RESUMEN
Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood-brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB-crossing adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer's disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH-B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV-VHH-B9 produces positive long-term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the AppNL-G-F Alzheimer's mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets.
Asunto(s)
Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas , Anticuerpos de Dominio Único , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/inmunología , Ácido Aspártico Endopeptidasas/metabolismo , Barrera Hematoencefálica , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos/uso terapéutico , Ratones , Ratones TransgénicosRESUMEN
Hair follicles (HFs) function as hubs for stem cells, immune cells, and commensal microbes, which must be tightly regulated during homeostasis and transient inflammation. Here we found that transmembrane endopeptidase ADAM10 expression in upper HFs was crucial for regulating the skin microbiota and protecting HFs and their stem cell niche from inflammatory destruction. Ablation of the ADAM10-Notch signaling axis impaired the innate epithelial barrier and enabled Corynebacterium species to predominate the microbiome. Dysbiosis triggered group 2 innate lymphoid cell-mediated inflammation in an interleukin-7 (IL-7) receptor-, S1P receptor 1-, and CCR6-dependent manner, leading to pyroptotic cell death of HFs and irreversible alopecia. Double-stranded RNA-induced ablation models indicated that the ADAM10-Notch signaling axis bolsters epithelial innate immunity by promoting ß-defensin-6 expression downstream of type I interferon responses. Thus, ADAM10-Notch signaling axis-mediated regulation of host-microbial symbiosis crucially protects HFs from inflammatory destruction, which has implications for strategies to sustain tissue integrity during chronic inflammation.
Asunto(s)
Proteína ADAM10/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Disbiosis/inmunología , Folículo Piloso/patología , Linfocitos/inmunología , Proteínas de la Membrana/inmunología , Receptores Notch/inmunología , Piel/microbiología , Alopecia/inmunología , Alopecia/patología , Animales , Corynebacterium , Disbiosis/patología , Femenino , Folículo Piloso/inmunología , Inmunidad Innata , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Ratones , Transducción de Señal/inmunología , Piel/inmunología , Piel/patologíaRESUMEN
Several genes in innate immunity have been implicated in Alzheimer's disease (AD). However, the effect of innate immunity on amyloid ß (Aß) production, which makes amyloid plaques in AD brains, was previously not known. Recently, the antiviral protein interferon-induced transmembrane protein 3 (IFITM3) has been identified as a novel γ-secretase modulatory protein for Aß production. In this review, the mechanisms of how innate immunity modulates Aß production via IFITM3-γ-secretase complexes and contributes to AD pathogenesis are discussed.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/patología , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/metabolismo , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/inmunología , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Membrana Celular/metabolismo , Humanos , Inmunidad Innata/inmunología , Inmunidad Innata/fisiología , Interferones/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/fisiología , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al ARN/fisiologíaRESUMEN
OBJECTIVE: Buerger's disease is a rare disease that causes critical limb ischemia; however, the underlying pathophysiological mechanism remains unclear. Therefore, we investigated the interaction between interleukin (IL)-17 and high-mobility group protein B 1 (HMGB1) and determined whether A disintegrin and metalloproteinase 10 (ADAM10) inhibit this interaction. PATIENTS AND METHODS: The study population included 15 patients with Buerger's disease and 10 healthy donors without a history of giving peripheral blood samples. Cytokine levels were measured using a luminex multiplex assay in plasma. Flow cytometry was used to analyze the subtypes of helper T (Th) cells among peripheral blood mononuclear cells (PBMCs). The effect of ADAM10 on PBMCs was analyzed in vitro. RESULTS: The levels of inflammatory cytokines and production of pathogenic Th cells were found to be higher in Korean patients with Buerger's disease. IL-17 treatment induced HMGB1 associated molecules. HMGB1 also induced IL-17 and Th17 associated transcription factors in Buerger's patients. We observed that ADAM10 regulates the interaction between IL-17 and HMGB1 via advanced glycation end products (RAGE)/nuclear factor-kappa B (NF-kB) pathway in patients with Buerger's disease. CONCLUSIONS: This study suggests that IL-17 and HMGB1 cytokines contribute to the pathogenesis of Buerger's disease. These results indicate that ADAM10 alleviates inflammation in Buerger's disease via the HMGB1 and RAGE/NF-κB signaling pathway and provides insights into the molecular basis of and a potential therapeutic strategy for Buerger's disease.
