RESUMEN
Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.
Asunto(s)
Fibrosis Quística , Modelos Animales de Enfermedad , Hurones , Pulmón , Transgenes , Animales , Humanos , Animales Modificados Genéticamente , Linaje de la Célula , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Hurones/genética , Hurones/fisiología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Pulmón/citología , Pulmón/metabolismo , Pulmón/patología , Tráquea/citología , Transgenes/genéticaRESUMEN
Rationale: CFTR (cystic fibrosis transmembrane conductance regulator) modulator drugs restore function to mutant channels in patients with cystic fibrosis (CF) and lead to improvements in body mass index and lung function. Although it is anticipated that early childhood treatment with CFTR modulators will significantly delay or even prevent the onset of advanced lung disease, lung neutrophils and inflammatory cytokines remain high in patients with CF with established lung disease despite modulator therapy, underscoring the need to identify and ultimately target the sources of this inflammation in CF lungs. Objectives: To determine whether CF lungs, like chronic obstructive pulmonary disease (COPD) lungs, harbor potentially pathogenic stem cell "variants" distinct from the normal p63/Krt5 lung stem cells devoted to alveolar fates, to identify specific variants that might contribute to the inflammatory state of CF lungs, and to assess the impact of CFTR genetic complementation or CFTR modulators on the inflammatory variants identified herein. Methods: Stem cell cloning technology developed to resolve pathogenic stem cell heterogeneity in COPD and idiopathic pulmonary fibrosis lungs was applied to end-stage lungs of patients with CF (three homozygous CFTR:F508D, one CFTR F508D/L1254X; FEV1, 14-30%) undergoing therapeutic lung transplantation. Single-cell-derived clones corresponding to the six stem cell clusters resolved by single-cell RNA sequencing of these libraries were assessed by RNA sequencing and xenografting to monitor inflammation, fibrosis, and mucin secretion. The impact of CFTR activity on these variants after CFTR gene complementation or exposure to CFTR modulators was assessed by molecular and functional studies. Measurements and Main Results: End-stage CF lungs display a stem cell heterogeneity marked by five predominant variants in addition to the normal lung stem cell, of which three are proinflammatory both at the level of gene expression and their ability to drive neutrophilic inflammation in xenografts in immunodeficient mice. The proinflammatory functions of these three variants were unallayed by genetic or pharmacological restoration of CFTR activity. Conclusions: The emergence of three proinflammatory stem cell variants in CF lungs may contribute to the persistence of lung inflammation in patients with CF with advanced disease undergoing CFTR modulator therapy.
Asunto(s)
Fibrosis Quística , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Preescolar , Animales , Ratones , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Pulmón/patología , Enfermedad Pulmonar Obstructiva Crónica/patología , Inflamación/metabolismoRESUMEN
Pulmonary ionocytes express high levels of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that is critical for hydration of the airways and mucociliary clearance. However, the cellular mechanisms that govern ionocyte specification and function remain unclear. We observed that increased abundance of ionocytes in cystic fibrosis (CF) airway epithelium was associated with enhanced expression of Sonic Hedgehog (SHH) effectors. In this study, we evaluated whether the SHH pathway directly impacts ionocyte differentiation and CFTR function in airway epithelia. Pharmacological HPI1-mediated inhibition of SHH signaling component GLI1 significantly impaired human basal cell specification of ionocytes and ciliated cells but significantly enhanced specification of secretory cells. By contrast, activation of the SHH pathway effector smoothened (SMO) with the chemical agonist SAG significantly enhanced ionocyte specification. The abundance of CFTR+ BSND+ ionocytes under these conditions had a direct relationship with CFTR-mediated currents in differentiated air-liquid interface (ALI) airway cultures. These findings were corroborated in ferret ALI airway cultures generated from basal cells in which the genes encoding the SHH receptor PTCH1 or its intracellular effector SMO were genetically ablated using CRISPR-Cas9, causing aberrant activation or suppression of SHH signaling, respectively. These findings demonstrate that SHH signaling is directly involved in airway basal cell specification of CFTR-expressing pulmonary ionocytes and is likely responsible for enhanced ionocyte abundance in the CF proximal airways. Pharmacologic approaches to enhance ionocyte and reduce secretory cell specification after CFTR gene editing of basal cells may have utility in the treatment of CF.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Proteínas Hedgehog , Animales , Humanos , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Epitelio/metabolismo , Hurones , Proteínas Hedgehog/metabolismoRESUMEN
