RESUMEN
The NLRP3 inflammasome is essential for caspase-1 activation and the release of interleukin (IL)-1ß, IL-18, and gasdermin-D in myeloid cells. However, research on species-specific NLRP3's physiological impact is limited. We engineer mice with the human NLRP3 gene, driven by either the human or mouse promoter, via syntenic replacement at the mouse Nlrp3 locus. Both promoters facilitate hNLRP3 expression in myeloid cells, but the mouse promoter responds more robustly to LPS. Investigating the disease impact of differential NLRP3 regulation, we introduce the D305N gain-of-function mutation into both humanized lines. Chronic inflammation is evident with both promoters; however, CNS outcomes vary significantly. Despite poor response to LPS, the human promoter results in D305N-associated aseptic meningitis, mirroring human pathology. The mouse promoter, although leading to increased CNS expression post-LPS, does not induce meningitis in D305N mutants. Therefore, human-like NLRP3 expression may be crucial for accurate modeling of its role in disease pathogenesis.
Asunto(s)
Enfermedades Autoinflamatorias Hereditarias , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Animales , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Lipopolisacáridos/farmacología , Inflamasomas/metabolismo , Inflamación , Síndrome , Interleucina-1beta/metabolismo , Caspasa 1/metabolismoRESUMEN
The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a central regulator of innate immunity, essential for processing and release of interleukin-1ß and pyroptotic cell death. As endogenous NLRP3 activating triggers are hallmarks of many human chronic inflammatory diseases, inhibition of NLRP3 has emerged as a therapeutic target. Here we identify NDT-19795 as a novel carboxylic acid-containing NLRP3 activation inhibitor in both human and mouse monocytes and macrophages. Remarkably, conversion of the carboxylate to an isopropyl-ester (NT-0796) greatly enhances NLRP3 inhibitory potency in human monocytes. This increase is attributed to the ester-containing pharmacophore being more cell-penetrant than the acid species and, once internalized, the ester being metabolized to NDT-19795 by carboxylesterase-1 (CES-1). Mouse macrophages do not express CES-1, and NT-0796 is ineffective in these cells. Mice also contain plasma esterase (Ces1c) activity which is absent in humans. To create a more human-like model, we generated a mouse line in which the genome was modified, removing Ces1c and replacing this segment of DNA with the human CES-1 gene driven by a mononuclear phagocyte-specific promoter. We show human CES-1 presence in monocytes/macrophages increases the ability of NT-0796 to inhibit NLRP3 activation both in vitro and in vivo. As NLRP3 is widely expressed by monocytes/macrophages, the co-existence of CES-1 in these same cells affords a unique opportunity to direct ester-containing NLRP3 inhibitors precisely to target cells of interest. Profiling NT-0796 in mice humanized with respect to CES-1 biology enables critical modeling of the pharmacokinetics and pharmacodynamics of this novel therapeutic candidate. SIGNIFICANCE STATEMENT: Inhibition of NLRP3 represents a desirable therapeutic strategy for the treatment of multiple human disorders. In this study pharmacological properties of a structurally-novel, ester-containing NLRP3 inhibitor NT-0796 are characterized. To study pharmacodynamics of NT-0796 in vivo, a mouse line was engineered possessing more human-like traits with respect to carboxylesterase biology. In the context of these hCES-1 mice, NT-0796 serves as a more effective inhibitor of NLRP3 activation than the corresponding acid, highlighting the full translational potential of the ester strategy.
