RESUMEN
Triggering receptor expressed on myeloid cells 2 (TREM2) sustains microglia response to brain injury stimuli including apoptotic cells, myelin damage, and amyloid ß (Aß). Alzheimer's disease (AD) risk is associated with the TREM2R47H variant, which impairs ligand binding and consequently microglia responses to Aß pathology. Here, we show that TREM2 engagement by the mAb hT2AB as surrogate ligand activates microglia in 5XFAD transgenic mice that accumulate Aß and express either the common TREM2 variant (TREM2CV) or TREM2R47H scRNA-seq of microglia from TREM2CV-5XFAD mice treated once with control hIgG1 exposed four distinct trajectories of microglia activation leading to disease-associated (DAM), interferon-responsive (IFN-R), cycling (Cyc-M), and MHC-II expressing (MHC-II) microglia types. All of these were underrepresented in TREM2R47H-5XFAD mice, suggesting that TREM2 ligand engagement is required for microglia activation trajectories. Moreover, Cyc-M and IFN-R microglia were more abundant in female than male TREM2CV-5XFAD mice, likely due to greater Aß load in female 5XFAD mice. A single systemic injection of hT2AB replenished Cyc-M, IFN-R, and MHC-II pools in TREM2R47H-5XFAD mice. In TREM2CV-5XFAD mice, however, hT2AB brought the representation of male Cyc-M and IFN-R microglia closer to that of females, in which these trajectories had already reached maximum capacity. Moreover, hT2AB induced shifts in gene expression patterns in all microglial pools without affecting representation. Repeated treatment with a murinized hT2AB version over 10 d increased chemokines brain content in TREM2R47H-5XFAD mice, consistent with microglia expansion. Thus, the impact of hT2AB on microglia is shaped by the extent of TREM2 endogenous ligand engagement and basal microglia activation.
Asunto(s)
Enfermedad de Alzheimer/genética , Encéfalo/metabolismo , Glicoproteínas de Membrana/genética , Microglía/metabolismo , Receptores Inmunológicos/genética , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Proliferación Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Cinética , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Microglía/clasificación , Microglía/efectos de los fármacos , Microglía/patología , Mutación , Unión Proteica , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/metabolismo , Factores SexualesRESUMEN
Thrombin cleavage alters the function of osteopontin (OPN) by exposing an integrin binding site and releasing a chemotactic C-terminal fragment. Here, we examined thrombin cleavage of OPN in the context of dendritic cell (DC) migration to define its functional domains. Full-length OPN (OPN-FL), thrombin-cleaved N-terminal fragment (OPN-R), thrombin- and carboxypeptidase B2-double-cleaved N-terminal fragment (OPN-L), and C-terminal fragment (OPN-CTF) did not have intrinsic chemotactic activity, but all potentiated CCL21-induced DC migration. OPN-FL possessed the highest potency, whereas OPNRAA-FL had substantially less activity, indicating the importance of RGD. We identified a conserved (168)RSKSKKFRR(176) sequence on OPN-FL that spans the thrombin cleavage site, and it demonstrated potent pro-chemotactic effects on CCL21-induced DC migration. OPN-FLR168A had reduced activity, and the double mutant OPNRAA-FLR168A had even lower activity, indicating that these functional domains accounted for most of the pro-chemotactic activity of OPN-FL. OPN-CTF also possessed substantial pro-chemotactic activity, which was fully expressed upon thrombin cleavage and its release from the intact protein, because OPN-CTF was substantially more active than OPNRAA-FLR168A containing the OPN-CTF sequence within the intact protein. OPN-R and OPN-L possessed similar potency, indicating that the newly exposed C-terminal SVVYGLR sequence in OPN-R was not involved in the pro-chemotactic effect. OPN-FL and OPN-CTF did not directly bind to the CD44 standard form or CD44v6. In conclusion, thrombin cleavage of OPN disrupts a pro-chemotactic sequence in intact OPN, and its loss of pro-chemotactic activity is compensated by the release of OPN-CTF, which assumes a new conformation and possesses substantial activity in enhancing chemokine-induced migration of DCs.
