oxysterol Signatures Distinguish Age-Related Macular Degeneration from Physiologic Aging.

Macrophage aging is pathogenic in numerous diseases, including age-related macular degeneration (AMD), a leading cause of blindness in older adults. Although prior studies have explored the functional consequences of macrophage aging, less is known about its cellular basis or what defines the transition from physiologic aging to disease. Here, we show that despite their frequent self-renewal, macrophages from old mice exhibited numerous signs of aging, such as impaired oxidative respiration. Transcriptomic profiling of aged murine macrophages revealed dysregulation of diverse cellular pathways, especially in cholesterol homeostasis, that manifested in altered oxysterol signatures. Although the levels of numerous oxysterols in human peripheral blood mononuclear cells and plasma exhibited age-associated changes, plasma 24-hydroxycholesterol levels were specifically associated with AMD. These novel findings demonstrate that oxysterol levels can discriminate disease from physiologic aging. Furthermore, modulation of cholesterol homeostasis may be a novel strategy for treating age-associated diseases in which macrophage aging is pathogenic.

WNT7A/B promote choroidal neovascularization.

Perturbations in WNT signaling are associated with congenital eye disorders, including familial exudative vitreoretinopathy and Norrie disease. More recently, activation of the WNT pathway has also been shown to be associated with age-related macular degeneration (AMD). In this study, we identified that in choroidal neovascular membranes from AMD patients, ß-catenin is activated specifically in the vascular endothelium, suggesting that WNT promotes pathologic angiogenesis by directly affecting vascular endothelial cells. WNT7B has been shown to be important during eye development for regression of the fetal hyaloid vasculature. However, it has not yet been established whether WNT7A and/or WNT7B are involved in neovascular AMD pathogenesis. Here, we show that WNT7A and WNT7B increase the proliferation of human dermal microvascular endothelial cells in a dose-dependent manner. Both WNT7A and WNT7B also stimulated vascular sprouting from mouse choroidal explants in vitro. To evaluate in vivo relevance, we generated mice systemically deficient in Wnt7a and/or Wnt7b. Genetic deletion of both Wnt7a and Wnt7b decreased the severity of laser injury-induced choroidal neovascularization (CNV), while individual deletion of either Wnt7a or Wnt7b did not have a significant effect on CNV, suggesting that WNT7A and WNT7B have redundant pro-angiogenic roles in vivo. Cumulatively, these findings identify specific WNT isoforms that may play a pathologic role in CNV as observed in patients with neovascular AMD. Although the source of increased WNT7A and/or WNT7B in CNV requires further investigation, WNT signaling may be a potential target for therapeutic intervention if these results are demonstrated to be relevant in human disease.

Disrupted cholesterol metabolism promotes age-related photoreceptor neurodegeneration.

Photoreceptors have high intrinsic metabolic demand and are exquisitely sensitive to metabolic perturbation. In addition, they shed a large portion of their outer segment lipid membranes in a circadian manner, increasing the metabolic burden on the outer retina associated with the resynthesis of cell membranes and disposal of the cellular cargo. Here, we demonstrate that deletion of both ABCA1 and ABCG1 in rod photoreceptors leads to age-related accumulation of cholesterol metabolites in the outer retina, photoreceptor dysfunction, degeneration of rod outer segments, and ultimately blindness. A high-fat diet significantly accelerates rod neurodegeneration and vision loss, further highlighting the role of lipid homeostasis in regulating photoreceptor neurodegeneration and vision.

Inflammation-Induced Photoreceptor Cell Death.

Neuroinflammation is an important aspect of many diseases of the eye, and experimental animal models have been widely used to determine its impact on retinal homeostasis and neuron survival. Physical separation of the neurosensory retina from the underlying retinal pigment epithelium (RPE) results in activation and infiltration of macrophages. Numerous studies have shown the critical role of macrophages in retinal disease processes. In retinal detachment, accumulation of macrophages in the subretinal space is associated with changes in cytokine and chemokine profile which lead to photoreceptor cell death. Targeted disruption of macrophage chemotaxis significantly reduces retinal detachment-induced photoreceptor degeneration. Apoptosis is the predominant mechanism of cell death; however regulated necrosis is also a contributor of photoreceptor loss. Therefore, effective neuroprotective approaches could integrate combined inhibition of both apoptotic and regulated necrosis pathways.

Macrophage microRNA-150 promotes pathological angiogenesis as seen in age-related macular degeneration.

