Airway mucus consists of various proteins such as long mucins MUC5AC and MUC5B, both of which contribute greatly to the proper gel-like consistency of this most essential bodily fluid.
UNC School of Medicine researchers led by mucin expert Mehmet Kesimer, Ph.D., had previously discovered that the total mucin concentrations in the lungs are associated with COPD disease progression and could be used as diagnostic markers of chronic bronchitis, a hallmark condition for patients with COPD.
The research, published in The Lancet Respiratory Medicine, shows that MUC5AC is found at elevated levels in smokers who had not yet developed COPD but whose lung function wound up decreasing over the course of the three-year study.
Former smokers at-risk for COPD, on the other hand, had normal levels of MUC5AC at the start of the study and maintained proper lung function over three years. MUC5AC hyperconcentration in the lungs may be a key factor in predicting the risks and rates of progression to more severe disease, according to the study.
“Currently, we cannot forecast which individuals in the at-risk smokers group will progress to COPD because we don’t have an objective biological marker to underpin the disease-causing pathways. Our research shows that MUC5AC could be a predictor of who will develop COPD from the large group of aging “at-risk” smokers,” said Kesimer, senior author of the study, professor in the UNC Department of Pathology and Laboratory Medicine, and member of the UNC Marsico Lung Institute. “We think MUC5AC could be a new biomarker for COPD prognosis and it could be a biomarker for testing the effectiveness of therapeutic strategies.”
MUC5AC could also become a target for pharmaceutical developers whose goal it is to halt COPD disease progression and help patients live more normal, active lives.
Symptoms include breathing difficulty, coughing, mucus production, and wheezing. It’s typically caused by long-term exposure to irritants, such as particulate matter like cigarette smoke. The two main conditions that contribute to COPD are chronic bronchitis, an inflammation of the lining of the bronchial tubes due to chronic mucin/mucus accumulation; and emphysema, when the tiny air sacs at the end of the smallest air passages of the lungs are destroyed.
There are some treatment options for COPD to attempt to slow disease progression and reduce symptoms, but treatments often don’t work well, especially during late stages of the condition, and there is no cure.
The Kesimer Lab in the UNC Marsico Lung Institute uses various techniques, including mass spectrometry, to identify and measure the different biological mechanisms involved in lung conditions. For this study, the UNC team of scientists were able to measure the concentrations of MUC5AC and MUC5B in different groups of people, including people who had never smoked cigarettes, who had quit smoking, and who continue to smoke with or without COPD.
Smoking cigarettes has long been known to be a major risk factor for COPD, but Kesimer’s work suggests that quiting smoking decreases the odds of developing COPD as we age.
“Our data indicate that increased MUC5AC concentrations in the airways may contribute to the initiation of COPD, as well as disease progression, symptom exacerbation, and how the disease progesses over time, in general,”Kesimer said. “We did not observe the same association with MUC5B.”
The best thing an aging person can do to avoid the inevitable decline associated COPD is quit smoking immediately before airway obstruction sets in due to mucin/mucus accumulation. Through Kesimer’s work, though, it might be possible to pinpoint which individuals are at the highest immediate risk for developing COPD soon.
A total 20 authors from 14 different institutions contributed to the study as a part of a nationwide COPD study called SPIROMICS.
Proteases are enzymes that catalyse the hydrolysis of peptide bonds within proteins, facilitating their cleavage; this hydrolysis can either activate, inactivate, or modulate the activity of the target protein. The identities of the amino acid residues that form the catalytic site have been used to group human proteases into serine, cysteine, matrix metallo-, aspartyl, and threonine protease classes. Within the lung, serine, cysteine and metalloproteases have received the most attention to date [1,2].
In healthy cells and tissues, both intracellular and extracellular protease activity is well managed by regulation at the transcriptional and translational levels, as well as by inhibitory pro-domains, modulatory factors (such as pH), and antiproteases at the protein level.
However, higher-than-normal protease levels and excessive protease activity are recognised as hallmarks in chronic lung diseases (CLDs) and we continue to gain a greater appreciation of how the protease burden contributes to pathology [3,4,5]. This review will focus on the contributions of proteases at the airway mucosal surface, including how they influence important aspects of airway function including mucus characteristics, mucociliary clearance (MCC) and immune cell recruitment and function.
Lung health is a product of many environmental and host factors, including exposure to toxins, particulates or pathogens, the mounting of appropriate immune responses to such stimuli, efficient ventilation mechanics and effective gas exchange. The mucosal surfaces of the airways are important interfaces for environmental and host factors, and alterations at this interface are a common feature in patients with CLD.
The mucosal surface of the airway is composed of epithelial cells, many of which are ciliated, and is coated with a thin apical layer of mucus, resident and recruited immune cells, and the inhaled contents of the airway lumen. In many CLDs, the most obvious clinical symptoms are related to airway mucus, its excessive production, and an inability to clear it.
