RESUMO
Antibacterial agents target the products of essential genes but rarely achieve complete target inhibition. Thus, the all-or-none definition of essentiality afforded by traditional genetic approaches fails to discern the most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs. In contrast, gene "vulnerability" is a continuous, quantifiable trait that relates the magnitude of gene inhibition to the effect on bacterial fitness. We developed a CRISPR interference-based functional genomics method to systematically titrate gene expression in Mycobacterium tuberculosis (Mtb) and monitor fitness outcomes. We identified highly vulnerable genes in various processes, including novel targets unexplored for drug discovery. Equally important, we identified invulnerable essential genes, potentially explaining failed drug discovery efforts. Comparison of vulnerability between the reference and a hypervirulent Mtb isolate revealed incomplete conservation of vulnerability and that differential vulnerability can predict differential antibacterial susceptibility. Our results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.
Assuntos
Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Mycobacterium tuberculosis/genética , Aminoacil-tRNA Sintetases/metabolismo , Antituberculosos/farmacologia , Teorema de Bayes , Evolução Biológica , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Variants of the CFH gene, encoding complement factor H (CFH), show strong association with age-related macular degeneration (AMD), a major cause of blindness. Here, we used murine models of AMD to examine the contribution of CFH to disease etiology. Cfh deletion protected the mice from the pathogenic subretinal accumulation of mononuclear phagocytes (MP) that characterize AMD and showed accelerated resolution of inflammation. MP persistence arose secondary to binding of CFH to CD11b, which obstructed the homeostatic elimination of MPs from the subretinal space mediated by thrombospsondin-1 (TSP-1) activation of CD47. The AMD-associated CFH(H402) variant markedly increased this inhibitory effect on microglial cells, supporting a causal link to disease etiology. This mechanism is not restricted to the eye, as similar results were observed in a model of acute sterile peritonitis. Pharmacological activation of CD47 accelerated resolution of both subretinal and peritoneal inflammation, with implications for the treatment of chronic inflammatory disease.
Assuntos
Antígeno CD47/imunologia , Fator H do Complemento/imunologia , Inflamação/imunologia , Degeneração Macular/imunologia , Animais , Fator H do Complemento/genética , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imuno-Histoquímica , Inflamação/genética , Degeneração Macular/genética , Camundongos , Camundongos Knockout , Peritonite/genética , Peritonite/imunologia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Retinal melanosome/melanolipofuscin-containing cells (MCCs), clinically visible as hyperreflective foci (HRF) and a highly predictive imaging biomarker for the progression of age-related macular degeneration (AMD), are widely believed to be migrating retinal pigment epithelial (RPE) cells. Using human donor tissue, we identify the vast majority of MCCs as melanophages, melanosome/melanolipofuscin-laden mononuclear phagocytes (MPs). Using serial block-face scanning electron microscopy, RPE flatmounts, bone marrow transplantation and in vitro experiments, we show how retinal melanophages form by the transfer of melanosomes from the RPE to subretinal MPs when the "don't eat me" signal CD47 is blocked. These melanophages give rise to hyperreflective foci in Cd47-/--mice in vivo, and are associated with RPE dysmorphia similar to intermediate AMD. Finally, we show that Cd47 expression in human RPE declines with age and in AMD, which likely participates in melanophage formation and RPE decline. Boosting CD47 expression in AMD might protect RPE cells and delay AMD progression.
Assuntos
Antígeno CD47 , Degeneração Macular , Humanos , Animais , Camundongos , Antígeno CD47/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Degeneração Macular/metabolismo , Retina/metabolismo , Tomografia de Coerência Óptica/métodosRESUMO
Photoreceptor degeneration in age-related macular degeneration (AMD) is associated with an infiltration and chronic accumulation of mononuclear phagocytes (MPs). We have previously shown that Cx3cr1-deficient mice develop age- and stress- related subretinal accumulation of MPs, which is associated with photoreceptor degeneration. Cx3cr1-deficient MPs have been shown to increase neuronal apoptosis through IL-1ß in neuroinflammation of the brain. The reason for increased IL-1ß secretion from Cx3cr1-deficient MPs, and whether IL-1ß is responsible for increased photoreceptor apoptosis in Cx3cr1-deficient mice, has not been elucidated. Here we show that Cx3cr1-deficient MPs express increased surface P2X7 receptor (P2RX7), which stimulates IL-1ß maturation and secretion. P2RX7 and IL-1ß inhibition efficiently blunted Cx3cr1-MP-dependent photoreceptor apoptosis in a monocyte/retina coculture system and in light-induced subretinal inflammation of Cx3cr1-deficient mice in vivo. Our results provide an explanation for increased CX3CR1-dependent IL-1ß secretion and suggest that IL-1ß or P2RX7 inhibition can help inhibit the inflammation-associated photoreceptor cell loss in late AMD, including geographic atrophy, for which no efficient treatment currently exists.
Assuntos
Interleucina-1beta/metabolismo , Degeneração Macular/metabolismo , Sistema Fagocitário Mononuclear/metabolismo , Células Fotorreceptoras/metabolismo , Receptores de Quimiocinas/deficiência , Receptores Purinérgicos P2X7/biossíntese , Animais , Receptor 1 de Quimiocina CX3C , Técnicas de Cocultura , Feminino , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sistema Fagocitário Mononuclear/patologia , Fagócitos/metabolismo , Fagócitos/patologia , Células Fotorreceptoras/patologia , Regulação para Cima/fisiologiaRESUMO
Contrary to Alzheimer's disease (AD), the APOE2 allele increases and the APOE4 allele reduces the risk to develop age-related macular degeneration (AMD) compared with the most common APOE3 allele. The underlying mechanism for this association with AMD and the reason for the puzzling difference with AD are unknown. We previously demonstrated that pathogenic subretinal mononuclear phagocytes (MPs) accumulate in Cx3cr1-deficient mice due to the overexpression of APOE, interleukin-6, and CC chemokine ligand 2 (CCL2). We here show using targeted replacement mice expressing the human APOE isoforms (TRE2, TRE3, and TRE4) that MPs of TRE2 mice express increased levels of APOE, interleukin-6, and CCL2 and develop subretinal MP accumulation, photoreceptor degeneration, and exaggerated choroidal neovascularization similar to AMD. Pharmacological inhibition of the cytokine induction inhibited the pathogenic subretinal inflammation. In the context of APOE-dependent subretinal inflammation in Cx3cr1(GFP/GFP) mice, the APOE4 allele led to diminished APOE and CCL2 levels and protected Cx3cr1(GFP/GFP) mice against harmful subretinal MP accumulation observed in Cx3cr1(GFP/GFP)TRE3 mice. Our study shows that pathogenic subretinal inflammation is APOE isoform-dependent and provides the rationale for the previously unexplained implication of the APOE2 isoform as a risk factor and the APOE4 isoform as a protective factor in AMD pathogenesis. SIGNIFICANCE STATEMENT: The understanding of how genetic predisposing factors, which play a major role in age-related macular degeneration (AMD), participate in its pathogenesis is an important clue to decipher the pathomechanism and develop efficient therapies. In this study, we used transgenic, targeted replacement mice that carry the three human APOE isoform-defining sequences at the mouse APOE chromosomal location and express the human APOE isoforms. Our study is the first to show how APOE2 provokes and APOE4 inhibits the cardinal AMD features, inflammation, degeneration, and exaggerated neovascularization. Our findings reflect the clinical association of the genetic predisposition that was recently confirmed in a major pooled analysis. They emphasize the role of APOE in inflammation and inflammation in AMD.
Assuntos
Envelhecimento , Apolipoproteínas E/metabolismo , Regulação da Expressão Gênica/genética , Inflamação/etiologia , Degeneração Macular/complicações , Degeneração Macular/genética , Isoformas de Proteínas/metabolismo , Animais , Apolipoproteínas E/genética , Receptor 1 de Quimiocina CX3C , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Proteínas do Olho/genética , Feminino , Humanos , Inflamação/genética , Lasers/efeitos adversos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/genética , Isoformas de Proteínas/genética , Receptores de Quimiocinas/genética , Retina/citologiaRESUMO
PURPOSE: Thinning of the RPE and the underlying vascular layer, the choroid, is observed with age in many human eye disorders. The reasons for this thinning are ill-defined. Here, we highlight the possible role of T lymphocyte recruitment in choroidoretinal thinning in aged and light-challenged mice. METHODS: In age and light challenge models, we measured chemokine concentrations using enzyme-linked immunosorbent assay and used flow cytometry to characterize lymphocyte populations. We quantified thinning in eye immunosections and RPE65 expression using quantitative PCR. RESULTS: Age and light challenge led to increased levels of the lymphotactic protein CXCL10 alone (aging) or in conjunction with CXCL9 (light challenge). Increased numbers of CD3+ T lymphocytes, most of them CD8+ cytotoxic T lymphocytes, were also observed in the choroid and retina of old mice and following light challenge. Influx of T lymphocytes was associated with RPE and choroidal thinning and diminished expression of RPE65 mRNA, an essential enzyme of the visual cycle. CONCLUSIONS: The observations from this study suggest that cytotoxic CD8(+) T lymphocytes might participate in choroidal and RPE degeneration and that modulation of T lymphocyte recruitment might be a novel strategy to reduce choroidoretinal dysfunctions observed with age and following photo-oxidative stress.
Assuntos
Envelhecimento/genética , Corioide/efeitos da radiação , RNA Mensageiro/genética , Epitélio Pigmentado da Retina/efeitos da radiação , Linfócitos T Citotóxicos/efeitos da radiação , cis-trans-Isomerases/genética , Envelhecimento/imunologia , Envelhecimento/patologia , Animais , Movimento Celular/efeitos da radiação , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Quimiocina CXCL9/genética , Quimiocina CXCL9/imunologia , Corioide/imunologia , Corioide/patologia , Regulação da Expressão Gênica , Humanos , Luz/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Processos Fotoquímicos , RNA Mensageiro/imunologia , Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/patologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , cis-trans-Isomerases/imunologiaRESUMO
Inflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1ß expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4(+/-) mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4(+/-) CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1ß expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV.
Assuntos
Neovascularização de Coroide/prevenção & controle , Endotélio Vascular/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Macrófagos Peritoneais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Western Blotting , Proteínas de Ligação ao Cálcio , Primers do DNA , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The pro-inflammatory cytokine IL-1ß has been shown to promote angiogenesis. It can have a neurotoxic or neuroprotective effect. Here, we have studied the expression of IL-1ß in vivo and the effect of the IL-1 receptor antagonist on choroidal neovascularization (CNV) and retinal degeneration (RD). IL-1ß expression significantly increased after laser injury (real time PCR) in C57BL/6 mice, in the C57BL/6 Cx3cr1(-/-) model of age-related macular degeneration (enzyme-linked immunoabsorbent assay), and in albino Wistar rats and albino BALB Cx3cr1(+/+) and Cx3cr1(-/-) mice (enzyme-linked immunoabsorbent assay) after light injury. IL-1ß was localized to Ly6G-positive, Iba1-negative infiltrating neutrophils in laser-induced CNV as determined by IHC. IL-1 receptor antagonist treatment significantly inhibited CNV but did not affect Iba1-positive macrophage recruitment to the injury site. IL-1ß significantly increased endothelial cell outgrowth in aortic ring assay independently of vascular endothelial growth factor, suggesting a direct effect of IL-1ß on choroidal endothelial cell proliferation. Inhibition of IL-1ß in light- and laser-induced RD models did not alter photoreceptor degeneration in Wistar rats, C57BL/6 mice, or RD-prone Cx3cr1(-/-) mice. Our results suggest that IL-1ß inhibition might represent a valuable and safe alternative to inhibition of vascular endothelial growth factor in the control of CNV in the context of concomitant photoreceptor degeneration as observed in age-related macular degeneration.
Assuntos
Neovascularização de Coroide/metabolismo , Interleucina-1beta/antagonistas & inibidores , Células Fotorreceptoras/metabolismo , Degeneração Retiniana/metabolismo , Animais , Neovascularização de Coroide/prevenção & controle , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Regeneração Nervosa/fisiologia , Células Fotorreceptoras/patologia , Ratos , Ratos Wistar , Degeneração Retiniana/prevenção & controle , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Netrins are secreted molecules with roles in axon guidance and angiogenesis. We identified Netrin-4 as a gene specifically overexpressed in VEGF-stimulated endothelial cells (EC) in vitro as well as in vivo. Knockdown of Netrin-4 expression in EC increased their ability to form tubular structures on Matrigel. To identify which receptor is involved, we showed by quantitative RT-PCR that EC express three of the six Netrin-1 cognate receptors: neogenin, Unc5B, and Unc5C. In contrast to Netrin-1, Netrin-4 bound only to neogenin but not to Unc5B or Unc5C receptors. Neutralization of Netrin-4 binding to neogenin by blocking antibodies abolished the chemotactic effect of Netrin-4. Furthermore, the silencing of either neogenin or Unc5B abolished Netrin-4 inhibitory effect on EC migration, suggesting that both receptors are essential for its function in vitro. Coimmunoprecipitation experiments demonstrated that Netrin-4 increased the association between Unc5B and neogenin on VEGF- or FGF-2-stimulated EC. Finally, we showed that Netrin-4 significantly reduced pathological angiogenesis in Matrigel and laser-induced choroidal neovascularization models. Interestingly, Netrin-4, neogenin, and Unc5B receptor expression was up-regulated in choroidal neovessel EC after laser injury. Moreover, Netrin-4 overexpression delayed tumor angiogenesis in a model of s.c. xenograft. We propose that Netrin-4 acts as an antiangiogenic factor through binding to neogenin and recruitment of Unc5B.
