RESUMO
Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1ß and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care.
Assuntos
Inflamassomos , Osteomielite , Humanos , Citocinas , Inflamassomos/genética , Inflamassomos/metabolismo , Osteomielite/genética , Potássio , Piroptose , Receptores Purinérgicos P2X7/genéticaRESUMO
PURPOSE OF REVIEW: To review recent trends in treatment and recent progress in developing outcome measures needed for chronic nonbacterial osteomyelitis (CNO) clinical trials. RECENT FINDINGS: CNO is an autoinflammatory bone disease. In a minority of patients, the disease is genetically driven, and diagnosis can be made by DNA sequencing. However, for nonsyndromic CNO there is no diagnostic test. The number of children with CNO appears to be increasing and damage is common. Increases in CNO diagnosis is due to raised awareness, increased availability of whole-body magnetic resonance imaging and rising incidence. Treatment remains empiric and it is unclear which second line treatment is superior. Tumor necrosis factor inhibitors (TNFi) and bisphosphonates continue to be used as second line agents for nonsteroidal anti-inflammatory drugs (NSAID) refractory CNO; newer immune modulatory medications are used if this fails. Validated classification criteria, clinical outcome measures and imaging scoring standards are needed for successful clinical trials. SUMMARY: Best treatment for NSAID refractory CNO remains unclear. Classification criteria, clinical outcomes measures and standardized imaging scoring have been developed or are near completion. This will facilitate robust clinical trials in CNO with the goal of having approved medications for this painful disease.
Assuntos
Anti-Inflamatórios não Esteroides , Osteomielite , Humanos , Osteomielite/diagnóstico , Osteomielite/tratamento farmacológico , Osteomielite/epidemiologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Imagem Corporal Total , Imageamento por Ressonância Magnética , Incidência , Avaliação de Resultados da Assistência ao Paciente , Resultado do TratamentoRESUMO
Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome is the most common periodic fever syndrome in children. The disease appears to cluster in families, but the pathogenesis is unknown. We queried two European-American cohorts and one Turkish cohort (total n = 231) of individuals with PFAPA for common variants previously associated with two other oropharyngeal ulcerative disorders, Behçet's disease and recurrent aphthous stomatitis. In a metaanalysis, we found that a variant upstream of IL12A (rs17753641) is strongly associated with PFAPA (OR 2.13, P = 6 × 10-9). We demonstrated that monocytes from individuals who are heterozygous or homozygous for this risk allele produce significantly higher levels of IL-12p70 upon IFN-γ and LPS stimulation than those from individuals without the risk allele. We also found that variants near STAT4, IL10, and CCR1-CCR3 were significant susceptibility loci for PFAPA, suggesting that the pathogenesis of PFAPA involves abnormal antigen-presenting cell function and T cell activity and polarization, thereby implicating both innate and adaptive immune responses at the oropharyngeal mucosa. Our results illustrate genetic similarities among recurrent aphthous stomatitis, PFAPA, and Behçet's disease, placing these disorders on a common spectrum, with recurrent aphthous stomatitis on the mild end, Behçet's disease on the severe end, and PFAPA intermediate. We propose naming these disorders Behçet's spectrum disorders to highlight their relationship. HLA alleles may be factors that influence phenotypes along this spectrum as we found new class I and II HLA associations for PFAPA distinct from Behçet's disease and recurrent aphthous stomatitis.
Assuntos
Síndrome de Behçet/genética , Febre/genética , Predisposição Genética para Doença , Linfadenite/genética , Faringite/genética , Estomatite Aftosa/genética , Alelos , Síndrome de Behçet/imunologia , Criança , Estudos de Coortes , Febre/imunologia , Genes MHC Classe I/genética , Genes MHC Classe I/imunologia , Genes MHC da Classe II/genética , Genes MHC da Classe II/imunologia , Loci Gênicos/imunologia , Humanos , Linfadenite/imunologia , Faringite/imunologia , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Estomatite Aftosa/imunologia , SíndromeRESUMO
Autoinflammatory syndromes are characterized by dysregulation of the innate immune response with subsequent episodes of acute spontaneous inflammation. Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder that presents with bone pain and localized swelling. Ali18 mice, isolated from a mutagenesis screen, exhibit a spontaneous inflammatory paw phenotype that includes sterile osteomyelitis and systemic reduced bone mineral density. To elucidate the molecular basis of the disease, positional cloning of the causative gene for Ali18 was attempted. Using a candidate gene approach, a missense mutation in the C-terminal region of Fgr, a member of Src family tyrosine kinases (SFKs), was identified. For functional confirmation, additional mutations at the N terminus of Fgr were introduced in Ali18 mice by CRISPR/Cas9-mediated genome editing. N-terminal deleterious mutations of Fgr abolished the inflammatory phenotype in Ali18 mice, but in-frame and missense mutations in the same region continue to exhibit the phenotype. The fact that Fgr null mutant mice are morphologically normal suggests that the inflammation in this model depends on Fgr products. Furthermore, the levels of C-terminal negative regulatory phosphorylation of Fgr Ali18 are distinctly reduced compared with that of wild-type Fgr. In addition, whole-exome sequencing of 99 CRMO patients including 88 trios (proband and parents) identified 13 patients with heterozygous coding sequence variants in FGR, including two missense mutant proteins that affect kinase activity. Our results strongly indicate that gain-of-function mutations in Fgr are involved in sterile osteomyelitis, and thus targeting SFKs using specific inhibitors may allow for efficient treatment of the disease.
Assuntos
Doenças Ósseas/genética , Mutação com Ganho de Função/genética , Inflamação/genética , Quinases da Família src/genética , Sequência de Aminoácidos , Animais , Humanos , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteomielite/genética , Fosforilação/genéticaRESUMO
BACKGROUND: Traumatic brain injury (TBI) is a leading cause of death and disability that lacks neuroprotective therapies. Following a TBI, secondary injury response pathways are activated and contribute to ongoing neurodegeneration. Microglia and astrocytes are critical neuroimmune modulators with early and persistent reactivity following a TBI. Although histologic glial reactivity is well established, a precise understanding of microglia and astrocyte function following trauma remains unknown. METHODS: Adult male C57BL/6J mice underwent either fluid percussion or sham injury. RNA sequencing of concurrently isolated microglia and astrocytes was conducted 7 days post-injury to evaluate cell-type-specific transcriptional responses to TBI. Dual in situ hybridization and immunofluorescence were used to validate the TBI-induced gene expression changes in microglia and astrocytes and to identify spatial orientation of cells expressing these genes. Comparative analysis was performed between our glial transcriptomes and those from prior reports in mild TBI and other neurologic diseases to determine if severe TBI induces unique states of microglial and astrocyte activation. RESULTS: Our findings revealed sustained, lineage-specific transcriptional changes in both microglia and astrocytes, with microglia showing a greater transcriptional response than astrocytes at this subacute time point. Microglia and astrocytes showed overlapping enrichment for genes related to type I interferon signaling and MHC class I antigen presentation. The microglia and astrocyte transcriptional response to severe TBI was distinct from prior reports in mild TBI and other neurodegenerative and neuroinflammatory diseases. CONCLUSION: Concurrent lineage-specific analysis revealed novel TBI-specific transcriptional changes; these findings highlight the importance of cell-type-specific analysis of glial reactivity following TBI and may assist with the identification of novel, targeted therapies.