Macrophage aging is pathogenic in diseases of the elderly, including age-related macular degeneration (AMD), a leading cause of blindness in older adults. However, the role of microRNAs, which modulate immune processes, in regulating macrophage dysfunction and thereby promoting age-associated diseases is underexplored. Here, we report that microRNA-150 (miR-150) coordinates transcriptomic changes in aged murine macrophages, especially those associated with aberrant lipid trafficking and metabolism in AMD pathogenesis. Molecular profiling confirmed that aged murine macrophages exhibit dysregulated ceramide and phospholipid profiles compared with young macrophages. Of translational relevance, upregulation of miR-150 in human peripheral blood mononuclear cells was also significantly associated with increased odds of AMD, even after controlling for age. Mechanistically, miR-150 directly targets stearoyl-CoA desaturase-2, which coordinates macrophage-mediated inflammation and pathologic angiogenesis, as seen in AMD, in a VEGF-independent manner. Together, our results implicate miR-150 as pathogenic in AMD and provide potentially novel molecular insights into diseases of aging.

GDF15 is elevated in mice following retinal ganglion cell death and in glaucoma patients.

Glaucoma is the second leading cause of blindness worldwide. Physicians often use surrogate endpoints to monitor the progression of glaucomatous neurodegeneration. These approaches are limited in their ability to quantify disease severity and progression due to inherent subjectivity, unreliability, and limitations of normative databases. Therefore, there is a critical need to identify specific molecular markers that predict or measure glaucomatous neurodegeneration. Here, we demonstrate that growth differentiation factor 15 (GDF15) is associated with retinal ganglion cell death. Gdf15 expression in the retina is specifically increased after acute injury to retinal ganglion cell axons and in a murine chronic glaucoma model. We also demonstrate that the ganglion cell layer may be one of the sources of secreted GDF15 and that GDF15 diffuses to and can be detected in aqueous humor (AH). In validating these findings in human patients with glaucoma, we find not only that GDF15 is increased in AH of patients with primary open angle glaucoma (POAG), but also that elevated GDF15 levels are significantly associated with worse functional outcomes in glaucoma patients, as measured by visual field testing. Thus, GDF15 maybe a reliable metric of glaucomatous neurodegeneration, although further prospective validation studies will be necessary to determine if GDF15 can be used in clinical practice.

NAMPT-Mediated NAD(+) Biosynthesis Is Essential for Vision In Mice.

Photoreceptor death is the endpoint of many blinding diseases. Identifying unifying pathogenic mechanisms in these diseases may offer global approaches for facilitating photoreceptor survival. We found that rod or cone photoreceptor-specific deletion of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the major NAD(+) biosynthetic pathway beginning with nicotinamide, caused retinal degeneration. In both cases, we could rescue vision with nicotinamide mononucleotide (NMN). Significantly, retinal NAD(+) deficiency was an early feature of multiple mouse models of retinal dysfunction, including light-induced degeneration, streptozotocin-induced diabetic retinopathy, and age-associated dysfunction. Mechanistically, NAD(+) deficiency caused metabolic dysfunction and consequent photoreceptor death. We further demonstrate that the NAD(+)-dependent mitochondrial deacylases SIRT3 and SIRT5 play important roles in retinal homeostasis and that NAD(+) deficiency causes SIRT3 dysfunction. These findings demonstrate that NAD(+) biosynthesis is essential for vision, provide a foundation for future work to further clarify the mechanisms involved, and identify a unifying therapeutic target for diverse blinding diseases.

Zika Virus Infection in Mice Causes Panuveitis with Shedding of Virus in Tears.

Zika virus (ZIKV) is an emerging flavivirus that causes congenital abnormalities and Guillain-Barré syndrome. ZIKV infection also results in severe eye disease characterized by optic neuritis, chorioretinal atrophy, and blindness in newborns and conjunctivitis and uveitis in adults. We evaluated ZIKV infection of the eye by using recently developed mouse models of pathogenesis. ZIKV-inoculated mice developed conjunctivitis, panuveitis, and infection of the cornea, iris, optic nerve, and ganglion and bipolar cells in the retina. This phenotype was independent of the entry receptors Axl or Mertk, given that Axl(-/-), Mertk(-/-), and Axl(-/-)Mertk(-/-) double knockout mice sustained levels of infection similar to those of control animals. We also detected abundant viral RNA in tears, suggesting that virus might be secreted from lacrimal glands or shed from the cornea. This model provides a foundation for studying ZIKV-induced ocular disease, defining mechanisms of viral persistence, and developing therapeutic approaches for viral infections of the eye.