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Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas NLR , Humanos , Animales , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Dominio Pirina , Inflamasomas/metabolismo , Caspasa 1/metabolismo , Ésteres , Hidrolasas de Éster Carboxílico/metabolismo , Interleucina-1beta/metabolismoRESUMEN
Angiotensin-converting enzyme 2 (ACE2), part of the renin-angiotensin system (RAS), serves as an entry point for SARS-CoV-2, leading to viral proliferation in permissive cell types. Using mouse lines in which the Ace2 locus has been humanized by syntenic replacement, we show that regulation of basal and interferon induced ACE2 expression, relative expression levels of different ACE2 transcripts, and sexual dimorphism in ACE2 expression are unique to each species, differ between tissues, and are determined by both intragenic and upstream promoter elements. Our results indicate that the higher levels of expression of ACE2 observed in the lungs of mice relative to humans may reflect the fact that the mouse promoter drives expression of ACE2 in populous airway club cells while the human promoter drives expression in alveolar type 2 (AT2) cells. In contrast to transgenic mice in which human ACE2 is expressed in ciliated cells under the control of the human FOXJ1 promoter, mice expressing ACE2 in club cells under the control of the endogenous Ace2 promoter show a robust immune response after infection with SARS-CoV-2, leading to rapid clearance of the virus. This supports a model in which differential expression of ACE2 determines which cell types in the lung are infected, and this in turn modulates the host response and outcome of COVID-19.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Receptores Virales , Animales , Humanos , Ratones , Enzima Convertidora de Angiotensina 2/genética , COVID-19/genética , Ratones Transgénicos , Receptores Virales/genética , SARS-CoV-2 , Tropismo ViralRESUMEN
BACKGROUND: Type 2 endotype asthma is driven by IL-4 and IL-13 signaling via IL-4Ra, which is highly expressed on airway epithelium, airway smooth muscle, and immunocytes in the respiratory mucosa, suggesting potential advantages of an inhalable antagonist. Lipocalin 1 (Lcn1), a 16 kDa protein abundant in human periciliary fluid, has a robust drug-like structure well suited to protein engineering, but it has never been used to make an inhaled Anticalin protein therapeutic. OBJECTIVES: We sought to reengineer Lcn1 into an inhalable IL-4Ra antagonist and assess its pharmacodynamic/kinetic profile. METHODS: Lcn1 was systematically modified by directed protein mutagenesis yielding a high-affinity, slowly dissociating, long-acting full antagonist of IL-4Ra designated PRS-060 with properties analogous to dupilumab, competitively antagonizing IL-4Ra-dependent cell proliferation, mucus induction, and eotaxin expression in vitro. Because PRS-060 displayed exquisite specificity for human IL-4Ra, with no cross-reactivity to rodents or higher primates, we created a new triple-humanized mouse model substituting human IL-4Ra, IL-4, and IL-13 at their correct syntenic murine loci to model clinical dosing. RESULTS: Inhaled PRS-060 strongly suppressed acute allergic inflammation indexes in triple-humanized mice with a duration of action longer than its bulk clearance, suggesting that it may act locally in the lung. CONCLUSION: Lcn1 can be reengineered into the Anticalin antagonist PRS-060 (elarekibep), exemplifying a new class of inhaled topical, long-acting therapeutic drugs with the potential to treat type 2 endotype asthma.
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Asma , Interleucina-13 , Animales , Humanos , Ratones , Asma/tratamiento farmacológico , Modelos Animales de Enfermedad , Interleucina-4/genética , Pulmón , Proteínas , Nebulizadores y Vaporizadores , Receptores de Interleucina-4/inmunologíaRESUMEN
Ozone (O3) is a prevalent air pollutant causing lung inflammation. Previous studies demonstrate that O3 oxidizes lipids, such as cholesterol, in the airway to produce oxysterols, such as secosterol A (SecoA), which are electrophiles that are capable of forming covalent linkages preferentially with lysine residues and that consequently modify protein function. The breadth of proteins modified by this oxysterol as well as the biological consequences in the lung are unknown. By using an alkynyl-tagged form of SecoA and shotgun proteomics, we identified 135 proteins as being modified in bronchial epithelial cells. Among them was NLRP2 (NLR family pyrin domain-containing protein 2), which forms an alkynyl-tagged SecoA-protein adduct at lysine residue 1019 (K1019) in the terminal leucine-rich repeat region, a known regulatory region for NLR proteins. NLRP2 expression in airway