Chemical nerve agents (CNA) are increasingly becoming a threat to both civilians and military personnel. CNA-induced acute effects on the nervous system have been known for some time and the long-term consequences are beginning to emerge. In this study, we used diisopropylfluorophosphate (DFP), a seizurogenic CNA to investigate the long-term impact of its acute exposure on the brain and its mitigation by an inducible nitric oxide synthase (iNOS) inhibitor, 1400W as a neuroprotectant in the rat model. Several experimental studies have demonstrated that DFP-induced seizures and/or status epilepticus (SE) causes permanent brain injury, even after the countermeasure medication (atropine, oxime, and diazepam). In the present study, DFP-induced SE caused a significant increase in iNOS and 3-nitrotyrosine (3-NT) at 24â¯h, 48â¯h, 7d, and persisted for a long-term (12â¯weeks post-exposure), which led to the hypothesis that iNOS is a potential therapeutic target in DFP-induced brain injury. To test the hypothesis, we administered 1400W (20â¯mg/kg, i.m.) or the vehicle twice daily for the first three days of post-exposure. 1400W significantly reduced DFP-induced iNOS and 3-NT upregulation in the hippocampus and piriform cortex, and the serum nitrite levels at 24â¯h post-exposure. 1400W also prevented DFP-induced mortality in <24â¯h. The brain immunohistochemistry (IHC) at 7d post-exposure revealed a significant reduction in gliosis and neurodegeneration (NeuN+ FJB positive cells) in the 1400W-treated group. 1400W, in contrast to the vehicle, caused a significant reduction in the epileptiform spiking and spontaneous recurrent seizures (SRS) during 12â¯weeks of continuous video-EEG study. IHC of brain sections from the same animals revealed a significant reduction in reactive gliosis (both microgliosis and astrogliosis) and neurodegeneration across various brain regions in the 1400W-treated group when compared to the vehicle-treated group. A multiplex assay from hippocampal lysates at 6â¯weeks post-exposure showed a significant increase in several key pro-inflammatory cytokines/chemokines such as IL-1α, TNFα, IL-1ß, IL-2, IL-6, IL-12, IL-17a, MCP-1, LIX, and Eotaxin, and a growth factor, VEGF in the vehicle-treated animals. 1400W significantly suppressed IL-1α, TNFα, IL-2, IL-12, and MCP-1 levels. It also suppressed DFP-induced serum nitrite levels at 6â¯weeks post-exposure. In the Morris water maze, the vehicle-treated animals spent significantly less time in the target quadrant in a probe trial at 9d post-exposure compared to their time spent in the same quadrant 11â¯days previously (i.e., 2â¯days prior to DFP exposure). Such a difference was not observed in the 1400W and control groups. However, learning and short-term memory were unaffected when tested at 10-16d and 28-34d post-exposure. Accelerated rotarod, horizontal bar test, and the forced swim test revealed no significant changes between groups. Overall, the findings from this study suggest that 1400W may be considered as a potential therapeutic agent as a follow-on therapy for CNA exposure, after controlling the acute symptoms, to prevent mortality and some of the long-term neurotoxicity parameters such as epileptiform spiking, SRS, neurodegeneration, reactive gliosis in some brain regions, and certain key proinflammatory cytokines and chemokine.
Asunto(s)
Amidinas/farmacología , Bencilaminas/farmacología , Encéfalo/efectos de los fármacos , Isoflurofato/toxicidad , Fármacos Neuroprotectores/farmacología , Síndromes de Neurotoxicidad/patología , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Masculino , Agentes Nerviosos/toxicidad , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/patología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Ratas , Ratas Sprague-DawleyRESUMEN
Organophosphate (OP) nerve agents are a threat to both the military and civilians. OP exposure causes cholinergic crisis and status epilepticus (SE) because of irreversible inhibition of acetylcholinesterase that can be life-threatening if left untreated. OP survivors develop long-term morbidity, such as cognitive impairment and motor dysfunction, because of oxidative stress and progressive neuroinflammation and neurodegeneration, which act as disease promoters. Current medical countermeasures (MCMs) do not mitigate these pathologies. Therefore, our goal was to target these disease promoters using diapocynin (DPO), an NADPH oxidase inhibitor, in addition to MCMs, in a rat diisopropylfluorophosphate (DFP) model. The DFP-intoxicated rats were treated with DPO (300 mg/kg, oral, six doses, 12-h intervals) or vehicle 2 h following behavioral SE termination with diazepam. The DPO treatment significantly rescued DFP-induced motor impairment and attenuated epileptiform spiking during the first 72 h after DFP exposure in severely seizing rats despite no difference in epileptiform spike rate between the vehicle and DPO groups in mild SE rats. DPO significantly reduced DFP-induced reactive astrogliosis, neurodegeneration, GP91phox , glutathiolated protein, serum nitrite, and proinflammatory cytokines and chemokines, such as interleukins (ILs) IL-1α, IL-6, IL-2, IL-17A, leptin, and IP-10, in the hippocampus. Collectively, these data support a neuroprotective role of DPO in an OP-induced neurotoxicity model.
Asunto(s)
Acetofenonas/farmacología , Compuestos de Bifenilo/farmacología , Inhibidores Enzimáticos/farmacología , Hipocampo/metabolismo , Isoflurofato/toxicidad , NADPH Oxidasas/antagonistas & inhibidores , Síndromes de Neurotoxicidad , Estado Epiléptico , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Hipocampo/patología , Masculino , Agentes Nerviosos/toxicidad , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/patología , Síndromes de Neurotoxicidad/prevención & control , Ratas , Ratas Sprague-Dawley , Estado Epiléptico/inducido químicamente , Estado Epiléptico/metabolismo , Estado Epiléptico/patología , Estado Epiléptico/prevención & controlRESUMEN
Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air-liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium.