RESUMO
Introduction: Anti-SSA antibodies target two unrelated proteins, Ro52 (E3 ligase) and Ro60 (RNA binding protein). Previous studies indicate that anti-Ro52 antibodies are frequently associated with various myositis-specific autoantibodies (MSAs)-including anti-tRNA synthetase antibodies-and that the coexistence of MSAs and anti-Ro52 antibodies may portend worse clinical outcomes. Although not well-described in the setting of myositis, work from our animal model of HRS (histidyl-tRNA synthetase)-induced myositis suggests that anti-Ro60 antibodies may also be linked to specific MSAs such as anti-HRS/Jo-1. We therefore aimed to demonstrate the prevalence and clinical characteristics of Ro52 and Ro60 antibody positivity in patients possessing Jo-1 antibodies. Methods: To establish the immunological link between anti-synthetase, anti-Ro52, and anti-Ro60 antibodies, we evaluated the relative titers of these antibodies in blood and bronchoalveolar lavage fluid (BALF) of mice following immunization with HRS/Jo-1. In parallel, we used ELISA-based approaches to assess sera from 177 anti-Jo1 antibody-positive patients for the presence of anti-Ro52 and/or anti-Ro60 antibodies. We then determined statistical associations between co-existing anti-Jo-1, anti-Ro52, and/or anti-Ro60 antibodies and clinical manifestations associated with the anti-synthetase syndrome. Results: Mice immunized with HRS had higher levels of anti-Ro52 and anti-Ro60 antibodies in serum and BALF than PBS-immunized mice. In 177 anti-Jo-1 antibody-positive patients, the prevalence of anti-Ro52 and anti-Ro60 antibodies was 36% and 15%, respectively. The frequency of dry eye/dry mouth, interstitial pneumonia, and pulmonary events over time differed between patients with various combinations of anti-Ro52 and anti-Ro60 antibodies. While anti-Ro52 antibodies generally correlated with statistically significant increases in each of these clinical manifestations, the presence of Ro60 antibodies alone was associated with decreased frequency of ILD. Discussion: Anti-Ro52 and/or anti-Ro60 antibodies are often co-expressed with anti-Jo1 antibodies, defining clinical subsets with different disease course/outcomes.
Assuntos
Miosite , Ribonucleoproteínas , Animais , Humanos , Ribonucleoproteínas/imunologia , Miosite/imunologia , Feminino , Camundongos , Masculino , Pessoa de Meia-Idade , Anticorpos Antinucleares/imunologia , Anticorpos Antinucleares/sangue , Autoanticorpos/sangue , Autoanticorpos/imunologia , Idoso , Adulto , Histidina-tRNA Ligase/imunologia , Modelos Animais de Doenças , Autoantígenos/imunologia , RNA Citoplasmático PequenoRESUMO
Introduction: Previous work in humans has demonstrated that both innate and adaptive immune signaling pathways contribute to the pathogenesis of idiopathic inflammatory myopathy (IIM), a systemic autoimmune disease targeting muscle as well as extra-muscular organs. To better define interactive signaling networks in IIM, we characterized the cellular phenotype and transcriptomic profiles of muscle-infiltrating cells in our established murine model of histidyl-tRNA synthetase (HRS)-induced myositis. Methods: Myositis was induced in wild type (WT) and various congenic/mutant strains of C57BL/6 mice through intramuscular immunization with recombinant HRS. Histopathological, immunohistochemical, flow cytometric, and transcriptomic assessments were used to characterize the functional relationship between muscle-infiltrating cell populations in these strains lacking different components of innate and/or adaptive immune signaling. Results: RAG1 KO mice developed markedly reduced muscle inflammation relative to WT mice, demonstrating a key requirement for T cells in driving HRS-induced myositis. While the reduction of mononuclear cell infiltrates in CD4-Cre.MyD88fl/fl conditional knockout mice and OT-II TCR transgenic mice highlighted roles for both innate and TCR-mediated/adaptive immune signaling in T cells, diminished inflammation in Lyz2-Cre.MyD88fl/fl conditional knockout mice underscored the importance of macrophage/myeloid cell populations in supporting T cell infiltration. Single cell RNA sequencing-based clustering of muscle-infiltrating subpopulations and associated pathway analyses showed that perturbations of T cell signaling/function alter the distribution and phenotype of macrophages, fibroblasts, and other non-lymphoid cell populations contributing to HRS-induced myositis. Discussion: Overall, HRS-induced myositis reflects the complex interplay between multiple cell types that collectively drive a TH1-predominant, pro-inflammatory tissue phenotype requiring antigen-mediated activation of both MyD88- and TCR-dependent T cell signaling pathways.
Assuntos
Histidina-tRNA Ligase , Miosite , Humanos , Camundongos , Animais , Linfócitos T , Camundongos Endogâmicos C57BL , Imunidade Adaptativa , Macrófagos , Inflamação , Camundongos Knockout , Receptores de Antígenos de Linfócitos TRESUMO
Ubiquitin carboxy terminal hydrolase L1 (UCHL1) is a protein highly expressed in neurons that may play important roles in the ubiquitin proteasome pathway (UPP) in neurons, axonal integrity, and motor function after traumatic brain injury (TBI). Binding of reactive lipid species to cysteine 152 of UCHL1 results in unfolding, aggregation, and inactivation of the enzyme. To test the role of this mechanism in TBI, mice bearing a cysteine to alanine mutation at site 152 (C152A mice) that renders UCHL1 resistant to inactivation by reactive lipids were subjected to the controlled cortical impact model (CCI) of TBI and compared to wild type (WT) controls. Alterations in protein ubiquitination and activation of autophagy pathway markers in traumatized brain were detected by immunoblotting. Cell death and axonal injury were determined by histological assessment and anti-amyloid precursor protein (APP) immunohistochemistry. Behavioral outcomes were determined using the beam balance and Morris water maze tests. C152A mice had reduced accumulation of ubiquitinated proteins, decreased activation of the autophagy markers Beclin-1 and LC3B, a decreased number of abnormal axons, decreased CA1 cell death, and improved motor and cognitive function compared to WT controls after CCI; no significant change in spared tissue volume was observed. These results suggest that binding of lipid substrates to cysteine 152 of UCHL1 is important in the pathogenesis of injury and recovery after TBI and may be a novel target for future therapeutic approaches.
Assuntos
Lesões Encefálicas Traumáticas , Ubiquitina Tiolesterase , Animais , Axônios/metabolismo , Sítios de Ligação , Morte Celular , Lipídeos , Camundongos , Mutação/genética , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
Ubiquitin (Ub) C-terminal hydrolase L1 (UCHL1) is a multifunctional protein that is expressed in neurons throughout brain at high levels. UCHL1 deletion is associated with axonal degeneration, progressive sensory motor ataxia, and premature death in mice. UCHL1 has been hypothesized to play a role in the pathogenesis of neurodegenerative diseases and recovery after neuronal injury. UCHL1 hydrolyzes Ub from polyubiquitinated (poly-Ub) proteins, but also may ligate Ub to select neuronal proteins, and interact with cytoskeletal proteins. These and other mechanisms have been hypothesized to underlie UCHL1's role in neurodegeneration and response to brain injury. A UCHL1 knockin mouse containing a C90A mutation (C90A) devoid of hydrolase activity was constructed. The C90A mouse did not develop the sensory and motor deficits, degeneration of the gracile nucleus and tract, or premature death as seen in UCHL1 deficient mice. C90A and wild type (WT) mice were subjected to the controlled cortical impact (CCI) model of traumatic brain injury (TBI), and cell death, axonal injury and behavioral outcome were assessed. C90A mice exhibited decreased spared tissue volume, greater loss of CA1 hippocampal neurons and greater axonal injury as detected using anti-amyloid precursor protein (APP) antibody and anti- non-phosphorylated neurofilament H (SMI-32) antibody immunohistochemistry after CCI compared to WT controls. Poly-Ub proteins and Beclin-1 were elevated after CCI in C90A mice compared to WT controls. Vestibular motor deficits assessed using the beam balance test resolved by day 5 after CCI in WT mice but not in C90A mice. These results suggest that the hydrolase activity of UCHL1 does not account for the progressive neurodegeneration and premature death seen in mice that do not express full length UCHL1. The hydrolase activity of UCHL1 contributes to the function of the ubiquitin proteasome pathway (UPP), ameliorates activation of autophagy, and improves motor recovery after CCI. Thus, UCHL1 hydrolase activity plays an important role in acute injury response after TBI.
