Acute Airway Acidification Induces Mucus Lung Obstruction and Impaired Mucus Transport

Guevara, Maria Valentina REZNIKOV LAB TABLE OF CONTENTS

ABSTRACT ...... 2 INTRODUCTION ...... 3 BACKGROUND AND SIGNIFICANCE ...... 4 RESEARCH PLAN ...... 6 RESULTS AND DISCUSSION ...... 8 GRANTS ...... 12 ACKNOWLEDGMENTS ...... 12 REFERENCES ...... 13

ABSTRACT

Mucin overproduction is a hallmark feature of airway diseases such as cystic fibrosis (CF) and asthma [7]. It is postulated that variations in the airway’s pH reduce airway host defenses, including the ability to fight bacterial infections, and thus worsen lung health [24]. However, the extent to which airway acidification modifies mucus properties is still being elucidated. We treated neonatal piglets with aerosolized solutions of 1% acetic acid or 1% saline solutions. In the present study, we examined mucus obstruction in lung tissues, as well as mucus transport in tracheal segments of acid-challenged piglets and saline controls. Our studies showed that acute airway acidification induced mucus obstruction in the lung and decreased mucus transport in piglets’ tracheas of acid-challenged animals. Thus, our data suggest that intra-airway acidification is sufficient to induce key features of asthma and CF, and therefore might have important connotations for disease pathogenesis and treatment.

INTRODUCTION

Respiratory diseases affects millions of people worldwide [43]. Airway diseases account for 10% of all deaths just in the United States. Distinctive features of airway disease vary, however, may are characterized by overproduction of mucus and inflammation [43-48]. For example, the airway mucus layer is especially tenacious in airway diseases like asthma and cystic fibrosis (CF), hindering drugs’ therapeutic effects. The airway mucus has gel-forming mucin glycoproteins such as MUC5AC and MUC5B, and small variations in the mucin content can be detrimental for lung health [6-9]. Airway acidification has been reported in airway diseases, including CF, asthma, and chronic obstructive pulmonary disease [49-55]. In CF, airway acidification is caused by impaired - bicarbonate (HCO3 ) transport [7, 21-27]. In asthma, airway acidification is caused by constant inflammatory processes, irregular ion channel activity, immune cell infiltration, and oxidative stress [54-57]. Several studies suggest that acidic airway environments reduce patients’ ability to fight pulmonary infections, causing accumulation of mucus and bacterial colonization in the airway [24]. Likewise, reduced water content and changes in mucins interactions are some of the known effects of acid in the airway mucus [7]. Given that acid is a contributor to airway diseases like asthma and CF, it is possible to think that airway acidification causes impaired mucus transport and mucus plugging. Therefore, we hypothesized that acute airway acidification in neonatal piglets might cause mucus obstruction in the lungs due to acid’s effect on the hydration level of the mucus layer. Further, we hypothesized that acid might change mucus viscoelasticity properties, causing decreased mucus transport in tracheal segments of piglets. We believe that our studies will provide valuable information to better understand acid’s contribution to asthma and CF, and thus, design and improved therapeutics.

BACKGROUND AND SIGNIFICANCE

Approximately 70,000 people worldwide suffer from CF, a progressive and hereditary airway disease, causing a life expectancy of just 37 years [17]. For other pulmonary diseases such as asthma, the affected population is even higher; more than 26 million people just in the US [18]. Thus, understanding of airway disease mediators for the development of novel therapeutics is critical.

Mucus: Overview

Mucus is a viscous gel layer found in mucosal surfaces of the airways, gastrointestinal tract, eyes, and female reproductive tract [1,2]. The primary function of mucus is to provide a protective film for the epithelium. The gel matrix traps foreign agents and rapidly removes them by clearance mechanisms. Likewise, mucus assists as a lubricant to reduce friction between organs and tissues, facilitating common processes such as blinking and eating [2,3,8].

