Loss of anion transport without increased sodium absorption characterizes newborn porcine cystic fibrosis airway epithelia.

Defective transepithelial electrolyte transport is thought to initiate cystic fibrosis (CF) lung disease. Yet, how loss of CFTR affects electrolyte transport remains uncertain. CFTR⁻(/)⁻ pigs spontaneously develop lung disease resembling human CF. At birth, their airways exhibit a bacterial host defense defect, but are not inflamed. Therefore, we studied ion transport in newborn nasal and tracheal/bronchial epithelia in tissues, cultures, and in vivo. CFTR⁻(/)⁻ epithelia showed markedly reduced Cl⁻ and HCO3⁻ transport. However, in contrast to a widely held view, lack of CFTR did not increase transepithelial Na(+) or liquid absorption or reduce periciliary liquid depth. Like human CF, CFTR⁻(/)⁻ pigs showed increased amiloride-sensitive voltage and current, but lack of apical Cl⁻ conductance caused the change, not increased Na(+) transport. These results indicate that CFTR provides the predominant transcellular pathway for Cl⁻ and HCO3⁻ in porcine airway epithelia, and reduced anion permeability may initiate CF airway disease.

Identification of cholinergic cells with chemosensory traits in the porcine uterus.

Chemosensory cells are specialized epithelial cells that act as sentinels near body entry sites. The majority of these cells express a cholinergic phenotype and utilize the taste signaling system to monitor the mucosal environment for potentially harmful substances, triggering protective reflexes. We report the identification of cells with a putative chemosensory role in the uterus. Presumptive chemosensory cells were immunoreactive to key components of the taste transduction, including the transient receptor potential channel M5 (TRPM5) and the phospholipase Cß2 (PLCB2). These cells localized to endometrial glandular and luminal epithelia, while absent from myometrium and perimetrium. Double immunofluorescence revealed co-expression of chemosensory cell markers with the acetylcholine (ACh) synthesizing enzyme, choline acetyltransferase (ChAT). Further, we investigated the regional distribution and expression of chemosensory cells at different stages of the estrous cycle. Uteri were collected postmortem from gilts and stages of the ovarian cycle were determined macroscopically. The uteri were classified into three groups: prepubertal (PB), follicular (FOL), or luteal (LUT). The number of ChAT-immunoreactive cells was increased in the luminal epithelium in the caudal compartment compared to the cranial region of the uterine horn, and at the LUT compared to PB and FOL stages. An increase in ChAT protein abundance in LUT uterine homogenates was noted, although not followed by an increase in ACh content. In summary, our study has identified a hitherto unrecognized cholinergic cell in the uterus that has chemosensory traits and may be involved in a multitude of biological processes.

Early Lung Disease Exhibits Bacteria-Dependent and -Independent Abnormalities in Cystic Fibrosis Pigs.

Rationale: Although it is clear that cystic fibrosis (CF) airway disease begins at a very young age, the early and subsequent steps in disease pathogenesis and the relative contribution of infection, mucus, and inflammation are not well understood. Objectives: As one approach to assessing the early contribution of infection, we tested the hypothesis that early and continuous antibiotics would decrease the airway bacterial burden. We believed that, if they do, this might reveal aspects of the disease that are more or less sensitive to decreasing infection. Methods: Three groups of pigs were studied from birth until ∼3 weeks of age: 1) wild-type, 2) CF, and 3) CF pigs treated continuously with broad-spectrum antibiotics from birth until study completion. Disease was assessed with chest computed tomography, histopathology, microbiology, and BAL. Measurements and Main Results: Disease was present by 3 weeks of age in CF pigs. Continuous antibiotics from birth improved chest computed tomography imaging abnormalities and airway mucus accumulation but not airway inflammation in the CF pig model. However, reducing bacterial infection did not improve two disease features already present at birth in CF pigs: air trapping and submucosal gland duct plugging. In the CF sinuses, antibiotics did not prevent the development of infection or disease or the number of bacteria but did alter the bacterial species. Conclusions: These findings suggest that CF airway disease begins immediately after birth and that early and continuous antibiotics impact some, but not all, aspects of CF lung disease development.

Influence of SARS-CoV-2 on airway mucus production: A review and proposed model.

Coronavirus disease 2019 (COVID-19) is a worldwide pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has affected millions of lives. Individuals who survive severe COVID-19 can experience sustained respiratory symptoms that persist for months after initial infection. In other airway diseases, abnormal airway mucus contributes to sustained airway symptoms. However, the impact of SARS-CoV-2 on airway mucus has received limited attention. In the current review, we assess literature describing the impact of SARS-CoV-2 on airway pathophysiology with specific emphasis on mucus production. Accumulating evidence suggests that the 2 major secreted airway mucin glycoproteins, MUC5AC and MUC5B, are abnormal in some patients with COVID-19. Aberrations in MUC5AC or MUC5B in response to SARS-CoV-2 infection are likely due to inflammation, though the responsible mechanisms have yet to be determined. Thus, we also provide a proposed model highlighting mechanisms that can contribute to acute and sustained mucus abnormalities in SARS-CoV-2, with an emphasis on inflammatory cells and mediators, including mast cells and histamine. Last, we bring to light the challenges of studying abnormal mucus production in SARS-CoV-2 infections and discuss the strengths and limitations of model systems commonly used to study COVID-19. The evidence to date suggests that ferrets, nonhuman primates, and cats may have advantages over other models to investigate mucus in COVID-19.

Identification of antiviral antihistamines for COVID-19 repurposing.

There is an urgent need to identify therapies that prevent SARS-CoV-2 infection and improve the outcome of COVID-19 patients. Although repurposed drugs with favorable safety profiles could have significant benefit, widely available prevention or treatment options for COVID-19 have yet to be identified. Efforts to identify approved drugs with in vitro activity against SARS-CoV-2 resulted in identification of antiviral sigma-1 receptor ligands, including antihistamines in the histamine-1 receptor binding class. We identified antihistamine candidates for repurposing by mining electronic health records of usage in population of more than 219,000 subjects tested for SARS-CoV-2. Usage of diphenhydramine, hydroxyzine and azelastine was associated with reduced incidence of SARS-CoV-2 positivity in subjects greater than age 61. We found diphenhydramine, hydroxyzine and azelastine to exhibit direct antiviral activity against SARS-CoV-2 in vitro. Although mechanisms by which specific antihistamines exert antiviral effects is not clear, hydroxyzine, and possibly azelastine, bind Angiotensin Converting Enzyme-2 (ACE2) and the sigma-1 receptor as off-targets. Clinical studies are needed to measure the effectiveness of diphenhydramine, hydroxyzine and azelastine for disease prevention, for early intervention, or as adjuvant therapy for severe COVID-19.

Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling.

Differentiated airway epithelia produce sonic hedgehog (SHH), which is found in the thin layer of liquid covering the airway surface. Although previous studies showed that vertebrate HH signaling requires primary cilia, as airway epithelia mature, the cells lose primary cilia and produce hundreds of motile cilia. Thus, whether airway epithelia have apical receptors for SHH has remained unknown. We discovered that motile cilia on airway epithelial cells have HH signaling proteins, including patched and smoothened. These cilia also have proteins affecting cAMP-dependent signaling, including Gαi and adenylyl cyclase 5/6. Apical SHH decreases intracellular levels of cAMP, which reduces ciliary beat frequency and pH in airway surface liquid. These results suggest that apical SHH may mediate noncanonical HH signaling through motile cilia to dampen respiratory defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses.

Acid exposure disrupts mucus secretion and impairs mucociliary transport in neonatal piglet airways.