AAV-mediated gene therapy in Dystrophin-Dp71 deficient mouse leads to blood-retinal barrier restoration and oedema reabsorption.

Dystrophin-Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells that provide a mechanical link at the Müller cell membrane by direct binding to actin and a transmembrane protein complex. Its absence has been related to blood-retinal barrier (BRB) permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels (1). We have previously shown that the adeno-associated virus (AAV) variant, ShH10, transduces Müller cells in the Dp71-null mouse retina efficiently and specifically (2,3). Here, we use ShH10 to restore Dp71 expression in Müller cells of Dp71 deficient mouse to study molecular and functional effects of this restoration in an adult mouse displaying retinal permeability. We show that strong and specific expression of exogenous Dp71 in Müller cells leads to correct localization of Dp71 protein restoring all protein interactions in order to re-establish a proper functional BRB and retina homeostasis thus preventing retina from oedema. This study is the basis for the development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular oedema such as diabetic retinopathy (DR).

Characteristics of chronic lymphocytic leukemia in Senegal.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a mature B-cell neoplasm characterized by the expansion of CD5-positive lymphocytes in peripheral blood. While CLL is the most common type of leukemia in Western populations, the disease is rare in Africans. Hence, clinical and laboratory data and studies of CLL in Sub Saharan populations have been limited. The aims of this study were to analyze the characteristics of senegalese patients with CLL at the time of the diagnosis and to identify the correlation between clinical characteristics (Binet stage) with age, gender, laboratory parameters and chromosomal abnormalities. METHODS: In this study, we investigated the clinical and laboratory characteristics of CLL in Senegal. A total of 40 patients who had been diagnosed with CLL during the period from July 2011 to April 2015 in Senegal were evaluated. Cytology and immunophenotype were performed in all patients to confirm the diagnosis. The prognosis factors such as Binet staging, CD38 and cytogenetic abnormalities were studied. The statistical analysis was performed using STATA version 13 (Stata college station Texas). Each patient signed a free and informed consent form before participating in the study. RESULTS: The mean age was 61 years ranged from 48 to 85. There were 31 males and only 9 females (sex ratio M : F = 3,44). At diagnosic, 82.5 % of the patients were classified as having advanced Binet stages B or C. The prognosis marker CD38 was positive in 28 patients. Cytogenetic abnormalities studied by FISH were performed in 25 patients, among them, 68 % (17 cases) had at least one cytogenetic abnormality and 28 % had 2 simultaneous cytogenetic abnormalities. CONCLUSION: Africans may present with CLL at a younger age and our data suggest that CLL in Senegal may be more aggressive than in Western populations.

Rosai Dorfman disease diagnosed by fine-needle aspiration cytology in a young man with HIV infection.

RDD (Rosai Dorfman disease) is a rare and benign histiocytic proliferative disorder of unknown etiology. FNAC (Fine-needle aspiration cytology) is a useful and reliable tool for the diagnosis of RDD, and as such, biopsy is avoidable.

IL10-driven STAT3 signalling in senescent macrophages promotes pathological eye angiogenesis.

Macrophage dysfunction plays a pivotal role during neovascular proliferation in diseases of ageing including cancers, atherosclerosis and blinding eye disease. In the eye, choroidal neovascularization (CNV) causes blindness in patients with age-related macular degeneration (AMD). Here we report that increased IL10, not IL4 or IL13, in senescent eyes activates STAT3 signalling that induces the alternative activation of macrophages and vascular proliferation. Targeted inhibition of both IL10 receptor-mediated signalling and STAT3 activation in macrophages reverses the ageing phenotype. In addition, adoptive transfer of STAT3-deficient macrophages into eyes of old mice significantly reduces the amount of CNV. Systemic and CD163(+) eye macrophages obtained from AMD patients also demonstrate STAT3 activation. Our studies demonstrate that impaired SOCS3 feedback leads to permissive IL10/STAT3 signalling that promotes alternative macrophage activation and pathological neovascularization. These findings have significant implications for our understanding of the pathobiology of age-associated diseases and may guide targeted immunotherapy.

Dystrophin Dp71 gene deletion induces retinal vascular inflammation and capillary degeneration.

We have previously shown that the deletion of the dystrophin Dp71 gene induces a highly permeable blood-retinal barrier (BRB). Given that BRB breakdown is involved in retinal inflammation and the pathophysiology of many blinding eye diseases, here we investigated whether the absence of Dp71 brings out retinal vascular inflammation and vessel loss by using specific Dp71-null mice. The expression of vascular endothelial growth factor (VEGF), quantified by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay methods, was higher in the retina of Dp71-null mice than in wild-type mice. In contrast, no differences were observed in VEGFR-2 and tumor necrosis factor-α expression. Moreover, mRNA expression of water channel, aquaporin 4 (AQP4) was increased after Dp71 deletion. The Dp71 deletion was also associated with the overexpression of intercellular adhesion molecule 1, which is expressed on endothelial cells surface to recruit leukocytes. Consistent with these findings, the total number of adherent leukocytes per retina, assessed after perfusion with fluorescein isothiocyanate-conjugated concanavalin A, was increased in the absence of Dp71. Finally, a significant increase in capillary degeneration quantified after retinal trypsin digestion was observed in mice lacking Dp71. These data illustrate for the first time that the deletion of Dp71 was associated with retinal vascular inflammation, vascular lesions with increased leukocyte adhesion and capillary degeneration. Thus, dystrophin Dp71 could play a critical role in retinal vascular inflammation disease, and therefore represent a potential therapeutic target.

Seeing through VEGF: innate and adaptive immunity in pathological angiogenesis in the eye.

The central role of vascular endothelial growth factor (VEGF) signaling in regulating normal vascular development and pathological angiogenesis has been documented in multiple studies. Ocular anti-VEGF therapy is highly effective for treating a subset of patients with blinding eye disorders such as diabetic retinopathy and neovascular age-related macular degeneration (AMD). However, chronic VEGF suppression can lead to adverse effects associated with poor visual outcomes due to the loss of prosurvival and neurotrophic capacities of VEGF. In this review, we discuss emerging evidence for immune-related mechanisms that regulate ocular angiogenesis in a VEGF-independent manner. These novel molecular and cellular pathways may provide potential therapeutic avenues for a multitarget strategy, preserving the neuroprotective functions of VEGF in those patients whose disease is unresponsive to VEGF neutralization.

Endothelial cell FGF signaling is required for injury response but not for vascular homeostasis.

Endothelial cells (ECs) express fibroblast growth factor receptors (FGFRs) and are exquisitely sensitive to FGF signals. However, whether the EC or another vascular cell type requires FGF signaling during development, homeostasis, and response to injury is not known. Here, we show that Flk1-Cre or Tie2-Cre mediated deletion of FGFR1 and FGFR2 (Fgfr1/2(Flk1-Cre) or Fgfr1/2(Tie2-Cre) mice), which results in deletion in endothelial and hematopoietic cells, is compatible with normal embryonic development. As adults, Fgfr1/2(Flk1-Cre) mice maintain normal blood pressure and vascular reactivity and integrity under homeostatic conditions. However, neovascularization after skin or eye injury was significantly impaired in both Fgfr1/2(Flk1-Cre) and Fgfr1/2(Tie2-Cre) mice, independent of either hematopoietic cell loss of FGFR1/2 or vascular endothelial growth factor receptor 2 (Vegfr2) haploinsufficiency. Also, impaired neovascularization was associated with delayed cutaneous wound healing. These findings reveal a key requirement for cell-autonomous EC FGFR signaling in injury-induced angiogenesis, but not for vascular homeostasis, identifying the EC FGFR signaling pathway as a target for diseases associated with aberrant vascular proliferation, such as age-related macular degeneration, and for modulating wound healing without the potential toxicity associated with direct manipulation of systemic FGF or VEGF activity.

Eyeballing cholesterol efflux and macrophage function in disease pathogenesis.

Disorders of lipid metabolism are strongly associated with cardiovascular disease. Recently, there has been significant focus on how tissues process lipid deposits. Impaired cholesterol efflux has been shown to be crucial in mediating lipid deposition in atherosclerosis. The inability of macrophages to effectively efflux cholesterol from tissues initiates inflammation, plaque neovascularization, and subsequent rupture. Recent studies suggest that inability to effectively efflux cholesterol from tissues may have global implications far beyond atherosclerosis, extending to the pathophysiology of unrelated diseases. We examine the unifying mechanisms by which impaired cholesterol efflux facilitates tissue-specific inflammation and disease progression in age-related macular degeneration (AMD), a blinding eye disease, and in atherosclerosis, a disease associated with significant cardiovascular morbidity.