Mucus is secreted from goblet cells or submucosal glands at mucous surfaces of the airway [3]. Its composition, regardless of the location, consists primarily of water (up to 95% by weight), highly cross-linked mucins, salts, proteins, lipids, cells, and cellular debris [1,2]. However, mucus thickness and pH vary significantly depending on the mucous membrane of origin. Acidic environments, such as those found in the gastrointestinal and vaginal tract, are characterized by an increase of the mucus viscoelasticity and significant variation in thickness (~ 200 휇m and ~ 50 휇m in the gastrointestinal and vaginal tract, respectively) [2,3]. In contrast, healthy lung and nasal mucus retains a generally neutral pH and studies suggest that airway mucus thickness can vary from ~ 5 to 55 휇m [2-4].

Viscosity and elasticity are common markers to describe the physical behavior of mucus. The bulk rheology of mucus is characterized by its viscoelasticity since it owns both flow (viscosity) and deformation (elasticity) behaviors [1,5]. Mucus is a non-Newtonian fluid, which its bulk rheology is critical for proper functioning, and changes in its properties greatly affect mucociliary clearance and selective barrier function. In diseases such as CF, mucociliary transport is reduced as a result of an increase in the viscoelasticity of mucus. The latter causing accumulation of mucus in the airway, bacterial overgrowth, and worsening of lung health [1]. Often at the time of lung transplantation, people with CF have severe mucus plugging of the small airways.

Airway Mucus: Mucins, Secretion, and Acidification

The selectivity and viscoelasticity of the airway mucus are rooted in its dense matrix of mucin fibers. Gel-forming mucins, such as MUC5AC and MUC5B, are the primary mucin polymer chains found in human airways. [6-9]. MUC5AC and MUC5B have a high molecular weight (5-50 MDa), with a high density of glycosylated (negatively charged) segments and hydrophobic interactions, creating a dense porous network [2,6,7,20]. Likewise, some studies suggest that the high degree of O-glycosylation and the presence of cysteine-rich domains results in a rigid mucin backbone and non-covalent interactions between polymers. Both factors being essential for gel formation and controlling mucus network properties [7].

▪ MUC5AC ▪ MUC5B

Healthy Disease Figure 1. Gel-forming mucin layer composed of MUC5AC (red) and MUC5B (blue).

MUC5AC and MUC5B are produced from superficial goblet cells and submucosal glands, respectively. (A) MUC5AC is in lesser amount than MUC5B in healthy airways. (B) In obstructive airway diseases such as cystic fibrosis, mucin production is increased, resulting in impaired mucus transport and bacterial colonization [7].

The location of MUC5AC and MUC5B is spatially separated in the airway. Recent studies in pigs have reported that MUC5AC is produced mainly by superficial airway goblet cells, while MUC5B is secreted by surface secretory cells and submucosal glands (Figure 1) [7,10-15]. The proportion of MUC5AC and MUC5B changes depending on health conditions. In healthy humans, mucin and water concentration of airway mucus is approximately 2% and 98% by weight, respectively. However, small changes in the mucin content can drastically alter the mucus structure and mobility [2,7,10]. CF is characterized by the overproduction of mucins (3-9% by weight), joined with a decrease in the water content. Increased mucin to water ratio results in increased viscoelasticity and osmotic pressure of the gel layer, impairing proper mucus transport [7,9,16].

Besides the increase of the airway mucin content, CF and other lung diseases such as asthma experience also airway acidification. In CF patients, the acidic airway environment is - caused by decreased HCO3 concentrations, which generates an airway surface liquid (ASL) with reduced pH and dehydration [7,21-27]. Some studies have reported that acidification of the airway surface compromises resistance against bacteria in CF pig models [24]. Likewise, acidic environments in the airway can disturb the conventional architecture of the mucus mesh [7]. Therefore, studying the effects of acid on the airway is of widespread interest to understand mucus transport, as well as mucus obstruction in the lungs.