Asunto(s)
Movimiento Celular/fisiología , Células Dendríticas/metabolismo , Proteínas Quimioatrayentes de Monocitos/metabolismo , Osteopontina/metabolismo , Proteolisis , Trombina/metabolismo , Secuencias de Aminoácidos , Animales , Células Dendríticas/citología , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Ratones , Ratones Noqueados , Proteínas Quimioatrayentes de Monocitos/genética , Osteopontina/genética , Trombina/genéticaRESUMEN
Osteopontin (OPN), which is highly expressed in malignant glioblastoma (GBM), possesses inflammatory activity modulated by proteolytic cleavage by thrombin and plasma carboxypeptidase B2 (CPB2) at a highly conserved cleavage site. Full-length OPN (OPN-FL) was elevated in cerebrospinal fluid (CSF) samples from all cancer patients compared with noncancer patients. However, thrombin-cleaved OPN (OPN-R) and thrombin/CPB2-double-cleaved OPN (OPN-L) levels were markedly increased in GBM and non-GBM gliomas compared with systemic cancer and noncancer patients. Cleaved OPN constituted â¼23 and â¼31% of the total OPN in the GBM and non-GBM CSF samples, respectively. OPN-R was also elevated in GBM tissues. Thrombin-antithrombin levels were highly correlated with cleaved OPN, but not OPN-FL, suggesting that the cleaved OPN fragments resulted from increased thrombin and CPB2 in this extracellular compartment. Levels of VEGF and CCL4 were increased in CSF of GBM and correlated with the levels of cleaved OPN. GBM cell lines were more adherent to OPN-R and OPN-L than OPN-FL. Adhesion to OPN altered gene expression, in particular genes involved with cellular processes, cell cycle regulation, death, and inflammation. OPN and its cleaved forms promoted motility of U-87 MG cells and conferred resistance to apoptosis. Although functional mutation of the RGD motif in OPN largely abolished these functions, OPN(RAA)-R regained significant cell binding and signaling function, suggesting that the SVVYGLR motif in OPN-R may substitute for the RGD motif if the latter becomes inaccessible. OPN cleavage contributes to GBM development by allowing more cells to bind in niches where they acquire anti-apoptotic properties.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Glioma/metabolismo , Glioma/patología , Osteopontina/metabolismo , Fragmentos de Péptidos/metabolismo , Trombina/metabolismo , Secuencia de Aminoácidos , Antitrombina III/metabolismo , Apoptosis/genética , Biomarcadores de Tumor/líquido cefalorraquídeo , Neoplasias Encefálicas/genética , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular/genética , Supervivencia Celular , Quimiocina CCL3/metabolismo , Quimiocina CCL4/metabolismo , Secuencia Conservada , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Oligopéptidos/metabolismo , Osteopontina/líquido cefalorraquídeo , Osteopontina/química , Péptido Hidrolasas/metabolismo , Proteolisis , Alineación de Secuencia , Estadísticas no Paramétricas , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Kallikrein is generated when the contact system is activated, subsequently cleaving high molecular weight kininogen to bradykinin (BK). BK binds to bradykinin receptor 2, causing vascular leakage. BK is inactivated by proteolysis by the plasma carboxypeptidase B2 and N (CPB2 and CPN). CPN is constitutively active but CPB2 is generated from its zymogen, proCPB2. OBJECTIVES: Determine the role of CPB2 and CPN in the regulation of vascular leakage. METHODS: Mice deficient in CPB2, CPN, or both (Cpb2-/- , Cpn-/- , and Cpb2-/- /Cpn-/- ) were compared with wild-type mice (WT) in a model of vascular leakage caused by skin irritation. In some experiments, mice were pretreated with antibodies that prevent activation of proCPB2. RESULTS: Skin irritation increased vascular leakage most in Cpb2-/- /Cpn-/- , less in Cpb2-/- and Cpn-/- , and least in WT mice. There was no difference in vascular leakage without the challenge. Antibodies inhibiting activation of proCPB2 by plasmin, but not by the thrombin/thrombomodulin complex, increased vascular leakage to the level seen in Cpb2-/- mice. There was no change in levels of markers of coagulation and fibrinolysis. CONCLUSIONS: Bradykinin is inactivated by both CPB2 and CPN independently. Plasmin is the activator of proCPB2 in this model. Mice lacking both plasma carboxypeptidases have more vascular leak than those lacking either alone. Although BK levels were not determined, BK is the likely substrate for CPB2 and CPN in this model.