epithelial cells was characterized, and CRISPR-Cas9 knockout (KO) and shRNA knockdown of NLRP2 were used to determine its function in O3-induced inflammation. No evidence for NLPR2 inflammasome formation or an NLRP2-dependent increase in caspase-1 activity in response to O3 was observed. O3-induced proinflammatory gene expression for CXCL2 and CXCL8/IL8 was further enhanced in NLRP2-KO cells, suggesting a negative regulatory role. Reconstitution of NLRP2-KO cells with the NLRP2 K1019 mutated to arginine partially blocked SecoA adduction and enhanced O3-induced IL-8 release as compared with wild-type NLRP2. Together, our findings uncover NLRP2 as a highly abundant, key component of proinflammatory signaling pathways in airway epithelial cells and as a novel mediator of O3-induced inflammation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Inflamación/metabolismo , Oxiesteroles/metabolismo , Ozono/efectos adversos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Sustitución de Aminoácidos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Bronquios/citología , Células Epiteliales , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Inflamasomas/metabolismo , Inflamación/inducido químicamente , Inflamación/patología , Interleucina-8/metabolismo , Oxiesteroles/químicaRESUMEN
Allergic asthma is characterized by elevated levels of IgE antibodies, type 2 cytokines such as interleukin-4 (IL-4) and IL-13, airway hyperresponsiveness (AHR), mucus hypersecretion and eosinophilia. Approved therapeutic monoclonal antibodies targeting IgE or IL-4/IL-13 reduce asthma symptoms but require costly lifelong administrations. Here, we develop conjugate vaccines against mouse IL-4 and IL-13, and demonstrate their prophylactic and therapeutic efficacy in reducing IgE levels, AHR, eosinophilia and mucus production in mouse models of asthma analyzed up to 15 weeks after initial vaccination. More importantly, we also test similar vaccines specific for human IL-4/IL-13 in mice expressing human IL-4/IL-13 and the related receptor, IL-4Rα, to find efficient neutralization of both cytokines and reduced IgE levels for at least 11 weeks post-vaccination. Our results imply that dual IL-4/IL-13 vaccination may represent a cost-effective, long-term therapeutic strategy for the treatment of allergic asthma as demonstrated in mouse models, although additional studies are warranted to assess its safety and feasibility.
Asunto(s)
Asma/terapia , Interleucina-13/antagonistas & inhibidores , Interleucina-4/antagonistas & inhibidores , Vacunación/métodos , Animales , Asma/inmunología , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/inmunología , Enfermedad Crónica/terapia , Modelos Animales de Enfermedad , Femenino , Humanos , Inyecciones Intramusculares , Interleucina-13/genética , Interleucina-13/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Ratones , Ratones Transgénicos , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/inmunologíaRESUMEN
BACKGROUND: Chronic exposure to inorganic arsenic (iAs) is a significant public health problem. Methylation of iAs by arsenic methyltransferase (AS3MT) controls iAs detoxification and modifies risks of iAs-induced diseases. Mechanisms underlying these diseases have been extensively studied using animal models. However, substantive differences between humans and laboratory animals in efficiency of iAs methylation have hindered the translational potential of the laboratory studies. OBJECTIVES: The goal of this study was to determine whether humanization of the As3mt gene confers a human-like pattern of iAs metabolism in mice. METHODS: We generated a mouse strain in which the As3mt gene along with the adjacent Borcs7 gene was humanized by syntenic replacement. We compared expression of the mouse As3mt and the human AS3MT and the rate and pattern of iAs metabolism in the wild-type and humanized mice. RESULTS: AS3MT expression in mouse tissues closely modeled that of human and differed substantially from expression of As3mt. Detoxification of iAs was much less efficient in the humanized mice than in wild-type mice. Profiles for iAs and its methylated metabolites in tissues and excreta of the humanized mice were consistent with those reported in humans. Notably, the humanized mice expressed both the full-length AS3MT that catalyzes iAs methylation and the human-specific AS3MTd2d3 splicing variant that has been linked to schizophrenia. CONCLUSIONS: These results suggest that AS3MT is the primary genetic locus responsible for the unique pattern of iAs metabolism in humans. Thus, the humanized mouse strain can be used to study the role of iAs methylation in the pathogenesis of iAs-induced diseases, as well as to evaluate the role of AS3MTd2d3 in schizophrenia. https://doi.org/10.1289/EHP6943.
