RESUMO
Mucopolysaccharidosis IIIB is a paediatric lysosomal storage disease caused by deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU), involved in the degradation of the glycosaminoglycan heparan sulphate. Absence of NAGLU leads to accumulation of partially degraded heparan sulphate within lysosomes and the extracellular matrix, giving rise to severe CNS degeneration with progressive cognitive impairment and behavioural problems. There are no therapies. Haematopoietic stem cell transplant shows great efficacy in the related disease mucopolysaccharidosis I, where donor-derived monocytes can transmigrate into the brain following bone marrow engraftment, secrete the missing enzyme and cross-correct neighbouring cells. However, little neurological correction is achieved in patients with mucopolysaccharidosis IIIB. We have therefore developed an ex vivo haematopoietic stem cell gene therapy approach in a mouse model of mucopolysaccharidosis IIIB, using a high-titre lentiviral vector and the myeloid-specific CD11b promoter, driving the expression of NAGLU (LV.NAGLU). To understand the mechanism of correction we also compared this with a poorly secreted version of NAGLU containing a C-terminal fusion to IGFII (LV.NAGLU-IGFII). Mucopolysaccharidosis IIIB haematopoietic stem cells were transduced with vector, transplanted into myeloablated mucopolysaccharidosis IIIB mice and compared at 8 months of age with mice receiving a wild-type transplant. As the disease is characterized by increased inflammation, we also tested the anti-inflammatory steroidal agent prednisolone alone, or in combination with LV.NAGLU, to understand the importance of inflammation on behaviour. NAGLU enzyme was substantially increased in the brain of LV.NAGLU and LV.NAGLU-IGFII-treated mice, with little expression in wild-type bone marrow transplanted mice. LV.NAGLU treatment led to behavioural correction, normalization of heparan sulphate and sulphation patterning, reduced inflammatory cytokine expression and correction of astrocytosis, microgliosis and lysosomal compartment size throughout the brain. The addition of prednisolone improved inflammatory aspects further. Substantial correction of lysosomal storage in neurons and astrocytes was also achieved in LV.NAGLU-IGFII-treated mice, despite limited enzyme secretion from engrafted macrophages in the brain. Interestingly both wild-type bone marrow transplant and prednisolone treatment alone corrected behaviour, despite having little effect on brain neuropathology. This was attributed to a decrease in peripheral inflammatory cytokines. Here we show significant neurological disease correction is achieved using haematopoietic stem cell gene therapy, suggesting this therapy alone or in combination with anti-inflammatories may improve neurological function in patients.
Assuntos
Encefalite/etiologia , Encefalite/terapia , Terapia Genética/métodos , Macrófagos/enzimologia , Mucopolissacaridose III , Células-Tronco/fisiologia , Animais , Encéfalo/enzimologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Gliose/terapia , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismo , Humanos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucopolissacaridose III/complicações , Mucopolissacaridose III/genética , Mucopolissacaridose III/patologia , Mucopolissacaridose III/terapia , Prednisolona/uso terapêutico , Baço/enzimologia , Sulfatases/genética , Sulfatases/metabolismoRESUMO
Recombinant adeno-associated viruses (AAVs) are popular in vivo gene transfer vehicles. However, vector doses needed to achieve therapeutic effect are high and some target tissues in the central nervous system remain difficult to transduce. Gene therapy trials using AAV for the treatment of neurological disorders have seldom led to demonstrated clinical efficacy. Important contributing factors are low transduction rates and inefficient distribution of the vector. To overcome these hurdles, a variety of capsid engineering methods have been utilized to generate capsids with improved transduction properties. Here we describe an alternative approach to capsid engineering, which draws on the natural evolution of the virus and aims to yield capsids that are better suited to infect human tissues. We generated an AAV capsid to include amino acids that are conserved among natural AAV2 isolates and tested its biodistribution properties in mice and rats. Intriguingly, this novel variant, AAV-TT, demonstrates strong neurotropism in rodents and displays significantly improved distribution throughout the central nervous system as compared to AAV2. Additionally, sub-retinal injections in mice revealed markedly enhanced transduction of photoreceptor cells when compared to AAV2. Importantly, AAV-TT exceeds the distribution abilities of benchmark neurotropic serotypes AAV9 and AAVrh10 in the central nervous system of mice, and is the only virus, when administered at low dose, that is able to correct the neurological phenotype in a mouse model of mucopolysaccharidosis IIIC, a transmembrane enzyme lysosomal storage disease, which requires delivery to every cell for biochemical correction. These data represent unprecedented correction of a lysosomal transmembrane enzyme deficiency in mice and suggest that AAV-TT-based gene therapies may be suitable for treatment of human neurological diseases such as mucopolysaccharidosis IIIC, which is characterized by global neuropathology.
Assuntos
Capsídeo/fisiologia , Terapia Genética/métodos , Engenharia de Proteínas/métodos , Animais , Dependovirus/genética , Feminino , Vetores Genéticos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucopolissacaridose III/genética , Mucopolissacaridose III/terapia , Células Fotorreceptoras/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Retina/fisiologia , Distribuição Tecidual , Transdução GenéticaRESUMO
Crescentic glomerulonephritis (Crgn) is a complex disease where the initial insult is often the glomerular deposition of antibodies against intrinsic or deposited antigens in the glomerulus. The role of Fc receptors in the induction and progression of Crgn is increasingly recognized, and our previous studies have shown that copy number variation in Fcgr3 partially explains the genetic susceptibility of the Wistar-Kyoto (WKY) rat to nephrotoxic nephritis, a rat model of Crgn. The Fcgr3-related sequence (Fcgr3-rs) is a novel rat-specific Fc receptor with a cytoplasmic domain 6 amino acids longer than its paralogue, Fcgr3. The Fcgr3-rs gene is deleted from the WKY rat genome, and this deletion is associated with enhanced macrophage activity in this strain. Here, we investigated the mechanism by which the deletion of Fcgr3-rs in the WKY strain leads to increased macrophage activation. By lentivirus-mediated gene delivery, we generated stably transduced U937 cells expressing either Fcgr3-rs or Fcgr3. In these cells, which lack endogenous Fcgr3 receptors, we show that Fcgr3-rs interacts with the common Fc-γ chain but that Fc receptor-mediated phagocytosis and signaling are defective. Furthermore, in primary macrophages, expression of Fcgr3-rs inhibits Fc receptor-mediated functions, because WKY bone marrow-derived macrophages transduced with Fcgr3-rs had significantly reduced phagocytic activity. This inhibitory effect on phagocytosis was mediated by the novel cytoplasmic domain of Fcgr3-rs. These results suggest that Fcgr3-rs may act to inhibit Fcgr3-mediated signaling and phagocytosis and could be considered as a novel mechanism in the modulation of Fc receptor-mediated cell activation in autoimmune diseases.
Assuntos
Glomerulonefrite/metabolismo , Macrófagos/metabolismo , Receptores de IgG/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular Tumoral , Citoplasma/metabolismo , Éxons/genética , Deleção de Genes , Duplicação Gênica , Glomerulonefrite/patologia , Humanos , Interleucina-1beta/metabolismo , Macrófagos/patologia , Camundongos , Dados de Sequência Molecular , Fagocitose , Filogenia , Estrutura Terciária de Proteína , Ratos , Receptores de IgG/química , Receptores de IgG/genética , Transdução de Sinais , Especificidade da Espécie , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: The oxidative burst is one of the major antimicrobial mechanisms adopted by macrophages. The WKY rat strain is uniquely susceptible to experimentally induced macrophage-dependent crescentic glomerulonephritis (Crgn). We previously identified the AP-1 transcription factor JunD as a determinant of macrophage activation in WKY bone marrow-derived macrophages (BMDMs). JunD is over-expressed in WKY BMDMs and its silencing reduces Fc receptor-mediated oxidative burst in these cells. RESULTS: Here we combined Jund RNA interference with microarray analyses alongside ChIP-sequencing (ChIP-Seq) analyses in WKY BMDMs to investigate JunD-mediated control of macrophage activation in basal and lipopolysaccharide (LPS) stimulated cells. Microarray analysis following Jund silencing showed that Jund activates and represses gene expression with marked differential expression (>3 fold) for genes linked with oxidative stress and IL-1ß expression. These results were complemented by comparing whole genome expression in WKY BMDMs with Jund congenic strain (WKY.LCrgn2) BMDMs which express lower levels of JunD. ChIP-Seq analyses demonstrated that the increased expression of JunD resulted in an increased number of binding events in WKY BMDMs compared to WKY.LCrgn2 BMDMs. Combined ChIP-Seq and microarray analysis revealed a set of primary JunD-targets through which JunD exerts its effect on oxidative stress and IL-1ß synthesis in basal and LPS-stimulated macrophages. CONCLUSIONS: These findings demonstrate how genetically determined levels of a transcription factor affect its binding sites in primary cells and identify JunD as a key regulator of oxidative stress and IL-1ß synthesis in primary macrophages, which may play a role in susceptibility to Crgn.
Assuntos
Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Estresse Oxidativo/genética , Proteínas Proto-Oncogênicas c-jun , Fator de Transcrição AP-1 , Animais , Sítios de Ligação , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Lipopolissacarídeos/toxicidade , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Cultura Primária de Células , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Genetic investigation of crescentic glomerulonephritis (Crgn) susceptibility in the Wistar Kyoto rat, a strain uniquely susceptible to nephrotoxic nephritis (NTN), allowed us to positionally clone the activator protein-1 transcription factor Jund as a susceptibility gene associated with Crgn. To study the influence of Jund deficiency (Jund(-/-)) on immune-mediated renal disease, susceptibility to accelerated NTN was examined in Jund(-/-) mice and C57BL/6 wild-type (WT) controls. Jund(-/-) mice showed exacerbated glomerular crescent formation and macrophage infiltration, 10 days after NTN induction. Serum urea levels were also significantly increased in the Jund(-/-) mice compared with the WT controls. There was no evidence of immune response differences between Jund(-/-) and WT animals because the quantitative immunofluorescence for sheep and mouse IgG deposition in glomeruli was similar. Because murine Jund was inactivated by replacement with a bacterial LacZ reporter gene, we then investigated its glomerular expression by IHC and found that the Jund promoter is mainly active in Jund(-/-) podocytes. Furthermore, cultured glomeruli from Jund(-/-) mice showed relatively increased expression of vascular endothelial growth factor A (Vegfa), Cxcr4, and Cxcl12, well-known HIF target genes. Accordingly, small-interfering RNA-mediated JUND knockdown in conditionally immortalized human podocyte cell lines led to increased VEGFA and HIF1A expression. Our findings suggest that deficiency of Jund may cause increased oxidative stress in podocytes, leading to altered VEGFA expression and subsequent glomerular injury in Crgn.
Assuntos
Glomerulonefrite/metabolismo , Glomerulonefrite/prevenção & controle , Podócitos/metabolismo , Proteínas Proto-Oncogênicas c-jun/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Western Blotting , Células Cultivadas , Feminino , Imunofluorescência , Glomerulonefrite/etiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas Imunoenzimáticas , Imunoglobulina G/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Podócitos/citologia , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ovinos , Fator de Transcrição AP-1 , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The Wistar Kyoto (WKY) rat is uniquely susceptible to experimentally induced crescentic glomerulonephritis. Two major quantitative trait loci (QTLs) on chromosomes 13 (Crgn1) and 16 (Crgn2) with logarithm of odds >8, as well as five other loci (Crgn3 through 7), largely explain this genetic susceptibility. To understand further the effects of Crgn1 and Crgn2, we generated a double-congenic strain by introgressing these loci from glomerulonephritis-resistant Lewis rats onto the WKY genetic background. Induction of nephrotoxic nephritis in the double-congenic rats (WKY.LCrgn1,2) produced markedly fewer glomerular crescents, reduced macrophage infiltration, and decreased expression of glomerular TNF-alpha and inducible nitric oxide synthase expression compared with control animals. Bone marrow and kidney transplantation studies between parental and WKY.LCrgn1,2 strains, together with in vitro experiments, demonstrated that Crgn1 and Crgn2 contribute exclusively to circulating cell-related glomerular injury by regulating macrophage infiltration and activation. The residual genetic susceptibility to crescentic glomerulonephritis in WKY.LCrgn1,2 rats associated with macrophage activity (especially with enhanced metalloelastase expression) rather than macrophage infiltration. Taken together, these results demonstrate that a genetic influence on macrophage activation, rather than number, determines glomerular damage in immune-mediated glomerulonephritis.
Assuntos
Loci Gênicos/genética , Loci Gênicos/fisiologia , Mesângio Glomerular/fisiopatologia , Glomerulonefrite/genética , Glomerulonefrite/fisiopatologia , Macrófagos/fisiologia , Animais , Transplante de Medula Óssea , Células Cultivadas , Modelos Animais de Doenças , Predisposição Genética para Doença/genética , Mesângio Glomerular/metabolismo , Mesângio Glomerular/patologia , Glomerulonefrite/patologia , Transplante de Rim , Macrófagos/patologia , Masculino , Metaloproteinase 12 da Matriz/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos WKY , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The pediatric lysosomal storage disorder mucopolysaccharidosis type II is caused by mutations in IDS, resulting in accumulation of heparan and dermatan sulfate, causing severe neurodegeneration, skeletal disease, and cardiorespiratory disease. Most patients manifest with cognitive symptoms, which cannot be treated with enzyme replacement therapy, as native IDS does not cross the blood-brain barrier. We tested a brain-targeted hematopoietic stem cell gene therapy approach using lentiviral IDS fused to ApoEII (IDS.ApoEII) compared to a lentivirus expressing normal IDS or a normal bone marrow transplant. In mucopolysaccharidosis II mice, all treatments corrected peripheral disease, but only IDS.ApoEII mediated complete normalization of brain pathology and behavior, providing significantly enhanced correction compared to IDS. A normal bone marrow transplant achieved no brain correction. Whilst corrected macrophages traffic to the brain, secreting IDS/IDS.ApoEII enzyme for cross-correction, IDS.ApoEII was additionally more active in plasma and was taken up and transcytosed across brain endothelia significantly better than IDS via both heparan sulfate/ApoE-dependent receptors and mannose-6-phosphate receptors. Brain-targeted hematopoietic stem cell gene therapy provides a promising therapy for MPS II patients.
Assuntos
Transplante de Medula Óssea , Terapia Genética , Glicoproteínas/genética , Mucopolissacaridose II/terapia , Transplante de Células-Tronco , Animais , Encéfalo/metabolismo , Feminino , Vetores Genéticos , Glicoproteínas/administração & dosagem , Glicoproteínas/uso terapêutico , Humanos , Lentivirus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Zygotic epigenetic reprogramming entails genome-wide DNA demethylation that is accompanied by Tet methylcytosine dioxygenase 3 (Tet3)-driven oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC; refs 1-4). Here we demonstrate using detailed immunofluorescence analysis and ultrasensitive LC-MS-based quantitative measurements that the initial loss of paternal 5mC does not require 5hmC formation. Small-molecule inhibition of Tet3 activity, as well as genetic ablation, impedes 5hmC accumulation in zygotes without affecting the early loss of paternal 5mC. Instead, 5hmC accumulation is dependent on the activity of zygotic Dnmt3a and Dnmt1, documenting a role for Tet3-driven hydroxylation in targeting de novo methylation activities present in the early embryo. Our data thus provide further insights into the dynamics of zygotic reprogramming, revealing an intricate interplay between DNA demethylation, de novo methylation and Tet3-driven hydroxylation.
Assuntos
5-Metilcitosina/metabolismo , Reprogramação Celular , Citosina/análogos & derivados , Metilação de DNA , Epigênese Genética , Zigoto/metabolismo , Animais , Biomarcadores/metabolismo , Cromatografia Líquida , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Técnicas de Cultura Embrionária , Fertilização in vitro , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Cinética , Espectrometria de Massas , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Macrophages can fuse to form osteoclasts in bone or multinucleate giant cells (MGCs) as part of the immune response. We use a systems genetics approach in rat macrophages to unravel their genetic determinants of multinucleation and investigate their role in both bone homeostasis and inflammatory disease. We identify a trans-regulated gene network associated with macrophage multinucleation and Kcnn4 as being the most significantly trans-regulated gene in the network and induced at the onset of fusion. Kcnn4 is required for osteoclast and MGC formation in rodents and humans. Genetic deletion of Kcnn4 reduces macrophage multinucleation through modulation of Ca(2+) signaling, increases bone mass, and improves clinical outcome in arthritis. Pharmacological blockade of Kcnn4 reduces experimental glomerulonephritis. Our data implicate Kcnn4 in macrophage multinucleation, identifying it as a potential therapeutic target for inhibition of bone resorption and chronic inflammation.
Assuntos
Artrite/metabolismo , Osso e Ossos/metabolismo , Núcleo Celular/fisiologia , Glomerulonefrite/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Macrófagos/metabolismo , Animais , Artrite/patologia , Reabsorção Óssea/metabolismo , Osso e Ossos/imunologia , Sinalização do Cálcio , Células Cultivadas , Redes Reguladoras de Genes , Glomerulonefrite/imunologia , Homeostase , Humanos , Camundongos Knockout , Ratos Endogâmicos Lew , Ratos Endogâmicos WKY , Receptores Imunológicos/metabolismoRESUMO
Crescentic glomerulonephritis (CRGN) is a major cause of human kidney failure, but the underlying mechanisms are not fully understood. Wistar Kyoto (WKY) rats are uniquely susceptible to CRGN following injection of nephrotoxic serum, whereas Lewis (LEW) rats are resistant. Our previous genetic studies of nephrotoxic nephritis (NTN), a form of CRGN induced by nephrotoxic serum, identified Fcgr3 and Jund as WKY genes underlying the two strongest quantitative trait loci for NTN phenotypes: Crgn1 and Crgn2, respectively. We also showed that introgression of WKY Crgn1 or Crgn2 individually into a LEW background did not lead to the formation of glomerular crescents. We have now generated a bicongenic strain, LEW.WCrgn1,2, in which WKY Crgn1 and Crgn2 are both introgressed into the LEW genetic background. These rats show development of NTN phenotypes, including glomerular crescents. Furthermore, we characterised macrophage function and glomerular cytokine profiles in this new strain. Additionally, we show that LEW.WCrgn1,2 rats are resistant to the development of glomerular crescents that is usually induced following immunisation with recombinant rat α3(IV)NC1, the specific Goodpasture autoantigen located in the glomerular basement membrane against which the immune response is directed in experimental autoimmune glomerulonephritis. Our results show that the new bicongenic strain responds differently to two distinct experimental triggers of CRGN. This is the first time that CRGN has been induced on a normally resistant rat genetic background and identifies the LEW.WCrgn1,2 strain as a new, potentially valuable model of macrophage-dependent glomerulonephritis.