RESUMEN
PURPOSE: A few de novo missense variants in the cytoplasmic FMRP-interacting protein 2 (CYFIP2) gene have recently been described as a novel cause of severe intellectual disability, seizures, and hypotonia in 18 individuals, with p.Arg87 substitutions in the majority. METHODS: We assembled data from 19 newly identified and all 18 previously published individuals with CYFIP2 variants. By structural modeling and investigation of WAVE-regulatory complex (WRC)-mediated actin polymerization in six patient fibroblast lines we assessed the impact of CYFIP2 variants on the WRC. RESULTS: Sixteen of 19 individuals harbor two previously described and 11 novel (likely) disease-associated missense variants. We report p.Asp724 as second mutational hotspot (4/19 cases). Genotype-phenotype correlation confirms a consistently severe phenotype in p.Arg87 patients but a more variable phenotype in p.Asp724 and other substitutions. Three individuals with milder phenotypes carry putative loss-of-function variants, which remain of unclear pathogenicity. Structural modeling predicted missense variants to disturb interactions within the WRC or impair CYFIP2 stability. Consistent with its role in WRC-mediated actin polymerization we substantiate aberrant regulation of the actin cytoskeleton in patient fibroblasts. CONCLUSION: Our study expands the clinical and molecular spectrum of CYFIP2-related neurodevelopmental disorder and provides evidence for aberrant WRC-mediated actin dynamics as contributing cellular pathomechanism.
Asunto(s)
Discapacidad Intelectual , Trastornos del Neurodesarrollo , Actinas/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Humanos , Discapacidad Intelectual/genética , Trastornos del Neurodesarrollo/genética , ConvulsionesRESUMEN
Early stages of inflammation are characterized by extensive oxidative insult by recruited and activated neutrophils. Secretion of peroxidases, including the main enzyme, myeloperoxidase, leads to the generation of reactive oxygen species. We show that this oxidative insult leads to polyunsaturated fatty acid (eg, docosahexaenoate), oxidation, and accumulation of its product 2-(ω-carboxyethyl)pyrrole (CEP), which, in turn, is capable of protein modifications. In vivo CEP is generated predominantly at the inflammatory sites in macrophage-rich areas. During thioglycollate-induced inflammation, neutralization of CEP adducts dramatically reduced macrophage accumulation in the inflamed peritoneal cavity while exhibiting no effect on the early recruitment of neutrophils, suggesting a role in the second wave of inflammation. CEP modifications were abundantly deposited along the path of neutrophils migrating through the 3-dimensional fibrin matrix in vitro. Neutrophil-mediated CEP formation was markedly inhibited by the myeloperoxidase inhibitor, 4-ABH, and significantly reduced in myeloperoxidase-deficient mice. On macrophages, CEP adducts were recognized by cell adhesion receptors, integrin αMß2 and αDß2 Macrophage migration through CEP-fibrin gel was dramatically augmented when compared with fibrin alone, and was reduced by ß2-integrin deficiency. Thus, neutrophil-mediated oxidation of abundant polyunsaturated fatty acids leads to the transformation of existing proteins into stronger adhesive ligands for αMß2- and αDß2-dependent macrophage migration. The presence of a carboxyl group rather than a pyrrole moiety on these adducts, resembling characteristics of bacterial and/or immobilized ligands, is critical for recognition by macrophages. Therefore, specific oxidation-dependent modification of extracellular matrix, aided by neutrophils, promotes subsequent αMß2- and αDß2-mediated migration/retention of macrophages during inflammation.
Asunto(s)
Antígenos CD11/metabolismo , Antígenos CD18/metabolismo , Movimiento Celular , Matriz Extracelular/metabolismo , Cadenas alfa de Integrinas/metabolismo , Antígeno de Macrófago-1/metabolismo , Macrófagos/metabolismo , Neutrófilos/metabolismo , Animales , Antígenos CD11/genética , Antígenos CD18/genética , Matriz Extracelular/genética , Matriz Extracelular/patología , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Cadenas alfa de Integrinas/genética , Antígeno de Macrófago-1/genética , Macrófagos/patología , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/metabolismo , Errores Innatos del Metabolismo/patología , Ratones , Ratones Noqueados , Neutrófilos/patología , Oxidación-ReducciónRESUMEN
Macrophage accumulation is a critical step during development of chronic inflammation, initiating progression of many devastating diseases. Leukocyte-specific integrin αDß2 (CD11d/CD18) is dramatically upregulated on macrophages at inflammatory sites. Previously we found that CD11d overexpression on cell surfaces inhibits in vitro cell migration due to excessive adhesion. In this study, we have investigated how inflammation-mediated CD11d upregulation contributes to macrophage retention at inflammatory sites during atherogenesis. Atherosclerosis was evaluated in CD11d-/-/ApoE-/- mice after 16 wk on a Western diet. CD11d deficiency led to a marked reduction in lipid deposition in aortas and isolated macrophages. Macrophage numbers in aortic sinuses of CD11d-/- mice were reduced without affecting their apoptosis and proliferation. Adoptive transfer of fluorescently labeled wild-type and CD11d-/- monocytes into ApoE-/- mice demonstrated similar recruitment from circulation, but reduced accumulation of CD11d-/- macrophages within the aortas. Furthermore, CD11d expression was significantly upregulated on macrophages in atherosclerotic lesions and M1 macrophages in vitro. Interestingly, expression of the related ligand-sharing integrin CD11b was not altered. This difference defines their distinct roles in the regulation of macrophage migration. CD11d-deficient M1 macrophages demonstrated improved migration in a three-dimensional fibrin matrix and during resolution of peritoneal inflammation, whereas migration of CD11b-/- M1 macrophages was not affected. These results prove the contribution of high densities of CD11d to macrophage arrest during atherogenesis. Because high expression of CD11d was detected in several inflammation-dependent diseases, we suggest that CD11d/CD18 upregulation on proinflammatory macrophages may represent a common mechanism for macrophage retention at inflammatory sites, thereby promoting chronic inflammation and disease development.