Assuntos
Axônios/patologia , Lesões Encefálicas Traumáticas/patologia , Morte Celular/efeitos dos fármacos , Neurônios/patologia , Ubiquitina Tiolesterase/genética , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Animais , Autofagia , Proteína Beclina-1/metabolismo , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/psicologia , Região CA1 Hipocampal/patologia , Morte Celular/genética , Técnicas de Introdução de Genes , Camundongos , Mutação/genética , Desempenho Psicomotor , Transdução de Sinais/genética , UbiquitinaçãoRESUMO
Ubiquitin C-terminal hydrolase L1 (UCHL1) is a unique brain-specific deubiquitinating enzyme. Mutations in and aberrant function of UCHL1 have been linked to many neurological disorders. UCHL1 activity protects neurons from hypoxic injury, and binding of stroke-induced reactive lipid species to the cysteine 152 (C152) of UCHL1 unfolds the protein and disrupts its function. To investigate the role of UCHL1 and its adduction by reactive lipids in inhibiting repair and recovery of function following ischemic injury, a knock-in (KI) mouse expressing the UCHL1 C152A mutation was generated. Neurons derived from KI mice had less cell death and neurite injury after hypoxia. UCHL1 C152A KI and WT mice underwent middle cerebral artery occlusion (MCAO) or sham surgery. White matter injury was significantly decreased in KI compared with WT mice 7 d after MCAO. Histological analysis revealed decreased tissue loss at 21 d after injury in KI mice. There was also significantly improved sensorimotor recovery in postischemic KI mice. K63- and K48-linked polyubiquitinated proteins were increased in penumbra of WT mouse brains but not in KI mouse brains at 24 h post MCAO. The UCHL1 C152A mutation preserved excitatory synaptic drive to pyramidal neurons and their excitability in the periinfarct zone; axonal conduction velocity recovered by 21 d post MCAO in KI mice in corpus callosum. These results demonstrate that UCHL1 activity is an important determinant of function after ischemia and further demonstrate that the C152 site of UCHL1 plays a significant role in functional recovery after stroke.
Assuntos
Axônios/enzimologia , Isquemia Encefálica/enzimologia , Isquemia Encefálica/fisiopatologia , Ubiquitina Tiolesterase/metabolismo , Animais , Isquemia Encefálica/genética , Morte Celular , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Mutação , Neurônios/citologia , Neurônios/enzimologia , Recuperação de Função Fisiológica , Ubiquitina Tiolesterase/genéticaRESUMO
Nuclear factor κB (NF-κB) is a transcription factor important for regulating innate and adaptive immunity, cellular proliferation, apoptosis, and senescence. Dysregulation of NF-κB and its upstream regulator IκB kinase (IKK) contributes to the pathogenesis of multiple inflammatory and degenerative diseases as well as cancer. An 11-amino acid peptide containing the NF-κB essential modulator (NEMO)-binding domain (NBD) derived from the C-terminus of ß subunit of IKK, functions as a highly selective inhibitor of the IKK complex by disrupting the association of IKKß and the IKKγ subunit NEMO. A structure-based pharmacophore model was developed to identify NBD mimetics by in silico screening. Two optimized lead NBD mimetics, SR12343 and SR12460, inhibited tumor necrosis factor α (TNF-α)- and lipopolysaccharide (LPS)-induced NF-κB activation by blocking the interaction between IKKß and NEMO and suppressed LPS-induced acute pulmonary inflammation in mice. Chronic treatment of a mouse model of Duchenne muscular dystrophy (DMD) with SR12343 and SR12460 attenuated inflammatory infiltration, necrosis and muscle degeneration, demonstrating that these small-molecule NBD mimetics are potential therapeutics for inflammatory and degenerative diseases.
