Mucins and their roles in asthma

Summary

Mucus is a crucial component of airway host defense. For optimal protection, its chief components – the mucins MUC5AC and MUC5B – need to be tightly regulated. Their expression localizes to specific secretory epithelial cell types capable of producing and secreting massive glycopolymers. In asthma, abnormal mucus is an important clinical problem that is effectively treated with therapies that directly target mucins. This review summarizes what is known about how mucin gene regulation, protein synthesis, and secretion are regulated in healthy and asthmatic lungs. Ultimately, a better understanding of these processes could help identify novel ways of preventing or reversing airway mucus dysfunction.

INTRODUCTION

Tissues of the respiratory tract are exposed to myriad environmental stimuli. Under normal conditions, the average adult breathes 8,000–12,000 liters of air each day (Figure 1A). Each liter contains 10–20 million particles, including microbes, such that respiratory surfaces are exposed daily to 100–200 billion particles whose accumulation is potentially harmful¹.

Figure 1. Exposures and barriers.

A. The primary role of conducting airways is to allow movement of inspired O₂ and expired CO₂. During normal breathing, the lungs are exposed to numerous types of particles including microbes and microbial products, allergens, smoke, and pollutants that could damage delicate gas exchange surfaces in the alveoli. To optimize gas exchange and airflow, the conducting airways are also primary sites of protection against inhaled particles and endogenous debris. B. The epithelium is the first line of airway defense. Cells atop a basal lamina have tight junctions whose functions are to keep basolateral spaces impenetrable. Additional protection is supported by mucus and mucociliary transport machinery.

As a first line of defense, epithelial cells that line the conducting airways perform barrier and clearance functions (Figure 1B). To prevent physiological detriment, defense is non-inflammatory under healthy homeostatic conditions. Protection is conferred by features such as basal lamina below epithelial cells, tight junctions that connect epithelial cells laterally, and apical glycoprotein-rich surface barriers such as mucus gels.

In conducting airways, mucus is crucial for trapping particles and reducing their direct contact with the epithelium². Mucus also mediates particle elimination via mucociliary clearance and cough mechanisms that direct transport towards the mouth for subsequent elimination by expectoration or swallowing.

Effective mucus function requires synthesis and maintenance of a viscoelastic hydrogel and the tight regulation of its macromolecular components. Mucin (MUC) glycoproteins are the principal components that form the polymer matrices of mucus hydrogels. In mammals, these polymeric mucins are encoded by five MUC genes, of which MUC5B/Muc5b and MUC5AC/Muc5ac predominate in the airways (‘MUC’ in humans, ‘Muc’ in mice).

In health, MUC5B is most abundant and is produced in mucous cells of submucosal glands found throughout the first ten airway generations³. In more distal bronchiolar generations, which lack submucosal glands, MUC5B is produced by secretory cells on the airway surface. The importance of MUC5B for homeostatic defense was first identified in mice, and recent studies have identified a similar requirement in humans^2,4–6. Its absence has revealed requirements for mucociliary clearance, protection from infection, and preservation of innate defense.

Muc5b deficient animals demonstrated constitutive up-regulation of Muc5ac. However, this was not sufficient for effective compensation for the lack of Muc5b. This striking difference is not yet understood mechanistically, but it correlates with well-known tissue-specific localization for all polymeric mucins^7–10. Emerging data suggest that mucin isoforms have functions that are specific to local host defense needs and may be mediated by protein and glycan-specific structural differences.

For example, intestinal tissues are ordinarily lined by mucous cells expressing the mucin isoform MUC2¹¹. This supports a mucus barrier that prevents epithelial contact with bacteria that could otherwise cause inflammation, epithelial damage, and invasive infection. During parasitic worm infection, Muc5ac is added to intestinal mucus. Muc5ac provides additional protection through direct effects on worm viability that are not observed with Muc2 alone^12,13.

Certain parasitic worms use the lungs as a critical portal access to the digestive tract during their lifecycles within infected hosts. These worms evoke immune responses that are mediated by type 2 cytokines such as interleukin (IL)-13, which is also a powerful activation signal for MUC5AC/Muc5ac expression in digestive and respiratory tract tissues.

