CFTR function in the airway and inflammation in cystic fibrosis

Learning Objectives

On completion of this session you should be able to

1. Understand the role of CFTR in airway physiology
2. Describe how CFTR dysfunction leads to airway inflammation in CF
3. Analyse the consequences of chronic airway inflammation in CF

CFTR in the Normal Bronchial Epithelium

UNIVERSITYOF PORTSMOUTH CFTR is expressed in the large airways, ie the bronchi, and so we are considering its function in bronchial epithelial cells. In the normal bronchial epithelium chloride ions are secreted at the apical membrane through CFTR and into the airway lumen. The channel is activated by phosphorylation. Increases in cAMP in the cell activate protein kinase A, an enzyme that phosphorylates a wide range of proteins including CFTR. Secretion also occurs thro alternate chloride channels, although most appears to be thro active CFTR. Regulatory protein networks link CFTR to the epithelial sodium channel, ENaC., thro which sodium ions are normally absorbed. Active CFTR negatively regulates ENaC and suppresses its activity, and limits sodium uptake. At the basal membrane, chloride ions are transported into the cell via the Na/K/Cl co-transporter, and electrochemical gradients push Cl out thro CFTR. Energy derived from the sodium/potassium pump drives the system, and keeps the sodium concentration in the cell low, so that sodium is able to move into the cell through ENaC down a concentration gradient. Overall this is a secretory epithelium. Sodium ions flow between the cells to neutralize the chloride ions secreted via CFTR, and water follows the movement of salt.

Na+ Cl- Cl- ENaC CFTR Cla - Na+ PKA H2O CAMP Cl- K+ Na+ K+ Na+ /K+ /Cl- Na+/K+ ATPase cotransporter

CFTR in Normal Airway Epithelial Cells

UNIVERSITYOF PORTSMOUTH In the normal human airway, CFTR is expressed both in the surface epithelium and more predominantly in the submucosal glands. In normal airways a thin film of fluid coats the entire surface of the epithelium. The volume and composition of the airway fluid is determined by the relative contribution of salt reabsorption and salt secretion processes.

0 Salt secretion is mainly due to the secretion of chloride ions through the CFTR ion channel. CFTR normally has a negative regulatory effect on the epithelial sodium channels, which limits the uptake of sodium ions via this route, and thus the amount of water that is reabsorbed. Salt reabsorption is mainly due to the passive diffusion of sodium down concentration gradients into the cell through the epithelial sodium channel (ENaC). The intracellular sodium concentration is maintained at low levels by the activity of the Na/K pump in the basolateral membrane of the cells which pumps sodium back out of the cell. Because sodium reabsorption is limited in the normal airway the epithelium that lines the airway is a secretory epithelium, ie there is net movement of water into the airway, to produce the watery fluid that lines the airway. The fluid layer is the height of the cilia that are present on the apical surface of airway epithelial cells and permits the synchronous beating of the cilia.

❑ Inhaled dirt and debris, bacteria and viruses, collect in a thin layer of mucus that is produced by goblet cells and the submucosal glands and sits on top of this fluid layer. The mucus is normally removed by the continuous beating of cilia on the surface of the epithelium which sweep the debris towards the mouth where it is swallowed.

mucus layer H2O fluid layer cilia epithelial layer _ Na+ Cl- ENaC CFTR goblet cell

CFTR in the CF Bronchial Epithelium

UNIVERSITYOF PORTSMOUTH O In the CF bronchial epithelial cell, CFTR is absent or defective. This leads to reduced chloride channel activity , and reduced chloride secretion thro CFTR into the airway lumen (although the alternate chloride channels are still present and active). The absence of a functional CFTR removes the negative regulatory effect of CFTR on the epithelial sodium channel, which leads to a massive increase in epithelial sodium absorption, estimated to be 10x greater than normal. This massive increase in sodium absorption is dealt with by a compensatory increase in the activity of the Na/K pump in the basal membrane. Therefore, although the primary genetic defect is in the chloride channel, the primary electrophysiological defect is in the sodium channel activity. Overall this is an absorptive epithelium, and chloride ions flow between cells to neutralize the excess uptake of sodium ions and water follows the movement of salt.

Na+ Cl- ENaC CFTR Cl, a Cl- Cl- H2O Cl- K+ K+ Na+ Na+ K+ Na+ /K+ /Cl- Na+/K+ ATPase cotransporter

Dehydrated Secretions and Infection in CF Airways

UNIVERSITYOF PORTSMOUTH Dehydrated secretions are retained in CF airways, inviting infection and inflammation

mucus bacterial infection hypersecretion (Pseudomonas aeruginosa) reabsorption of excessive amounts of water Cl- O Na+ O inflammation; neutrophils are recruited to fight infection

H2O. The link between the defect in the CFTR in the airways epithelium and infections in the CF lung has only recently been recognised, mainly because of the difficulty in sampling the fluid lining the lower airways. The CFTR is expressed both in the surface epithelium and more predominantly in the submucosal glands which are enlarged, or hyperplastic, in CF patients. Mucus is secreted by the goblet cells of the bronchial epithelium and by the submucosal glands, which are the primary source and produce viscoelastic secretions which accumulate in the airways. Airway epithelial cells also have altered ion transport properties.

• The absence or defective functioning of CFTR results in reduced chloride secretion into the airways and therefore a reduced movement of water into the airways. CFTR normally has a negative regulatory influence on the ENaC and when this is removed, as it is in CF, there is a massive increase in the uptake of sodium through ENaC, and with it reabsorption of excessive amounts of water. The relative dehydration of the secreted mucus results in thick, sticky secretions that invite infection. Recurrent and chronic respiratory infections are responsible for most of the morbidity and mortality of the disease. When CF was described in 1938 most patients died by the age of 5years due to staphylococcus aureus infection. As antibiotic therapy improved, with penicillin to treat staphylococcus aureus, so did the life expectancy of the patients. In the last two decades the most prevalent pathogen in the airways has been Pseudomonas aeruginosa and CF patients are chronically infected with this organism. The altered secretions of the respiratory tract leading to the accumulation of thick mucus is thought to be responsible for these persistent infections. Accumulations of thick mucus have even been described in foetuses with CF and pulmonary infection in CF children as young as 3 months. An altered composition of the airway fluid may also favour bacterial growth.
• A neutrophilic inflammatory response is mounted to try to fight the infection.

Polybacterial Infection in CF Airways

UNIVERSITYOF PORTSMOUTH Polybacterial infection in the CF airways ...

• Staphylococcus aureus
• Haemophilus influenza (less common)

Early Infection Pathogens

• Pseudomonas aeruginosa - inevitably
• Burkholderia cepacia complex
• Stenotrophomonas maltophilia

Later Infection Pathogens

- inherently multiresistant

UNIVERSITYOF PORTSMOUTH Soon after birth, initial infection of the bronchi, the large airways, with bacterial pathogens starts. CF has a unique set of bacterial pathogens that are often acquired in an age-dependent sequence. Apart from S. aureus, these organisms are not normally pathogenic, and are considered opportunistic pathogens, that is they are only pathogenic under certain conditions but are normally harmless. Early infections are most frequently caused by S aureus and H.influenza. P aeruginosa is the most significant pathogen in CF, and up to 80% of patients are eventually infected with this organism. Acquisition of Pseudomonas is associated with rapid decline in lung function, higher sputum volume and more intense inflammation. Obstruction and destruction of the airways leads to rapid clinical clinical decline. Pseudomonas is acquired from environmental water reservoirs, showers, sinks, toilets, humidifiers etc. The Burkholderia complex is a group of at least nine closely related species, and emerges in the later course of the disease. Other organisms include S maltophilia as well as fungi including aspergillus and nontuberculosis mycobacteria. Of these the B cepacia is most serious as it is associated with high fever, bacteremia, rapid progression to severe necrotizing pneumonia, pulmonary deterioration and death. S maltophilia is seen more commonly than B cepacia, but is less virulent. Respiratory viruses have also been recognised, and respiratory viral disease can be more severe in patients with CF and contribute to the progression of the disease.

Events Leading to Chronic Infection

UNIVERSITYOF PORTSMOUTH

a NL 60 um/s po, Q = NL b CF D um's PO, Q =11 Na+ CI- HO C po2 d po, e PO2 f po,

Normal Airway Epithelia

a) Normal airway epithelia-Thin mucus layer on top of watery periciliary fluid facilitates MCC. Normal epithelial O2 consumption (QO2 ).

CF Epithelium Changes

b) CF epithelium-Depletion of periciliary fluid, mucus transport slows/stops. Increased O2 consumption with increased ion channel activity.

Mucus Layer and Oxygen Gradients

C) Continued mucus secretion increases the height of the mucus layer and raised QO2 generates steep O2 gradients.

Pseudomonas Penetration

d) Pseudomonas deposited on mucus surface penetrates into hypoxic zones within the mucus.

Pseudomonas Adaptation

e) Pseudomonas adapts to hypoxic niche with increased alginate production and formation of macrocolonies.

Macrocolony Resistance and Hypoxia

f) Macrocolonies resist secondary defences, including neutrophils, setting the stage for chronic infection. Increased numbers of bacteria and neutrophils render the mucus hypoxic.In the early acute stages of pseudomonas infection, the organism can be eradicated by antibiotic therapy, but the CF airways quickly become chronically infected with pseudomonas which becomes impossible to eradicate with antibiotic therapy alone. A; In normal airways the airway surface fluid above the epithelial surface consists of two layers, a periciliary liquid layer with a height of the extended cilium (~7um) and a mucus layer above it. The periciliary fluid promotes efficient mucociliary clearance. A normal rate of epithelial oxygen consumption (QO2) results in no gradient in the partial pressure of oxygen (p O2) within the airway surface fluid. B; In the CF airway, volume depletion of the periciliary fluid results in reduced mucociliary transport. C; Persistent mucus hypersecretion from mucus glands and goblet cells increases the height of the mucus layer with time. Increased metabolic activity of CF epithelial cells generates steep hypoxic gradients in the thick mucus layer. D; Pseudomonas bacteria deposited on the surface of mucus plugs in the planktonic form penetrate into the hypoxic interior. E; Pseudomonas adapts in the hypoxic environment with increased expression of alginate and form macrocolonies F; The increased numbers of bacteria and neutrophils renders the mucus layer more hypoxic. Pseudomonas resists lung defences, including neutrophils and results in chronic infection.