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Arsénico/metabolismo , Metiltransferasas/metabolismo , Animales , Arsenicales , Humanos , Metiltransferasas/genética , RatonesRESUMEN
Environmental and endogenous electrophiles cause tissue damage through their high reactivity with endogenous nucleophiles such as DNA, proteins, and lipids. Protection against damage is mediated by glutathione (GSH) conjugation, which can occur spontaneously or be facilitated by the glutathione S-transferase (GST) enzymes. To determine the role of GST enzymes in protection against electrophiles as well as the role of specific GST families in mediating this protection, we exposed mutant mouse lines lacking the GSTP, GSTM, and/or GSTT enzyme families to the model electrophile acrylamide, a ubiquitous dietary contaminant known to cause adverse effects in humans. An analysis of urinary metabolites after acute acrylamide exposure identified the GSTM family as the primary mediator of GSH conjugation to acrylamide. However, surprisingly, mice lacking only this enzyme family did not show increased toxicity after an acute acrylamide exposure. Therefore, GSH conjugation is not the sole mechanism by which GSTs protect against the toxicity of this substrate. Given the prevalence of null GST polymorphisms in the human population (approximately 50% for GSTM1 and 20-50% for GSTT1), a substantial portion of the population may also have impaired acrylamide metabolism. However, our study also defines a role for GSTP and/or GSTT in protection against acrylamide mediated toxicity. Thus, while the canonical detoxification function of GSTs may be impaired in GSTM null individuals, disease risk secondary to acrylamide exposure may be mitigated through non-canonical pathways involving members of the GSTP and/or GSTT families.
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Acrilamida/toxicidad , Compuestos Epoxi/toxicidad , Eliminación de Gen , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Hígado/patología , Animales , Modelos Animales de Enfermedad , Femenino , Glutatión/orina , Humanos , Inactivación Metabólica , Isoenzimas/genética , Isoenzimas/metabolismo , Hígado/enzimología , Masculino , Ratones , Pruebas de MutagenicidadRESUMEN
Lysosomes play a critical role in maintenance of the integrity of neuronal function, and mutations in genes that contribute to lysosome formation, transport, and activity are associated with neurodegenerative disorders. Recently, the multisubunit complex, BLOC-one-related complex (BORC), has been shown to be involved in positioning lysosomes within the cytoplasm, although the consequences of altered BORC function in adult animals have not been established. We show that a spontaneous truncation mutation in the mouse Borcs7 gene, identified through whole-genome sequencing followed by genetic complementation, results in progressive axonal dystrophy with dramatic impairment of motor function. Furthermore, mice homozygous for deletion of the entire Borcs7 coding sequence die shortly after birth, and neurons cultured from these animals show impaired centrifugal transport of lysosomes. This identifies BORCS7 as a central factor in axonal transport of lysosomes and a possible target for improving disease-related disturbances in this important function.
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Transporte Axonal , Proteínas de Membrana de los Lisosomas/genética , Distrofias Neuroaxonales/genética , Animales , Femenino , Locomoción , Mutación con Pérdida de Función , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neuronas/metabolismoRESUMEN
The NLRP3 inflammasome plays a critical role in host defense by facilitating caspase I activation and maturation of IL-1ß and IL-18, whereas dysregulation of inflammasome activity results in autoinflammatory disease. Factors regulating human NLRP3 activity that contribute to the phenotypic heterogeneity of NLRP3-related diseases have largely been inferred from the study of Nlrp3 mutant mice. By generating a mouse line in which the NLRP3 locus is humanized by syntenic replacement, we show the functioning of the human NLRP3 proteins in vivo, demonstrating the ability of the human inflammasome to orchestrate immune reactions in response to innate stimuli. Humanized mice expressing disease-associated mutations develop normally but display acute sensitivity to endotoxin and develop progressive and debilitating arthritis characterized by granulocytic infiltrates, elevated cytokines, erosion of bones, and osteoporosis. This NLRP3-dependent arthritis model provides a platform for testing therapeutic reagents targeting the human inflammasome.