Due to structural similarities with parasites, inhaled allergens also stimulate IL-13-mediated MUC5AC expression under conditions where worm trapping and expulsion are irrelevant¹⁴. The aberrant formation of an abnormal mucin matrix results in mucus with heightened viscoelasticity that impairs mucociliary clearance and promotes airway lumen obstruction. This cost-without-benefit scenario is an important pathological feature of numerous lung diseases, including asthma.

In people with asthma, fatal exacerbations are characterized by widespread mucus obstruction that is likely a cause of respiratory failure^15–18. Excessive mucus is also found in mild to moderate asthma¹⁹. This may reflect persistent re-programming of airway epithelia in airways that become chronically obstructed^18,20–23. However, there are currently no effective therapies to directly reverse or prevent mucus dysfunction in asthma. This is an important treatment gap.

Ongoing efforts seek to close this gap by identifying mechanisms involved in mucin expression, glycoprotein assembly, secretion, and post-secretory fates. Determining how each of these steps affects pathologic mucus in asthma could reveal novel treatment strategies. Here we provide an overview of known and emerging concepts related to mechanisms of mucin production, secretion, and function that may help identify new intervention targets.

Airway mucous cell ontogeny and mucin gene expression

In tracheobronchial airways, the main progenitors for mature airway epithelia are basal cells. Undifferentiated basal cells undergo fate specification and differentiation programs that are driven by the activities of Notch receptors, their ligands (such as Jagged (JAG) proteins), and other signaling molecules. Cells that express JAG-1 to −3 instruct adjacent cells expressing Notch-1 and −3 to undergo secretory differentiation (Figure 2). Cells that escape Notch signaling differentiate into ciliated cells²⁴.

Figure 2. Epithelial barrier formation.

Tracheobronchial mucociliary airway epithelial cells arise from basal cell progenitors. As basal cells begin to differentiate, they express Notch receptor isoforms or its ligands, including Jagged (JAG). JAG-expressing cells send signals that instruct Notch-expressing receiving cells to undergo secretory differentiation.

Notch activation then drives secretory cell fate determination and ultimately to mature differentiation typified by club and mucous cell phenotypes^25–29. There are incomplete (and sometimes conflicting) concepts around signals that mediate mucous phenotypes induced in cells that arise from basal cells or club cell progenitors. Part of this stems from the need to distinguish normal (healthy) processes at baseline from abnormal (diseased) processes resulting from external triggers such as infection, injury, and the inflammatory responses they evoke.

In health, bone morphogenic protein (BMP), Wnt/β-catenin, and epidermal growth factor signaling pathways also promote steady state mucous secretory phenotypes^30–32. During inflammation, these pathways can be reactivated, they can contribute to epithelial remodeling by changing numbers and distributions of multiciliated and secretory cells. In the latter, mucous phenotypes are driven by cytokine-mediated transcriptional regulation of mucous gene expression.

Control of Mucus Production in Health and During Allergic Inflammation

Mucin gene expression is tightly controlled spatially and temporally^7,10,33. In addition to cell-specific mechanisms that specify mucous and multiciliated cell fates, mucin expression in the airways is also tissue-specific^7,9. At a macroscopic level, large airways such as the trachea and bronchi possess submucosal glands that are major sources of mucus components. These tracheobronchial generations have greater abundance of mucous cells and polymeric mucin gene expression than smaller bronchiolar airways do. In health, there is a gradient such that numbers of cells and expression levels of MUC5AC and MUC5B decrease dramatically in terminal and respiratory bronchioles and are essentially absent in alveoli^7,9,34.

The mechanisms that regulate tissue-specific localization of airway mucous cell differentiation are poorly understood. By contrast, in the intestine, there is a master regulator for secretory cell fate that has a direct impact on mucous cell differentiation. The basic helix-loop-helix transcription factor atonal homolog 1 (ATOH1) is required for secretory cell fate determination in intestinal crypts. In Atoh1 knockout mice, apical secretory Paneth and goblet cells are absent³⁵. A similar relationship may exist for club cells and mucous cells in the lungs, but a specific mechanism has not been described.

