An Adverse Outcome Pathway for Decreased Lung Function Focusing on Mechanisms of Impaired Mucociliary Clearance Following Inhalation Exposure

Adverse outcome pathways (AOPs) help to organize available mechanistic information related to an adverse outcome into key events (KEs) spanning all organizational levels of a biological system(s). AOPs, therefore, aid in the biological understanding of a particular pathogenesis and also help with linking exposures to eventual toxic effects. In the regulatory context, knowledge of disease mechanisms can help design testing strategies using in vitro methods that can measure or predict KEs relevant to the biological effect of interest. The AOP described here evaluates the major processes known to be involved in regulating efficient mucociliary clearance (MCC) following exposures causing oxidative stress. MCC is a key aspect of the innate immune defense against airborne pathogens and inhaled chemicals and is governed by the concerted action of its functional components, the cilia and airway surface liquid (ASL). The AOP network described here consists of sequences of KEs that culminate in the modulation of ciliary beat frequency and ASL height as well as mucus viscosity and hence, impairment of MCC, which in turn leads to decreased lung function.

Neuropeptide Y Reduces Nasal Epithelial T2R Bitter Taste Receptor–Stimulated Nitric Oxide Production

Bitter taste receptors (T2Rs) are G-protein-coupled receptors (GPCRs) expressed on the tongue but also in various locations throughout the body, including on motile cilia within the upper and lower airways. Within the nasal airway, T2Rs detect secreted bacterial ligands and initiate bactericidal nitric oxide (NO) responses, which also increase ciliary beat frequency (CBF) and mucociliary clearance of pathogens. Various neuropeptides, including neuropeptide tyrosine (neuropeptide Y or NPY), control physiological processes in the airway including cytokine release, fluid secretion, and ciliary beating. NPY levels and/or density of NPYergic neurons may be increased in some sinonasal diseases. We hypothesized that NPY modulates cilia-localized T2R responses in nasal epithelia. Using primary sinonasal epithelial cells cultured at air–liquid interface (ALI), we demonstrate that NPY reduces CBF through NPY2R activation of protein kinase C (PKC) and attenuates responses to T2R14 agonist apigenin. We find that NPY does not alter T2R-induced calcium elevation but does reduce T2R-stimulated NO production via a PKC-dependent process. This study extends our understanding of how T2R responses are modulated within the inflammatory environment of sinonasal diseases, which may improve our ability to effectively treat these disorders.

Impaired Ciliary Beat Frequency and Ciliogenesis Alteration during Airway Epithelial Cell Differentiation in COPD

Chronic obstructive pulmonary disease (COPD) is a frequent respiratory disease. However, its pathophysiology remains partially elucidated. Epithelial remodeling including alteration of the cilium is a major hallmark of COPD, but specific assessments of the cilium have been rarely investigated as a diagnostic tool in COPD. Here we explore the dysregulation of the ciliary function (ciliary beat frequency (CBF)) and differentiation (multiciliated cells formation in air-liquid interface cultures) of bronchial epithelial cells from COPD (n = 17) and non-COPD patients (n = 15). CBF was decreased by 30% in COPD (11.15 +/− 3.37 Hz vs. 7.89 +/− 3.39 Hz, p = 0.037). Ciliary differentiation was altered during airway epithelial cell differentiation from COPD patients. While the number of multiciliated cells decreased (p < 0.005), the number of primary ciliated cells increased (p < 0.05) and primary cilia were shorter (p < 0.05). Altogether, we demonstrate that COPD can be considered as a ciliopathy through both primary non-motile cilia modifications (related to airway epithelial cell repair and remodeling) and motile cilia function impairment (associated with decrease sputum clearance and clinical respiratory symptoms). These observations encourage considering cilia-associated features in the complex COPD physiopathology and highlight the potential of cilia-derived biomarkers for diagnosis.

Efficacy and safety evaluation of a hypertonic seawater solution enriched with manganese and copper salts

Background: Nasal irrigation is commonly recommended as an adjuvant treatment for blocked nose. In the present study, the safety and efficacy profile of Stérimar Blocked Nose (SBN), a hypertonic seawater solution enriched with manganese and copper salts, has been evaluated on nasal epithelium, in vitro. Methodology: 3D reconstituted human nasal epithelium tissue model, MucilAir™, has been used to investigate the safety of SBN on nasal epithelium by measuring trans-epithelial electrical resistance (TEER), cytotoxicity (lactate dehydrogenase (LDH) release) and phlogosis-related effects (interleukin-8 secretion). Efficacy assessment was measured by ciliary beat frequency (CBF), mucociliary clearance (MCC) and antimicrobial activities (against Staphylococcus aureus and Pseudomonas aeruginosa). Results: Four-day SBN treatment did not compromise the nasal epithelium integrity as TEER values were over the tissue integrity limit. SBN treatment did not exert cytotoxic (LDH release) or pro-inflammatory effects (IL-8 secretion). SBN treatment significantly increased the CBF and MCC rates compared to untreated cells. SBN treatment exerted a bactericidal effect on S. aureus and P. aeruginosa cultures, whereas seawater not enriched in copper and manganese had only a bacteriostatic effect. Conclusions: The results demonstrate that SBN is a safe formula for use on human nasal epithelium. The results also suggest a better potential therapeutic role for SBN in comparison to not-enriched seawater when used to control nasal congestion and inhibit bacterial growth which may cause nasal congestion.

The impact of zinc supplementation on the outcome of endoscopic sinus surgery and ciliary function of the nasal mucosa in children with chronic rhinosinusitis

Несмотря на указания на роль цинка в функции реснитчатого эпителия, данные относительно влияния коррекции обмена цинка на цилиарную функцию у пациентов с хроническим риносинуситом отсутствуют. Целью настоящего исследования явилась оценка эффективности хирургического лечения, активности мукоцилиарного аппарата, а также местной воспалительной реакции слизистой оболочки носа в послеоперационном периоде у детей с хроническим риносинуситом на фоне применения цинка. Методы. Обследовано 192 ребенка с хроническим риносинуситом, в лечении которых применялась эндоскопическая риносинусохирургия. Из них 131 ребенок в послеоперационном периоде получал стандартные назначения, тогда как 61 ребенок дополнительно получал цинк в суточной дозе 10 мг в течение 90 дней. Оценка концентрации цинка в сыворотке крови осуществлялась методом ИСП-МС. Для определения функции цилий использована видеоцитоморфометрия. Определение эффективности лечения осуществлялось с использованием опросника SNOT-20, а также шкал Лунд-Кеннеди и Лунд-Маккей. Результаты. Проведение эндоскопической операции приводило к достоверному снижению значений шкал SNOT-20, Лунд-Кеннеди и Лунд-Маккей у пациентов вне зависимости от приема цинка. В то же время, пациенты, принимавшие цинк, характеризовались достоверно меньшим риском ревизионной хирургии. Цинк также оказывал значительное влияние на функционирование реснитчатого эпителия. В частности, к 12-му месяцу наблюдения количество клеток с подвижными цилиями, частота биения цилий, длина цилий и выживаемость клеток у пациентов, принимавших цинк, превышала соответствующие показатели в контрольной группе. Повышение уровня цинка в организме также сопровождалось многократным снижением количества инфильтрирующих слизистую оболочку нейтрофилов и лимфоцитов. Заключение. Предполагается, что цинк ускоряет репаративные процессы в слизистой оболочке носа и обладает противовоспалительным действием. Despite the presence of certain indications of the role of zinc in ciliated epithelium functioning, data on the potential effect of Zn supplementation in ciliary function in patients with chronic rhinosinusitis are absent. The aim of this study was to evaluate the efficiency of surgical treatment, ciliary activity, as well as local inflammation of nasal mucosa in children with chronic rhinosinusitis undergoing functional endoscopic surgery. Methods. 192 pediatric patients with chronic rhinosinusitis were examined. 131 patients received standard postoperative prescriptions, whereas 61 children received 10 mg Zn daily for 90 days. Serum Zn concentrations were measured by inductively coupled plasma mass spectrometry. Ciliary function was evaluated by video cytomorphometry. Surgery efficiency was assessed with a SNOT-20 questionnaire, as well as with Lund-Kennedy and Lund-Mackay scales. Results. Functional endoscopic sinus surgery resulted in a significant decrease in total SNOT-20, Lund-Kennedy, and Lund-Mackay scores independent of Zn supplementation. At the same time, Zn supplemented patients had a significantly lower risk of revision surgery. Zn also had a significant impact on ciliary function. Specifically, up to the 12th mo postoperatively, Zn supplementation resulted in higher number of cells with motile cilia, ciliary beat frequency, ciliary length, and cell viability as compared to controls. Increased Zn status was also associated with a manyfold decrease in mucosal neutrophil and lymphocyte infiltration. Conclusions. It is proposed that Zn enhances reparative processes in nasal mucosa and possesses anti-inflammatory activity.

Glycated Albumin Triggers an Inflammatory Response in the Human Airway Epithelium and Causes an Increase in Ciliary Beat Frequency

The role of inflammation in airway epithelial cells and its regulation are important in several respiratory diseases. When disease is present, the barrier between the pulmonary circulation and the airway epithelium is damaged, allowing serum proteins to enter the airways. We identified that human glycated albumin (GA) is a molecule in human serum that triggers an inflammatory response in human airway epithelial cultures. We observed that single-donor human serum induced IL-8 secretion from primary human airway epithelial cells and from a cystic fibrosis airway cell line (CF1-16) in a dose-dependent manner. IL-8 secretion from airway epithelial cells was time dependent and rapidly increased in the first 4 h of incubation. Stimulation with GA promoted epithelial cells to secrete IL-8, and this increase was blocked by the anti-GA antibody. The IL-8 secretion induced by serum GA was 10–50-fold more potent than TNFα or LPS stimulation. GA also has a functional effect on airway epithelial cells in vitro, increasing ciliary beat frequency. Our results demonstrate that the serum molecule GA is pro-inflammatory and triggers host defense responses including increases in IL-8 secretion and ciliary beat frequency in the human airway epithelium. Although the binding site of GA has not yet been described, it is possible that GA could bind to the receptor for advanced glycated end products (RAGE), known to be expressed in the airway epithelium; however, further experiments are needed to identify the mechanism involved. We highlight a possible role for GA in airway inflammation.

Ciliary function and sinonasal mucosal cytology in pediatric patients with chronic rhinosinusitis during a year after functional endoscopic sinus surgery

Background: The objective of the study is evaluation of ciliary function and mucosal cytology after endoscopic sinus surgery in children with chronic rhinosinusitis (CRS). Methodology: A total of 132 children with CRS who underwent endoscopic sinus surgery, as well as 15 healthy controls were involved in the study. In this follow-up study patients were examined preoperatively, as well as 3, 6, 9, and 12 months after endoscopic sinus surgery. Assessment of ciliary function and sinonasal mucosal cytology was performed using high-speed videomicroscopy. Lund-Kennedy, Lund-Mackay, and sinonasal outcome test 20 (SNOT20) scores were also evaluated. Results: Total SNOT-20, Lund-Mackay, and Lund-Kennedy values significantly decreased after sinus surgery. In contrast, ciliary function and mucosal cytology only tended to improve after 6 months. 9 months after surgery the number of ciliated cells, ciliary beat frequency, cell viability, and ciliary length were significantly higher than preoperatively. The most significant improvement of ciliary function and cell height was observed 12 months after operation, whereas epithelial dystrophy and neutrophil infiltration were significantly reduced. Conclusions: Substantial improvement was observed only in a year after surgery, whereas 0 to 3 months after the surgery ciliary function was severely impaired thus predisposing to recurrent sinusitis or other complications.

nNOS regulates ciliated cell polarity, ciliary beat frequency, and directional flow in mouse trachea

Clearance of the airway is dependent on directional mucus flow across the mucociliary epithelium, and deficient flow is implicated in a range of human disorders. Efficient flow relies on proper polarization of the multiciliated cells and sufficient ciliary beat frequency. We show that NO, produced by nNOS in the multiciliated cells of the mouse trachea, controls both the planar polarity and the ciliary beat frequency and is thereby necessary for the generation of the robust flow. The effect of nNOS on the polarity of ciliated cells relies on its interactions with the apical networks of actin and microtubules and involves RhoA activation. The action of nNOS on the beat frequency is mediated by guanylate cyclase; both NO donors and cGMP can augment fluid flow in the trachea and rescue the deficient flow in nNOS mutants. Our results link insufficient availability of NO in ciliated cells to defects in flow and ciliary activity and may thereby explain the low levels of exhaled NO in ciliopathies.

Differential effect of anesthetics on mucociliary clearance in vivo in mice

Respiratory mucociliary clearance (MCC) is a key defense mechanism that functions to entrap and transport inhaled pollutants, particulates, and pathogens away from the lungs. Previous work has identified a number of anesthetics to have cilia depressive effects in vitro. Wild-type C57BL/6 J mice received intra-tracheal installation of 99mTc-Sulfur colloid, and were imaged using a dual-modality SPECT/CT system at 0 and 6 h to measure baseline MCC (n = 8). Mice were challenged for one hour with inhalational 1.5% isoflurane, or intraperitoneal ketamine (100 mg/kg)/xylazine (20 mg/kg), ketamine (0.5 mg/kg)/dexmedetomidine (50 mg/kg), fentanyl (0.2 mg/kg)/1.5% isoflurane, propofol (120 mg/Kg), or fentanyl/midazolam/dexmedetomidine (0.025 mg/kg/2.5 mg/kg/0.25 mg/kg) prior to MCC assessment. The baseline MCC was 6.4%, and was significantly reduced to 3.7% (p = 0.04) and 3.0% (p = 0.01) by ketamine/xylazine and ketamine/dexmedetomidine challenge respectively. Importantly, combinations of drugs containing fentanyl, and propofol in isolation did not significantly depress MCC. Although no change in cilia length or percent ciliation was expected, we tried to correlate ex-vivo tracheal cilia ciliary beat frequency and cilia-generated flow velocities with MCC and found no correlation. Our results indicate that anesthetics containing ketamine (ketamine/xylazine and ketamine/dexmedetomidine) significantly depress MCC, while combinations containing fentanyl (fentanyl/isoflurane, fentanyl/midazolam/dexmedetomidine) and propofol do not. Our method for assessing MCC is reproducible and has utility for studying the effects of other drug combinations.