RESUMO
Glutathione peroxidase 4 (GPx4) is known for its unique function in the direct detoxification of lipid peroxides in the cell membrane and as a key regulator of ferroptosis, a form of lipid peroxidation-induced nonapoptotic cell death. However, the cytosolic isoform of GPx4 is considered to play a major role in inhibiting ferroptosis in somatic cells, whereas the roles of the mitochondrial isoform of GPx4 (mGPx4) in cell survival are not yet clear. In the present study, we found that mGPx4 KO mice exhibit a cone-rod dystrophy-like phenotype in which loss of cone photoreceptors precedes loss of rod photoreceptors. Specifically, in mGPx4 KO mice, cone photoreceptors disappeared prior to their maturation, whereas rod photoreceptors persisted through maturation but gradually degenerated afterward. Mechanistically, we demonstrated that vitamin E supplementation significantly ameliorated photoreceptor loss in these mice. Furthermore, LC-MS showed a significant increase in peroxidized phosphatidylethanolamine esterified with docosahexaenoic acid in the retina of mGPx4 KO mice. We also observed shrunken and uniformly condensed nuclei as well as caspase-3 activation in mGPx4 KO photoreceptors, suggesting that apoptosis was prevalent. Taken together, our findings indicate that mGPx4 is essential for the maturation of cone photoreceptors but not for the maturation of rod photoreceptors, although it is still critical for the survival of rod photoreceptors after maturation. In conclusion, we reveal novel functions of mGPx4 in supporting development and survival of photoreceptors in vivo.
Assuntos
Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Células Fotorreceptoras Retinianas Cones , Células Fotorreceptoras Retinianas Bastonetes , Animais , Sobrevivência Celular/genética , Camundongos , Mitocôndrias/enzimologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologiaAssuntos
Biofarmácia , Doença/etiologia , Ecotoxicologia , Alimentos/efeitos adversos , Nutrientes/efeitos adversos , Ciências da Nutrição , Medicina Preventiva , Animais , Cádmio/metabolismo , Proteínas de Transporte de Cátions , Ácidos Graxos Ômega-3 , Análise de Alimentos , Glutationa/análogos & derivados , Humanos , Lisofosfolipídeos , Manganês/metabolismo , Camundongos , Compostos Organosselênicos , Esfingosina/análogos & derivados , Ácidos Graxos trans/efeitos adversos , Vitamina K , Vitamina K 2/análogos & derivadosRESUMO
Nonalcoholic steatohepatitis (NASH) is a metabolic liver disease that progresses from simple steatosis to the disease state of inflammation and fibrosis. Previous studies suggest that apoptosis and necroptosis may contribute to the pathogenesis of NASH, based on several murine models. However, the mechanisms underlying the transition of simple steatosis to steatohepatitis remain unclear, because it is difficult to identify when and where such cell deaths begin to occur in the pathophysiological process of NASH. In the present study, our aim is to investigate which type of cell death plays a role as the trigger for initiating inflammation in fatty liver. By establishing a simple method of discriminating between apoptosis and necrosis in the liver, we found that necrosis occurred prior to apoptosis at the onset of steatohepatitis in the choline-deficient, ethionine-supplemented (CDE) diet model. To further investigate what type of necrosis is involved in the initial necrotic cell death, we examined the effect of necroptosis and ferroptosis inhibition by administering inhibitors to wild-type mice in the CDE diet model. In addition, necroptosis was evaluated using mixed lineage kinase domain-like protein (MLKL) knockout mice, which is lacking in a terminal executor of necroptosis. Consequently, necroptosis inhibition failed to block the onset of necrotic cell death, while ferroptosis inhibition protected hepatocytes from necrotic death almost completely, and suppressed the subsequent infiltration of immune cells and inflammatory reaction. Furthermore, the amount of oxidized phosphatidylethanolamine, which is involved in ferroptosis pathway, was increased in the liver sample of the CDE diet-fed mice. These findings suggest that hepatic ferroptosis plays an important role as the trigger for initiating inflammation in steatohepatitis and may be a therapeutic target for preventing the onset of steatohepatitis.
Assuntos
Ferroptose , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Animais , Apoptose/efeitos dos fármacos , Tetracloreto de Carbono/toxicidade , Cromanos/farmacologia , Citocinas/metabolismo , Dieta , Etionina , Ferroptose/efeitos dos fármacos , Hepatite/imunologia , Hepatite/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Quelantes de Ferro/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necroptose/efeitos dos fármacos , Necrose , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/imunologia , Linfócitos T Citotóxicos/imunologia , Resinas Acrílicas/administração & dosagem , Administração Intranasal , Transferência Adotiva , Animais , Modelos Animais de Doenças , Citometria de Fluxo , Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Lecitinas/administração & dosagem , Lecitinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologiaRESUMO
Singlet oxygen causes the cytotoxic process of tumour cells in photodynamic therapy. The mechanism by which singlet oxygen damages cells is, however, not fully understood. To address this issue, we synthesized and used two types of endoperoxides, MNPE (1-methylnaphthalene-4-propionate endoperoxide) and NDPE (naphthalene-1,4-dipropionate endoperoxide), that generate defined amounts of singlet oxygen at 37 degrees C with similar half lives. MNPE, which is more hydrophobic than NDPE, induced the release of cytochrome c from mitochondria into the cytosol and exhibited cytotoxicity, but NDPE did not. RBL cells, a rat basophil leukaemia-derived line, that overexpress phospholipid hydroperoxide glutathione peroxidase in mitochondria were found to be highly resistant to the cytotoxic effect of MNPE. MNPE treatment induced much less DNA ladder formation and nuclear fragmentation in cells than etoposide treatment, even though these treatments induced a similar extent of cellular damage. Singlet oxygen inhibited caspase 9 and 3 activities directly and also suppressed the activation of the caspase cascade. Collectively, these data suggest that singlet oxygen triggers an apoptotic pathway by releasing cytochrome c from mitochondria via the peroxidation of mitochondrial components and results in cell death that is different from typical apoptosis, because of the abortive apoptotic pathway caused by impaired caspase activation.
Assuntos
Apoptose , Inibidores de Caspase , Oxigênio Singlete/metabolismo , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Sistema Livre de Células , Citocromos c/metabolismo , Ativação Enzimática/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peróxidos Lipídicos/farmacologia , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Estrutura Molecular , Naftóis/farmacologia , Propionatos/farmacologia , Selênio/metabolismo , Oxigênio Singlete/química , Tocoferóis , Vitamina E/análogos & derivados , Vitamina E/metabolismo , beta Caroteno/metabolismoRESUMO
Reactive oxygen species (ROS) are known mediators of intracellular signal cascades. Excessive production of ROS may lead to oxidative stress, loss of cell function, and cell death by apoptosis or necrosis. Lipid hydroperoxides are one type of ROS whose biological function has not yet been clarified. Phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a unique antioxidant enzyme that can directly reduce phospholipid hydroperoxide in mammalian cells. This contrasts with most antioxidant enzymes, which cannot reduce intracellular phospholipid hydroperoxides directly. In this review, we focus on the structure and biological functions of PHGPx in mammalian cells. Recently, molecular techniques have allowed overexpression of PHGPx in mammalian cell lines, from which it has become clear that lipid hydroperoxides also have an important function as activators of lipoxygenase and cyclooxygenase, participate in inflammation, and act as signal molecules for apoptotic cell death and receptor-mediated signal transduction at the cellular level.