RESEARCH PLAN

Specific Aim 1: Will acute airway acidification induce mucus obstruction? Rationale: Airway mucus obstruction is present in CF and other obstructive pulmonary diseases. Accumulation of mucus in the lung produces bacteria overgrowth and airflow obstruction. Acidification of the airway causes dehydration in the airway surface liquid (ASL), resulting in increased mucus concentration [37]. Thus, we hypothesized that intra-airway acidification will induce mucus obstruction in the lungs. Increased concentration of mucus will cause mucus adhesion to the airway surface, making it less difficult to clear and leading to mucus plugging [37]. Specific Aim 2: Will acute airway acidification decrease mucus transport? Rationale: Remodeled mucus is a feature in CF patients. The airway surface liquid (ASL) in CF is characterized by abnormal dehydration and reduced pH, causing impaired respiratory defense - - [32, 33]. Likewise, reduction of HCO3 and Cl secretions in CF alters mucus mobility out of the airway. Therefore, we hypothesized that intra-airway acidification will decrease mucus transport due to alterations in the mucus mesh electrostatic interactions. MUC5AC and MUC5B, the primary mucins present in mucus, are held in proximity by electrostatic bonds between such polymers. [32, 34, 35]. Since electrostatic bonds are sensitive to changes in pH, we believed that acidic environments in the airway will strength such electrostatic interactions, perturbing rheological properties of mucus and reducing mucociliary transport [36].

Experimental Strategy for Specific Aim 1 and 2 -Animals: 24 piglets (Yorkshire Landrace breed, 2-3 days of age) were feed with milk replacer (Soweena Litter Life). Piglets were allowed a 36-48-hour acclimation cycle before interventions. All procedures were approved by the University of Florida Animal Care and Use Committee. -Airway Instillation: Piglets were anesthetized with 8% SevoThesia (Henry Schein Animal Health). The airway was accessed with a laryngoscope, a laryngotracheal atomizer (MADgic) [28]. We aerosolized piglets’ airway with 500 휇L 0.9% saline solution (Control animals) or 500 휇L 1% of acetic acid. This technique results in a widespread delivery of both aerosolized solutions in the animals’ airway. -FlexiVent Studies: Piglets were anesthetized with ketamine (20 mg/Kg) and xylazine (2.0 mg/Kg), as well as intravenous propofol (2 mg/Kg) (Henry Schein Animal Health) after 48 hours of airway instillation. A tracheostomy was performed, and piglets were connected to the FlexiVent instrument (SCIREQ) as previously described [28, 29]. Piglets were ventilated at a volume of 10 ml/kg at 60 breaths/min. Piglets received an intravenous dose of methacholine in order to induce a mechanical stimulus of the airway. -Histology (Experimental Design for Aim 1): Following euthanasia, left lung segments were collected and fixed in 10% neutral buffered formalin for 7-10 days, and later paraffin-embedded. Lung sections (~ 5 nm) were stained with Periodic-acid-Schiff (PAS) to characterized glycoproteins [28]. Zeiss Axio Zoom V16 microscope was used to collect digital images of the lung sections. Lung obstruction scores were assigned as previously described [28, 30]. The scoring

6 parameters for mucus lung obstruction are as follow: 1 = within normal limits; 2= accumulation within airway (Minor mucus aggregates) (< 33%); 3 = accumulation within airway (34-66%), and 4 = accumulation within the airway (>67%). Scoring occurred once during the imaging process and only two lung sections were scored per piglet [28]. -Mucus Transport Assay (Experimental Design for Aim 2): Following euthanasia, 2-3 rings of piglets’ trachea were collected. Mucociliary transport was studied as previously mentioned [31]. The transport assay visualized mobile mucus in the entire tracheal segment. Tracheal tissues were cut along the ventral surface and pinned flat to facilitate the cilia to propel mucus (Figure 2B) [31]. Tracheal tissues were covered with 5 mL of a stock solution at 40℃. The stock solution consisted of a phosphate buffered saline solution with 10 mM of HEPES, and a 1:1000 concentration of FluoSpheres (Life Technology). After 5 minutes of baseline period, 5 휇L of methacholine were added to the tracheal tissues (Figure 2). Zeiss Axio Zoom V16 microscope was used to collect images every minute for 35 minutes in order to create a live trachea image. Fluorescence-labelled mucus was analyzed using IMARIS software, which allowed us to track mucus transport through computer assigned particles. Particles are assigned according to signal intensity.