Asunto(s)
Citocinas/inmunología , Proteína HMGB1/inmunología , Tromboangitis Obliterante/inmunología , Proteína ADAM10/inmunología , Adulto , Secretasas de la Proteína Precursora del Amiloide/inmunología , Células Cultivadas , Citocinas/sangre , Citocinas/genética , Femenino , Proteína HMGB1/sangre , Proteína HMGB1/genética , Humanos , Leucocitos Mononucleares/inmunología , Masculino , Proteínas de la Membrana/inmunología , Persona de Mediana Edad , FN-kappa B/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Receptor para Productos Finales de Glicación Avanzada/genética , Tromboangitis Obliterante/sangre , Tromboangitis Obliterante/genéticaRESUMEN
The imbalance between production and clearance of amyloid ß (Aß) peptides and their resulting accumulation in the brain is an early and crucial step in the pathogenesis of Alzheimer's disease (AD). Therefore, Aß is strongly positioned as a promising and extensively validated therapeutic target for AD. Investigational disease-modifying approaches aiming at reducing cerebral Aß concentrations include prevention of de novo production of Aß through inhibition of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), and clearance of Aß deposits via passive Aß immunotherapy. We have developed a novel, high affinity antibody against Aß peptides bearing a pyroglutamate residue at amino acid position 3 (3pE), an Aß species abundantly present in plaque deposits in AD brains. Here, we describe the preclinical characterization of this antibody, and demonstrate a significant reduction in amyloid burden in the absence of microhemorrhages in different mouse models with established plaque deposition. Moreover, we combined antibody treatment with chronic BACE1 inhibitor treatment and demonstrate significant clearance of pre-existing amyloid deposits in transgenic mouse brain, without induction of microhemorrhages and other histopathological findings. Together, these data confirm significant potential for the 3pE-specific antibody to be developed as a passive immunotherapy approach that balances efficacy and safety. Moreover, our studies suggest further enhanced treatment efficacy and favorable safety after combination of the 3pE-specific antibody with BACE1 inhibitor treatment.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/antagonistas & inhibidores , Anticuerpos Monoclonales/administración & dosificación , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inmunización Pasiva/métodos , Fragmentos de Péptidos/antagonistas & inhibidores , Placa Amiloide/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/inmunología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Ácido Aspártico Endopeptidasas/inmunología , Ácido Aspártico Endopeptidasas/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Placa Amiloide/inmunología , Placa Amiloide/metabolismo , Resultado del TratamientoRESUMEN
The transmembrane chemokine pathways CXCL16/CXCR6 and CX3CL1/CX3CR1 are strongly implicated in inflammation and angiogenesis. We investigated the involvement of these chemokine pathways and their processing metalloproteinases ADAM10 and ADAM17 in the pathophysiology of proliferative diabetic retinopathy (PDR). Vitreous samples from 32 PDR and 24 non-diabetic patients, epiretinal membranes from 18 patients with PDR, rat retinas, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. In vitro angiogenesis assays were performed and the adherence of leukocytes to CXCL16-stimulated HRMECs was assessed. CXCL16, CX3CL1, ADAM10, ADAM17 and vascular endothelial growth factor (VEGF) levels were significantly increased in vitreous samples from PDR patients. The levels of CXCL16 were 417-fold higher than those of CX3CL1 in PDR vitreous samples. Significant positive correlations were found between the levels of VEGF and the levels of CXCL16, CX3CL1, ADAM10 and ADAM17. Significant positive correlations were detected between the numbers of blood vessels expressing CD31, reflecting the angiogenic activity of PDR epiretinal membranes, and the numbers of blood vessels and stromal cells expressing CXCL16, CXCR6, ADAM10 and ADAM17. CXCL16 induced upregulation of phospho-ERK1/2, p65 subunit of NF-κB and VEGF in cultured Müller cells and tumor necrosis factor-α induced upregulation of soluble CXCL16 and ADAM17 in Müller cells. Treatment of HRMECs with CXCL16 resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and increased leukocyte adhesion to HRMECs. CXCL16 induced HRMEC proliferation, formation of sprouts from HRMEC spheroids and phosphorylation of ERK1/2. Intravitreal administration of CXCL16 in normal rats induced significant upregulation of the p65 subunit of NF-κB, VEGF and ICAM-1 in the retina. Our findings suggest that the chemokine axis CXCL16/CXCR6 and the processing metalloproteinases ADAM10 and ADAM17 might serve a role in the initiation and progression of PDR.