Asunto(s)
Carboxipeptidasa B2 , Animales , Carboxipeptidasas/genética , Fibrinolisina/metabolismo , Fibrinólisis , Lisina Carboxipeptidasa/genética , RatonesRESUMEN
Migration to draining lymph nodes is a critical requirement for dendritic cells (DCs) to control T-cell-mediated immunity. The calcium-activated potassium channel KCa3.1 has been shown to be involved in regulating cell migration in multiple cell types. In this study, KCa3.1 expression and its functional role in lung DC migration were examined. Fluorescence-labeled antigen was intranasally delivered into mouse lungs to label lung Ag-carrying DCs. Lung CD11c(high)CD11b(low) and CD11c(low)CD11b(high) DCs from PBS-treated and ovalbumin (OVA)-sensitized mice were sorted using MACS and FACS. Indo-1 and DiBAC4(3) were used to measure intracellular Ca(2+) and membrane potential, respectively. The mRNA expression of KCa3.1 was examined using real-time PCR. Expression of KCa3.1 protein and CCR7 was measured using flow cytometry. Migration of two lung DC subsets to lymphatic chemokines was examined using TransWell in the absence or presence of the KCa3.1 blocker TRAM-34. OVA sensitization up-regulated mRNA and protein expression of KCa3.1 in lung DCs, with a greater response by the CD11c(high)CD11b(low) than CD11c(low)CD11b(high) DCs. Although KCa3.1 expression in Ag-carrying DCs was higher than that in non-Ag-carrying DCs in OVA-sensitized mice, the difference was not as prominent. However, Ag-carrying lung DCs expressed significantly higher CCR7 than non-Ag-carrying DCs. CCL19, CCL21, and KCa3.1 activator 1-EBIO induced an increase in intracellular calcium in both DC subsets. In addition, 1-EBIO-induced calcium increase was suppressed by TRAM-34. In vitro blockade of KCa3.1 with TRAM-34 impaired CCL19/CCL21-induced transmigration. In conclusion, KCa3.1 expression in lung DCs is up-regulated by OVA sensitization in both lung DC subsets, and KCa3.1 is involved in lung DC migration to lymphatic chemokines.
Asunto(s)
Movimiento Celular/inmunología , Células Dendríticas/inmunología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/inmunología , Pulmón/inmunología , Animales , Bencimidazoles/farmacología , Antígenos CD11/inmunología , Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Quimiocinas/inmunología , Células Dendríticas/efectos de los fármacos , Femenino , Citometría de Flujo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/agonistas , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/biosíntesis , Pulmón/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Pirazoles/farmacología , Receptores CCR7/inmunologíaRESUMEN
Fms-like tyrosine kinase 3 ligand (Flt3L) reverses the features of allergic airway inflammation and increases a Th2-suppressive regulatory lung CD11c(high)CD11b(low) dendritic cell (DC) subset in a mouse model. We examined the migratory pattern and Ag uptake efficiency of lung DC subsets in the therapeutic effect of Flt3L. Lung CD11c(high)CD11b(low) and CD11c(low)CD11b(high) DCs from PBS-treated, OVA-sensitized, and Flt3L-treated/OVA-sensitized BALB/c mice were sorted using MACS and FACS for phenotype analysis. Lymphatic chemokine expression in thoracic lymph nodes was determined by immunohistochemistry. Migration of two lung DC subsets to lymphatic chemokines was examined in vitro using a Transwell chemotaxis assay. Labeled Ag was intranasally delivered into mouse lung to track the migration and Ag uptake of lung DCs. The in vitro cytokine secretion of mediastinal lymph node cells was determined using ELISA. CD11c(low)CD11b(high) DCs have higher expression of CCR5, CCR6, and CCR7, but lower expression of CCR2 than CD11c(high)CD11b(low) DCs. CD11c(low)CD11b(high) DCs in Flt3L-treated/OVA-sensitized mice demonstrated a less mature phenotype, inefficiency in Ag uptake, and impaired migration in vitro to lymphatic chemokine than those in OVA-sensitized mice. Administration of Flt3L decreased the expression of CCR5 and CCR7 in CD11c(low)CD11b(high) DCs in OVA-sensitized mice. Fewer Ag-carrying cells were detected in the lungs and lymph nodes in Flt3L-treated/OVA-sensitized mice than OVA-sensitized mice with a greater decrease in CD11c(low)CD11b(high) DCs. Mediastinal lymph node cells from Flt3L-treated mice secreted higher levels of Th1 cytokines and IL-10 than OVA-sensitized mice in vitro. In conclusion, Flt3L-generated lung immunogenic CD11c(low)CD11b(high) DCs have a less mature phenotype, impaired Ag uptake, and impaired migration to draining lymph nodes.