A B

Figure 2: Representation of Mucus Transport Assay

A – B Tracheas collected from acid and saline-challenged piglets are cut along the ventral surface and pinned flat in a petridish. The tracheal segments are covered with a fluorospheres stock solution to track mucus movement over time. After 5 minutes of baseline period, tracheal segments are treated with methacholine to induce mucus secretion and airway smooth muscle contraction. Images are taken every minute for 35 minutes in order to observe mucus transport across time [31].

RESULTS AND DISCUSSION

Acute Airway Acidification and Lung Obstruction

Acid is a potent stimulus of airway afferent nerve activity [38] and a potential contributor to airway diseases like asthma and CF. Thus, airways of neonatal piglets were atomized with 1% acetic acid or 1% saline. Forty-eight hours after airway instillation, piglets were connected to a FlexiVent instrument [28, 29] in order to assess airway mechanics. A similar baseline airway resistance was noticed among both treatment groups (Figure 3). However, it was observed that male acid-challenged piglets had an increase in airway resistance compared to the saline-challenged animals after receiving an intravenous dose of methacholine Figure 3: Basal Airway Resistance on acid- [42]. Female acid-challenged and saline-challenged piglets challenge and saline-challenge piglets. P did not demonstrate changes in airway resistance upon (treatments) = 0.69. [42] methacholine intervention. Methacholine is typically used to diagnose asthma and other obstructive pulmonary diseases since this drug provokes airway narrowing, as well as mucus secretion [39]. Airway narrowing, which consequently causes increased airway resistance, was expected in all treatment groups after methacholine intervention. Particularly, we were expecting a significant increase in airway resistance in acid-challenged piglets due to acid’s effect on bronchoconstriction and mucus secretion (Figure 4) [40]. Therefore, our study indicated that intra-airway acidification induced key features of asthma, such as airway narrowing, only in male piglets.

A B

Figure 4: Airway narrowing in male acid-challenged piglets Lung sections were stained with Periodic-acid-Schiff (PAS) to characterized glycoproteins (Mucus) [28]. Zeiss Axio Zoom V16 microscope was used to collect digital images of the lung tissues. A) Male saline-challenged pig and B) Male acid-challenge pigs. Scale bar: 1,000휇m. Airways are identified with an arrow.

Airway obstruction is also a hallmark feature of CF, asthma, and other obstructive pulmonary diseases. We completed a histological scoring of the intrapulmonary airways in order to measure airway mucus obstruction. Lung tissues were stained with Periodic-acid Schiff (PAS). Obstruction scores indicated that intra-airway acidification induced mucus plugging in the small airways of both female and male acid-challenged piglets (Figure 5). Acidification in airway diseases like CF is characterized by the dehydration of the airway surface liquid (ASL) [7,21-27]. Therefore, mucus obstruction in acid-challenged animals was expected Figure 5: Mean Airway Obstruction Score due to predicted rise in mucus concentration. Elevated mucus content will cause mucus adhesion Obstruction score: 1 = within normal limits; 2= accumulation within airway (< 33%); 3 = accumulation to the airway surface, as well as an increase in the within airway (34-66%), and 4 = accumulation within the osmotic pressure of the gel layer [7, 9, 16]. Both airway (>67%). Scoring occurred once during the factors contribute to impaired mucus clearance by imaging process and only two lung sections were scored cilia, leading to mucus plugging [37]. per piglet [42]

Our histological analyses did not show any injury of the lung tissue due to airway acidification. Nevertheless, it is possible that local airway narrowing, as well as mucus obstruction, could have been caused by acid injury. During the initial airway instillation, the acidic vapor reaches the trachea and the lung of the piglets. Local accumulation of acid in regions of the small airways is not yet known, although previous studies indicate that the method of delivery, we used in our studies delivers solutions to the entire lung. However, we believed that intra-airway acidification indeed caused mucus obstruction in both female and male piglets.

A B

Figure 6: Airway Obstruction in acid-challenged pigs

Small airways are identified with an arrow, while the airway lumen is noted with an asterisk. A) Lung tissue of a male saline- challenged piglet, and B) lung tissue of a male acid-challenge piglet. Scale bar: 1,000 휇 [42].

Acute Airway Acidification and Mucus Transport

A B

Figure 7: Mucus Transport Assay

Tracheal segments were collected and pinned flat. Methacholine was added after 5 minutes of baseline period [31]. Zeiss Axio Zoom V16 microscope was used to collect live images of the tracheal segments for 35 minutes. A) Mobile mucus labeled with fluorescence nanospheres. B) Particles are assigned according to signal intensity by IMARIS software. Computer assigned particles allowed us to track mucus movement across time.

Patients with CF or asthma often experience impaired mucus transport and their mucus defense systems are compromised. Therefore, understanding the effects of acid on mucus clearance mechanisms is of widespread interest. Mucociliary transport was studied by collecting tracheal segments of acid-challenged and saline-challenged piglets [31]. With our transport assay, we were able to visualize mobile mucus in the tracheal section for 35 minutes. The trachea tissues were covered with a stock solution, containing fluorescence nanospheres. After 5 minutes, methacholine was added in order to induce airway hyperreactivity. IMARIS software was used to analyze mucus movement over a 35-minute period, and it was found that average distance traveled of mucus assigned particles was decreased in male acid-challenged piglets compared to saline controls. Consequently, the average and maximum mucus assigned particle speed was decreased as well. This effect was observed in male piglets and studies in female piglets are ongoing (Figure 8). A C B

* *

Figure 8: Effects of acid on mucus transport in male piglets.

A) Length of particle track, B) mean particle speed, and C) max particle speed were parameters studied in mucociliary transport in tracheal segments of male acid-challenged piglets. 10

The airway mucus layer is particularly tenacious in airway diseases such as asthma and CF, and it has been postulated that cilia activity is crucial for efficient mucus clearance in healthy lungs. Likewise, effective cilia function is correlated to the physical properties and structure of the mucus layer [32, 36, 41]. Acidification of the airway was expected to induce abnormal viscoelastic properties, affecting the ability of cilia to expulse mucus. Therefore, decreased mucus transport after intra-airway acidification was anticipated.

We believe that reduced mucus movement in the tracheal segments occurred due to changes in the electrostatic forces present in the mucin fibers of mucus. MUC5AC and MUC5B are held by non-covalent interactions between polymers. Our studies could suggest that the acidic environment reinforced electrostatic interactions between the negatively charged mucin fibers and acid, producing abnormal mucus adhesion to the airway surface. Likewise, we can interpret that such atypical mucus adhesion reduced the distance traveled of mucus, impairing mucociliary transport. Thus, our study indicated that intra-airway acidification provoked changes in mucus physical properties.

GRANTS

This work was funded by the National Heart Lung and Blood Institute, Grant HL119560, and the National Institutes of Health Common Fund 0T2TR001983-02.

ACKNOWLEDGMENTS

I would like to thank Dr. Leah Reznikov for helpful suggestions and guidance throughout this project. The author also thanks Dr. Ignacio Aguirre and members of Reznikov Lab for providing resources in histological analysis and contribution in data collection. Likewise, I would like to thank Juan Carlos Velasquez for designing the images present in this study.

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