RESUMEN
In cystic fibrosis (CF) lungs, epithelial Na+ channel (ENaC) hyperactivity causes a reduction in airway surface liquid volume, leading to decreased mucocilliary clearance, chronic bacterial infection, and lung damage. Inhibition of ENaC is an attractive therapeutic option. However, ENaC antagonists have failed clinically because of off-target effects in the kidney. The S18 peptide is a naturally occurring short palate lung and nasal epithelial clone 1 (SPLUNC1)-derived ENaC antagonist that restores airway surface liquid height for up to 24 h in CF human bronchial epithelial cultures. However, its efficacy and safety in vivo are unknown. To interrogate the potential clinical efficacy of S18, we assessed its safety and efficacy using human airway cultures and animal models. S18-mucus interactions were tested using superresolution microscopy, quartz crystal microbalance with dissipation, and confocal microscopy. Human and murine airway cultures were used to measure airway surface liquid height. Off-target effects were assessed in conscious mice and anesthetized rats. Morbidity and mortality were assessed in the ß-ENaC-transgenic (Tg) mouse model. Restoration of normal mucus clearance was measured in cystic fibrosis transmembrane conductance regulator inhibitor 172 [CFTR(inh)-172]-challenged sheep. We found that S18 does not interact with mucus and rapidly penetrated dehydrated CF mucus. Compared with amiloride, an early generation ENaC antagonist, S18 displayed a superior ability to slow airway surface liquid absorption, reverse CFTR(inh)-172-induced reduction of mucus transport, and reduce morbidity and mortality in the ß-ENaC-Tg mouse, all without inducing any detectable signs of renal toxicity. These data suggest that S18 is the first naturally occurring ENaC antagonist to show improved preclinical efficacy in animal models of CF with no signs of renal toxicity.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Glicoproteínas/metabolismo , Enfermedades Pulmonares/tratamiento farmacológico , Péptidos/farmacología , Fosfoproteínas/metabolismo , Mucosa Respiratoria/efectos de los fármacos , Animales , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Transporte Iónico , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Masculino , Ratones , Ratones Transgénicos , Ratas , Ratas Sprague-Dawley , Mucosa Respiratoria/metabolismoRESUMEN
Airway dehydration causes mucus stasis and bacterial overgrowth in cystic fibrosis and chronic bronchitis (CB). Rehydration by hypertonic saline is efficacious but suffers from a short duration of action. We tested whether epithelial sodium channel (ENaC) inhibition would rehydrate normal and dehydrated airways to increase mucociliary clearance (MCC) over a significant time frame. For this, we used a tool compound (Compound A), which displays nanomolar ENaC affinity and retention in the airway surface liquid (ASL). Using normal human bronchial epithelial cultures (HBECs) grown at an air-liquid interface, we evaluated in vitro potency and efficacy using short-circuit current (I(sc)) and ASL height measurements where it inhibited I(sc) and increased ASL height by â¼ 50% (0.052 µM at 6 h), respectively. The in vivo efficacy was investigated in a modified guinea pig tracheal potential difference model, where we observed an effective dose (ED50) of 5 µg/kg (i.t.), and by MCC measures in rats and sheep, where we demonstrated max clearance rates at 100 µg/kg (i.t.) and 75 µg/kg (i.t.), respectively. Acute cigarette smoke-induced ASL height depletion in HBECs was used to mimic the situation in patients with CB, and pretreatment prevented both cigarette smoke-induced ASL dehydration and lessened the decrease in ciliary beat frequency. Furthermore, when added after cigarette smoke exposure, Compound A increased the rate of ASL rehydration. In conclusion, Compound A demonstrated significant effects and a link between increased airway hydration, ciliary function, and MCC. These data support the hypothesis that ENaC inhibition may be efficacious in the restoration of mucus hydration and transport in patients with CB.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Moco/metabolismo , Mucosa Respiratoria/metabolismo , Fumar/metabolismo , Animales , Transporte Biológico Activo , Células Cultivadas , Cilios/metabolismo , Cilios/patología , Femenino , Cobayas , Humanos , Ratas , Ratas Wistar , Mucosa Respiratoria/patología , Ovinos , Fumar/efectos adversos , Fumar/patologíaRESUMEN
OBJECTIVES/HYPOTHESIS: Motile cilia of airway epithelial cells help to expel harmful inhaled material. Activation of bitterant-responsive G protein-coupled receptors (GPCRs) is believed to potentiate cilia beat frequency and mucociliary clearance. In this study, we investigated whether regulator of G protein signaling-21 (RGS21) has the potential to modulate signaling pathways connected to airway mucociliary clearance, given that RGS proteins modulate GPCR signaling by acting as GTPase-accelerating proteins (GAPs) for the Gα subunits of heterotrimeric G proteins. STUDY DESIGN: This is a pilot investigation to determine if RGS21, a potential tastant specific RGS gene, is expressed in sinonasal mucosa, and to determine its specific Gα substrate using in vitro biochemical assays with purified proteins. METHODS: Rgs21 expression in sinonasal mucosa was determined using quantitative, real-time PCR and a transgenic mouse expressing RFP from the Rgs21 promoter. Rgs21 was cloned, over-expressed, and purified using multistep protein chromatography. Biochemical and biophysical assays were used to determine if RGS21 could bind and accelerate the hydrolysis of GTP on heterotrimeric Gα subunits. RESULTS: Rgs21 was expressed in sinonasal mucosa and lingual epithelium. Purified recombinant protein directly bound and accelerated GTP hydrolysis on Gα subunits. CONCLUSIONS: Rgs21 is expressed in sinonasal mucosa, is amenable to purification as a recombinant protein, and can bind to Gα(i/o/q) subunits. Furthermore, RGS21 can accelerate the hydrolysis rate of GTP on Gαi subunits. This provides evidence that RGS21 may be a negative regulator of bitterant responses. Future studies will be needed to determine the physiological role of this protein in mucociliary clearance.
Asunto(s)
Regulación de la Expresión Génica , Depuración Mucociliar/genética , Proteínas RGS/genética , Gusto/genética , Animales , Ratones , Ratones Transgénicos , Modelos Animales , Proyectos Piloto , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Transducción de Señal/genéticaRESUMEN
The ability to maintain proper airway surface liquid (ASL) volume homeostasis is vital for mucus hydration and clearance, which are essential aspects of the mammalian lung's innate defense system. In cystic fibrosis (CF), one of the most common life-threatening genetic disorders, ASL dehydration leads to mucus accumulation and chronic infection. In normal airways, the secreted protein short palate lung and nasal epithelial clone 1 (SPLUNC1) effectively inhibits epithelial Na(+) channel (ENaC)-dependent Na(+) absorption and preserves ASL volume. In CF airways, it has been hypothesized that increased ENaC-dependent Na(+) absorption contributes to ASL depletion, and hence increased disease. However, this theory is controversial, and the mechanism for abnormal ENaC regulation in CF airways has remained elusive. Here, we show that SPLUNC1 is a pH-sensitive regulator of ENaC and is unable to inhibit ENaC in the acidic CF airway environment. Alkalinization of CF airway cultures prevented CF ASL hyperabsorption, and this effect was abolished when SPLUNC1 was stably knocked down. Accordingly, we resolved the crystal structure of SPLUNC1 to 2.8 Å. Notably, this structure revealed two pH-sensitive salt bridges that, when removed, rendered SPLUNC1 pH-insensitive and able to regulate ASL volume in acidic ASL. Thus, we conclude that ENaC hyperactivity is secondary to reduced CF ASL pH. Together, these data provide molecular insights into the mucosal dehydration associated with a range of pulmonary diseases, including CF, and suggest that future therapy be directed toward alkalinizing the pH of CF airways.