Assuntos
Células Endoteliais/citologia , Proteínas de Membrana/metabolismo , Neovascularização Patológica , Fatores de Crescimento Neural/fisiologia , Receptores de Superfície Celular/metabolismo , Animais , Bovinos , Linhagem Celular Tumoral , Células Cultivadas , Quimiotaxia , Feminino , Humanos , Lasers/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/irrigação sanguínea , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Receptores de Netrina , Netrinas , Neoplasias da Próstata/patologia , Ligação Proteica/fisiologia , Proteínas Recombinantes/farmacologia , Transplante Heterólogo , Regulação para Cima/genéticaRESUMO
The role of retinal microglial cells (MCs) in age-related macular degeneration (AMD) is unclear. Here we demonstrated that all retinal MCs express CX3C chemokine receptor 1 (CX3CR1) and that homozygosity for the CX3CR1 M280 allele, which is associated with impaired cell migration, increases the risk of AMD. In humans with AMD, MCs accumulated in the subretinal space at sites of retinal degeneration and choroidal neovascularization (CNV). In CX3CR1-deficient mice, MCs accumulated subretinally with age and albino background and after laser impact preceding retinal degeneration. Raising the albino mice in the dark prevented both events. The appearance of lipid-bloated subretinal MCs was drusen-like on funduscopy of senescent mice, and CX3CR1-dependent MC accumulation was associated with an exacerbation of experimental CNV. These results show that CX3CR1-dependent accumulation of subretinal MCs evokes cardinal features of AMD. These findings reveal what we believe to be a novel pathogenic process with important implications for the development of new therapies for AMD.
Assuntos
Degeneração Macular/etiologia , Microglia/patologia , Receptores de Quimiocinas/genética , Retina/patologia , Alelos , Animais , Receptor 1 de Quimiocina CX3C , Movimento Celular/genética , Neovascularização de Coroide/genética , Neovascularização de Coroide/patologia , Homozigoto , Humanos , Degeneração Macular/genética , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Knockout , Microglia/metabolismo , Polimorfismo Genético , Retina/metabolismo , Drusas Retinianas/genética , Drusas Retinianas/patologiaRESUMO
Background: CD36, a member of the class B scavenger receptor family, participates in Toll-like receptor signaling on mononuclear phagocytes (MP) and can promote sterile pathogenic inflammation. We here analyzed the effect of CD36 deficiency on retinal inflammation and photoreceptor degeneration, the hallmarks of age-related macular degeneration (AMD), that characterize Cx3cr1-/-mice. Methods: We analyzed subretinal MP accumulation, and cone- and rod-degeneration in light-challenged and aged, CD36 competent or deficient, hyper-inflammatory Cx3cr1-/- mice, using histology and immune-stained retinal flatmounts. Monocytes (Mo) were subretinally adoptively transferred to evaluate their elimination rate from the subretinal space and Interleukin 6 (IL-6) secretion from cultured Mo-derived cells (MdCs) of the different mouse strains were analyzed. Results: CD36 deficient Cx3cr1-/- mice were protected against age- and light-induced subretinal inflammation and associated cone and rod degeneration. CD36 deficiency in Cx3cr1-/- MPs inhibited their prolonged survival in the immune-suppressive subretinal space and reduced the exaggerated IL-6 secretion observed in Cx3cr1-/- MPs that we previously showed leads to increased subretinal MP survival. Conclusion:Cd36 deficiency significantly protected hyperinflammatory Cx3cr1-/- mice against subretinal MP accumulation and associated photoreceptor degeneration. The observed CD36-dependent induction of pro-inflammatory IL-6 might be at least partially responsible for the prolonged MP survival in the immune-suppressive environment and its pathological consequences on photoreceptor homeostasis.