Asunto(s)
Presentación de Antígeno/inmunología , Quimiotaxis de Leucocito/inmunología , Células Dendríticas/inmunología , Proteínas de la Membrana/inmunología , Neumonía/inmunología , Animales , Antígenos/inmunología , Antígenos/metabolismo , Citocinas/biosíntesis , Citocinas/inmunología , Células Dendríticas/citología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Hipersensibilidad/inmunología , Hipersensibilidad/metabolismo , Inmunohistoquímica , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Fenotipo , Neumonía/metabolismoRESUMEN
We recently reported that the adoptive transfer of T-regulatory cells (Tregs) isolated from lung and spleen tissue of green fluorescent protein-transgenic mice reversed airway hyperresponsiveness and airway inflammation. Because Programmed Death-1 (PD-1) is a pivotal receptor regulating effector T-cell activation by Tregs, we evaluated whether PD-1 is involved in the therapeutic effect of naturally occurring Tregs (NTregs) and inducible Tregs (iTregs) in cockroach (CRA)-sensitized and challenged mice. The CD4(+)CD25(+) NTregs and CD4(+)CD25(-) iTregs isolated from the lungs and spleens of BALB/c mice were adoptively transferred into CRA-sensitized and CRA-challenged mice with and without anti-PD-1 antibody (100 µg/mice). The CD4(+)CD25(+) T cells in the lung were phenotyped after adoptive transfer. Concentrations of IL-4, IL-5, IL-10, IFN-γ, and IL-13 in bronchoalveolar lavage fluid (BALF) were measured using ELISA. The NTregs and iTregs from either lung or spleen tissue reversed airway hyperresponsiveness for at least 4 wk. However, the therapeutic effect was blocked by administering the anti-PD-1 antibody. The administration of Tregs-recipient mice with anti-PD-1 antibody significantly decreased cytotoxic T-lymphocyte antigen-4 expression, with low concentrations of Forkhead-winged transcriptional factor box 3 (Foxp3) mRNA transcripts in lung CD4(+)CD25(+) T cells. These mice had substantially higher concentrations of BALF IL-4, IL-5, and IL-13, but significantly decreased levels of BALF IL-10. Adoptive therapy recipients without the anti-PD-1 antibody exhibited high levels of CTLA-4 expression and Foxp3 transcripts in lung CD4(+)CD25(+) T cells, with a significant decrease in BALF IL-4, IL-5, and IL-13 concentrations and a substantial increase in BALF IL-10 concentrations. These data suggest that the reversal of airway hyperresponsiveness and airway inflammation by Tregs is mediated in part by PD-1, because other costimulatory molecules (e.g., inducible costimulatory molecule [ICOS] or CTLA-4) have been shown to play a role in Treg-mediated suppression.
Asunto(s)
Alérgenos/inmunología , Anticuerpos/inmunología , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Asma/inmunología , Cucarachas/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Anticuerpos/farmacología , Antígenos CD/metabolismo , Antígenos de Plantas , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Asma/patología , Asma/fisiopatología , Hiperreactividad Bronquial/complicaciones , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/patología , Líquido del Lavado Bronquioalveolar/citología , Antígenos CD4/metabolismo , Antígeno CTLA-4 , Separación Celular , Citocinas/metabolismo , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Receptor de Muerte Celular Programada 1 , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
We previously reported that Fms-like tyrosine kinase 3 ligand (Flt3-L) reversed airway hyperresponsiveness (AHR) and airway inflammation, and increased the number of regulatory CD11c(high)CD8α(high)CD11b(low) dendritic cells and CD4(+)CD25(+)ICOS(+)Foxp3(+)IL-10(+) T-regulatory cells in the lung of allergen-sensitized and -challenged mice. In this study, we evaluated the effect of Flt3-L on Th17 cells and expression of suppressors of cytokine signaling (SOCS) proteins in the lungs of house dust mite (HDM)-sensitized and -challenged mice. BALB/c mice were sensitized and challenged with HDM, and AHR to methacholine was established. Mice were treated with Flt3-L (5 µg, intraperitoneal) daily for 10 days. Levels of IL-4, -5, -6, -8, and -13, and transforming growth factor (TGF)-ß in the bronchoalveolar lavage fluid (BALF) were examined by ELISA. Flt3-L treatment reversed existing AHR to methacholine and substantially decreased eosinophils, neutrophils, IL-5, -6, -8, and IL-13, and TGF-ß levels in the BALF. HDM-sensitized and -challenged mice showed a significant increase in lung CD4(+)IL-17(+)IL-23R(+)CD25â» T cells with high expression of retinoic acid-related orphan receptor (ROR)-γt transcripts. However, administration of Flt3-L substantially decreased the number of lung CD4(+)IL-17(+)IL-23R(+)CD25â» T cells, with significantly decreased expression of ROR-γt mRNA in these cells. HDM sensitization caused a significant increase in the expression of SOCS-1, -3, and -5 in the lung. Flt3-L treatment abolished the increase in SOCS-1 and SOCS-3 proteins, whereas SOCS-5 expression was significantly reduced. These data suggest that the therapeutic effect of Flt3-L in reversing the hallmarks of allergic asthma in a mouse model is mediated by decreasing IL-6 and TGF-ß levels in the BALF, which, in turn, decrease CD4(+)IL-17(+)IL-23R(+)ROR-γt(+)CD25â» T cells and the expression of SOCS-1 and SOCS-3 in the lung of HDM-sensitized and -challenged mice.