Asunto(s)
Proteína ADAM10/inmunología , Proteína ADAM17/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Receptor 1 de Quimiocinas CX3C/inmunología , Quimiocina CX3CL1/inmunología , Quimiocina CXCL16/inmunología , Retinopatía Diabética/inmunología , Proteínas de la Membrana/inmunología , Animales , Retinopatía Diabética/patología , Humanos , Masculino , RatasRESUMEN
Introduction: Coronary artery disease originates from the blockage of the inner walls of the coronary arteries due to a plaque buildup. Accumulating studies have highlighted the role of microRNAs (miRs) delivered by exosomes in the progression of coronary artery disease. Thus, the current study was to elucidate the role and mechanism by which miR-25-3p influences oxidized low density lipoprotein (ox-LDL)-induced coronary vascular endothelial cell (CVEC) inflammation. Methods: Primarily isolated CVECs were treated with ox-LDL to induce inflammation. Atherosclerosis models were induced in ApoE-/- mice and the peripheral blood platelet exosomes (PLT-Exo) were extracted and induced by thrombin, followed by co-culture with CVECs. The relationship between miR-25-3p and A disintegrin and metalloprotease 10 (Adam10) as well as the involvement of the NF-κB signaling pathway was evaluated. In order to evaluate the effect of PLT-Exo containing miR-25-3p on ox-LDL-induced CVEC inflammation, lipid accumulation and fibrosis, miR-25-3p mimic/inhibitor (in vitro), miR-25-3p agomir (in vivo), and si-Adam10 were delivered. Results: MiR-25-3p was expressed poorly in ox-LDL-induced CVECs and vascular tissues but exhibited high levels of expression in thrombin-induced PLT-Exo of atherosclerosis models of ApoE-/- mice. CVECs endocytosed PLT-Exo upregulated the miR-25-3p expression. Adam10 was identified as a target gene of miR-25-3p. The thrombin-induced activated PLT-Exo carrying miR-25-3p reduced Adam10 expression to inhibit ox-LDL-induced CVEC inflammation and lipid deposition through downregulating levels of α-smooth muscle actin, Collagen I a1, Collagen III a1, triglycerides, total cholesterol, interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α. Furthermore, the NF-κB signaling pathway participated in the inhibitory effect of PLT-Exo carrying miR-25-3p. Conclusion: Collectively, PLT-Exo overexpressing miR-25-3p attenuates ox-LDL-induced CVEC inflammation in ApoE-/- mouse models of atherosclerosis.
Asunto(s)
Proteína ADAM10/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Enfermedad de la Arteria Coronaria/inmunología , Vasos Coronarios , Células Endoteliales/inmunología , Proteínas de la Membrana/inmunología , MicroARNs/inmunología , FN-kappa B/inmunología , Transducción de Señal/inmunología , Proteína ADAM10/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Enfermedad de la Arteria Coronaria/genética , Vasos Coronarios/inmunología , Vasos Coronarios/patología , Citocinas/genética , Citocinas/inmunología , Células Endoteliales/patología , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados para ApoE , MicroARNs/genética , FN-kappa B/genética , Transducción de Señal/genéticaRESUMEN
Otitis media with effusion (OME) often occurs in infants and young children, which is related to allergic reactions. Notch signaling pathway plays an important role in allergic responses. In this study, we aimed to investigate the role of Notch signaling pathway in the ovalbumin (OVA)-mediated allergic OME in vivo. OVA-induced OME rats were treated with a control vehicle or a γ-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) suppressing the Notch signaling. We studied the effect of Notch signaling pathway in OME model, including histopathological assessment, the expression of Th1 cytokines (IFN-γ), Th2 cytokines (IL-4, IL-5), key transcription factors (T-bet, GATA-3) by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the level of Notch ligand (Jagged1) and the downstream target gene Hes1 were also evaluated by qRT-PCR and immunofluorescent staining. We observed that the production of Th2 cytokines was increased, the level of Th1 cytokines was decreased in OME experimental model. Likewise, Th2-cytokine(IL-4)level was reduced, but the level of Th1 cytokines(IFN-γ) was no changes. Additionally, administration of DAPT induced a decrease in the expression of GATA-3 mRNA, however, no influence on T-bet mRNA production. These results suggest that there is an imbalance with Th1/Th2 in OVA-mediated allergic OME. DAPT treatment can block the Notch signaling pathway and relieve the middle ear inflammation through modulating the level of Th2 responses in OVA-induced allergic OME.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Antiinflamatorios/uso terapéutico , Diaminas/uso terapéutico , Otitis Media/tratamiento farmacológico , Tiazoles/uso terapéutico , Alérgenos/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Animales , Antiinflamatorios/farmacología , Citocinas/inmunología , Diaminas/farmacología , Factor de Transcripción GATA3/genética , Proteína Jagged-1/genética , Masculino , Otitis Media/genética , Otitis Media/inmunología , Ovalbúmina/inmunología , Ratas Sprague-Dawley , Células Th2/efectos de los fármacos , Células Th2/inmunología , Tiazoles/farmacología , Factor de Transcripción HES-1/genéticaRESUMEN