Assuntos
Antígenos CD36/deficiência , Receptor 1 de Quimiocina CX3C/deficiência , Suscetibilidade a Doenças , Degeneração Retiniana/etiologia , Retinite/etiologia , Animais , Biomarcadores , Citocinas/metabolismo , Modelos Animais de Doenças , Predisposição Genética para Doença , Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Camundongos , Camundongos Knockout , Fagócitos/imunologia , Fagócitos/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Retinite/metabolismo , Retinite/patologiaRESUMO
BACKGROUND: In the Western world, a major cause of blindness is age-related macular degeneration (AMD). Recent research in angiogenesis has furthered the understanding of choroidal neovascularization, which occurs in the "wet" form of AMD. In contrast, very little is known about the mechanisms of the predominant, "dry" form of AMD, which is characterized by retinal atrophy and choroidal involution. The aim of this study is to elucidate the possible implication of the scavenger receptor CD36 in retinal degeneration and choroidal involution, the cardinal features of the dry form of AMD. METHODS AND FINDINGS: We here show that deficiency of CD36, which participates in outer segment (OS) phagocytosis by the retinal pigment epithelium (RPE) in vitro, leads to significant progressive age-related photoreceptor degeneration evaluated histologically at different ages in two rodent models of CD36 invalidation in vivo (Spontaneous hypertensive rats (SHR) and CD36-/- mice). Furthermore, these animals developed significant age related choroidal involution reflected in a 100%-300% increase in the avascular area of the choriocapillaries measured on vascular corrosion casts of aged animals. We also show that proangiogenic COX2 expression in RPE is stimulated by CD36 activating antibody and that CD36-deficient RPE cells from SHR rats fail to induce COX2 and subsequent vascular endothelial growth factor (VEGF) expression upon OS or antibody stimulation in vitro. CD36-/- mice express reduced levels of COX2 and VEGF in vivo, and COX2-/- mice develop progressive choroidal degeneration similar to what is seen in CD36 deficiency. CONCLUSIONS: CD36 deficiency leads to choroidal involution via COX2 down-regulation in the RPE. These results show a novel molecular mechanism of choroidal degeneration, a key feature of dry AMD. These findings unveil a pathogenic process, to our knowledge previously undescribed, with important implications for the development of new therapies.
Assuntos
Antígenos CD36/genética , Doenças da Coroide/enzimologia , Ciclo-Oxigenase 2/metabolismo , Regulação para Baixo/genética , Animais , Antígenos CD36/fisiologia , Células Cultivadas , Doenças da Coroide/genética , Ciclo-Oxigenase 2/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Degeneração Retiniana/enzimologia , Degeneração Retiniana/genéticaRESUMO
Photo-transduction in cone segments (CS) is crucial for high acuity daytime vision. For ill-defined reasons, CS degenerate in retinitis pigmentosa (RP) and in the transitional zone (TZ) of atrophic zones (AZ), which characterize geographic atrophy (GA). Our experiments confirm the loss of cone segments (CS) in the TZ of patients with GA and show their association with subretinal CD14(+)mononuclear phagocyte (MP) infiltration that is also reported in RP. Using human and mouse MPs in vitro and inflammation-prone Cx3cr1(GFP/GFP) mice in vivo, we demonstrate that MP-derived IL-1ß leads to severe CS degeneration. Our results strongly suggest that subretinal MP accumulation participates in the observed pathological photoreceptor changes in these diseases. Inhibiting subretinal MP accumulation or Il-1ß might protect the CS and help preserve high acuity daytime vision in conditions characterized by subretinal inflammation, such as AMD and RP.
Assuntos
Atrofia Geográfica/patologia , Atrofia Geográfica/fisiopatologia , Interleucina-1beta/metabolismo , Fagócitos/imunologia , Retina/patologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Animais , Humanos , CamundongosRESUMO
Physiologically, the retinal pigment epithelium (RPE) expresses immunosuppressive signals such as FAS ligand (FASL), which prevents the accumulation of leukocytes in the subretinal space. Age-related macular degeneration (AMD) is associated with a breakdown of the subretinal immunosuppressive environment and chronic accumulation of mononuclear phagocytes (MPs). We show that subretinal MPs in AMD patients accumulate on the RPE and express high levels of APOE. MPs of Cx3cr1(-/-) mice that develop MP accumulation on the RPE, photoreceptor degeneration, and increased choroidal neovascularization similarly express high levels of APOE. ApoE deletion in Cx3cr1(-/-) mice prevents pathogenic age- and stress-induced subretinal MP accumulation. We demonstrate that increased APOE levels induce IL-6 in MPs via the activation of the TLR2-CD14-dependent innate immunity receptor cluster. IL-6 in turn represses RPE FasL expression and prolongs subretinal MP survival. This mechanism may account, in part, for the MP accumulation observed in Cx3cr1(-/-) mice. Our results underline the inflammatory role of APOE in sterile inflammation in the immunosuppressive subretinal space. They provide rationale for the implication of IL-6 in AMD and open avenues toward therapies inhibiting pathogenic chronic inflammation in late AMD.