Assuntos
Materiais Biomiméticos/farmacologia , Quinase I-kappa B/antagonistas & inibidores , Distrofia Muscular de Duchenne/tratamento farmacológico , Pneumonia/tratamento farmacológico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Materiais Biomiméticos/química , Linhagem Celular , Feminino , Células HEK293 , Humanos , Quinase I-kappa B/química , Quinase I-kappa B/metabolismo , Inflamação/tratamento farmacológico , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Necrose/tratamento farmacológico , Domínios Proteicos , Células RAW 264.7RESUMO
In Duchenne muscular dystrophy (DMD) patients and the mdx mouse model of DMD, chronic activation of the classical nuclear factor-κB (NF-κB) pathway contributes to the pathogenesis that causes degeneration of muscle fibers, inflammation and fibrosis. Prior studies demonstrate that inhibition of inhibitor of κB kinase (IKK)-mediated NF-κB activation using L-isomer NF-κB essential modulator (NEMO)-binding domain (NBD) peptide-based approaches reduce muscle pathology in the mdx mouse. For our studies, the NBD peptide is synthesized as a fusion peptide with an eight-lysine (8K) protein transduction domain to facilitate intracellular delivery. We hypothesized that the d-isoform peptide could have a greater effect than the naturally occurring L-isoform peptide due to the longer persistence of the D-isoform peptide in vivo. In this study, we compared systemic treatment with low (1 mg/kg) and high (10 mg/kg) doses of L- and D-isomer 8K-wild-type-NBD peptide in mdx mice. Treatment with both L- or D-isoform 8K-wild-type-NBD peptide resulted in decreased activation of NF-κB and improved histology in skeletal muscle of the mdx mouse. However, we observed kidney toxicity (characterized by proteinuria), increased serum creatinine, activation of NF-κB and pathological changes in kidney cortex that were most severe with treatment with the D-isoform of 8K-wild-type-NBD peptide. The observed toxicity was also seen in normal mice.
Assuntos
Substituição de Aminoácidos/genética , Distrofia Muscular de Duchenne/tratamento farmacológico , NF-kappa B/genética , Peptídeos/administração & dosagem , Animais , Modelos Animais de Doenças , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , NF-kappa B/antagonistas & inibidores , Peptídeos/genética , Transdução de Sinais/efeitos dos fármacos , EstereoisomerismoRESUMO
The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB-activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging.
Assuntos
Envelhecimento/efeitos dos fármacos , Senescência Celular , Dano ao DNA , Quinase I-kappa B/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Envelhecimento/genética , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Quinase I-kappa B/metabolismo , Camundongos , Camundongos Transgênicos , Estresse Oxidativo , Peptídeos/farmacologia , Fosforilação , Progéria/tratamento farmacológico , Progéria/patologia , Ligação Proteica , Transdução de Sinais , Fator de Transcrição RelA/genética , Ativação TranscricionalRESUMO
Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmd(mdx)/J (mdx) mice, the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery.
Assuntos
Diafragma/fisiologia , Distrofina/genética , Músculo Esquelético/fisiologia , NF-kappa B/metabolismo , Peptídeos/farmacologia , Animais , Dependovirus , Distrofina/metabolismo , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Distrofia Muscular de DuchenneRESUMO
The activation of nuclear factor κB (NF-κB) contributes to muscle degeneration that results from dystrophin deficiency in human Duchenne muscular dystrophy (DMD) and in the mdx mouse. In dystrophic muscle, NF-κB participates in inflammation and failure of muscle regeneration. Peptides containing the NF-κB Essential Modulator (NEMO) binding domain (NBD) disrupt the IκB kinase complex, thus blocking NF-κB activation. The NBD peptide, which is linked to a protein transduction domain to achieve in vivo peptide delivery to muscle tissue, was systemically delivered to mdx mice for 4 or 7 weeks to study NF-κB activation, histological changes in hind limb and diaphragm muscle and ex vivo function of diaphragm muscle. Decreased NF-κB activation, decreased necrosis and increased regeneration were observed in hind limb and diaphragm muscle in mdx mice treated systemically with NBD peptide, as compared to control mdx mice. NBD peptide treatment resulted in improved generation of specific force and greater resistance to lengthening activations in diaphragm muscle ex vivo. Together these data support the potential of NBD peptides for the treatment of DMD by modulating dystrophic pathways in muscle that are downstream of dystrophin deficiency.
Assuntos
Quinase I-kappa B/administração & dosagem , Quinase I-kappa B/farmacocinética , Peptídeos e Proteínas de Sinalização Intracelular/administração & dosagem , Peptídeos e Proteínas de Sinalização Intracelular/farmacocinética , Músculo Esquelético/fisiologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Peptídeos/uso terapêutico , Animais , Diafragma/patologia , Diafragma/fisiologia , Modelos Animais de Doenças , Distrofina/deficiência , Distrofina/genética , Quinase I-kappa B/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/fisiologia , Necrose/prevenção & controle , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Peptídeos/administração & dosagem , Peptídeos/farmacocinética , Estrutura Terciária de Proteína/genéticaRESUMO
Adenoviral-associated viral vectors (AAV) have shown significant promise for efficient gene delivery to multiple tissues. Studies of different serotypes of AAV revealed different expression patterns provided by gene delivery in postnatal mice. Previous in utero gene delivery studies of AAV serotype 2 (AAV2) demonstrated efficient gene expression in certain fetal tissues depending on route of administration. We studied the pattern of gene expression from AAV serotype 1 (AAV1) using intramuscular, intraperitoneal, and intravascular routes of administration in embryonic day 16 C57BL/6 mice. Limb skeletal muscle transduction was only achieved with AAV1 by intramuscular administration. The levels of gene expression were 20-fold higher than a comparable administration of AAV2. Diaphragm muscle transduction by AAV1 was achieved at the highest level by intraperitoneal administration, and to a lesser degree by intravascular administration. All delivery routes resulted in transgene expression in the lung. Our results indicate that AAV1 can offer higher transgene expression in fetal skeletal muscle than AAV2 with intramuscular administration. The transgene expression pattern in different tissues, which depends on vector serotype and route of administration, will need to be considered in planning therapeutic studies for specific disorders.
Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Músculo Esquelético/embriologia , Animais , Feminino , Expressão Gênica , Vetores Genéticos/genética , Injeções Intramusculares , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/fisiologia , Gravidez , Distribuição Tecidual , Transgenes , beta-Galactosidase/genética , beta-Galactosidase/farmacocinéticaRESUMO
Adenoviral (Ad) vectors feature attractive characteristics for gene therapy of a wide variety of diseases. In many cases, the Ad vector must be administered using catheters and other plastic medical devices. Although poly(vinyl chloride) is one of the most frequently used catheter materials, it is relatively rigid and requires the addition of a plasticizer such as di-2-ethylhexyl phthalate (DEHP) to increase its flexibility. In this study, we demonstrated that exposure to a DEHP-containing catheter decreased the infectivity of Ad vectors but not the total particle number of the vector. Loss of Ad vector infectivity was directly related to the time of exposure to the DEHP-containing catheter, but it was not due to simple leaching of the chemical from the plastic. The loss of Ad vector infectivity could be prevented by preflushing the tube with albumin. Careful consideration of the compatibility between gene therapy vectors and medical delivery devices will be critical to the success of human gene therapy applications.