Although an upstream switch is unknown, there are several candidates for downstream regulation of airway mucin expression. These include Nirenberg and Kim-2 homeobox-1 (Nkx2–1, thyroid transcription factor (TTF)-1) and forkhead box A2 (Foxa2), whose expression in the steady state represses mucous differentiation^32,36,37. It is possible that a baseline mucous phenotype is driven by removal of these inhibitory molecules, thereby optimizing defense (or creating excessive mucus) by de-repressing these.

Upon injury, infection, or inflammatory challenge, airway epithelia may undergo dramatic structural remodeling. Depending on the nature of each stimulus, immune signals will vary with respect to cytokine milieu and inflammatory cell profiles. The best characterized stimuli for mucin overproduction in the airways include proinflammatory, allergic (type 2), and type 17 cytokine signals.

In asthma, type 2 allergic inflammation is mediated by T helper 2 (Th2) lymphocytes, eosinophils, mast cells, and type 2 innate lymphoid cells (ILC2s)³⁸. IL-13 plays a pivotal role in inducing goblet cell metaplasia through engagement of IL-13 receptor α−1 and IL-4 receptor α (IL4RA) heterodimers. Signaling occurs through IL4RA via Janus kinase (JAK)-mediated activation of signal transducer and activator of transcription-6 (STAT6)³⁹.

Upon activation, STAT6 homodimers enter the nucleus and bind to canonical TTC-N4-GAA DNA motifs⁴⁰. Epithelial STAT6 expression is required for IL-13-induced MUC5AC expression⁴¹. However, STAT6 binding sites have not been identified in regulatory domains of either the human MUC5AC and MUC5B genes or their orthologs. Rather, there is clear evidence that type 2 pathway induction operates through additional mechanisms.

IL-13 induces expression of SPDEF in epithelial cells, which in turn activates MUC5AC/Muc5ac gene transcription^42,43. In humans, IL-13 also strongly inhibits MUC5B production even though MUC5B is positively regulated by SPDEF^44,45. These seemingly incongruent mechanisms are not fully understood, but they may be explained by additional layers of control provided by an upstream enhancer element in SPDEF (SPDEFe)⁴⁶. SPDEFe controls IL-13-driven MUC5AC transcription. However, SPDEFe has no effect on IL-1β-driven MUC5AC or MUC5B gene induction even though both are SPDEF dependent.

In contrast to type 2 inflammation in allergic asthma, non-type 2 disease is characterized by neutrophilic inflammation and reduced presence of classical Th2 cytokines. Mucus hypersecretion in non-type 2 asthma is often resistant to corticosteroids, making it difficult to manage⁴⁷. Non-type 2 asthma is associated with heterogeneous profiles that include innate proinflammatory cytokines IL-1β and TNF, as well as IL-17A⁴⁸.

IL-1β is a potent driver of airway remodeling and mucus hypersecretion that occur in non-type 2 asthma, and during acute injury and infection⁴⁹. IL-1β stimulates transcription of MUC5AC and MUC5B the airway epithelium via CREB-, NF-κB-, and SPDEF-dependent pathways^39,50,51. Similarly, TNF can also induce mucus gene expression in murine and human cells. TNF has been shown to upregulate MUC5AC expression in airway epithelial cells, primarily by activating the NF-κB and mitogen-activated protein kinase (MAPK) pathways^52–54.

IL-17A directly upregulates MUC5AC expression by activating downstream signaling pathways, including via MAPK and NF-κB^51,55. IL-17A induces goblet cell metaplasia and increases mucin secretion, independent of STAT signaling, underscoring its potential significance in non-allergic asthma phenotypes⁵⁶.