Asunto(s)
Artropatías/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Osteoporosis/genética , Animales , Modelos Animales de Enfermedad , Humanos , Inflamasomas/genética , Artropatías/patología , Ratones , Mutación , Osteoporosis/patologíaRESUMEN
UDP-Glucuronosyltransferases (UGTs) conjugate a glucuronyl group from glucuronic acid to a wide range of lipophilic substrates to form a hydrophilic glucuronide conjugate. The glucuronide generally has decreased bioactivity and increased water solubility to facilitate excretion. Glucuronidation represents an important detoxification pathway for both endogenous waste products and xenobiotics, including drugs and harmful industrial chemicals. Two clinically significant families of UGT enzymes are present in mammals: UGT1s and UGT2s. Although the two families are distinct in gene structure, studies using recombinant enzymes have shown considerable overlap in their ability to glucuronidate many substrates, often obscuring the relative importance of the two families in the clearance of particular substrates in vivo. To address this limitation, we have generated a mouse line, termed ΔUgt2, in which the entire Ugt2 gene family, extending over 609 kilobase pairs, is excised. This mouse line provides a means to determine the contributions of the two UGT families in vivo. We demonstrate the utility of these animals by defining for the first time the in vivo contributions of the UGT1 and UGT2 families to glucuronidation of the environmental estrogenic agent bisphenol A (BPA). The highest activity toward this chemical is reported for human and rodent UGT2 enzymes. Surprisingly, our studies using the ΔUgt2 mice demonstrate that, while both UGT1 and UGT2 isoforms can conjugate BPA, clearance is largely dependent on UGT1s.
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Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/genética , Microsomas Hepáticos/metabolismo , Xenobióticos/metabolismo , Animales , Compuestos de Bencidrilo/metabolismo , Compuestos de Bencidrilo/farmacología , Inactivación Metabólica/fisiología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microsomas Hepáticos/efectos de los fármacos , Fenoles/metabolismo , Fenoles/farmacología , Xenobióticos/farmacologíaRESUMEN
Glutathione S-transferases (GSTs) form a superfamily defined by their ability to catalyze the conjugation of glutathione with electrophilic substrates. These enzymes are proposed to play a critical role in protection of cellular components from damage mediated by reactive metabolites. Twenty-two cytosolic GSTs, grouped into seven families, are recognized in mice. This complexity hinders the assignment of function to a subset or family of these genes. We report generation of a mouse line in which the locus encoding three GST gene families is deleted. This includes the four Gstt genes spanning 65 kb on chromosome 10 and the seven Gstm genes found on a 150 kb segment of DNA chromosome 3. In addition, we delete two Gstp genes on chromosome 19 as well as a third related gene located 15 kb telomeric to Gstp1 and Gstp2, which we identify as a potential new member of this gene family. We show that, despite the loss of up to 75% of total GST activity in some tissues from these animals, the mice are healthy and fertile, with normal life expectancy. The normal development and health of these animals make them an appropriate model for defining the role of these families in redox homeostasis and metabolism of drugs and environmental pollutants.
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Sitios Genéticos/genética , Gutatión-S-Transferasa pi/genética , Glutatión Transferasa/genética , Secuencia de Aminoácidos , Animales , Femenino , Gutatión-S-Transferasa pi/deficiencia , Glutatión Transferasa/deficiencia , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia MolecularRESUMEN
The prostanoid thromboxane A2 has been implicated to contribute to the pathogenesis of many cardiovascular diseases, including hypertension. To study the role of vascular thromboxane-prostanoid (TP) receptors in blood pressure regulation, we generated mice with cell-specific deletion of TP receptors in smooth muscle using Cre/Loxp technology. We crossed the KISM22α-Cre transgenic mouse line expressing Cre recombinase in smooth muscle cells with a mouse line bearing a conditional allele of the Tbxa2r gene (Tp(flox)). In KISM22α-Cre(+)Tp(flox/flox) (TP-SMKO) mice, TP receptors were efficiently deleted from vascular smooth muscle cells. In TP-SMKOs, acute vasoconstrictor responses to the TP agonist U46619 were attenuated to a similar extent in both the peripheral and renal circulations. Yet, acute vascular responses to angiotensin II were unaffected at baseline and after chronic angiotensin II administration. Infusion of high-dose U46619 caused circulatory collapse and death in a majority of control mice but had negligible hemodynamic effects in TP-SMKOs, which were completely protected from U46619-induced sudden death. Baseline blood pressures were normal in TP-SMKOs. However, the absence of TP receptors in vascular smooth muscle cells was associated with significant attenuation of angiotensin II-induced hypertension and diminished vascular remodeling. This was also associated with reduced urinary thromboxane production after chronic angiotensin II. Thus, TP receptors in vascular smooth muscle cells play a major role in mediating the actions of thromboxane A(2) in TP agonist-induced shock, hypertension, and vascular remodeling of the aorta.
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Aorta/metabolismo , Hipertensión/genética , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptores de Tromboxanos/genética , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Aorta/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Muerte Súbita , Hipertensión/metabolismo , Ratones , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Receptores de Tromboxanos/metabolismo , Tromboxano A2/farmacología , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacologíaRESUMEN
Acute inflammation in response to both exogenous and endogenous danger signals can lead to the assembly of cytoplasmic inflammasomes that stimulate the activation of caspase-1. Subsequently, caspase-1 facilitates the maturation and release of cytokines and also, under some circumstances, the induction of cell death by pyroptosis. Using a mouse line lacking expression of NLRP1, we show that assembly of this inflammasome in cells is triggered by a toxin from anthrax and that it initiates caspase-1 activation and release of IL-1ß. Furthermore, NLRP1 inflammasome activation also leads to cell death, which escalates over 3 d following exposure to the toxin and culminates in acute lung injury and death of the mice. We show that these events are not dependent on production of IL-1ß by the inflammasome but are dependent on caspase-1 expression. In contrast, muramyl dipeptide-mediated inflammasome formation is not dependent on NLRP1 but NLRP3. Taken together, our findings show that assembly of the NLRP1 inflammasome is sufficient to initiate pyroptosis, which subsequently leads to a self-amplifying cascade of cell injury within the lung from which the lung cannot recover, eventually resulting in catastrophic consequences for the organism.
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Lesión Pulmonar Aguda/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Inflamasomas/metabolismo , Lesión Pulmonar Aguda/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/inmunología , Caspasa 1/metabolismo , Citometría de Flujo , Inflamasomas/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Actions of thromboxane (TXA(2)) to alter airway resistance were first identified over 25 years ago. However, the mechanism underlying this physiological response has remained largely undefined. Here we address this question using a novel panel of mice in which expression of the thromboxane receptor (TP) has been genetically manipulated. We show that the response of the airways to TXA(2) is complex: it depends on expression of other G protein-coupled receptors but also on the physiological context of the signal. In the healthy airway, TXA(2)-mediated airway constriction depends on expression of TP receptors by smooth muscle cells. In contrast, in the inflamed lung, the direct actions of TXA(2) on smooth muscle cell TP receptors no longer contribute to bronchoconstriction. Instead, in allergic lung disease, TXA(2)-mediated airway constriction depends on neuronal TP receptors. Furthermore, this mechanistic switch persists long after resolution of pulmonary inflammation. Our findings demonstrate the powerful ability of lung inflammation to modify pathways leading to airway constriction, resulting in persistent changes in mechanisms of airway reactivity to key bronchoconstrictors. Such alterations are likely to shape the pathogenesis of asthmatic lung disease.
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Bronquios/inervación , Broncoconstricción , Miocitos del Músculo Liso , Neuronas Aferentes , Receptores de Tromboxanos/metabolismo , Tromboxano A2/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Resistencia de las Vías Respiratorias/efectos de los fármacos , Animales , Asma/patología , Bronquios/metabolismo , Broncoconstricción/efectos de los fármacos , Broncoconstricción/fisiología , Células Cultivadas , Hipersensibilidad/patología , Ratones , Ratones Transgénicos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Neumonía/fisiopatología , Receptores de Tromboxanos/genética , Receptores de Tromboxano A2 y Prostaglandina H2/efectos de los fármacos , Sistema Respiratorio/metabolismo , Sistema Respiratorio/patología , Tromboxano A2/análogos & derivados , Vasoconstrictores/farmacologíaRESUMEN
Hypertension affects more than 1.5 billion people worldwide but the precise cause of elevated blood pressure (BP) cannot be determined in most affected individuals. Nonetheless, blockade of the renin-angiotensin system (RAS) lowers BP in the majority of patients with hypertension. Despite its apparent role in hypertension pathogenesis, the key cellular targets of the RAS that control BP have not been clearly identified. Here we demonstrate that RAS actions in the epithelium of the proximal tubule have a critical and nonredundant role in determining the level of BP. Abrogation of AT(1) angiotensin receptor signaling in the proximal tubule alone is sufficient to lower BP, despite intact vascular responses. Elimination of this pathway reduces proximal fluid reabsorption and alters expression of key sodium transporters, modifying pressure-natriuresis and providing substantial protection against hypertension. Thus, effectively targeting epithelial functions of the proximal tubule of the kidney should be a useful therapeutic strategy in hypertension.