Tenacious mucus produced by tracheal and bronchial submucosal glands is a defining feature of several airway diseases, including cystic fibrosis (CF). Airway acidification as a driving force of CF airway pathology has been controversial. Here we tested the hypothesis that transient airway acidification produces pathologic mucus and impairs mucociliary transport. We studied pigs challenged with intra-airway acid. Acid had a minimal effect on mucus properties under basal conditions. However, cholinergic stimulation in acid-challenged pigs revealed retention of mucin 5B (MUC5B) in the submucosal glands, decreased concentrations of MUC5B in the lung lavage fluid, and airway obstruction. To more closely mimic a CF-like environment, we also examined mucus secretion and transport following cholinergic stimulation under diminished bicarbonate and chloride transport conditions ex vivo. Under these conditions, airways from acid-challenged pigs displayed extensive mucus films and decreased mucociliary transport. Pretreatment with diminazene aceturate, a small molecule with ability to inhibit acid detection through blockade of the acid-sensing ion channel (ASIC) at the doses provided, did not prevent acid-induced pathologic mucus or transport defects but did mitigate airway obstruction. These findings suggest that transient airway acidification early in life has significant impacts on mucus secretion and transport properties. Furthermore, they highlight diminazene aceturate as an agent that might be beneficial in alleviating airway obstruction.

Airway cholinergic history modifies mucus secretion properties to subsequent cholinergic challenge in diminished chloride and bicarbonate conditions.

NEW FINDINGS: What is the central question of this study? What is the impact of airway cholinergic history on the properties of airway mucus secretion in a cystic fibrosis-like environment? What is the main finding and its importance? Prior cholinergic challenge slightly modifies the characteristics of mucus secretion in response to a second cholinergic challenge in a diminished bicarbonate and chloride transport environment. Such modifications might lead to retention of mucus on the airway surface, thereby potentiating exacerbations of airway disease. ABSTRACT: Viral infections precipitate exacerbations in many airway diseases, including asthma and cystic fibrosis. Although viral infections increase cholinergic transmission, few studies have examined how cholinergic history modifies subsequent cholinergic responses in the airway. In our previous work, we found that airway resistance in response to a second cholinergic challenge was increased in young pigs with a history of airway cholinergic stimulation. Given that mucus secretion is regulated by the cholinergic nervous system and that abnormal airway mucus contributes to exacerbations of airway disease, we hypothesized that prior cholinergic challenge would also modify subsequent mucus responses to a secondary cholinergic challenge. Using our established cholinergic challenge-rechallenge model in pigs, we atomized the cholinergic agonist bethanechol or saline control to pig airways. Forty-eight hours later, we removed tracheas and measured mucus secretion properties in response to a second cholinergic stimulation. The second cholinergic stimulation was conducted in conditions of diminished chloride and bicarbonate transport to mimic a cystic fibrosis-like environment. In pigs previously challenged with bethanechol, a second cholinergic stimulation produced a mild increase in sheet-like mucus films; these films were scarcely observed in animals originally challenged with saline control. The subtle increase in mucus films was not associated with changes in mucociliary transport. These data suggest that prior cholinergic history might modify mucus secretion characteristics with subsequent stimulation in certain environmental conditions or disease states. Such modifications and/or more repetitive stimulation might lead to retention of mucus on the airway surface, thereby potentiating exacerbations of airway disease.

Sex-specific airway hyperreactivity and sex-specific transcriptome remodeling in neonatal piglets challenged with intra-airway acid.

Acute airway acidification is a potent stimulus of sensory nerves and occurs commonly with gastroesophageal reflux disease, cystic fibrosis, and asthma. In infants and adults, airway acidification can acutely precipitate asthma-like symptoms, and treatment-resistant asthma can be associated with gastroesophageal reflux disease. Airway protective behaviors, such as mucus secretion and airway smooth muscle contraction, are often exaggerated in asthma. These behaviors are manifested through activation of neural circuits. In some populations, the neural response to acid might be particularly important. For example, the immune response in infants is relatively immature compared with adults. Infants also have a high frequency of gastroesophageal reflux. Thus, in the current study, we compared the transcriptomes of an airway-nervous system circuit (e.g., tracheal epithelia, nodose ganglia, and brain stem) in neonatal piglets challenged with intra-airway acid. We hypothesized that the identification of parallel changes in the transcriptomes of two neutrally connected tissues might reveal the circuit response, and, hence, molecules important for the manifestation of asthma-like features. Intra-airway acid induced airway hyperreactivity and airway obstruction in male piglets. In contrast, female piglets displayed airway obstruction without airway hyperreactivity. Pairwise comparisons revealed parallel changes in genes directly implicated in airway hyperreactivity ( scn10a) in male acid-challenged piglets, whereas acid-challenged females exhibited parallel changes in genes associated with mild asthma ( stat 1 and isg15). These findings reveal sex-specific responses to acute airway acidification and highlight distinct molecules within a neural circuit that might be critical for the manifestation of asthma-like symptoms in pediatric populations.

Strategies for measuring airway mucus and mucins.

Mucus secretion and mucociliary transport are essential defense mechanisms of the airways. Deviations in mucus composition and secretion can impede mucociliary transport and elicit airway obstruction. As such, mucus abnormalities are hallmark features of many respiratory diseases, including asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD). Studying mucus composition and its physical properties has therefore been of significant interest both clinically and scientifically. Yet, measuring mucus production, output, composition and transport presents several challenges. Here we summarize and discuss the advantages and limitations of several techniques from five broadly characterized strategies used to measure mucus secretion, composition and mucociliary transport, with an emphasis on the gel-forming mucins. Further, we summarize advances in the field, as well as suggest potential areas of improvement moving forward.

The vagal ganglia transcriptome identifies candidate therapeutics for airway hyperreactivity.

Mainstay therapeutics are ineffective in some people with asthma, suggesting a need for additional agents. In the current study, we used vagal ganglia transcriptome profiling and connectivity mapping to identify compounds beneficial for alleviating airway hyperreactivity (AHR). As a comparison, we also used previously published transcriptome data from sensitized mouse lungs and human asthmatic endobronchial biopsies. All transcriptomes revealed agents beneficial for mitigating AHR; however, only the vagal ganglia transcriptome identified agents used clinically to treat asthma (flunisolide, isoetarine). We also tested one compound identified by vagal ganglia transcriptome profiling that had not previously been linked to asthma and found that it had bronchodilator effects in both mouse and pig airways. These data suggest that transcriptome profiling of the vagal ganglia might be a novel strategy to identify potential asthma therapeutics.

Neuropeptides in asthma, chronic obstructive pulmonary disease and cystic fibrosis.

The nervous system mediates key airway protective behaviors, including cough, mucus secretion, and airway smooth muscle contraction. Thus, its involvement and potential involvement in several airway diseases has become increasingly recognized. In the current review, we focus on the contribution of select neuropeptides in three distinct airway diseases: asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. We present data on some well-studied neuropeptides, as well as call attention to a few that have not received much consideration. Because mucus hypersecretion and mucus obstruction are common features of many airway diseases, we place special emphasis on the contribution of neuropeptides to mucus secretion. Finally, we highlight evidence implicating involvement of neuropeptides in mucus phenotypes in asthma, COPD and cystic fibrosis, as well as bring to light knowledge that is still lacking in the field.

Solitary Cholinergic Stimulation Induces Airway Hyperreactivity and Transcription of Distinct Pro-inflammatory Pathways.

