RESUMO
Age-related macular degeneration (AMD) is associated with an overactive complement system and an increase in circulating antibodies. Our search for potential neoantigens that can trigger complement activation in disease has led us to investigate elastin. A loss of the elastin layer (EL) of Bruch's membrane (BrM) has been reported in aging and AMD together with an increase of serum elastin-derived peptides and α-elastin antibodies. In the mouse model of cigarette smoke exposure (CSE), damage in BrM, loss of the EL, and vision loss are dependent on complement activation. We have examined the hypothesis that CSE generates immunogenic elastin neoepitopes that trigger an increase in α-elastin IgG and IgM antibodies, which can then bind to the neoepitopes in the target cells or membranes, triggering complement activation. Specifically, we showed that immunization with elastin peptide oxidatively modified by cigarette smoke (ox-elastin) exacerbated ocular pathology and vision loss in CSE mice. In contrast, mice receiving peptide immunotherapy (PIT) with ox-elastin did not lose vision over the smoking period and exhibited a more preserved BrM. Immunization and PIT correlated with humoral immunity and complement activation and IgG/IgM deposition in the RPE/BrM/choroid. Finally, PIT modulated immune markers IFNγ and IL-4. The data further support the hypothesis that complement activation, triggered by immune complex formation in target tissues, plays a role in ocular damage in the CSE model. As PIT with ox-elastin peptides reduces damage, we discuss the possibility that AMD progression might be preventable.
Assuntos
Lâmina Basilar da Corioide , Degeneração Macular , Camundongos , Animais , Lâmina Basilar da Corioide/patologia , Elastina/metabolismo , Imunização , Degeneração Macular/metabolismo , Imunoglobulina M , Imunoglobulina GRESUMO
PURPOSE: Age-related macular degeneration (AMD), the leading cause of blindness in western populations, is associated with an overactive complement system, and an increase in circulating antibodies against certain epitopes, including elastin. As loss of the elastin layer of Bruch's membrane (BrM) has been reported in aging and AMD, we previously showed that immunization with elastin peptide oxidatively modified by cigarette smoke (ox-elastin), exacerbated ocular pathology in the smoke-induced ocular pathology (SIOP) model. Here we asked whether ox-elastin peptide-based immunotherapy (PIT) ameliorates damage. METHODS: C57BL/6J mice were injected with ox-elastin peptide at two doses via weekly subcutaneous administration, while exposed to cigarette smoke for 6 months. FcγR-/- and uninjected C57BL/6J mice served as controls. Retinal morphology was assessed by electron microscopy, and complement activation, antibody deposition and mechanisms of immunological tolerance were assessed by Western blotting and ELISA. RESULTS: Elimination of Fcγ receptors, preventing antigen/antibody-dependent cytotoxicity, protected against SIOP. Mice receiving PIT with low dose ox-elastin (LD-PIT) exhibited reduced humoral immunity, reduced complement activation and IgG/IgM deposition in the RPE/choroid, and largely a preserved BrM. While there is no direct evidence of ox-elastin pathogenicity, LD-PIT reduced IFNγ and increased IL-4 within RPE/choroid. High dose PIT was not protective. CONCLUSIONS: These data further support ox-elastin role in ocular damage in part via elastin-specific antibodies, and support the corollary that PIT with ox-elastin attenuates ocular pathology. Overall, damage is associated with complement activation, antibody-dependent cell-mediated cytotoxicity, and altered cytokine signature.
Assuntos
Fumar Cigarros/efeitos adversos , Elastina/imunologia , Imunoterapia/métodos , Degeneração Macular/terapia , Peptídeos/uso terapêutico , Receptores de IgG/efeitos dos fármacos , Fumaça/efeitos adversos , Animais , Ativação do Complemento , Modelos Animais de Doenças , Elastina/metabolismo , Degeneração Macular/induzido quimicamente , Degeneração Macular/diagnóstico , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Peptídeos/imunologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/ultraestruturaRESUMO
Purpose: The risk for age-related macular degeneration has been tied to an overactive complement system. Despite combined attempts by academia and industry to develop therapeutics that modulate the complement response, particularly in the late geographic atrophy form of advanced AMD, to date, there is no effective treatment. We have previously demonstrated that pathology in the smoke-induced ocular pathology (SIOP) model, a model with similarities to dry AMD, is dependent on activation of the alternative complement pathway and that a novel complement activation site targeted inhibitor of the alternative pathway can be delivered to ocular tissues via an adeno-associated virus (AAV). Methods: Two different viral vectors for specific tissue targeting were compared: AAV5-VMD2-CR2-fH for delivery to the retinal pigment epithelium (RPE) and AAV2YF-smCBA-CR2-fH for delivery to retinal ganglion cells (RGCs). Efficacy was tested in SIOP (6 months of passive smoke inhalation), assessing visual function (optokinetic responses), retinal structure (optical coherence tomography), and integrity of the RPE and Bruch's membrane (electron microscopy). Protein chemistry was used to assess complement activation, CR2-fH tissue distribution, and CR2-fH transport across the RPE. Results: RPE- but not RGC-mediated secretion of CR2-fH was found to reduce SIOP and complement activation in RPE/choroid. Bioavailability of CR2-fH in RPE/choroid could be confirmed only after AAV5-VMD2-CR2-fH treatment, and inefficient, adenosine triphosphate-dependent transport of CR2-fH across the RPE was identified. Conclusions: Our results suggest that complement inhibition for AMD-like pathology is required basal to the RPE and argues in favor of AAV vector delivery to the RPE or outside the blood-retina barrier.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Inativadores do Complemento/administração & dosagem , Degeneração Macular/tratamento farmacológico , Epitélio Pigmentado da Retina/patologia , Animais , Corioide , Modelos Animais de Doenças , Injeções Intravítreas , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Camundongos , Camundongos Endogâmicos C57BL , Retina , Epitélio Pigmentado da Retina/efeitos dos fármacos , Tomografia de Coerência ÓpticaRESUMO
Purpose: Age-related macular degeneration (AMD) is the leading cause of blindness in Western populations. While an overactive complement system has been linked to pathogenesis, mechanisms contributing to its activation are largely unknown. In aged and AMD eyes, loss of the elastin layer (EL) of Bruch's membrane (BrM) has been reported. Elastin antibodies are elevated in patients with AMD, the pathogenic significance of which is unclear. Here we assess the role of elastin antibodies using a mouse model of smoke-induced ocular pathology (SIOP), which similarly demonstrates EL loss. Methods: C57BL/6J mice were immunized with elastin or elastin peptide oxidatively modified by cigarette smoke (ox-elastin). Mice were then exposed to cigarette smoke or air for 6 months. Visual function was assessed by optokinetic response, retinal morphology by spectral-domain optical coherence tomography and electron microscopy, and complement activation and antibody deposition by Western blot. Results: Ox-elastin IgG and IgM antibodies were elevated in ox-elastin immunized mice following 6 months of smoke, whereas elastin immunization had a smaller effect. Ox-elastin immunization exacerbated smoke-induced vision loss, with thicker BrM and more damaged retinal pigment epithelium (RPE) mitochondria compared with mice immunized with elastin or nonimmunized controls. These changes were correlated with increased levels of IgM, IgG2, IgG3, and complement activation products in RPE/choroid. Conclusions: These data demonstrate that SIOP mice generate elastin-specific antibodies and that immunization with ox-elastin exacerbates ocular pathology. Elastin antibodies represented complement fixing isotypes that, together with the increased presence of complement activation seen in immunized mice, suggest that elastin antibodies exert pathogenic effects through mediating complement activation.
Assuntos
Autoanticorpos/sangue , Lâmina Basilar da Corioide/patologia , Modelos Animais de Doenças , Elastina/imunologia , Atrofia Geográfica/etiologia , Epitélio Pigmentado da Retina/patologia , Fumar/efeitos adversos , Animais , Western Blotting , Ativação do Complemento/fisiologia , Proteínas do Sistema Complemento/metabolismo , Sensibilidades de Contraste/fisiologia , Ensaio de Imunoadsorção Enzimática , Atrofia Geográfica/imunologia , Atrofia Geográfica/patologia , Imunização , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Camundongos , Camundongos Endogâmicos C57BL , Nistagmo Optocinético/fisiologia , Oxirredução , Produtos do Tabaco , Acuidade Visual/fisiologiaRESUMO
PURPOSE: Extracellular vesicles (EVs) can mediate long-distance communication in polarized RPE monolayers. Specifically, EVs from oxidatively stressed donor cells (stress EVs) rapidly reduced barrier function (transepithelial resistance, TER) in naïve recipient monolayers, when compared to control EVs. This effect on TER was dependent on dynamin-mediated EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Here, we further determined molecular mechanisms involved in uptake of EVs by naïve RPE cells. METHODS: RPE cells were grown as monolayers in media supplemented with 1% FBS followed by transfer to FBS-free media. Cultures were used to collect control or stress EVs upon treatment with H2O2, others served as naïve recipient cells. In recipient monolayers, TER was used to monitor EV-uptake-based activity, live-cell imaging confirmed uptake. EV surface proteins were quantified by protein chemistry. RESULTS: Clathrin-independent, lipid raft-mediated internalization was excluded as an uptake mechanism. Known ligand-receptor interactions involved in clathrin-dependent endocytosis include integrins and proteoglycans. Desialylated glycans and integrin-receptors on recipient cells were necessary for EV uptake and subsequent reduction of TER in recipient cells. Protein quantifications confirmed elevated levels of ligands and neuraminidase on stress EVs. However, control EVs could confer activity in the TER assay if exogenous neuraminidase or additional ligand was provided. CONCLUSIONS: In summary, while EVs from both stressed cells and control contain cargo to communicate stress messages to naive RPE cells, stress EVs contain surface ligands that confer rapid uptake by recipient cells. We propose that EVs potentially contribute to RPE dysfunction in aging and disease.