While each of the examples above has been demonstrated in isolation, it is important to note that human asthma involves complex profiles. Patients may be concurrently exposed to bacterial, fungal, viral, and chemical irritants. Moreover, immune profiles may be altered by systemic diseases and metabolic diseases such as diabetes and obesity⁵⁷. Accordingly, mixtures of cytokines and endocrine factors may result in mixed inflammatory phenotypes.

Although Th lymphocytes are most commonly recognized in asthma pathogenesis, innate immune cells including macrophages, eosinophils, and neutrophils also play critical roles, particularly in non-type 2 inflammation⁵⁸. These effector cell types produce numerous mediators that contribute to injury and may thus perpetuate inflammation. They may also evoke tissue repair signals that include Notch and epidermal growth factor pathways^59,60. The latter has been shown to be a necessary and sufficient parallel signal transduction mechanism involved in activation of MUC5AC transcription and translation^61,62.

Translation and assembly of mucin polymers

MUC5AC and MUC5B are massive proteins individually comprising 3000 to over 5000 amino acids. The defining characteristic of mucins is their extensive glycosylation within long, repetitive stretches of proline, threonine, and serine (PTS) residues^63,64. These PTS domains are flanked by amino (N-) and carboxyl (C-) termini that are structural homologs of the hemostasis protein von Willebrand factor (vWF)^65–67.(

Like other secretory proteins, translation of MUC5AC and MUC5B begins in the cytosol. The first stretches of amino acids encode N-terminal signal-peptides that facilitate ribosomal recruitment to the endoplasmic reticulum (ER) where mucin protein backbones (apoproteins) are elongated, folded, and partially assembled^68,69. As mucin proteins are being threaded through translocon complexes into the ER lumen, chaperones mediate proper folding, formation of >100 intra-molecular disulfide bonds, and ultimately dimerization via formation of inter-molecular disulfide bonds^70–72.

At their N-termini, MUC5AC and MUC5B possess vWF-like VWD1, D2, D’, and D3 domains. These globular regions comprise mixtures of α helices and β sheets that are stabilized by over 50 intramolecular cysteine-cysteine disulfide bonds^14,73. These internal disulfides are formed in the ER, but a small number of VWD3 domain cysteines of are used for formation of inter-molecular disulfides later in the Golgi apparatus⁷⁴. How these cysteines are reserved, or paired and rearranged, is still unclear and is an area of active investigation.

The C-terminal regions of MUC5AC and MUC5B are also conserved with vWF. They contain an additional VWD domain (D4), VWC domains, and cysteine knot (CK) domains⁷⁵. The VWC domains are important for non-covalent, calcium-dependent associations between two individual mucin molecules. Using cryo-electron microscopy and small-angle X-ray scattering for MUC5B, Ridley et al. demonstrated that these associations help compact mucin chains to enable them to fit into secretory granules⁷⁶.

The CK domain is required for the formation of disulfide bonds that link the chains together covalently. Ultrastructure studies of vWF, reveal a CK formed by linking two long monomers that each have two β sheet ribbons joined at the tips by four intrachain disulfides. The monomers then interact to form three intermolecular disulfides that create a stable, highly reinforced dimer⁷⁵. MUC2 has an analogous architecture, and both MUC5AC and MUC5B are expected to be similar as well. Protein disulfide isomerase (PDI) enzymes usually mediate disulfide bond formation, and PDIA1 is implicated for vWF⁷⁷. Exactly which PDI mediates MUC5AC or MUC5B intra- and intermolecular disulfide bond formation is unknown.

Between the N- and C-termini- is the PTS regions are stretches of protein that are ~1,600–3,600 amino acids long⁷⁸. In MUC5AC and MUC5B, small segments of ~100 amino acids are dispersed along the apoprotein backbone thereby breaking the PTS domain into modular segments of ~30–1,000 residues. While in the ER, PTS modules are non-glycosylated and unstructured. Thus, nascent mucin protomers present cells with high concentrations of protein that can trigger ER stress and activation of the unfolded protein response (UPR)⁷⁹.

The UPR is induced when protein volume in the ER exceeds translation or folding capacities, or under conditions where mis-folded or intrinsically unfolded proteins are abundant. Initially, the UPR is a self-protective mechanism that controls protein translation rates and folding efficiencies in order to restore proteostasis⁸⁰. Prolonged ER stress can result in a UPR that leads to more severe outcomes, including cell death.

The need for sustained expression of mucins possessing complex N- and C-termini separated by intrinsically unfolded PTS domains presents mucous cells with substantial challenges⁸⁰. Three transmembrane UPR proteins act as ER stress sensors that can activate the UPR. They include PKR-like eukaryotic initiating factor α kinase, activating transcription factor 6, and inositol-requiring enzyme 1 (IRE-1)^81,82.

IRE-1 is a master controller of the UPR that senses unfolded proteins that displace the chaperone BiP from IRE-1. This activates a cytoplasmic facing RNAse that mediates non-canonical splicing of mRNA encoding the transcription factor XBP-1. Mucous cells have evolved processes to evade detrimental UPRs by subverting IRE-1 function (Figure 3).

Figure 3. IRE-1 components of the UPR in mucin biosynthesis.

During synthesis, MUC5AC MUC5B mucins have structured N- and C-termini (white and gray, respectively) that flank long stretches of unfolded PTS domain segments (black). These unfolded regions bind IRE-1 ER stress sensor proteins (α, purple and β green), resulting in displacement of BiP and activation of intrinsic kinase activities in IRE-1. Phosphorylation of IRE-1 activates cytosolic RNase activity that cleaves XBP1, and this splicing (S) creates a frameshifted mRNA that encodes an activated form or XBP1 called XBPI(S). RNase activity can also cause regulated IRE-1 dependent decay (RIDD), which blocks expression of numerous other target mRNAs. IRE-1α is the ubiquitous form present in nearly all cells. IRE-1β is structurally similar to the α isoform, but it has weaker activity that can dampen IRE-1α dependent signals. Note: The mucin molecule depicted is not drawn to scale.

IRE-1 has two isoforms – α and β. IRE-1α is ubiquitously expressed, and it is the isoform responsible for XBP-1 splicing in most cells. However, mucous cells also express IRE-1β, an isoform with weaker RNAse activity that allows it to act as a dominant-negative repressor of IRE-1α function⁸³. This permits mucous cells to tolerate the high levels of ER stress evoked by the translation and assembly of polymeric mucins. By preserving the integrity of cells required for barrier function in microbe-rich environments like the colon, IRE-1β promotes sustainable defense in a setting where having a dense mucus barrier is protective⁸⁴.

IRE-1β is also found in human and mouse airway mucous cells. It facilitates mucin production via XBP-1 splicing and the coordinated regulation of other chaperones and UPR components^83,84, and it can also promote transcription of human MUC5B⁸⁵. IRE-1β expression increases in airway epithelia after IL-1β or IL-13 exposure, and its absence suppresses mucin excessive mRNA and protein production in inflamed airways where mucus adhesion or aggregation can disrupt respiratory function^83,85.

Collectively, it appears that dampening ER stress promotes mucin production, but this could lead to excessive mucus is organ dependent. What may be beneficial in the digestive tract could be detrimental in the airways under healthy conditions. After injury to digestive tissue, altered expression of UPR components could skew the ability of IRE-1β to maintain tolerable levels of mucins needed for a healthy barrier. On the other hand, loss of IRE-1β mediated suppression of the potentially cytotoxic UPR effects could be considered an option for preventing excessive mucin production in airway diseases like asthma. Additional work is needed to determine anatomic, temporal, and inflammatory contexts that underlie how IRE-1 isoform specific activities control of the UPR and mucin-mediated defenses.

The extent and complexity of regulation required for mucin biosynthesis underscores the importance of the apoprotein backbone in mucus function. Reducing agents such as dithiothreitol (DTT) or tris(2-carboxyethyl) phosphine (TCEP) can break disulfide bonds within and between mucin chains, resulting in disruption of the underlying matrices of mucus gels⁸⁶. This is being as mucolytic strategy to treat airflow obstruction in asthma and other lung diseases. Reducing mucin chain length under diseased conditions can improve mucus viscoelasticity by lowering elastic moduli^87,88.

Mucus viscoelasticity can also be affected by proteases that have direct effects on mucin protein backbones. These include endogenous proteases that can cleave peptides at specific target sites or regions^89,90. In addition, neutrophil elastase has been shown to degrade mucins⁹¹. Its specificity is incompletely characterized, and the balance between improved versus impaired clearance functions should be considered within contexts of location, disease state, and degrees of degradation.

Proteases from exogenous sources have also been shown to cleave mucins. These are especially relevant in the gut where bacteria are plentiful^92,93. Among these, a recently identified class of proteases has been shown to target mucins with a high level of selectivity due to preferences for amino acid targets in mucins^94–98. These ‘mucinases’ have combined effects on mucus elasticity and viscosity due to their targeting of O-glycosylated mucin PTS domains.

GLYCOSYLATION

Heavy O-glycosylation is a defining characteristic of MUC5AC and MUC5B, but it is poorly understood mechanistically. In part, this is due to challenges intrinsic to the nature of these massive proteins. Based on primary structures alone (5,762 and 5,654 amino acids respectively), MUC5AC and MUC5B are the 14^th and 15^th largest proteins in humans. Through disulfide-mediated CK domain dimerization and VWD3 domain multimerization, they become even more massive, such that polymers are among the top five largest human proteins^99,100.

In the Golgi, this is amplified further as they are further post-translationally modified through glycosylation (Figure 4). Mucin glycosylation occurs through stepwise processes that define the order and types of glycans added. This is determined by the expression and location of glycosyltransferase (GT) enzymes, the availability and presentation of donor and acceptor substrates, and the processes that underlie cargo transport through Golgi compartments.

Figure 4. Mucin biosynthesis and secretory trafficking.

MUC5AC and MUC5B are transcribed in the nucleus, and mRNA is transported to the cytoplasm (arrow). Co-translational import into the ER is followed by folding, internal disulfide bond formation, and C-terminal dimerization. Mucin protomers traffic through cis, medial, and trans-Golgi compartments where glycans are added in serial steps. Late in the Golgi, mucins undergo N-terminal multimerization and are then packaged into secretory granules (SGs). Mucous cells retain these granules until stimuli induce Ca²⁺ dependent regulated exocytosis via Synaptotagmin (Syt) calcium sensors and SNAREs.

Typical illustrations depict Golgi structures as stacks comprising cis-, medial-, and trans-Golgi cisternae and dispersed trans-Golgi network (TGN)¹⁰¹. However, with mucin polymers as cargo and the fraction of cytoplasmic volume occupied by mucin secretory granules, this visual representation faces significant challenges. Gaining a better description of mucous cell morphology and how it is mechanistically related to glycopolymer synthesis is an active area of investigation.

Irrespective of Golgi morphology, glycosylation follows an ordered path beginning with elaboration of carbohydrates to serine (Ser, S) and threonine (Thr, T) residues in mucin tandem repeat domains (Figure 5). This begins with attachment of N-acetylgalactosamine (GalNAc) to S/T hydroxyl groups, thus creating ‘O’ linkages in the cis-Golgi¹⁰². It is followed by addition of galactose (Gal) and/or N-acetylglucosamine (GlcNAc) residues that form Core and extended glycan structures. Glycans can then be elaborated further by addition of Gal and GlcNAc extensions that often terminate upon addition of sialic acid (Sia) or fucose (Fuc) to Gal^103–105. Internal structures can also be augmented with branch-forming units, adjunct glycans such as α1,3- or α1,4-linked Fuc to GlcNAc, and sulfates added to Gal or GlcNAc.

Figure 5. Enzymes and locations involved in mucin O-glycosylation.

