RESUMO
UNLABELLED: Photooxidative damage and chronic innate immune activation have been implicated in retinal pigment epithelium (RPE) dysfunction, a process that underlies blinding diseases such as age-related macular degeneration (AMD). To identify a potential molecular link between these mechanisms, we investigated whether lipofuscin-mediated phototoxicity activates the NLRP3 inflammasome in RPE cells in vitro. We found that blue light irradiation (dominant wavelength 448 nm, irradiance 0.8 mW/cm(2), duration 6 h) of lipofuscin-loaded primary human RPE cells and ARPE-19 cells induced photooxidative damage, lysosomal membrane permeabilization (79.5 % of cells vs. 3.8 % in nonirradiated controls), and cytosolic leakage of lysosomal enzymes. This resulted in activation of the inflammasome with activation of caspase-1 and secretion of interleukin-1ß (14.6 vs. 0.9 pg/ml in nonirradiated controls) and interleukin-18 (87.7 vs. 0.2 pg/ml in nonirradiated controls). Interleukin secretion was dependent on the activity of NLRP3, caspase-1, and lysosomal proteases cathepsin B and L. These results demonstrate that accumulation of lipofuscin-like material in vitro renders RPE cells susceptible to phototoxic destabilization of lysosomes, resulting in NLRP3 inflammasome activation and secretion of inflammatory cytokines. This new mechanism of inflammasome activation links photooxidative damage and innate immune activation in RPE pathology and may provide novel targets for therapeutic intervention in retinal diseases such as AMD. KEY MESSAGE: ⢠Visible light irradiation of lipofuscin-loaded RPE cells activates inflammasome. ⢠Inflammasome activation results from lysosomal permeabilization and enzyme leakage. ⢠Inflammasome activation induces secretion of inflammatory cytokines by RPE cells. ⢠Photooxidative damage by visible light as new mechanism of inflammasome activation. ⢠Novel link between hallmark pathogenetic features of retinal degenerative diseases.
Assuntos
Proteínas de Transporte/imunologia , Inflamassomos/imunologia , Luz , Lipofuscina/imunologia , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/efeitos da radiação , Caspase 1/imunologia , Células Cultivadas , Humanos , Interleucina-18/imunologia , Interleucina-1beta/imunologia , Lisossomos/imunologia , Lisossomos/patologia , Degeneração Macular/etiologia , Degeneração Macular/imunologia , Degeneração Macular/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Epitélio Pigmentado da Retina/imunologiaRESUMO
Physiological processes such as ovarian follicle atresia generate large amounts of unnecessary cells or tissue detritus, which needs to be disposed of rapidly. IL-33 is a member of the IL-1 cytokine gene family. Constitutive expression of IL-33 in a wide range of tissues has hinted at its role beyond immune defense. We have previously reported a close correlation between IL-33 expression patterns and ovarian atresia. In this study, we demonstrated that IL-33 is required for disposal of degenerative tissue during ovarian atresia using Il33(-/-) mice. Deletion of the Il33 gene impaired normal disposal of atretic follicles, resulting in massive accumulations of tissue wastes abundant with aging-related catabolic wastes such as lipofuscin. Accumulation of tissue wastes in Il33(-/-) mice, in turn, accelerated ovarian aging and functional decline. Thus, their reproductive life span was shortened to two thirds of that for Il33(+/-) littermates. IL-33 orchestrated disposal mechanism through regulation of autophagy in degenerating tissues and macrophage migration into the tissues. Our study provides direct evidence supporting an expanded role of IL-33 in tissue integrity and aging through regulating disposal of unnecessary tissues or cells.
Assuntos
Fertilidade/imunologia , Atresia Folicular/imunologia , Interleucinas/imunologia , Folículo Ovariano/imunologia , Animais , Autofagia , Senescência Celular/imunologia , Feminino , Atresia Folicular/genética , Deleção de Genes , Regulação da Expressão Gênica , Interleucina-33 , Interleucinas/deficiência , Interleucinas/genética , Lipofuscina/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Folículo Ovariano/patologia , Transdução de Sinais , Fatores de TempoRESUMO
Age-related macular degeneration is an outer retinal disease that involves aging and immune dysfunction. In the aging retina, microglia aggregate in the outer retina and acquire intracellular autofluorescent lipofuscin deposits. In this study, we investigated whether accumulation of A2E, a key bisretinoid constituent of ocular lipofuscin, alters the physiology of retinal microglia in pathologically relevant ways. Our findings show that sublethal accumulations of intracellular A2E in cultured retinal microglia increased microglial activation and decreased microglial neuroprotection of photoreceptors. Increased A2E accumulation also lowered microglial expression of chemokine receptors and suppressed microglial chemotaxis, suggesting that lipofuscin accumulation may potentiate subretinal microglial accumulation. Significantly, A2E accumulation altered microglial complement regulation by increasing complement factor B and decreasing complement factor H expression, favoring increased complement activation and deposition in the outer retina. Taken together, our findings highlight the role of microglia in the local control of complement activation in the retina and present the age-related accumulation of ocular lipofuscin in subretinal microglia as a cellular mechanism capable of driving outer retinal immune dysregulation in age-related macular degeneration pathogenesis.
Assuntos
Envelhecimento , Ativação do Complemento , Lipofuscina , Degeneração Macular , Microglia , Compostos de Piridínio/metabolismo , Retina , Retinoides/metabolismo , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/imunologia , Envelhecimento/metabolismo , Animais , Receptor 1 de Quimiocina CX3C , Inibição de Migração Celular/imunologia , Células Cultivadas , Fator B do Complemento/metabolismo , Fator H do Complemento/metabolismo , Humanos , Lipofuscina/imunologia , Lipofuscina/metabolismo , Degeneração Macular/imunologia , Degeneração Macular/metabolismo , Camundongos , Camundongos Transgênicos , Microglia/citologia , Microglia/imunologia , Microglia/metabolismo , Pessoa de Meia-Idade , Receptores de Quimiocinas/genética , Retina/citologia , Retina/imunologia , Retina/metabolismoRESUMO
The nucleus tuberalis lateralis (NTL) is located in the basolateral part of the hypothalamus and is only present as a well-delineated nucleus in human and higher primates. In Alzheimer's disease (AD), NTL neurons show strong early cytoskeletal alterations, as revealed by the antibody Alz-50, but practically no senile plaques or neurofibrillary tangles. To study whether the activity of NTL neurons decreases when cytoskeletal changes appear, i.e., during aging and in AD, we applied a polyclonal antibody raised against the medial cisternae of the Golgi apparatus (GA). The size of the GA and the cell profile of NTL neurons, two established parameters for neuronal activity, were measured by an image analysis system. No significant change in the size of the profiles of the GA or of the neurons was observed in this nucleus during aging or AD. Earlier studies have shown that there is no decrease in cell number in the NTL in AD. We conclude that in the NTL an early hallmark of AD, i.e., cytoskeletal changes as stained by Alz-50, does not correlate with decreased neuronal activity, as reflected by the size of the GA, nor with a decrease in cell number. In addition, we found that the very early occurring and abundant presence of lipofuscin in NTL neurons does not go together with decreased neuronal activity.
Assuntos
Doença de Alzheimer/diagnóstico , Anticorpos Monoclonais/imunologia , Citoesqueleto/metabolismo , Hipotálamo/metabolismo , Adulto , Idoso , Doença de Alzheimer/metabolismo , Antígenos/imunologia , Autopsia , Encéfalo/metabolismo , Feminino , Complexo de Golgi/metabolismo , Humanos , Imunoglobulina G/imunologia , Imuno-Histoquímica , Lipofuscina/imunologia , Masculino , Pessoa de Meia-Idade , Neuritos/imunologiaRESUMO
Monoclonal antibodies generated against a synthetic peptide corresponding to amino acids 1 to 24 of cerebrovascular amyloid beta-protein do not only stain amyloidotic blood vessels and the amyloid deposits of the (senile) neuritic plaques, but also the neuronal pigment lipofuscin. Staining of lipofuscin is observed in both cerebral and cerebellar cortices, subcortical nuclei as well as the brain stem, and is identical in Alzheimer and normal control brain. Western blots of a lipofuscin enriched fraction show an anti-beta-protein reactive polypeptide migrating at approximately 31 kDa position on SDS-polyacrylamide gel electrophoresis. These results suggest that this polypeptide is associated with lipofuscin and is most likely derived from the predicted amyloid precursor protein. This implicates that, unlike in Alzheimer's disease where this protein is also processed extraneuronally in a manner to release an amyloid fiber forming fragment, the end point of its processing in the nerve cell seems to accumulate on a lipopigment characteristic for normal aging.
Assuntos
Doença de Alzheimer/metabolismo , Amiloide/imunologia , Anticorpos Monoclonais/imunologia , Encéfalo/metabolismo , Lipofuscina/imunologia , Proteínas do Tecido Nervoso/imunologia , Pigmentos Biológicos/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/fisiologia , Amiloide/análise , Peptídeos beta-Amiloides , Western Blotting , Química Encefálica , Criança , Pré-Escolar , Feminino , Humanos , Imuno-Histoquímica , Lactente , Masculino , Pessoa de Meia-Idade , Neurônios/imunologiaRESUMO
In experiments on rabbits immunized with soluble protein antigen immune reactions were found to be accompanied by the production of lipofuscin in macrophages. This process was the morphological manifestation of the digestion of antigen by macrophages which thus acquired the ability to migrate in the organ and to form lymphoid follicules in the medullary zone of lymph nodes. The newly formed follicules seem to be the basis of pronounced specific immune response. In staphylococcal bacteriemia the phagocytic activity of macrophages was delayed, thus causing disturbances in lipofuscin production; as a result, the subsequent phases of immune response also lagged somewhat behind in time.