Asunto(s)
Inmunización , Pulmón/inmunología , Pulmón/parasitología , Proteínas de la Membrana/farmacología , Pyroglyphidae/inmunología , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/patología , Líquido del Lavado Bronquioalveolar/citología , Antígenos CD4/metabolismo , Recuento de Células , Separación Celular , Citocinas/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Pulmón/efectos de los fármacos , Pulmón/patología , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos BALB C , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Pyroglyphidae/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Células Th2/efectos de los fármacos , Células Th2/inmunologíaRESUMEN
Advances have been made in defining the mechanisms for the control of allergic airway inflammation in response to inhaled antigens. Several genes, including ADAM33, DPP10, PHF11, GPRA, TIM-1, PDE4D, OPN3, and ORMDL3, have been implicated in the pathogenesis and susceptibility to atopy and asthma. Growing evidence associates asthma with a systemic propensity for allergic T-helper type 2 cytokines. Disordered coagulation and fibrinolysis also exacerbate asthma symptoms. Balance among functionally distinct dendritic cell subsets contributes to the outcome of T-cell-mediated immunity. Allergen-specific T-regulatory cells play a pivotal role in the development of tolerance to allergens and immune suppression. The major emphasis on immunotherapy for asthma during the past decade has been to direct the immune response to a type 1 response, or immune tolerance. In this review, we discuss the current information on the pathogenesis of allergic airway inflammation and potential immunotherapy, which could be beneficial in the treatment of airway inflammation, allergy, and asthma.
Asunto(s)
Asma , Predisposición Genética a la Enfermedad , Sistema Respiratorio/inmunología , Alérgenos/inmunología , Animales , Asma/etiología , Asma/genética , Asma/inmunología , Asma/terapia , Células Dendríticas/inmunología , Fibrinólisis/inmunología , Humanos , Tolerancia Inmunológica/inmunología , Factores Inmunológicos/inmunología , Factores Inmunológicos/uso terapéutico , Inmunoterapia/métodos , Inflamación/inmunología , Interleucinas/inmunología , Ratones , Linfocitos T Reguladores/inmunología , Células Th2/inmunologíaRESUMEN
BACKGROUND: Dendritic cell (DC) subsets display different functional roles in regulating immune responses and lead to various outcomes, including T(H)1 versus T(H)2 or regulatory versus immunologic responses. Administration of Fms-like tyrosine kinase 3 (Flt3) ligand prevents and reverses allergic airway inflammation and airway hyperresponsiveness in a mouse model. However, the underlying mechanisms are unclear. OBJECTIVE: We characterized and examined the role of lung DC subsets in the therapeutic effect of Flt3 ligand. METHODS: DCs were isolated from the lungs of ovalbumin (OVA)-sensitized and OVA-challenged mice treated with recombinant human Flt3 ligand. Two populations of CD11c+ cells labeled with fluorochrome-conjugated antibodies were sorted. The ability of the purified cells to stimulate T-cell proliferation and cytokine secretion patterns by different DC subsets was examined. Also, DCs were adoptively transferred in mice to examine their effect on pulmonary function. RESULTS: Two DC populations, CD11c(high)CD11b(low) and CD11c(low)CD11b(high), were identified in the lungs of naive and OVA-sensitized and OVA-challenged mice with and without treatment with Flt3 ligand. The expression levels of CD8alpha, B220, CD19, F4/80, MHC II, CCR7, CD40, programmed death ligand 1, programmed death ligand 2, CD80, and CD86 were distinctly different between the 2 DC populations, which supports the notion that CD11c(high)CD11b(low) and CD11c(low)CD11b(high) DCs potentially have regulatory and immunogenic properties, respectively. Administration of Flt3 ligand increased the DCs with regulatory potential in the lungs of antigen-sensitized mice, and CD11c(high)CD11b(low) DCs acquired a maximum degree of regulatory capacity after Flt3 ligand treatment. CONCLUSION: These data suggest that Flt3 ligand reverses airway hyperresponsiveness by regulating the function of lung DCs in a mouse model of allergic airway inflammation.