Critical for diverse biological processes, proteases represent one of the largest families of pharmaceutical targets. To inhibit pathogenic proteases with desired selectivity, monoclonal antibodies (mAbs) hold great promise as research tools and therapeutic agents. However, identification of mAbs with inhibitory functions is challenging because current antibody discovery methods rely on binding rather than inhibition. This study developed a highly efficient selection method for protease inhibitory mAbs by coexpressing 3 recombinant proteins in the periplasmic space of Escherichia coli-an antibody clone, a protease of interest, and a ß-lactamase modified by insertion of a protease cleavable peptide sequence. During functional selection, inhibitory antibodies prevent the protease from cleaving the modified ß-lactamase, thereby allowing the cell to survive in the presence of ampicillin. Using this method to select from synthetic human antibody libraries, we isolated panels of mAbs inhibiting 5 targets of 4 main protease classes: matrix metalloproteinases (MMP-14, a predominant target in metastasis; MMP-9, in neuropathic pain), ß-secretase 1 (BACE-1, an aspartic protease in Alzheimer's disease), cathepsin B (a cysteine protease in cancer), and Alp2 (a serine protease in aspergillosis). Notably, 37 of 41 identified binders were inhibitory. Isolated mAb inhibitors exhibited nanomolar potency, exclusive selectivity, excellent proteolytic stability, and desired biological functions. Particularly, anti-Alp2 Fab A4A1 had a binding affinity of 11 nM and inhibition potency of 14 nM, anti-BACE1 IgG B2B2 reduced amyloid beta (Aß40) production by 80% in cellular assays, and IgG L13 inhibited MMP-9 but not MMP-2/-12/-14 and significantly relieved neuropathic pain development in mice.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Péptido Hidrolasas/genética , Inhibidores de Proteasas/inmunología , Proteínas Recombinantes/inmunología , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/terapia , Secuencia de Aminoácidos/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/inmunología , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/farmacología , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/inmunología , Aspergilosis/inmunología , Aspergilosis/terapia , Catepsina B/genética , Catepsina B/inmunología , Escherichia coli/genética , Humanos , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/inmunología , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/inmunología , Inhibidores de la Metaloproteinasa de la Matriz/inmunología , Inhibidores de la Metaloproteinasa de la Matriz/metabolismo , Ratones , Neoplasias/inmunología , Neoplasias/terapia , Péptido Hidrolasas/química , Péptido Hidrolasas/inmunología , Periplasma/genética , Inhibidores de Proteasas/farmacología , Proteolisis/efectos de los fármacos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Serina Proteasas/genética , Serina Proteasas/inmunologíaRESUMEN
Conventional dendritic cells (cDCs) derive from bone marrow (BM) precursors that undergo cascades of developmental programs to terminally differentiate in peripheral tissues. Pre-cDC1s and pre-cDC2s commit in the BM to each differentiate into CD8α+/CD103+ cDC1s and CD11b+ cDC2s, respectively. Although both cDCs rely on the cytokine FLT3L during development, mechanisms that ensure cDC accessibility to FLT3L have yet to be elucidated. Here, we generated mice that lacked a disintegrin and metalloproteinase (ADAM) 10 in DCs (Itgax-cre × Adam10-fl/fl; ADAM10∆DC) and found that ADAM10 deletion markedly impacted splenic cDC2 development. Pre-cDC2s accumulated in the spleen with transcriptomic alterations that reflected their inability to differentiate and exhibited abrupt failure to survive as terminally differentiated cDC2s. Induced ADAM10 ablation also led to the reduction of terminally differentiated cDC2s, and restoration of Notch signaling, a major pathway downstream of ADAM10, only modestly rescued them. ADAM10∆DC BM failed to generate cDC2s in BM chimeric mice with or without cotransferred ADAM10-sufficient BM, indicating that cDC2 development required cell-autonomous ADAM10. We determined cDC2s to be sources of soluble FLT3L, as supported by decreased serum FLT3L concentration and the retention of membrane-bound FLT3L on cDC2 surfaces in ADAM10∆DC mice, and by demonstrating the release of soluble FLT3L by cDC2 in ex vivo culture supernatants. Through in vitro studies utilizing murine embryonic fibroblasts, we determined FLT3L to be a substrate for ADAM10. These data collectively reveal cDC2s as FLT3L sources and highlight a cell-autonomous mechanism that may enhance FLT3L accessibility for cDC2 development and survival.
Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Diferenciación Celular/inmunología , Células Dendríticas/fisiología , Proteínas de la Membrana/metabolismo , Bazo/citología , Proteína ADAM10/genética , Proteína ADAM10/inmunología , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/inmunología , Animales , Trasplante de Médula Ósea , Membrana Celular/inmunología , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/inmunología , Micropartículas Derivadas de Células/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Fibroblastos , Células Madre Hematopoyéticas/fisiología , Inmunidad Celular , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Bazo/inmunología , Quimera por TrasplanteRESUMEN
The role of ICOS and its ligand (ICOSL) have both been shown to be essential for proper humoral responses as well as autoimmune Ab development in mouse models of lupus. In this paper, we report a specific role for the metalloprotease ADAM10 on B cells in regulating both ICOSL and ICOS in a mouse model of increased humoral immunity using B6mir146a-/- mice and a model of lymphoproliferative disease using the well-characterized lpr model. B6lpr mice lacking ADAM10 on B cells (A10Blpr) have decreased nodal proliferation and T cell accumulation compared with control B6lpr mice. Additionally, A10Blpr mice have a drastic reduction in autoimmune anti-dsDNA Ab production. In line with this, we found a significant reduction in follicular helper T cells and germinal center B cells in these mice. We also show that lymphoproliferation in this model is closely tied to elevated ICOS levels and decreased ICOSL levels. Overall, our data not only show a role of B cell ADAM10 in control autoimmunity but also increase our understanding of the regulation of ICOS and ICOSL in the context of autoimmunity.
Asunto(s)
Proteína ADAM10/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Linfocitos B/inmunología , Inmunidad Humoral , Ligando Coestimulador de Linfocitos T Inducibles/genética , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Lupus Eritematoso Sistémico/inmunología , Proteínas de la Membrana/genética , Proteína ADAM10/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Animales , Autoanticuerpos/sangre , Autoinmunidad , Proliferación Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación de la Expresión Génica , Proteínas de la Membrana/inmunología , Ratones , Ratones Noqueados , MicroARNs/genéticaRESUMEN
Proteolysis is an irreversible physiological process that can result in the termination or activation of protein function. Many transmembrane proteins that are involved in the cellular communication between immune cells and structural cells - for example, Notch, CD23, CD44, and membrane-anchored cytokines and their receptors - are cleaved by the ADAM (a disintegrin and metalloproteinase) family of enzymes. Here, we review recent insights into the molecular activation, substrate specificity and function of ADAM proteins in the development and regulation of the immune system, with a particular focus on the roles of ADAM10 and ADAM17.
Asunto(s)
Proteína ADAM10/inmunología , Proteína ADAM17/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Sistema Inmunológico/fisiología , Proteínas de la Membrana/inmunología , Proteolisis , Receptores Notch/metabolismo , Linfocitos B/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Células Mieloides/inmunología , Linfocitos T/inmunologíaRESUMEN
Background: The pathogenesis of hidradenitis suppurativa (HS) is not fully understood. This systematic review examined the latest evidence for molecular inflammatory pathways involved in HS as a chronic inflammatory skin disease. Methods: A systematic literature search was performed in PubMed/Medline and EMBASE from January 2013 through September 2017, according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA). Findings on HS pathogenesis were also compared with those of other immune-mediated inflammatory diseases (IMIDs) in a non-systematic review. In addition, current therapeutic options for HS are briefly discussed on the basis of the findings for the inflammatory pathways involved in HS. Results: A total of 32 eligible publications were identified by the systematic search; these were supplemented with three additional publications. The extracted data indicated that four key themes underlie the pathogenesis of HS and related syndromic conditions. First, nicastrin (NCSTN) and PSTPIP1 mutations are directly associated with auto-inflammatory disease. Secondly, the up-regulation of several cytokines including tumor necrosis factor-α and T helper-17/interleukin-23 are connected to auto-inflammatory mechanisms in the pathogenesis of HS. Thirdly, the microbiome of lesional skin differs significantly vs. normal-appearing skin. Fourthly, HS risk is enhanced through physiological and environmental factors such as smoking, obesity, and mechanical friction. There is significant overlap between the pathogenesis of HS, its syndromic forms and other IMIDs, particularly with respect to aberrations in the innate immune response. Conclusions: The evidence presented in this review supports HS as an auto-inflammatory skin disorder associated with alterations in the innate immune system. Based on these most recent data, an integrative viewpoint is presented on the pathogenesis of HS. Current management strategies on HS consist of anti-inflammatory therapies, surgical removal of chronic lesions, and lifestyle changes such as smoking cessation and weight loss. As large gaps remain in the understanding of the pathogenesis of HS, further research is warranted to ultimately improve the management and treatment of patients with HS and related syndromic conditions.
Asunto(s)
Hidradenitis Supurativa/inmunología , Inflamación/inmunología , Modelos Inmunológicos , Transducción de Señal/inmunología , Piel/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/inmunología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/inmunología , Proteínas del Citoesqueleto/metabolismo , Hidradenitis Supurativa/genética , Hidradenitis Supurativa/metabolismo , Humanos , Inflamación/genética , Inflamación/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Mutación , Transducción de Señal/genética , Piel/microbiología , Piel/patologíaRESUMEN
BACKGROUND AND PURPOSE: The potential for therapeutic antibody treatment of neurological diseases is limited by poor penetration across the blood-brain barrier. I.c.v. delivery is a promising route to the brain; however, it is unclear how efficiently antibodies delivered i.c.v. penetrate the cerebrospinal spinal fluid (CSF)-brain barrier and distribute throughout the brain parenchyma. EXPERIMENTAL APPROACH: We evaluated the pharmacokinetics and pharmacodynamics of an inhibitory monoclonal antibody against ß-secretase 1 (anti-BACE1) following continuous infusion into the left lateral ventricle of healthy adult cynomolgus monkeys. KEY RESULTS: Animals infused with anti-BACE1 i.c.v. showed a robust and sustained reduction (~70%) of CSF amyloid-ß (Aß) peptides. Antibody distribution was near uniform across the brain parenchyma, ranging from 20 to 40 nM, resulting in a ~50% reduction of Aß in the cortical parenchyma. In contrast, animals administered anti-BACE1 i.v. showed no significant change in CSF or cortical Aß levels and had a low (~0.6 nM) antibody concentration in the brain. CONCLUSION AND IMPLICATIONS: I.c.v. administration of anti-BACE1 resulted in enhanced BACE1 target engagement and inhibition, with a corresponding dramatic reduction in CNS Aß concentrations, due to enhanced brain exposure to antibody.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/farmacocinética , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/inmunología , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/líquido cefalorraquídeo , Ácido Aspártico Endopeptidasas/inmunología , Encéfalo/metabolismo , Femenino , Infusiones Intraventriculares , Macaca fascicularisRESUMEN
The cytokine Interleukin-11 (IL-11) signals through the membrane-bound IL-11 receptor (IL-11R), which is expressed in a cell-type specific manner. We have recently shown that the metalloprotease ADAM10 can cleave the IL-11R. The liberated soluble IL-11R (sIL-11R) ectodomain can bind its ligand, and the resulting IL-11/sIL-11R complex can activate cells that do not express the IL-11R (trans-signaling). In this study, we show that the remaining C-terminal fragment (CTF1) after ADAM10-mediated cleavage is subsequently cleaved within the membrane by the gamma-secretase complex, and that the resulting shorter CTF2 is further degraded by the proteasome. In contrast to other transmembrane receptors, e.g. Notch, we find no evidence that the IL-11R CTF can translocate into the nucleus to act as a transcription factor, suggesting that regulated intramembrane proteolysis of the IL-11R CTF acts as a mechanism to clear the plasma membrane from remaining protein fragments after cleavage of its ectodomain.
Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Interleucina-11/metabolismo , Proteínas de la Membrana/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores de Interleucina-11/metabolismo , Proteína ADAM10/genética , Proteína ADAM10/inmunología , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/inmunología , Animales , Línea Celular Tumoral , Membrana Celular/inmunología , Membrana Celular/metabolismo , Expresión Génica , Interleucina-11/genética , Interleucina-11/inmunología , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/inmunología , Unión Proteica , Proteolisis , Receptores de Interleucina-11/genética , Receptores de Interleucina-11/inmunología , Transducción de SeñalRESUMEN
The recruitment of blood leukocytes across the endothelium to sites of tissue infection is central to inflammation, but also promotes chronic inflammatory diseases. A disintegrin and metalloproteinase 10 (ADAM10) is a ubiquitous transmembrane molecular scissor that is implicated in leukocyte transmigration by proteolytically cleaving its endothelial substrates. These include VE-cadherin, a homotypic adhesion molecule that regulates endothelial barrier function, and transmembrane chemokines CX3CL1 and CXCL16, which have receptors on leukocytes. However, a definitive role for endothelial ADAM10 in transmigration of freshly isolated primary leukocytes under flow has not been demonstrated, and the relative importance of distinct ADAM10 substrates is unknown. Emerging evidence suggests that ADAM10 can be regarded as six different molecular scissors with different substrate specificities, depending on which of six TspanC8 tetraspanins it is associated with, but TspanC8s remain unstudied in leukocyte transmigration. In the current study, ADAM10 knockdown on primary HUVECs was found to impair transmigration of freshly isolated human peripheral blood T lymphocytes, but not neutrophils or B lymphocytes, in an in vitro flow assay. This impairment was due to delayed transmigration rather than a complete block, and was overcome in the presence of neutrophils. Transmigration of purified lymphocytes was dependent on ADAM10 regulation of VE-cadherin, but not CX3CL1 and CXCL16. Tspan5 and Tspan17, the two most closely related TspanC8s by sequence, were the only TspanC8s that regulated VE-cadherin expression and were required for lymphocyte transmigration. Therefore endothelial Tspan5- and Tspan17-ADAM10 complexes may regulate inflammation by maintaining normal VE-cadherin expression and promoting T lymphocyte transmigration.
Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Antígenos CD/genética , Cadherinas/genética , Proteínas de la Membrana/metabolismo , Linfocitos T/fisiología , Tetraspaninas/metabolismo , Migración Transendotelial y Transepitelial , Proteína ADAM10/deficiencia , Proteína ADAM10/genética , Proteína ADAM10/inmunología , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/inmunología , Antígenos CD/metabolismo , Linfocitos B/inmunología , Linfocitos B/fisiología , Cadherinas/metabolismo , Células Cultivadas , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/inmunología , Quimiocina CXCL16 , Quimiocinas CXC/genética , Quimiocinas CXC/inmunología , Células Endoteliales/inmunología , Células Endoteliales/fisiología , Humanos , Inflamación/inmunología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Neutrófilos/inmunología , Neutrófilos/fisiología , Receptores Depuradores/genética , Receptores Depuradores/inmunología , Linfocitos T/inmunología , Tetraspaninas/genética , Tetraspaninas/inmunologíaRESUMEN
Tspan5 is a member of a subgroup of tetraspanins referred to as TspanC8. These tetraspanins directly interact with the metalloprotease ADAM10, regulate its exit from the endoplasmic reticulum and subsequent trafficking, and differentially regulate its ability to cleave various substrates and activate Notch signaling. The study of Tspan5 has been limited by the lack of good antibodies. This study provides new insights into Tspan5 using new monoclonal antibodies (mAbs), including two mAbs recognizing both Tspan5 and the highly similar tetraspanin Tspan17. Using these mAbs, we show that endogenous Tspan5 associates with ADAM10 in human cell lines and in mouse tissues where it is the most abundant, such as the brain, the lung, the kidney, or the intestine. We also uncover two TspanC8-specific motifs in the large extracellular domain of Tspan5 that are important for ADAM10 interaction and exit from the endoplasmic reticulum. One of the anti-Tspan5 mAbs does not recognize Tspan5 associated with ADAM10, providing a convenient way to measure the fraction of Tspan5 not associated with ADAM10. This fraction is minor in the cell lines tested, and it increases upon transfection of cells with TspanC8 tetraspanins such as Tspan15 or Tspan33 that inhibit Notch signaling. Finally, two antibodies inhibit ligand-induced Notch signaling, and this effect is stronger in cells depleted of the TspanC8 tetraspanin Tspan14, further indicating that Tspan5 and Tspan14 can compensate for each other in Notch signaling.
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Anticuerpos Monoclonales de Origen Murino/química , Retículo Endoplásmico/metabolismo , Transducción de Señal/fisiología , Tetraspaninas/metabolismo , Proteína ADAM10/genética , Proteína ADAM10/inmunología , Proteína ADAM10/metabolismo , Secuencias de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/inmunología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Línea Celular Tumoral , Retículo Endoplásmico/genética , Retículo Endoplásmico/inmunología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Dominios Proteicos , Receptores Notch/genética , Receptores Notch/inmunología , Receptores Notch/metabolismo , Tetraspaninas/genética , Tetraspaninas/inmunologíaRESUMEN
Deregulated gp130-dependent STAT3 signalling by the pleiotropic cytokine interleukin (IL)-11 has been implicated in the pathogenesis of gastric cancer (GC), the third most common cancer worldwide. While the IL-11-gp130-STAT3 signalling axis has traditionally been thought to exclusively use the membrane-bound IL-11 receptor (mIL-11R), recent evidence suggests that mIL-11R can be proteolytically cleaved to generate a soluble form (sIL-11R) which can elicit trans-signalling. Since the role of IL-11 trans-signalling in disease pathogenesis is unknown, here we have employed the IL-11-driven gp130F/F spontaneous model of GC to determine whether IL-11 trans-signalling promotes gastric tumourigenesis. sIL-11R protein was detectable in gastric tissue from GC patients, and sIL-11R levels were elevated in tumours of gp130F/F mice compared to matched non-tumours. Among candidate proteases associated with the generation of sIL-11R, ADAM10 and the related metalloprotease ADAM17 were significantly upregulated in tumours of both gp130F/F mice and GC patients compared to matched non-tumour tissues. The genetic blockade of IL-11 trans-signalling in gp130F/F mice upon the transgenic over-expression of the trans-signalling antagonist, sgp130Fc, failed to suppress gastric inflammation and associated tumour growth, and also had no effect on reducing hyper-activated STAT3 levels. Furthermore, a non-essential role for ADAM17 in IL-11-driven gastric tumourigenesis was supported by the observation that the tumour burden was unaffected in gp130F/F:Adam17ex/ex mice in which ADAM17 expression levels have been substantially reduced. Collectively, these findings suggest that classic signalling rather than trans-signalling is the mode by which IL-11 promotes gastric tumourigenesis.
Asunto(s)
Interleucina-11/inmunología , Proteínas de Neoplasias/inmunología , Transducción de Señal/inmunología , Neoplasias Gástricas/inmunología , Proteína ADAM10/genética , Proteína ADAM10/inmunología , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/inmunología , Animales , Receptor gp130 de Citocinas/genética , Receptor gp130 de Citocinas/inmunología , Interleucina-11/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Transgénicos , Proteínas de Neoplasias/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Transducción de Señal/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologíaRESUMEN
The study of membrane protein function and structure requires their successful detection, expression, solubilization, and/or reconstitution, which poses a challenging task and relies on the availability of suitable tools. Several research groups have successfully applied Nanobodies in the purification, as well as the functional and structural characterization of membrane proteins. Nanobodies are small, single-chain antibody fragments originating from camelids presenting on average a longer CDR3 which enables them to bind in cavities and clefts (such as active and allosteric sites). Notably, Nanobodies generally bind conformational epitopes making them very interesting tools to stabilize, dissect, and characterize specific protein conformations. In the clinic, several Nanobodies are under evaluation either as potential drug candidates or as diagnostic tools. In recent years, we have successfully generated high-affinity, conformation-sensitive anti-γ-secretase Nanobodies. γ-Secretase is a multimeric membrane protease involved in processing of the amyloid precursor protein with high clinical relevance as mutations in its catalytic subunit (Presenilin) cause early-onset Alzheimer's disease. Advancing our knowledge on the mechanisms governing γ-secretase intramembrane proteolysis through various strategies may lead to novel therapeutic avenues for Alzheimer's disease. In this chapter, we present the strategies we have developed and applied for the screening and characterization of anti-γ-secretase Nanobodies. These protocols could be of help in the generation of Nanobodies targeting other membrane proteins.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide/química , Biología Molecular/métodos , Presenilinas/química , Anticuerpos de Dominio Único/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Secretasas de la Proteína Precursora del Amiloide/uso terapéutico , Dominio Catalítico/genética , Epítopos/inmunología , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Mutación , Presenilinas/genética , Presenilinas/inmunología , Presenilinas/uso terapéutico , Conformación Proteica , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/uso terapéutico , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/uso terapéutico , Relación Estructura-ActividadRESUMEN
Macrophages play a key role in the pathogenesis of liver granuloma and fibrosis in schistosomiasis. However, the underlying mechanisms have not been fully characterized. This study revealed that the macrophages infiltrating the liver tissues in a murine model of Schistosoma japonica infection exhibited M2 functional polarization, and Notch1/Jagged1 signaling was significantly upregulated in the M2 polarized macrophages in vivo and in vitro. Furthermore, the blockade of Notch signaling pathway by a γ-secretase inhibitor could reverse macrophage M2 polarization in vitro and alleviate liver granuloma and fibrosis in the murine model of schistosomiasis. These results implied that the Notch1/Jagged1 signaling-dependent M2 polarization of macrophages might play an important role in liver granuloma and fibrosis in schistosomiasis, and the inhibition of Notch1/Jagged1 signaling might provide a novel therapeutic approach to administrate patients with schistosomiasis.