Asunto(s)
Fibrosis Quística/patología , Deshidratación/metabolismo , Canales Epiteliales de Sodio/metabolismo , Glicoproteínas/química , Modelos Moleculares , Moco/química , Fosfoproteínas/química , Mucosa Respiratoria/química , Adulto , Análisis de Varianza , Células Cultivadas , Cristalización , Fibrosis Quística/complicaciones , Deshidratación/etiología , Deshidratación/patología , Técnicas de Silenciamiento del Gen , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , North Carolina , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patologíaRESUMEN
Interleukin-2 (IL-2) is a critical cytokine for the homeostasis and function of forkhead box p3-expressing regulatory T cells (Foxp3(+)Tregs). Dysregulation of the IL-2-IL-2 receptor axis is associated with aberrant Foxp3(+)Tregs and T cell-mediated autoimmune diseases such as type 1 diabetes. Treatment with recombinant IL-2 has been reported to enhance Foxp3(+)Tregs and suppress different models of autoimmunity. However, efficacy of IL-2 therapy is dependent on achieving sufficient levels of IL-2 to boost tissue-resident Foxp3(+)Tregs while avoiding the potential toxic effects of systemic IL-2. With this in mind, adeno-associated virus (AAV) vector gene delivery was used to localize IL-2 expression to the islets of NOD mice. Injection of a double-stranded AAV vector encoding IL-2 driven by a mouse insulin promoter (dsAAVmIP-IL2) increased Foxp3(+)Tregs in the islets but not the draining pancreatic lymph nodes. Islet Foxp3(+)Tregs in dsAAVmIP-IL2-treated NOD mice exhibited enhanced fitness marked by increased expression of Bcl-2, proliferation, and suppressor function. In contrast, ectopic IL-2 had no significant effect on conventional islet-infiltrating effector T cells. Notably, ß-cell-specific IL-2 expression suppressed late preclinical type 1 diabetes in NOD mice. Collectively, these findings demonstrate that ß-cell-specific IL-2 expands an islet-resident Foxp3(+)Tregs pool that effectively suppresses ongoing type 1 diabetes long term.
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Factores de Transcripción Forkhead/metabolismo , Interleucina-2/uso terapéutico , Islotes Pancreáticos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Dependovirus/inmunología , Diabetes Mellitus Tipo 1/prevención & control , Femenino , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Interleucina-2/biosíntesis , Islotes Pancreáticos/metabolismo , Ratones , Ratones Endogámicos NODRESUMEN
Mucus clearance is an important component of the lung's innate defense system. A failure of this system brought on by mucus dehydration is common to both cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Mucus clearance rates are regulated by the volume of airway surface liquid (ASL) and by ciliary beat frequency (CBF). Chronic treatment with macrolide antibiotics is known to be beneficial to both CF and COPD patients. However, chronic macrolide usage may induce bacterial resistance. We have developed a novel macrolide, 2'-desoxy-9-(S)-erythromycylamine (GS-459755), that has significantly diminished antibiotic activity against Staphylococcus aureus, Streptococcus pneumonia, Moraxella catarrhalis, and Haemophilus influenzae. Since neutrophilia frequently occurs in chronic lung disease and human neutrophil elastase (HNE) induces mucus stasis by activating the epithelial sodium channel (ENaC), we tested the ability of GS-459755 to protect against HNE-induced mucus stasis. GS-459755 had no effect on HNE activity. However, GS-459755 pretreatment protected against HNE-induced ASL volume depletion in human bronchial epithelial cells (HBECs). The effect of GS-459755 on ASL volume was dose dependent (IC50 ~3.9 µM) and comparable to the antibacterial macrolide azithromycin (IC50 ~2.4 µM). Macrolides had no significant effect on CBF or on transepithelial water permeability. However, the amiloride-sensitive transepithelial voltage, a marker of ENaC activity, was diminished by macrolide pretreatment. We conclude that GS-459755 may limit HNE-induced activation of ENaC and may be useful for the treatment of mucus dehydration in CF and COPD without inducing bacterial resistance.
Asunto(s)
Canales Epiteliales de Sodio/efectos de los fármacos , Eritromicina/análogos & derivados , Elastasa de Leucocito/antagonistas & inhibidores , Macrólidos/farmacología , Moco/fisiología , Azitromicina/farmacología , Eritromicina/farmacología , Humanos , Elastasa de Leucocito/metabolismo , Moco/efectos de los fármacos , Mucosa Respiratoria/efectos de los fármacos , Sistema Respiratorio/metabolismoRESUMEN
The gustatory system detects tastants and transmits signals to the brain regarding ingested substances and nutrients. Although tastant receptors and taste signaling pathways have been identified, little is known about their regulation. Because bitter, sweet, and umami taste receptors are G protein-coupled receptors (GPCRs), we hypothesized that regulators of G protein signaling (RGS) proteins may be involved. The recent cloning of RGS21 from taste bud cells has implicated this protein in the regulation of taste signaling; however, the exact role of RGS21 has not been precisely defined. Here, we sought to determine the role of RGS21 in tastant responsiveness. Biochemical analyses confirmed in silico predictions that RGS21 acts as a GTPase-accelerating protein (GAP) for multiple G protein α subunits, including adenylyl cyclase-inhibitory (Gα(i)) subunits and those thought to be involved in tastant signal transduction. Using a combination of in situ hybridization, RT-PCR, immunohistochemistry, and immunofluorescence, we demonstrate that RGS21 is not only endogenously expressed in mouse taste buds but also in lung airway epithelial cells, which have previously been shown to express components of the taste signaling cascade. Furthermore, as shown by reverse transcription-PCR, the immortalized human airway cell line 16HBE was found to express transcripts for tastant receptors, RGS21, and downstream taste signaling components. Over- and underexpression of RGS21 in 16HBE cells confirmed that RGS21 acts to oppose bitter tastant signaling to cAMP and calcium second messenger changes. Our data collectively suggests that RGS21 modulates bitter taste signal transduction.
Asunto(s)
Señalización del Calcio/fisiología , AMP Cíclico/metabolismo , Reguladores de Proteínas de Unión al GTP/biosíntesis , Mucosa Respiratoria/metabolismo , Papilas Gustativas/metabolismo , Gusto/fisiología , Animales , Células COS , Calcio/metabolismo , Chlorocebus aethiops , AMP Cíclico/genética , Reguladores de Proteínas de Unión al GTP/genética , Humanos , Ratones , Ratones Transgénicos , Proteínas RGS , Mucosa Respiratoria/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Papilas Gustativas/citologíaRESUMEN
Adiponectin is a hormone that lowers glucose production by increasing liver insulin sensitivity. Insulin blocks the generation of biochemical intermediates for glucose production by inhibiting autophagy. However, autophagy is stimulated by an essential mediator of adiponectin action, AMPK. This deadlock led to our hypothesis that adiponectin inhibits autophagy through a novel mediator. Mass spectrometry revealed a novel protein that we call suppressor of glucose by autophagy (SOGA) in adiponectin-treated hepatoma cells. Adiponectin increased SOGA in hepatocytes, and siRNA knockdown of SOGA blocked adiponectin inhibition of glucose production. Furthermore, knockdown of SOGA increased late autophagosome and lysosome staining and the secretion of valine, an amino acid that cannot be synthesized or metabolized by liver cells, suggesting that SOGA inhibits autophagy. SOGA decreased in response to AICAR, an activator of AMPK, and LY294002, an inhibitor of the insulin signaling intermediate, PI3K. AICAR reduction of SOGA was blocked by adiponectin; however, adiponectin did not increase SOGA during PI3K inhibition, suggesting that adiponectin increases SOGA through the insulin signaling pathway. SOGA contains an internal signal peptide that enables the secretion of a circulating fragment of SOGA, providing a surrogate marker for intracellular SOGA levels. Circulating SOGA increased in parallel with adiponectin and insulin activity in both humans and mice. These results suggest that adiponectin-mediated increases in SOGA contribute to the inhibition of glucose production.
Asunto(s)
Adiponectina/sangre , Adiponectina/farmacología , Glucemia/metabolismo , Hipoglucemiantes/sangre , Insulina/sangre , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hígado/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Autofagia , Proteínas Relacionadas con la Autofagia , Clonación Molecular , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Hígado/citología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Obesos , Ratones Transgénicos , Persona de Mediana Edad , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Conejos , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto JovenRESUMEN
Activation of the innate immune system promotes polyclonal antibody secretion to eliminate invading pathogens. Inherent in this process is the potential to activate autoreactive B cells and induce autoimmunity. We showed previously that TLR-stimulated dendritic cells and macrophages regulate B cell tolerance to Smith antigen, in part through the secretion of interleukin-6 (IL-6). In this manuscript, we show that neutralization of IL-6 fails to abrogate macrophage-mediated repression and identify soluble CD40 ligand (CD40L) as a second repressive factor secreted by macrophages. CD40L selectively repressed Ig secretion by chronically antigen-experienced (anergic) immunoglobulin transgenic and nontransgenic B cells but not by transiently stimulated B cells. The importance of macrophages in maintaining B cell tolerance was apparent in lupus-prone MRL/lpr mice. Compared with C57BL/6 mice, macrophages from MRL/lpr mice were significantly less efficient at repressing immunoglobulin secretion coincident with diminished IL-6 and CD40 ligand production. These data indicate that macrophages regulate autoreactive B cells by secreting repressive factors that prohibit terminal differentiation of B cells. The regulation of autoreactive B cells by macrophages is diminished in lupus-prone mice suggesting a role in autoimmunity.