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Presión Sanguínea/fisiología , Túbulos Renales Proximales/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Animales , Hipertensión/patología , Ratones , Receptor de Angiotensina Tipo 1/genética , Sistema Renina-Angiotensina/efectos de los fármacos , Transducción de Señal , Sodio/metabolismo , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12RESUMEN
A genetic contribution to asthma susceptibility is well recognized, and linkage studies have identified a large number of genes associated with asthma pathogenesis. Recently, a locus encoding a seven-transmembrane protein was shown to be associated with asthma in founder populations. The expression of the protein GPRA (G protein-coupled receptor for asthma susceptibility) in human airway epithelia and smooth muscle, and its increased expression in a mouse model of asthma, suggested that a gain-of-function mutation in this gene increased the disease risk. However, we report here that the development of allergic lung disease in GPRA-deficient mice is unaltered. A possible explanation for this finding became apparent upon reexamination of the expression of this gene. In contrast to initial studies, our analyses failed to detect expression of GPRA in human lung tissue or in mice with allergic lung disease. We identify a single parameter that distinguishes GPRA-deficient and wild-type mice. Whereas the change in airway resistance in response to methacholine was identical in control and GPRA-deficient mice, the mutant animals showed an attenuated response to thromboxane, a cholinergic receptor-dependent bronchoconstricting agent. Together, our studies fail to support a direct contribution of GPRA to asthma pathogenesis. However, our data suggest that GPRA may contribute to the asthmatic phenotype by altering the activity of other pathways, such as neurally mediated mechanisms, that contribute to disease. This interpretation is supported by high levels of GPRA expression in the brain and its recent identification as the neuropeptide S receptor.
Asunto(s)
Asma/fisiopatología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Enfermedad Aguda , Anafilaxia/inmunología , Anafilaxia/metabolismo , Anafilaxia/fisiopatología , Animales , Asma/inmunología , Asma/metabolismo , Broncoconstrictores/farmacología , Modelos Animales de Enfermedad , Expresión Génica/inmunología , Humanos , Hipotálamo/fisiología , Lipopolisacáridos/farmacología , Pulmón/citología , Pulmón/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Músculo Liso/fisiología , Ovalbúmina/inmunología , Ovalbúmina/farmacología , Fenotipo , Neumonía/inmunología , Neumonía/metabolismo , Neumonía/fisiopatología , Mecánica Respiratoria , Retina/fisiologíaRESUMEN
A mouse strain has been developed that expresses low levels of the NR1 subunit of the NMDA receptor. These mice are a model of chronic developmental NMDA receptor hypofunction and may therefore have relevance to the hypothesized NMDA receptor hypofunction in schizophrenia. Many schizophrenia patients show exaggerated behavioral and neuronal responses to amphetamine compared to healthy subjects. Studies were designed to determine if the NR1-deficient mice would exhibit enhanced sensitivity to amphetamine. Effects of amphetamine on behavioral activation and Fos induction were compared between the NR1-deficient mice and wild-type controls. The NR1 hypomorphic mice and controls exhibited similar locomotor activation after administration of amphetamine at 2 mg/kg. The mutant mice showed slightly reduced peak locomotor activity and slightly increased stereotypy after 4 mg/kg amphetamine. There were no differences in Fos induction in response to amphetamine in the caudate putamen, nucleus accumbens, medial or central amygdala nuclei, or bed nucleus of the stria terminalis. However, amphetamine-induced Fos was substantially attenuated in the medial frontal (infralimbic) and cingulate cortices, basolateral amygdala, and in the lateral septum of the mutant mice. The results suggest a neuroanatomically selective activation deficit to amphetamine challenge in the NR1-deficient mice.