Airway hyperreactivity is a hallmark feature of asthma and can be precipitated by airway insults, such as ozone exposure or viral infection. A proposed mechanism linking airway insults to airway hyperreactivity is augmented cholinergic transmission. In the current study, we tested the hypothesis that acute potentiation of cholinergic transmission is sufficient to induce airway hyperreactivity. We atomized the cholinergic agonist bethanechol to neonatal piglets and forty-eight hours later measured airway resistance. Bethanechol-treated piglets displayed increased airway resistance in response to intravenous methacholine compared to saline-treated controls. In the absence of an airway insult, we expected to find no evidence of airway inflammation; however, transcripts for several asthma-associated cytokines, including IL17A, IL1A, and IL8, were elevated in the tracheas of bethanechol-treated piglets. In the lungs, prior bethanechol treatment increased transcripts for IFNγ and its downstream target CXCL10. These findings suggest that augmented cholinergic transmission is sufficient to induce airway hyperreactivity, and raise the possibility that cholinergic-mediated regulation of pro-inflammatory pathways might contribute.

Cystic Fibrosis Transmembrane Conductance Regulator in Sarcoplasmic Reticulum of Airway Smooth Muscle. Implications for Airway Contractility.

RATIONALE: An asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine. OBJECTIVES: Airway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility. METHODS: Newborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity. MEASUREMENTS AND MAIN RESULTS: We found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation. CONCLUSIONS: Loss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing.

Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala.

Stimulating presynaptic terminals can increase the proton concentration in synapses. Potential receptors for protons are acid-sensing ion channels (ASICs), Na(+)- and Ca(2+)-permeable channels that are activated by extracellular acidosis. Those observations suggest that protons might be a neurotransmitter. We found that presynaptic stimulation transiently reduced extracellular pH in the amygdala. The protons activated ASICs in lateral amygdala pyramidal neurons, generating excitatory postsynaptic currents. Moreover, both protons and ASICs were required for synaptic plasticity in lateral amygdala neurons. The results identify protons as a neurotransmitter, and they establish ASICs as the postsynaptic receptor. They also indicate that protons and ASICs are a neurotransmitter/receptor pair critical for amygdala-dependent learning and memory.

pH modulates the activity and synergism of the airway surface liquid antimicrobials ß-defensin-3 and LL-37.

The pulmonary airways are continuously exposed to bacteria. As a first line of defense against infection, the airway surface liquid (ASL) contains a complex mixture of antimicrobial factors that kill inhaled and aspirated bacteria. The composition of ASL is critical for antimicrobial effectiveness. For example, in cystic fibrosis an abnormally acidic ASL inhibits antimicrobial activity. Here, we tested the effect of pH on the activity of an ASL defensin, human ß-defensin-3 (hBD-3), and the cathelicidin-related peptide, LL-37. We found that reducing pH from 8.0 to 6.8 reduced the ability of both peptides to kill Staphylococcus aureus. An acidic pH also attenuated LL-37 killing of Pseudomonas aeruginosa. In addition, we discovered synergism between hBD-3 and LL-37 in killing S. aureus. LL-37 and lysozyme were also synergistic. Importantly, an acidic pH reduced the synergistic effects of combinations of ASL antibacterials. These results indicate that an acidic pH reduces the activity of individual ASL antimicrobials, impairs synergism between them, and thus may disrupt an important airway host defense mechanism.

Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes life-threatening disease. Dipeptidyl peptidase 4 (DPP4) is the receptor for cell binding and entry. There is a need for small-animal models of MERS, but mice are not susceptible to MERS because murine dpp4 does not serve as a receptor. We developed transgenic mice expressing human DPP4 (hDPP4) under the control of the surfactant protein C promoter or cytokeratin 18 promoter that are susceptible to infection with MERS-CoV. Notably, mice expressing hDPP4 with the cytokeratin 18 promoter developed progressive, uniformly fatal disease following intranasal inoculation. High virus titers were present in lung and brain tissues 2 and 6 days after infection, respectively. MERS-CoV-infected lungs revealed mononuclear cell infiltration, alveolar edema, and microvascular thrombosis, with airways generally unaffected. Brain disease was observed, with the greatest involvement noted in the thalamus and brain stem. Animals immunized with a vaccine candidate were uniformly protected from lethal infection. These new mouse models of MERS-CoV should be useful for investigation of early disease mechanisms and therapeutic interventions.