Assuntos
Astrócitos/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Interferon Tipo I/biossíntese , Microglia/metabolismo , Transcriptoma/fisiologia , Animais , Astrócitos/patologia , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Interferon Tipo I/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/patologiaRESUMO
PURPOSE OF REVIEW: To describe the main clinical differences of children and adults with chronic nonbacterial osteomyelitis (CNO). RECENT FINDINGS: CNO is a severe systemic autoinflammatory syndrome characterized by multiple bone lesions because of inflammatory osteitis. Delay to diagnosis of CNO can lead to functional impairment, fractures, and chronic pain. Key clinical aspects and disease patterns differ in children and adults, including onset and time to diagnosis, symptom localization, associated comorbidities (i.e. skin, joints), bone lesion distribution pattern, and treatment approach. Novel biomarkers, such as urine N-terminal telopeptide in children and serum IgG4 in adults, are being studied for possible future use in improving diagnosis and guiding treatment. Despite recent advances in our understanding of CNO, many children and adults have a high disease burden and poor long-term outcomes. Recent findings suggest that adults with CNO tend to have a more chronic disease course and are less likely to achieve remission in follow-up. SUMMARY: The clinical presentation of CNO differs in children and adults, highlighting the importance of these key features for the accurate diagnosis and early treatment in CNO.
Assuntos
Osteomielite/diagnóstico , Adulto , Fatores Etários , Criança , Doença Crônica , Efeitos Psicossociais da Doença , Progressão da Doença , Humanos , Osteomielite/tratamento farmacológico , Índice de Gravidade de DoençaRESUMO
Chronic recurrent multifocal osteomyelitis (CRMO) is a pediatric autoinflammatory disorder that is characterized by multiple sterile inflammatory bone lesions with a relapsing and remitting course. CRMO belongs to the autoinflammatory family of rheumatologic disorders based on absence of significant titers of autoantibodies and autoreactive T-lymphocytes. In absence of pathognomonic clinical, radiographic or pathological features, diagnosis can be challenging. CRMO shares imaging features with other diseases. It is important for radiologists to be able to differentiate other diseases from CRMO because prognosis varies from completely benign to frankly malignant. In this article we first present the clinical and imaging features of CRMO to help readers gain an understanding of the disease process, then discuss our imaging approach to CRMO and review other disease processes that sometimes share similar imaging findings to CRMO and review differentiating features to help avoid misdiagnoses.
Assuntos
Imageamento por Ressonância Magnética/métodos , Osteomielite/diagnóstico por imagem , Radiografia/métodos , Criança , Diagnóstico Diferencial , Feminino , Humanos , MasculinoRESUMO
Compound heterozygotes occur when different variants at the same locus on both maternal and paternal chromosomes produce a recessive trait. Here we present the tool VarCount for the quantification of variants at the individual level. We used VarCount to characterize compound heterozygous coding variants in patients with epileptic encephalopathy and in the 1000 Genomes Project participants. The Epi4k data contains variants identified by whole exome sequencing in patients with either Lennox-Gastaut Syndrome (LGS) or infantile spasms (IS), as well as their parents. We queried the Epi4k dataset (264 trios) and the phased 1000 Genomes Project data (2504 participants) for recessive variants. To assess enrichment, transcript counts were compared between the Epi4k and 1000 Genomes Project participants using minor allele frequency (MAF) cutoffs of 0.5 and 1.0%, and including all ancestries or only probands of European ancestry. In the Epi4k participants, we found enrichment for rare, compound heterozygous variants in six genes, including three involved in neuronal growth and development - PRTG (p = 0.00086, 1% MAF, combined ancestries), TNC (p = 0.022, 1% MAF, combined ancestries) and MACF1 (p = 0.0245, 0.5% MAF, EU ancestry). Due to the total number of transcripts considered in these analyses, the enrichment detected was not significant after correction for multiple testing and higher powered or prospective studies are necessary to validate the candidacy of these genes. However, PRTG, TNC and MACF1 are potential novel recessive epilepsy genes and our results highlight that compound heterozygous variants should be considered in sporadic epilepsy.
Assuntos
Epilepsia/genética , Epilepsia/metabolismo , Análise de Sequência de DNA/métodos , Adulto , Alelos , Exoma , Feminino , Frequência do Gene/genética , Genes Recessivos/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Heterozigoto , Humanos , Lactente , Recém-Nascido , Síndrome de Lennox-Gastaut/genética , Síndrome de Lennox-Gastaut/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Mutação , Fenótipo , Estudos Prospectivos , Espasmos Infantis/genética , Espasmos Infantis/metabolismo , Tenascina/genéticaRESUMO
PURPOSE OF REVIEW: To summarize the current advances in our understanding or the genetic basis of nonbacterial osteomyelitis. RECENT FINDINGS: Chronic recurrent multifocal osteomyelitis (CRMO) is a complex genetic disorder. Past discoveries identified several single gene defects (LPIN2, Pstpip2 and IL1RN) that cause IL-1-mediated sterile multifocal osteomyelitis. Recently Lorden et al.'s studies show that LIPIN2 deficiency can activate the NLRP3 inflammasome through alterations in the function of P2X7 receptor providing evidence that Majeed syndrome is an NLRP3 inflammasomopathy. New gene discoveries include the identification of FBLIM1 as a CRMO susceptibility gene. Mutations in FBLIM1 were found in a consanguineous family with CRMO. Fblim1 is one of the most significantly differentially expressed gene in bone from chronic multifocal osteomyelitis (cmo) mice, plays a role in IL-10-driven anti-inflammatory responses, and is involved in the physiology of bone remodeling. Lastly, new data on the putative CRMO susceptibility locus on chromosome 18 is presented here. Using Sanger sequencing, rather than microsatellite analysis, the DS18S60 susceptibility region could not be replicated in a larger cohort. SUMMARY: CRMO occurs in humans, nonhuman primates, dogs and mice. There is a genetic component to disease but the genetic basis has only been identified for a small percentage of all cases.
Assuntos
Osteomielite/genética , Animais , Moléculas de Adesão Celular/genética , Cromossomos Humanos Par 18/genética , Doença Crônica , Proteínas do Citoesqueleto/genética , Predisposição Genética para Doença , Humanos , Inflamassomos/genética , Camundongos , MutaçãoRESUMO
Polymorphonuclear leukocytes (PMN) achieve an intermediate or primed state of activation following stimulation with certain agonists. Primed PMN have enhanced responsiveness to subsequent stimuli, which can be beneficial in eliminating microbes but may cause host tissue damage in certain disease contexts, including sepsis. As PMN priming by TLR4 agonists is well described, we hypothesized that ligation of TLR2/1 or TLR2/6 would prime PMN. Surprisingly, PMN from only a subset of donors were primed in response to the TLR2/1 agonist, Pam3CSK4, although PMN from all donors were primed by the TLR2/6 agonist, FSL-1. Priming responses included generation of intracellular and extracellular reactive oxygen species, MAPK phosphorylation, integrin activation, secondary granule exocytosis, and cytokine secretion. Genotyping studies revealed that PMN responsiveness to Pam3CSK4 was enhanced by a common single-nucleotide polymorphism (SNP) in TLR1 (rs5743618). Notably, PMN from donors with the SNP had higher surface levels of TLR1 and were demonstrated to have enhanced association of TLR1 with the endoplasmic reticulum chaperone gp96. We analyzed TLR1 genotypes in a pediatric sepsis database and found that patients with sepsis or septic shock who had a positive blood culture and were homozygous for the SNP associated with neutrophil priming had prolonged pediatric intensive care unit length of stay. We conclude that this TLR1 SNP leads to excessive PMN priming in response to cell stimulation. Based on our finding that septic children with this SNP had longer pediatric intensive care unit stays, we speculate that this SNP results in hyperinflammation in diseases such as sepsis.
Assuntos
Ativação de Neutrófilo/genética , Receptor 1 Toll-Like/genética , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Genótipo , Humanos , Immunoblotting , Unidades de Terapia Intensiva , Tempo de Internação , Neutrófilos/imunologia , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse , Receptor 1 Toll-Like/imunologiaRESUMO
Epilepsy is a common disabling disease with complex, multifactorial genetic and environmental etiology. The small fraction of epilepsies subject to Mendelian inheritance offers key insight into epilepsy disease mechanisms; and pathologies brought on by mutations in a single gene can point the way to generalizable therapeutic strategies. Mutations in the PRICKLE genes can cause seizures in humans, zebrafish, mice, and flies, suggesting the seizure-suppression pathway is evolutionarily conserved. This pathway has never been targeted for novel anti-seizure treatments. Here, the mammalian PRICKLE-interactome was defined, identifying prickle-interacting proteins that localize to synapses and a novel interacting partner, USP9X, a substrate-specific de-ubiquitinase. PRICKLE and USP9X interact through their carboxy-termini; and USP9X de-ubiquitinates PRICKLE, protecting it from proteasomal degradation. In forebrain neurons of mice, USP9X deficiency reduced levels of Prickle2 protein. Genetic analysis suggests the same pathway regulates Prickle-mediated seizures. The seizure phenotype was suppressed in prickle mutant flies by the small-molecule USP9X inhibitor, Degrasyn/WP1130, or by reducing the dose of fat facets a USP9X orthologue. USP9X mutations were identified by resequencing a cohort of patients with epileptic encephalopathy, one patient harbored a de novo missense mutation and another a novel coding mutation. Both USP9X variants were outside the PRICKLE-interacting domain. These findings demonstrate that USP9X inhibition can suppress prickle-mediated seizure activity, and that USP9X variants may predispose to seizures. These studies point to a new target for anti-seizure therapy and illustrate the translational power of studying diseases in species across the evolutionary spectrum.
Assuntos
Convulsões/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Drosophila melanogaster , Humanos , Espectrometria de Massas , Camundongos , Convulsões/tratamento farmacológico , Ubiquitina Tiolesterase/genéticaRESUMO
PURPOSE OF REVIEW: We focus on recent advances in the understanding of the genetic, molecular, immunologic, and environmental factors implicated in the pathogenesis of autoinflammatory bone diseases including the syndromic and non-syndromic forms of chronic recurrent multifocal osteomyelitis (CRMO). RECENT FINDINGS: Evidence implicating the IL-1 pathway in the pathogenesis of the Mendelian forms of CRMO is growing. LIPIN2 can regulate the NLRP3 inflammasome by affecting P2X7 receptor activation, and intracellular cholesterol can modulate P2X7R currents. Work in a mouse model of CRMO demonstrates that dietary manipulation can alter the microbiome and protect these mice from the development of sterile osteomyelitis in vivo. Although the genetic and immunologic basis of non-syndromic CRMO remains only partially understood, the IL-1 pathway is central to the pathogenesis in the syndromic autoinflammatory bone disorders. Recent work implicates lipids and the microbiome in sterile osteomyelitis.
Assuntos
Doenças Hereditárias Autoinflamatórias/etiologia , Osteomielite/etiologia , Anemia Diseritropoética Congênita/etiologia , Animais , Moléculas de Adesão Celular/genética , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Humanos , Síndromes de Imunodeficiência , Inflamassomos/fisiologia , Interleucina-1/imunologia , Camundongos , Microbiota , Proteínas Nucleares/fisiologiaRESUMO
PURPOSE OF REVIEW: Chronic non-bacterial osteomyelitis (CNO) with its most severe form chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder. We summarize the clinical presentation, diagnostic approaches, most recent advances in understanding the pathophysiology, and available treatment options and outcomes in CNO/CRMO. RECENT FINDINGS: Though the exact molecular pathophysiology of CNO/CRMO remains somewhat elusive, it appears likely that variable defects in the TLR4/MAPK/inflammasome signaling cascade result in an imbalance between pro- and anti-inflammatory cytokine expressions in monocytes from CNO/CRMO patients. In this context, we present previously unpublished data on cytokine and chemokine expression in monocytes and tissues. CNO/CRMO is an autoinflammatory bone disorder resulting from imbalanced cytokine expression from innate immune cells. Though the exact molecular pathophysiology remains unclear, variable molecular defects appear to result in inflammasome activation and pro-inflammatory cytokine expression in monocytes from CNO/CRMO patients. Recent advances suggest signaling pathways and single molecules as biomarkers for CNO/CRMO as well as future treatment targets.
Assuntos
Citocinas/imunologia , Inflamassomos/imunologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , Monócitos/imunologia , Osteomielite/imunologia , Receptor 4 Toll-Like/imunologia , Corticosteroides/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Conservadores da Densidade Óssea/uso terapêutico , Quimiocina CCL2/imunologia , Quimiocina CCL4/imunologia , Quimiocina CCL5/imunologia , Quimiocinas/imunologia , Difosfonatos/uso terapêutico , Modelos Animais de Doenças , Humanos , Imunossupressores/uso terapêutico , Interleucina-12/imunologia , Interleucina-6/imunologia , Metotrexato/uso terapêutico , Camundongos , Osteomielite/diagnóstico , Osteomielite/terapia , Receptores de Interleucina-2/imunologia , Transdução de Sinais , Sulfassalazina/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Chronic recurrent multifocal osteomyelitis (CRMO) is a human autoinflammatory disorder that primarily affects bone. Missense mutation (L98P) of proline-serine-threonine phosphatase-interacting protein 2 (Pstpip2) in mice leads to a disease that is phenotypically similar to CRMO called chronic multifocal osteomyelitis (cmo). Here we show that deficiency of IL-1RI in cmo mice resulted in a significant reduction in the time to onset of disease as well as the degree of bone pathology. Additionally, the proinflammatory cytokine IL-1ß, but not IL-1α, played a critical role in the pathology observed in cmo mice. In contrast, disease in cmo mice was found to be independent of the nucleotide-binding domain, leucine-rich repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome as well as caspase-1. Neutrophils, but not bone marrow-derived macrophages, from cmo mice secreted increased IL-1ß in response to ATP, silica, and Pseudomonas aeruginosa compared with neutrophils from WT mice. This aberrant neutrophil response was sensitive to inhibition by serine protease inhibitors. These results demonstrate an inflammasome-independent role for IL-1ß in disease progression of cmo and implicate neutrophils and neutrophil serine proteases in disease pathogenesis. These data provide a rationale for directly targeting IL-1RI or IL-1ß as a therapeutic strategy in CRMO.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica , Interleucina-1beta/metabolismo , Osteomielite/imunologia , Animais , Células da Medula Óssea/citologia , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Inflamassomos/metabolismo , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Mutação de Sentido Incorreto , Neutrófilos/citologia , Neutrófilos/metabolismo , Osteomielite/genética , Estrutura Terciária de Proteína , Receptores de Interleucina-1/genéticaRESUMO
PSTPIP1 is a cytoskeletal adaptor and F-BAR protein that has been implicated in autoinflammatory disease, most notably in the PAPA syndrome: pyogenic sterile arthritis, pyoderma gangrenosum, and acne. However, the mechanism by which PSTPIP1 regulates the actin cytoskeleton and contributes to disease pathogenesis remains elusive. Here, we show that endogenous PSTPIP1 negatively regulates macrophage podosome organization and matrix degradation. We identify a novel PSTPIP1-R405C mutation in a patient presenting with aggressive pyoderma gangrenosum. Identification of this mutation reveals that PSTPIP1 regulates the balance of podosomes and filopodia in macrophages. The PSTPIP1-R405C mutation is in the SRC homology 3 (SH3) domain and impairs Wiskott-Aldrich syndrome protein (WASP) binding, but it does not affect interaction with protein-tyrosine phosphatase (PTP)-PEST. Accordingly, WASP inhibition reverses the elevated F-actin content, filopodia formation, and matrix degradation induced by PSTPIP1-R405C. Our results uncover a novel role for PSTPIP1 and WASP in orchestrating different types of actin-based protrusions. Our findings implicate the cytoskeletal regulatory functions of PSTPIP1 in the pathogenesis of pyoderma gangrenosum and suggest that the cytoskeleton is a rational target for therapeutic intervention in autoinflammatory disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Macrófagos/metabolismo , Acne Vulgar/metabolismo , Actinas/metabolismo , Algoritmos , Artrite Infecciosa/metabolismo , Quimiotaxia , Citoesqueleto/metabolismo , DNA/metabolismo , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Inflamação/metabolismo , Microscopia de Fluorescência , Mutação , Fenótipo , Estrutura Terciária de Proteína , Pseudópodes/metabolismo , Pioderma Gangrenoso/metabolismo , RNA Interferente Pequeno/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Neural tube defects (NTDs) is a general term for central nervous system malformations secondary to a failure of closure or development of the neural tube. The resulting pathologies may involve the brain, spinal cord and/or vertebral column, in addition to associated structures such as soft tissue or skin. The condition is reported among the more common birth defects in humans, leading to significant infant morbidity and mortality. The etiology remains poorly understood but genetic, nutritional, environmental factors, or a combination of these, are known to play a role in the development of NTDs. The variable conditions associated with NTDs occur naturally in dogs, and have been previously reported in the Weimaraner breed. Taking advantage of the strong linkage-disequilibrium within dog breeds we performed genome-wide association analysis and mapped a genomic region for spinal dysraphism, a presumed NTD, using 4 affected and 96 unaffected Weimaraners. The associated region on canine chromosome 8 (pgenome â=3.0 × 10(-5)), after 100,000 permutations, encodes 18 genes, including NKX2-8, a homeobox gene which is expressed in the developing neural tube. Sequencing NKX2-8 in affected Weimaraners revealed a G to AA frameshift mutation within exon 2 of the gene, resulting in a premature stop codon that is predicted to produce a truncated protein. The exons of NKX2-8 were sequenced in human patients with spina bifida and rare variants (rs61755040 and rs10135525) were found to be significantly over-represented (p=0.036). This is the first documentation of a potential role for NKX2-8 in the etiology of NTDs, made possible by investigating the molecular basis of naturally occurring mutations in dogs.
Assuntos
Mapeamento Cromossômico , Estudo de Associação Genômica Ampla , Proteínas de Homeodomínio/genética , Defeitos do Tubo Neural/genética , Fatores de Transcrição/genética , Animais , Cães , Éxons/genética , Ácido Fólico/genética , Ácido Fólico/metabolismo , Predisposição Genética para Doença , Humanos , Desequilíbrio de Ligação , Mutação , Defeitos do Tubo Neural/patologiaRESUMO
Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Convulsões/etiologia , Proteínas Supressoras de Tumor/genética , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Encéfalo/metabolismo , Cálcio/metabolismo , Drosophila melanogaster/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Epilepsias Mioclônicas/genética , Feminino , Heterozigoto , Humanos , Técnicas Imunoenzimáticas , Hibridização In Situ , Proteínas com Domínio LIM , Masculino , Camundongos , Camundongos Knockout , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Convulsões/metabolismo , Peixe-Zebra/genéticaRESUMO
Traumatic Brain Injury (TBI) induces neuroinflammatory response that can initiate epileptogenesis, which develops into epilepsy. Recently, we identified anti-convulsive effects of naltrexone, a mu-opioid receptor (MOR) antagonist, used to treat drug addiction. While blocking opioid receptors can reduce inflammation, it is unclear if post-TBI seizures can be prevented by blocking MORs. Here, we tested if naltrexone prevents neuroinflammation and/or seizures post-TBI. TBI was induced by a modified Marmarou Weight-Drop (WD) method on 4-week-old C57BL/6J male mice. Mice were placed in two groups: non-telemetry assessing the acute effects or in telemetry monitoring for interictal events and spontaneous seizures both following TBI and naltrexone. Molecular, histological and neuroimaging techniques were used to evaluate neuroinflammation, neurodegeneration and fiber track integrity at 8 days and 3 months post-TBI. Peripheral immune responses were assessed through serum chemokine/cytokine measurements. Our results show an increase in MOR expression, nitro-oxidative stress, mRNA expression of inflammatory cytokines, microgliosis, neurodegeneration, and white matter damage in the neocortex of TBI mice. Video-EEG revealed increased interictal events in TBI mice, with 71% mice developing post-traumatic seizures (PTS). Naltrexone treatment ameliorated neuroinflammation, neurodegeneration, reduced interictal events and prevented seizures in all TBI mice, which makes naltrexone a promising candidate against PTS, TBI-associated neuroinflammation and epileptogenesis in a WD model of TBI.