Asunto(s)
Asma/inmunología , Células Dendríticas/efectos de los fármacos , Pulmón/inmunología , Proteínas de la Membrana/farmacología , Animales , Asma/tratamiento farmacológico , Hiperreactividad Bronquial/tratamiento farmacológico , Antígeno CD11b/análisis , Antígeno CD11c/análisis , Citocinas/biosíntesis , Células Dendríticas/inmunología , Femenino , Inmunofenotipificación , Activación de Linfocitos/efectos de los fármacos , Proteínas de la Membrana/uso terapéutico , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Linfocitos T/inmunología , Células Th2/inmunologíaRESUMEN
The real-time quantification of target engagement (TE) by small-molecule ligands in living cells remains technically challenging. Systematic quantification of such interactions in a high-throughput setting holds promise for identification of target-specific, potent small molecules within a pathophysiological and biologically relevant cellular context. The salt-inducible kinases (SIKs) belong to a subfamily of the AMP-activated protein kinase (AMPK) family and are composed of three isoforms in humans (SIK1, SIK2, and SIK3). They modulate the production of pro- and anti-inflammatory cytokines in immune cells. Although pan-SIK inhibitors are sufficient to reverse SIK-dependent inflammatory responses, the apparent toxicity associated with SIK3 inhibition suggests that isoform-specific inhibition is required to realize therapeutic benefit with acceptable safety margins. Here, we used the NanoBRET TE intracellular kinase assay, a sensitive energy transfer technique, to directly measure molecular proximity and quantify TE in HEK293T cells overexpressing SIK2 or SIK3. Our 384-well high-throughput screening of 530 compounds demonstrates that the NanoBRET TE intracellular kinase assay was sensitive and robust enough to reveal differential engagement of candidate compounds with the two SIK isoforms and further highlights the feasibility of high-throughput implementation of NanoBRET TE intracellular kinase assays for target-driven small-molecule screening.
Asunto(s)
Fosfotransferasas/aislamiento & purificación , Isoformas de Proteínas/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Fosfotransferasas/genética , Isoformas de Proteínas/antagonistas & inhibidores , Proteínas Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidoresRESUMEN
Chloride channels are involved in many different physiological processes such as cell migration, proliferation and apoptosis. The importance of the CLC family of chloride channels in these cellular functions has been recognized only recently. Infiltration of inflammatory cells, such as eosinophils, T cells, mast cells and neutrophils, is a hallmark of allergy and asthma. Indeed, chronic asthma is associated with widespread damage to the bronchial epithelium, due to excessive apoptosis, and with defective epithelial repair. However, the relationship between the immune cells of allergic airway diseases and chloride channels has not been clearly elucidated. In this review, characteristics of CLC channels are mainly discussed based on their function and presence in different immune cells in airway diseases. Not only are chloride channels involved in the recruitment of immune cells, they also play a role in the activation of these cells. Thus, understanding the role of CLC channels in the immune cells would provide unique insights to the pathophysiologic process of chronic asthma and the means to prevent or reverse the disease.