Assuntos
Apolipoproteínas E/imunologia , Degeneração Macular/imunologia , Fagócitos/citologia , Animais , Apolipoproteínas E/genética , Receptor 1 de Quimiocina CX3C , Sobrevivência Celular , Neovascularização de Coroide , Proteína Ligante Fas/imunologia , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Degeneração Macular/genética , Degeneração Macular/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Fagócitos/imunologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia , Epitélio Pigmentado da Retina/imunologiaRESUMO
Recent evidence suggests that transient hyperglycemia in extremely low birth weight infants is strongly associated with the occurrence of retinopathy of prematurity (ROP). We propose a new model of Neonatal Hyperglycemia-induced Retinopathy (NHIR) that mimics many aspects of retinopathy of prematurity. Hyperglycemia was induced in newborn rat pups by injection of streptozocine (STZ) at post natal day one (P1). At various time points, animals were assessed for vascular abnormalities, neuronal cell death and accumulation and activation of microglial cells. We here report that streptozotocin induced a rapid and sustained increase of glycemia from P2/3 to P6 without affecting rat pups gain weight or necessitating insulin treatment. Retinal vascular area was significantly reduced in P6 hyperglycemic animals compared to control animals. Hyperglycemia was associated with (i) CCL2 chemokine induction at P6, (ii) a significant recruitment of inflammatory macrophages and an increase in total number of Iba+ macrophages/microglia cells in the inner nuclear layer (INL), and (iii) excessive apoptosis in the INL. NHIR thereby reproduces several aspects of ischemic retinopathies, including ROP and diabetic retinopathies, and might be a useful model to decipher hyperglycemia-induced cellular and molecular mechanisms in the small rodent.
Assuntos
Hiperglicemia/complicações , Retinopatia da Prematuridade/etiologia , Retinopatia da Prematuridade/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/genética , Apoptose/fisiologia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Hiperglicemia/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/metabolismo , Neovascularização Fisiológica/fisiologia , Ratos , Retina/metabolismo , Retina/patologia , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Estreptozocina/toxicidadeRESUMO
Atrophic age-related macular degeneration (AMD) is associated with the subretinal accumulation of mononuclear phagocytes (MPs). Their role in promoting or inhibiting retinal degeneration is unknown. We here show that atrophic AMD is associated with increased intraocular CCL2 levels and subretinal CCR2(+) inflammatory monocyte infiltration in patients. Using age- and light-induced subretinal inflammation and photoreceptor degeneration in Cx3cr1 knockout mice, we show that subretinal Cx3cr1 deficient MPs overexpress CCL2 and that both the genetic deletion of CCL2 or CCR2 and the pharmacological inhibition of CCR2 prevent inflammatory monocyte recruitment, MP accumulation and photoreceptor degeneration in vivo. Our study shows that contrary to CCR2 and CCL2, CX3CR1 is constitutively expressed in the retina where it represses the expression of CCL2 and the recruitment of neurotoxic inflammatory CCR2(+) monocytes. CCL2/CCR2 inhibition might represent a powerful tool for controlling inflammation and neurodegeneration in AMD.
Assuntos
Degeneração Macular/imunologia , Monócitos/imunologia , Células Fotorreceptoras de Vertebrados/imunologia , Receptores CCR2/imunologia , Receptores de Quimiocinas/deficiência , Animais , Receptor 1 de Quimiocina CX3C , Quimiocina CCL2/imunologia , Feminino , Humanos , Inflamação/genética , Inflamação/imunologia , Degeneração Macular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos , Receptores CCR2/genética , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologiaRESUMO
PURPOSE: Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or "wet" Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice. METHODS: The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with "wet" AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8(+/-) mice expressing ß-galactosidase. Aged Mfge8(+/-) and Mfge8(-/-) mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV. RESULTS: rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8(-/-) mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch's membrane (BM) was slightly but significantly thicker in Mfge8(-/-) mice as compared to controls. CONCLUSIONS: Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8(-/-) mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD.