RESUMO
GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.
Assuntos
Produtos Biológicos/uso terapêutico , Encéfalo/metabolismo , Doenças por Armazenamento dos Lisossomos/terapia , Progranulinas/uso terapêutico , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Endossomos/metabolismo , Feminino , Demência Frontotemporal/sangue , Demência Frontotemporal/líquido cefalorraquidiano , Gliose/complicações , Gliose/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/patologia , Metabolismo dos Lipídeos , Lipofuscina/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Degeneração Neural/patologia , Fenótipo , Progranulinas/deficiência , Progranulinas/metabolismo , Receptores Imunológicos/metabolismo , Receptores da Transferrina/metabolismo , Distribuição TecidualRESUMO
Triggering receptor expressed on myeloid cells 2 (TREM2) is essential for the transition of homeostatic microglia to a disease-associated microglial state. To enhance TREM2 activity, we sought to selectively increase the full-length protein on the cell surface via reducing its proteolytic shedding by A Disintegrin And Metalloproteinase (i.e., α-secretase) 10/17. We screened a panel of monoclonal antibodies against TREM2, with the aim to selectively compete for α-secretase-mediated shedding. Monoclonal antibody 4D9, which has a stalk region epitope close to the cleavage site, demonstrated dual mechanisms of action by stabilizing TREM2 on the cell surface and reducing its shedding, and concomitantly activating phospho-SYK signaling. 4D9 stimulated survival of macrophages and increased microglial uptake of myelin debris and amyloid ß-peptide in vitro. In vivo target engagement was demonstrated in cerebrospinal fluid, where nearly all soluble TREM2 was 4D9-bound. Moreover, in a mouse model for Alzheimer's disease-related pathology, 4D9 reduced amyloidogenesis, enhanced microglial TREM2 expression, and reduced a homeostatic marker, suggesting a protective function by driving microglia toward a disease-associated state.
Assuntos
Anticorpos Monoclonais/farmacologia , Glicoproteínas de Membrana/imunologia , Microglia , Mieloma Múltiplo , Receptores Imunológicos/imunologia , Peptídeos beta-Amiloides , Animais , Linhagem Celular Tumoral , Feminino , Macrófagos , Camundongos , Microglia/patologia , Ratos , Ratos WistarRESUMO
Loss-of-function (LOF) variants of TREM2, an immune receptor expressed in microglia, increase Alzheimer's disease risk. TREM2 senses lipids and mediates myelin phagocytosis, but its role in microglial lipid metabolism is unknown. Combining chronic demyelination paradigms and cell sorting with RNA sequencing and lipidomics, we find that wild-type microglia acquire a disease-associated transcriptional state, while TREM2-deficient microglia remain largely homeostatic, leading to neuronal damage. TREM2-deficient microglia phagocytose myelin debris but fail to clear myelin cholesterol, resulting in cholesteryl ester (CE) accumulation. CE increase is also observed in APOE-deficient glial cells, reflecting impaired brain cholesterol transport. This finding replicates in myelin-treated TREM2-deficient murine macrophages and human iPSC-derived microglia, where it is rescued by an ACAT1 inhibitor and LXR agonist. Our studies identify TREM2 as a key transcriptional regulator of cholesterol transport and metabolism under conditions of chronic myelin phagocytic activity, as TREM2 LOF causes pathogenic lipid accumulation in microglia.
Assuntos
Encéfalo/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Microglia/metabolismo , Bainha de Mielina/metabolismo , Fagocitose/genética , Receptores Imunológicos/genética , Acetil-CoA C-Acetiltransferase/antagonistas & inibidores , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Ésteres do Colesterol/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Células-Tronco Pluripotentes Induzidas , Metabolismo dos Lipídeos/genética , Lipidômica , Receptores X do Fígado/agonistas , Camundongos , Camundongos Knockout , Camundongos Knockout para ApoE , RNA-SeqRESUMO
The presence of foreign DNA in the cytosol of mammalian cells elicits a potent antiviral interferon response. Recently, cytosolic DNA was proposed to induce the synthesis of cyclic GMP-AMP (cGAMP) upon binding to an enzyme called cGAMP synthase (cGAS). cGAMP activates an interferon response by binding to a downstream receptor called STING. Here, we identify natural variants of human STING (hSTING) that are poorly responsive to cGAMP yet, unexpectedly, are normally responsive to DNA and cGAS signaling. We explain this paradox by demonstrating that the cGAS product is actually a noncanonical cyclic dinucleotide, cyclic [G(2'-5')pA(3'-5')p], which contains a single 2'-5' phosphodiester bond. Cyclic [G(2'-5')pA(3'-5')p] potently activates diverse hSTING receptors and, therefore, may be a useful adjuvant or immunotherapeutic. Our results indicate that hSTING variants have evolved to distinguish conventional (3'-5') cyclic dinucleotides, known to be produced mainly by bacteria, from the noncanonical cyclic dinucleotide produced by mammalian cGAS.
Assuntos
Proteínas de Membrana/metabolismo , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/metabolismo , Oligonucleotídeos/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Células HEK293 , Humanos , Imunidade Inata , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Nucleotídeos Cíclicos/química , Oligonucleotídeos/química , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
The innate immune system detects infection by using germline-encoded receptors that are specific for conserved microbial molecules. The recognition of microbial ligands leads to the production of cytokines, such as type I interferons (IFNs), that are essential for successful pathogen elimination. Cytosolic detection of pathogen-derived DNA is one major mechanism of inducing IFN production, and this process requires signalling through TANK binding kinase 1 (TBK1) and its downstream transcription factor, IFN-regulatory factor 3 (IRF3). In addition, a transmembrane protein called STING (stimulator of IFN genes; also known as MITA, ERIS, MPYS and TMEM173) functions as an essential signalling adaptor, linking the cytosolic detection of DNA to the TBK1-IRF3 signalling axis. Recently, unique nucleic acids called cyclic dinucleotides, which function as conserved signalling molecules in bacteria, have also been shown to induce a STING-dependent type I IFN response. However, a mammalian sensor of cyclic dinucleotides has not been identified. Here we report evidence that STING itself is an innate immune sensor of cyclic dinucleotides. We demonstrate that STING binds directly to radiolabelled cyclic diguanylate monophosphate (c-di-GMP), and we show that unlabelled cyclic dinucleotides, but not other nucleotides or nucleic acids, compete with c-di-GMP for binding to STING. Furthermore, we identify mutations in STING that selectively affect the response to cyclic dinucleotides without affecting the response to DNA. Thus, STING seems to function as a direct sensor of cyclic dinucleotides, in addition to its established role as a signalling adaptor in the IFN response to cytosolic DNA. Cyclic dinucleotides have shown promise as novel vaccine adjuvants and immunotherapeutics, and our results provide insight into the mechanism by which cyclic dinucleotides are sensed by the innate immune system.
Assuntos
GMP Cíclico/análogos & derivados , Imunidade Inata/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Adjuvantes Imunológicos , Sequência de Aminoácidos , Animais , GMP Cíclico/imunologia , DNA/imunologia , Células HEK293 , Humanos , Interferons/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas de Membrana/genética , Camundongos , Dados de Sequência MolecularRESUMO
The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed that Nlrx1(-/-) mice exhibited increased expression of antiviral signaling molecules IFN-ß, STAT2, OAS1, and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1(-/-) mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.
Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Proteínas Mitocondriais/imunologia , Infecções por Orthomyxoviridae/imunologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Interferon beta/biossíntese , Interferon beta/imunologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Macrófagos/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/deficiência , NF-kappa B/imunologia , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular , Fator 6 Associado a Receptor de TNF/imunologia , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
Type I interferons (IFNs) are central regulators of the innate and adaptive immune responses to viral and bacterial infections. Type I IFNs are induced upon cytosolic detection of microbial nucleic acids, including DNA, RNA, and the bacterial second messenger cyclic-di-GMP (c-di-GMP). In addition, a recent study demonstrated that the intracellular bacterial pathogen Listeria monocytogenes stimulates a type I IFN response due to cytosolic detection of bacterially secreted c-di-AMP. The transmembrane signaling adaptor Sting (Tmem173, Mita, Mpys, Eris) has recently been implicated in the induction of type I IFNs in response to cytosolic DNA and/or RNA. However, the role of Sting in response to purified cyclic dinucleotides or during in vivo L. monocytogenes infection has not been addressed. In order to identify genes important in the innate immune response, we have been conducting a forward genetic mutagenesis screen in C57BL/6 mice using the mutagen N-ethyl-N-nitrosourea (ENU). Here we describe a novel mutant mouse strain, Goldenticket (Gt), that fails to produce type I IFNs upon L. monocytogenes infection. By genetic mapping and complementation experiments, we found that Gt mice harbor a single nucleotide variant (T596A) of Sting that functions as a null allele and fails to produce detectable protein. Analysis of macrophages isolated from Gt mice revealed that Sting is absolutely required for the type I interferon response to both c-di-GMP and c-di-AMP. Additionally, Sting is required for the response to c-di-GMP and L. monocytogenes in vivo. Our results provide new functions for Sting in the innate interferon response to pathogens.
Assuntos
GMP Cíclico/análogos & derivados , Fosfatos de Dinucleosídeos/metabolismo , Interferon Tipo I/metabolismo , Listeria monocytogenes/imunologia , Listeriose/imunologia , Proteínas de Membrana/fisiologia , Alelos , Animais , Linhagem Celular , GMP Cíclico/metabolismo , Etilnitrosoureia/toxicidade , Feminino , Regulação da Expressão Gênica/fisiologia , Teste de Complementação Genética , Humanos , Interferon Tipo I/genética , Listeriose/metabolismo , Macrófagos/microbiologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Mutação , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Legionella pneumophila is a gram-negative bacterial pathogen that replicates in host macrophages and causes a severe pneumonia called Legionnaires' Disease. The innate immune response to L. pneumophila remains poorly understood. Here we focused on identifying host and bacterial factors involved in the production of type I interferons (IFN) in response to L. pneumophila. It was previously suggested that the delivery of L. pneumophila DNA to the host cell cytosol is the primary signal that induces the type I IFN response. However, our data are not easily reconciled with this model. We provide genetic evidence that two RNA-sensing proteins, RIG-I and MDA5, participate in the IFN response to L. pneumophila. Importantly, these sensors do not seem to be required for the IFN response to L. pneumophila DNA, whereas we found that RIG-I was required for the response to L. pneumophila RNA. Thus, we hypothesize that bacterial RNA, or perhaps an induced host RNA, is the primary stimulus inducing the IFN response to L. pneumophila. Our study also identified a secreted effector protein, SdhA, as a key suppressor of the IFN response to L. pneumophila. Although viral suppressors of cytosolic RNA-sensing pathways have been previously identified, analogous bacterial factors have not been described. Thus, our results provide new insights into the molecular mechanisms by which an intracellular bacterial pathogen activates and also represses innate immune responses.
Assuntos
Citosol/metabolismo , RNA Helicases DEAD-box/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Interferon Tipo I/metabolismo , Legionella pneumophila/patogenicidade , Doença dos Legionários/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Modelos Animais de Doenças , Imunidade Inata/fisiologia , Helicase IFIH1 Induzida por Interferon , Doença dos Legionários/fisiopatologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/fisiologiaRESUMO
The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di-guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor kappaB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid-sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.