Asunto(s)
Canales de Cloruro/fisiología , Hipersensibilidad Respiratoria/inmunología , Animales , Apoptosis , Movimiento Celular , Proliferación Celular , Humanos , Macrófagos/fisiología , Mastocitos/fisiología , Neutrófilos/fisiología , Linfocitos T/fisiologíaRESUMEN
It is well known that the architecture of the extreme learning machine (ELM) significantly affects its performance and how to determine a suitable set of hidden neurons is recognized as a key issue to some extent. The leave-one-out cross-validation (LOO-CV) is usually used to select a model with good generalization performance among potential candidates. The primary reason for using the LOO-CV is that it is unbiased and reliable as long as similar distribution exists in the training and testing data. However, the LOO-CV has rarely been implemented in practice because of its notorious slow execution speed. In this paper, an efficient LOO-CV formula and an efficient LOO-CV-based ELM (ELOO-ELM) algorithm are proposed. The proposed ELOO-ELM algorithm can achieve fast learning speed similar to the original ELM without compromising the reliability feature of the LOO-CV. Furthermore, minimal user intervention is required for the ELOO-ELM, thus it can be easily adopted by nonexperts and implemented in automation processes. Experimentation studies on benchmark datasets demonstrate that the proposed ELOO-ELM algorithm can achieve good generalization with limited user intervention while retaining the efficiency feature.
RESUMEN
Atherosclerosis is a chronic inflammatory disease with atherosclerotic plaques containing inflammatory cells, including T-lymphocytes, dendritic cells (DCs) and macrophages that are responsible for progression and destabilization of atherosclerotic plaques. Stressed cells undergoing necrosis release molecules that act as endogenous danger signals to alert and activate innate immune cells. In atherosclerotic tissue the number of DCs increases with the progression of the lesion and produce several inflammatory cytokines and growth factors. Triggering receptor expressed on myeloid cells (TREM)-1 plays a crucial role in inflammation. However, relationship of DCs and the role of TREM-1 with the stability of atherosclerotic plaques have not been examined. In this study, we investigated the heterogeneity of the plaque DCs, myeloid (mDC1 and mDC2) and plasmacytoid (pDCs), and examined the expression of TREM-1 and their co-localization with DCs in the plaques from symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. We found increased expression of HLA-DR, fascin, and TREM-1 and decreased expression of TREM-2 and α-smooth muscle actin in S compared to AS atherosclerotic carotid plaques. Both TREM-1 and fascin were co-localized suggesting increased expression of TREM-1 in plaque DCs of S compared to AS patients. These data were supported by increased mRNA transcripts of TREM-1 and decreased mRNA transcripts of TREM-2 in carotid plaques of S compared to AS patients. There was higher density of both CD1c+ mDC1 and CD141+ mDC2 in the carotid plaques from AS compared to S patients, where as the density of CD303+ pDCs were higher in the carotid plaques of S compared to AS patients. These findings suggest a potential role of pDCs and TREM-1 in atherosclerotic plaque vulnerability. Thus, newer therapies could be developed to selectively block TREM-1 for stabilizing atherosclerotic plaques.
Asunto(s)
Estenosis Carotídea/inmunología , Células Dendríticas/inmunología , Actinas/metabolismo , Estenosis Carotídea/metabolismo , Estenosis Carotídea/cirugía , Estudios de Casos y Controles , Endarterectomía Carotidea , Femenino , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Placa Aterosclerótica/patología , ARN Mensajero/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptor Activador Expresado en Células Mieloides 1RESUMEN
The role of ion channels is largely unknown in chemokine-induced migration in nonexcitable cells such as dendritic cells (DCs). Here, we examined the role of intermediate-conductance calcium-activated potassium channel (KCa3.1) and chloride channel (CLC3) in lymphatic chemokine-induced migration of DCs. The amplitude and kinetics of chemokine ligand (CCL19/CCL21)-induced Ca(2+) influx were associated with chemokine receptor 7 expression levels, extracellular-free Ca(2+) and Cl(-), and independent of extracellular K(+). Chemokines (CCL19 and CCL21) and KCa3.1 activator (1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) induced plasma membrane hyperpolarization and K(+) efflux, which was blocked by 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, suggesting that KCa3.1 carried larger conductance than the inward calcium release-activated calcium channel. Blockade of KCa3.1, low Cl(-) in the medium, and low dose of 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) impaired CCL19/CCL21-induced Ca(2+) influx, cell volume change, and DC migration. High doses of DIDS completely blocked DC migration possibly by significantly disrupting mitochondrial membrane potential. In conclusion, KCa3.1 and CLC3 are critical in human DC migration by synergistically regulating membrane potential, chemokine-induced Ca(2+) influx, and cell volume.