Asunto(s)
Núcleo Caudado/metabolismo , Sistema Límbico/metabolismo , Modelos Genéticos , Núcleo Accumbens/metabolismo , Proteínas Oncogénicas v-fos/metabolismo , Receptores de N-Metil-D-Aspartato/fisiología , Anfetamina/farmacología , Animales , Conducta Animal/efectos de los fármacos , Núcleo Caudado/efectos de los fármacos , Recuento de Células/métodos , Inhibidores de Captación de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Inmunohistoquímica/métodos , Sistema Límbico/anatomía & histología , Sistema Límbico/efectos de los fármacos , Ratones , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/deficiencia , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
Reduced NMDA receptor function is hypothesized to contribute to the pathophysiology of schizophrenia. In order to model chronic and developmental NMDA receptor hypofunction, a mouse line was developed that expresses low levels of the NMDA R1 subunit (NR1) of the NMDA receptor. The present study tested the hypothesis that these NR1 hypomorphic mice would exhibit deficits in sensorimotor and conspecific interactions, analogous to deficits observed in schizophrenic patients. F1 hybrid mice homozygous for the NR1 hypomorphic mutation (NR1 -/-) were generated by crossing heterozygous mice (NR1 +/-) from C57BL/6 and 129 Sv/Ev backgrounds. To assess sensorimotor gating, mice were tested in the paradigm of prepulse inhibition of acoustic startle. The NR1 hypomorphic mice exhibited increased acoustic startle responses and also showed deficits in prepulse inhibition. Startle responses were differentially altered by predator odor exposure in the male NR1 -/- mice, in comparison to control mice. In a test of social affiliation, the wild type mice spent significantly more time investigating a novel mouse in comparison to the NR1 -/- mice. In a resident-intruder test, marked deficits were found in sex-specific aggressive behavior between the wild type and mutant mice. These data support the contention that the NR1 hypomorphic mice exhibit alterations in sensorimotor gating and typical conspecific interactions, reminiscent of behavioral disturbances associated with schizophrenia. The NR1 hypomorphic mice could represent a model system to explore novel treatment and preventative strategies for certain symptoms of schizophrenia.
Asunto(s)
Atención/fisiología , Modelos Animales de Enfermedad , Modelos Genéticos , Corteza Motora/fisiopatología , Receptores de N-Metil-D-Aspartato/genética , Reflejo de Sobresalto/genética , Esquizofrenia/genética , Conducta Social , Corteza Somatosensorial/fisiopatología , Conducta Agonística/fisiología , Animales , Nivel de Alerta/genética , Nivel de Alerta/fisiología , Miedo/fisiología , Femenino , Giro del Cíngulo/fisiopatología , Hipocampo/fisiopatología , Humanos , Masculino , Ratones , Ratones Endogámicos , Ratones Mutantes Neurológicos , Motivación , Inhibición Neural/genética , Inhibición Neural/fisiología , Corteza Prefrontal/fisiopatología , Receptores de N-Metil-D-Aspartato/fisiología , Reflejo de Sobresalto/fisiología , Umbral Sensorial/fisiología , Medio SocialRESUMEN
Mast cell activation induced by aggregation of Fc epsilon RI receptors with immunoglobulin E and antigen is mediated through the activation of multiple protein kinase cascades. Here we report that the regulatory protein RabGEF1 bound to Ras and negatively regulated Ras activation and its 'downstream' effector pathways in Fc epsilon RI-dependent mast cell activation. RabGEF1-deficient mast cells showed enhanced degranulation and release of lipid mediators and cytokines in response to Fc epsilon RI aggregation. RabGEF1-deficient mice developed severe skin inflammation and had increased numbers of mast cells. Thus, RabGEF1 is a negative regulator of Fc epsilon RI-dependent mast cell activation, and a lack of RabGEF1 results in the development of skin inflammation in vivo.