Asunto(s)
Aterosclerosis/inmunología , Vasos Sanguíneos/patología , Antígenos CD11/genética , Antígenos CD18/genética , Cadenas alfa de Integrinas/genética , Macrófagos/inmunología , Animales , Aorta/inmunología , Aorta/patología , Apolipoproteínas E/deficiencia , Aterosclerosis/etiología , Aterosclerosis/patología , Vasos Sanguíneos/inmunología , Antígenos CD11/inmunología , Antígenos CD18/inmunología , Dieta Occidental , Humanos , Inflamación/patología , Mediadores de Inflamación/metabolismo , Cadenas alfa de Integrinas/deficiencia , Cadenas alfa de Integrinas/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Noqueados , Peritonitis/inmunología , Peritonitis/patología , Activación Transcripcional , Regulación hacia ArribaRESUMEN
UNLABELLED: The liver is considered to be an immune-privileged organ that favors the induction of tolerance. The underlying mechanisms are not completely understood. Interestingly, liver transplants are spontaneously accepted in several animal models, but hepatocyte transplants are acutely rejected, suggesting that liver nonparenchymal cells may effectively protect the parenchymal cells from immune attack. We have shown the profound T cell inhibitory activity of hepatic stellate cells (HSCs). Thus, cotransplantation with HSCs effectively protects islet allografts from rejection in mice. In this study, using T cell receptor transgenic and gene knockout approaches, we provided definitive evidence that HSCs protected cotransplanted islet allografts by exerting comprehensive inhibitory effects on T cells, including apoptotic death in graft-infiltrating antigen-specific effector T cells and marked expansion of CD4(+) Forkhead box protein (Foxp)3(+) T regulatory (Treg) cells. All these effects required an intact interferon-gamma (IFN-gamma) signaling in HSCs, demonstrated by using HSCs isolated from IFN-gamma receptor 1 knockout mice. B7-H1 expression on HSCs, a product molecule of IFN-gamma signaling, was responsible for induction of T cells apoptosis, but had no effect on expansion of Treg cells, suggesting that undetermined effector molecules produced by IFN-gamma signaling is involved in this process. CONCLUSION: Upon inflammatory stimulation, specific organ stromal cells (such as HSCs in the liver) demonstrate potent immune regulatory activity. Understanding of the mechanisms involved may lead to development of novel strategies for clinical applications in transplantation and autoimmune diseases.
Asunto(s)
Células Estrelladas Hepáticas/inmunología , Interferón gamma/fisiología , Transducción de Señal/fisiología , Animales , Apoptosis , Antígeno B7-1/genética , Antígeno B7-1/fisiología , Antígeno B7-H1 , Linfocitos T CD8-positivos/inmunología , Factores de Transcripción Forkhead/fisiología , Trasplante de Islotes Pancreáticos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos , Péptidos/genética , Péptidos/fisiología , Receptores de Interferón/fisiología , Linfocitos T Reguladores/fisiología , Receptor de Interferón gammaRESUMEN
BACKGROUND: The prostate gland represents a multifaceted system in which prostate epithelia and stroma have distinct physiological roles. To understand the interaction between stroma and glandular epithelia, it is essential to delineate the gene expression profiles of these two tissue types in prostate cancer. Most studies have compared tumor and normal samples by performing global expression analysis using a mixture of cell populations. This report presents the first study of prostate tumor tissue that examines patterns of differential expression between specific cell types using laser capture microdissection (LCM). METHODS: LCM was used to isolate distinct cell-type populations and identify their gene expression differences using oligonucleotide microarrays. Ten differentially expressed genes were then analyzed in paired tumor and non-neoplastic prostate tissues by quantitative real-time PCR. Expression patterns of the transcription factors, WT1 and EGR1, were further compared in established prostate cell lines. WT1 protein expression was also examined in prostate tissue microarrays using immunohistochemistry. RESULTS: The two-step method of laser capture and microarray analysis identified nearly 500 genes whose expression levels were significantly different in prostate epithelial versus stromal tissues. Several genes expressed in epithelial cells (WT1, GATA2, and FGFR-3) were more highly expressed in neoplastic than in non-neoplastic tissues; conversely several genes expressed in stromal cells (CCL5, CXCL13, IGF-1, FGF-2, and IGFBP3) were more highly expressed in non-neoplastic than in neoplastic tissues. Notably, EGR1 was also differentially expressed between epithelial and stromal tissues. Expression of WT1 and EGR1 in cell lines was consistent with these patterns of differential expression. Importantly, WT1 protein expression was demonstrated in tumor tissues and was absent in normal and benign tissues. CONCLUSIONS: The prostate represents a complex mix of cell types and there is a need to analyze distinct cell populations to better understand their potential interactions. In the present study, LCM and microarray analysis were used to identify novel gene expression patterns in prostate cell populations, including identification of WT1 expression in epithelial cells. The relevance of WT1 expression in prostate cancer was confirmed by analysis of tumor tissue and cell lines, suggesting a potential role for WT1 in prostate tumorigenesis.