Asunto(s)
Canales de Cloruro/metabolismo , Células Dendríticas/fisiología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Bencimidazoles/farmacología , Señalización del Calcio , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Movimiento Celular/fisiología , Quimiocina CCL19/metabolismo , Quimiocina CCL21/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/agonistas , Potenciales de la Membrana/efectos de los fármacos , Pirazoles/farmacología , Receptores CCR7/metabolismo , Investigación Biomédica TraslacionalRESUMEN
Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin(+) dendritic cells are tolerogenic while thrombomodulin(-) dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin(+) and thrombomodulin(-) dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while expression of cell cycle genes were increased in thrombomodulin-treated and thrombomodulin(+) dendritic cells compared to control dendritic cells and thrombomodulin(-) dendritic cells. Thrombomodulin-treated and thrombomodulin(+) dendritic cells had higher expression of 15-lipoxygenase suggesting increased synthesis of lipoxins. Thrombomodulin(+) dendritic cells produced more lipoxins than thrombomodulin(-) dendritic cells, as measured by ELISA, confirming that this pathway was upregulated. There was more phosphorylation of several cell cycle kinases in thrombomodulin(+) dendritic cells while phosphorylation of kinases involved with pro-inflammatory cytokine signaling was reduced. Cultures of thrombomodulin(+) dendritic cells contained more cells actively dividing than those of thrombomodulin(-) dendritic cells. Production of IL-10 is increased in thrombomodulin(+) dendritic cells. Antagonism of IL-10 with a neutralizing antibody inhibited the effects of thrombomodulin treatment of dendritic cells suggesting a mechanistic role for IL-10. The surface of thrombomodulin(+) dendritic cells supported activation of protein C and procarboxypeptidase B2 in a thrombomodulin-dependent manner. Thus thrombomodulin treatment increases the number of thrombomodulin(+) dendritic cells, which have significantly altered gene expression compared to thrombomodulin(-) dendritic cells in key immune function pathways.
Asunto(s)
Antígenos de Superficie/genética , Células Dendríticas/metabolismo , Regulación de la Expresión Génica , Trombomodulina/genética , Animales , Antígenos de Superficie/metabolismo , Ácido Araquidónico/metabolismo , Biomarcadores/metabolismo , Carboxipeptidasa B2/metabolismo , Ciclo Celular/genética , Diferenciación Celular , Análisis por Conglomerados , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Perfilación de la Expresión Génica , Hemostasis/genética , Humanos , Inmunofenotipificación , Inflamación/genética , Inflamación/metabolismo , Interleucina-10/antagonistas & inhibidores , Redes y Vías Metabólicas , Ratones , MicroARNs/genética , Fosforilación , Unión Proteica , Proteína C/metabolismo , Trombomodulina/metabolismoRESUMEN
Widespread damage of airway epithelium and defective epithelial repair are hallmarks of chronic asthma. Growth factors and cytokines spatially and temporally regulate epithelial shedding and repair. Within this context, a key function is exerted by transforming growth factor (TGF)-beta. Recent growing evidence suggests that chloride (Cl(-)) channels are critical to cell apoptosis. We examined the effects of TGF-beta1 on Cl(-) channel expression and activity and its relationship with apoptosis in human bronchial epithelial cells (HBECs). The small interfering RNA (siRNA) approach was used to investigate the potential role of CLC-3, a member of the volume-regulated Cl(-) channel family, in apoptosis of HBECs. TGF-beta1 significantly induced HBEC apoptosis, which paralleled to a significant decrease in the endogenous expression of CLC-3 protein and mRNA transcripts. Outward rectifying and voltage-dependent CLC-3-like Cl(-) currents in HBECs were diminished by TGF-beta1. siRNA for CLC-3 abolished Cl(-) current and enhanced TGF-beta1-induced cell apoptosis. Overexpression of CLC-3 in HBECs inhibited TGF-beta1-induced cell apoptosis. Bcl-2 was also downregulated after TGF-beta stimulation. TGF-beta1-induced cell apoptosis was suppressed in Bcl-2-transfected HBECs. Our data demonstrate that CLC-3-like voltage-gated chloride channels play a critical role in TGF-beta-induced apoptosis of human airway epithelial cells.