MCC is a vital feature of the innate immune system in the airways [6,7]. A number of processes are essential to maintain effective MCC including regulation of ion channel activity, ciliary beat frequency (CBF), mucin expression and secretion and mucus viscosity [8].
Mucus is a hydrogel composed of water, salts, large mucin polymers, non-mucin proteins, lipids, and cellular debris [9,10]. Under normal conditions, water makes up 97–98% of mucus, producing a loose and mobile gel that ably protects the airway surface from inhaled pathogens and toxins, which are removed from the airways by ciliary beat and cough.
However, in many CLDs, and especially the so-called ‘muco-obstructive’ lung diseases (chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), primary ciliary dyskinesia (PCD) and non-CF bronchiectasis), mucus composition is radically altered, producing a hyper-concentrated mucus layer [10,11,12,13].
The osmotic pressure of this hyper-concentrated mucus layer can exceed that of the subjacent periciliary layer, causing compression and flattening of the cilia, resulting in impaired ciliary beating and reduced mucus clearance. This leads to mucostasis and the build-up of mucus plaques and plugs in the airway lumen, producing muco-obstructive lung disease.
The inciting causes of these original changes in the airways, mucus composition and MCC, vary between the different muco-obstructive lung diseases (environmental factors, recurrent infection, genetic mutations to ion channels etc.), but they share pathological mechanisms, many of which are mediated or modulated by proteases.
Mucin Expression
The role of proteases in the regulation of mucin gene expression has been examined in several studies, largely focusing on the regulation of MUC5AC expression, with little assessment of the regulation of MUC5B. This is likely a result of the current dogma that MUC5AC upregulation is the driving force behind mucus phenotypes in CLDs, while MUC5B is required for maintaining normal MCC [58].
The serine protease NE induces MUC5AC messenger ribonucleic acid (mRNA) and protein in airway epithelial cells (AECs) through increased mRNA stability or via a retinoic acid receptor-dependent mechanism [59,60]. Furthermore, induction of oxidative stress by NE has been shown to increase MUC5AC expression [61,62]. Changes in MUC5AC expression were not observed upon exposure of AECs to cysteine or metalloproteases in this study, suggesting these mechanisms may be specific to serine proteases [60].
However, in a separate study, a disintegrin and metalloprotease 17 (ADAM-17) and matrix metalloprotease 9 (MMP-9) induced MUC5AC expression through the activation of epidermal growth factor receptor (EGFR) [63]. Another serine protease, human airway trypsin-like protease (HAT) indirectly induced mucin gene expression in AECs through a similar mechanism [64]. Treatment of AECs with HAT induced expression and secretion of the EGFR ligand amphiregulin, leading to EGFR pathway activation and increased MUC5AC expression [64].
Interestingly, protease-mediated changed in CFTR and ENaC activity may also impact mucin production. For example, changes in these ion channels have been shown to lower intracellular Zn2+ concentrations by inducing alternative splicing of the zinc importer, ZIP2, which in turn drives MUC5AC hypersecretion [65].
In addition to human proteases, fungal proteases also regulate mucin expression. Notably, proteases released by Aspergillus fumigatus, a fungus that is highly prevalent in the early CF lung, induce MUC5AC expression [66,67]. A more recent study identified a Ras/Raf1/extracellular signal-regulated kinase (ERK) signalling pathway through which mucin expression was induced by fungal proteases [68]. Upregulation of MUC5AC by NE and other proteases in CLD will alter the MUC5AC/MUC5B ratio in favour of MUC5AC. This is important, as a higher MUC5AC/MUC5B ratio has been observed in pathogenic conditions including asthma [69].
The reason for the more pathogenic nature of MUC5AC is not fully understood. However, the tendency of MUC5AC to form sheets, and increased tethering to the airway epithelium, may play a part in impairing MCC to promote disease [70,71]. Impairing MCC would also be of benefit to fungal species trying to colonise the airway, giving an evolutionary advantage to those that induce MUC5AC expression.
Future studies providing a clearer understanding of how proteases regulate the expression of MUC5B will be important not only in muco-obstructive lung disease, due to its role in MCC [58], but also the wider field of CLD including in idiopathic pulmonary fibrosis where a MUC5B promoter polymorphism and impaired MCC are associated with disease development [72,73].
Mucin Secretion
Following translation, mucins are packaged in a dehydrated form in secretory granules. Upon exocytosis the mucins are hydrated, absorbing more than 100 times their volume in water and, in the process, expand and acquire the correct viscoelastic properties to allow effective MCC [74].
Secretion of mucins is an incredibly rapid process occurring within a few hundred milliseconds [75]. Additionally, this secretory process is highly inducible, increasing over 1000-fold in response to certain stimuli [76,77]. Mucus hypersecretion is a major component of muco-obstructive lung diseases associated with declining lung function [78,79].
Metalloproteases including ADAM-10, meprin-α, and MMP-9, as well as the neutrophil serine proteases NE, cathepsin G and proteinase 3, are potent mucus secretagogues, inducing goblet cell degranulation and secretion of mucins from airway submucosal glands [80,81,82,83].
The specific mechanisms through which proteases induce mucin secretion are not fully understood. A number of key pathways have been highlighted in the literature. A study by Takeyama et al. demonstrated that cell-bound NE, but not free NE, could induce goblet cell degranulation, suggesting that a secondary signal may be required from the intercellular adhesion molecule (ICAM)-1 on the neutrophil cell surface to induce degranulation [84].
The intracellular signalling pathways that may be involved in this process were not elucidated in this study. More recently, NE was shown to induce mucin secretion via a protein kinase C (PKC)-dependent mechanism involving phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), a PKC target and key regulator of mucin secretion [85].
Additionally, miR-146a negatively regulates NE-induced MUC5AC secretion from AECs through the inactivation of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) signaling [86]. Much like mucin expression, it is not only human proteases that regulate mucin secretion. Bacterial proteases including Pseudomonas elastase B, alkaline protease, and protease IV have all been shown to induce mucin secretion [87].
Mucus Viscoelastic Properties
Once secreted, gel-forming mucins MUC5AC and MUC5B form part of the mucus gel layer. The concentration of mucins in this layer contributes to its viscoelastic properties. Healthy mucus contains approximately 3% solids, having the consistency of egg whites [9].
However, in chronic lung disease this can increase to up to 15% solids as a result of airway dehydration coupled with increased mucin expression and hypersecretion [9].
However, it is not only the solid content of mucus that determines its viscoelastic properties; a number of other factors influence mucus viscosity including pH, extracellular deoxyribonucleic acid (DNA) content and the presence of mucin crosslinking, which occurs via the formation of disulphide bonds between mucins during oxidative stress [40,57,88].
Besides regulating mucin expression and secretion, proteases also regulate mucus viscoelastic properties by directly acting on secreted mucin proteins. In vitro studies have demonstrated that serine proteases are capable of degrading mucins [89]. While this would seem to suggest that protease activity may decrease mucus viscosity, this has not been directly measured. Importantly MUC5B is required for MCC and therefore its degradation could in fact hinder airway clearance [58]. Furthermore, proteases regulate the release of neutrophil extracellular traps (NETs) [90].
Induction of NET formation and subsequent increases in extracellular DNA may contribute to increased mucus viscosity. NETs also provide a protective lattice around proteases preventing access and inhibition by their natural inhibitors [91,92]. Bacterial species in the airway use mucolytic proteases to promote colonisation by inhibiting entrapment in the mucus layer and to gain access to the airway epithelium. P. aeruginosa-derived elastase B (pseudolysin) degrades both MUC5AC and MUC5B [89].
Mucins in the airways are highly sulphated, a mechanism to protect against degradation from bacterial proteases. However, P. aeruginosa has evolved the ability to secrete sulfatases, allowing it to bypass this protective barrier [93]. Fungal species including A. fumigatus break down mucins, not only to promote colonisation, but also to utilise it as a nutrient source [94]. A summary of the effects of proteases on mucus and MCC in muco-obstructive lung disease can be found in Figure 1.
The effect of proteases on mucus and mucociliary clearance in the chronically inflamed airway. Proteases contribute to CLD pathogenesis through their impact on every step of the MCC mechanism. Elevated protease activity leads to (A) activation of ENaC and (B) loss of CFTR at the epithelial surface contributing to airway surface dehydration. (C) Protease-dependent damage to ciliated epithelial cells and cleavage of ciliary proteins leads to ineffective mucus clearance. This clearance defect is compounded by (D) protease-mediated increases in mucin expression and secretion from goblet cells and submucosal glands resulting in a highly viscous mucus layer that can no longer be cleared effectively. (E) Proteases can degrade mucins and (F) induce release of NETs, which may further alter mucus viscoelastic properties. Together, protease-dependent mucin/mucus hypersecretion and mucus dehydration produce highly viscous mucus, setting the stage for mucus plugging in the airways of patients with muco-obstructive lung disease.
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125985/
More information: Giorgia Radicioni et al, Airway mucin MUC5AC and MUC5B concentrations and the initiation and progression of chronic obstructive pulmonary disease: an analysis of the SPIROMICS cohort, The Lancet Respiratory Medicine (2021). DOI: 10.1016/S2213-2600(21)00079-5