Mucin type glycosylation begins in the cis Golgi with the addition of GalNAc onto serine (S) and threonine (T) residues onto oxygen (O) in their hydroxyl side chains. In medial compartments, O-GalNAc linkages are then elaborated by the addition of Gal and/or GlcNAc glycans to form Cores (types 1 and 2 most commonly in mucins), and these are elaborated further through extensions and branching. In the trans-Golgi, the last steps of glycosylation include the additions of terminating sugars such as Sia or Fuc onto Gal residues through α1–2, α2–3, and α2–6 linkages that prevent further glycan attachments. Additional modifications include SO2 and Fuc linkages internally.

Among glycans found on airway mucins, fucosylation may play a significant role in the mucus dysfunction observed in asthma. Fucose is a non-polar glycan relative to others on airway mucins. For example, Fuc has a polar surface area of 90 Ų with only 5 H-bond acceptors, which is ~1/2 of Sia’s polar surface area of 177 Ų and its 9 H-bond acceptors. Fucose content correlates with excessive mucus viscoelasticity that can lead to aggregation in airspaces¹⁰⁶. In addition to differences in their effects on biophysical properties of mucus, mucin glycans may also play significant roles in interactions between parenchymal and immune cells in mucosal tissues. These are discussed further below.

In asthma, expression of the fucosyltransferase enzyme FUT2 is associated with disease severity¹⁰⁷. Plugged airways in fatal asthma and mucous metaplastic airways in mouse models of asthma show that α1,2-fucosylated mucin is prominent¹⁰⁸. FUT2 is required for formation of Fuc(α1–2)Gal linkages that terminate glycosylation on mucins^107–109. In mice, Fut2-mediated mucin α1,2-fuocsylation is required for airway hyperreactivity and mucus plugging in an allergic asthma model¹⁰⁸. Determining how mucin fucosylation is mediated could reveal strategies for mucus interventions.

Due to their massive sizes and complex glycosylation, mucins epitomize the need for translation and post-translational modifications to be tightly regulated^110,111. Precisely where and how these steps occur in mucous cells are not known. Further work elucidating how GTs are targeted to appropriate sites in the Golgi may reveal means for controlling MUC5AC and MUC5B glycosylation and function when secreted into airspaces.

TRAFFICKING AND SECRETION

Mucin secretagogues

In addition to controlling airway smooth muscle contraction, neuronal signaling is also a significant contributor to mucus production in asthma. Vagal sensory and parasympathetic neurons promote goblet cell metaplasia and mucin secretion^112,113. Activation of vagal reflexes is required for airway hyperreactivity in mice, and stimulating mucin secretion alone is insufficient to induce hyperreactivity¹¹⁴. Thus, their combined effects on physical disruption of airflow are synergistic.

Neuronal inputs also affect inflammatory and epithelial remodeling processes. Ablation of pulmonary nociceptor neurons in murine models of allergic airway inflammation reduces type 2 inflammation, goblet cell metaplasia, and bronchial hyperresponsiveness¹¹². This effect was observed even in Rag2^−/− mice¹¹³, suggesting that neuronal regulation of mucin production operates independently of adaptive immune cells. Substance P and calcitonin gene-related peptide (CGRP) and other mediators are also reported to drive mucin release^113,115, further implicating neuronal and neuroepithelial signaling in airway mucus regulation. Recent work from Tamari et al. showed that JAK1 signaling specific to vagal sensory neurons promotes anti-inflammatory responses in Alternaria-induced allergic inflammation, including mucus production¹¹⁶.

Additional non-neuronal sources of neurotransmitters involved in mucin secretion include acetylcholine (ACh) from epithelial tuft cells, which can drive goblet cell degranulation and MUC5AC secretion in intestinal type 2 inflammation¹¹⁷. Gamma-aminobutyric acid (GABA) signaling has also been implicated in mucus overproduction^118,119. Pulmonary neuroendocrine cells (PNECs) are a primary source of GABA in the airways^118,120, and GABA is required for IL-13-induced MUC5AC expression in cultured epithelial cells and for goblet cell metaplasia in OVA-challenged animal models¹¹⁹. Altogether, these studies show that neuronal signaling contributes to mucin production in allergic inflammation, which may open new targeting strategies to control mucus hypersecretion in asthma.

Several other molecules associated with inflammation and danger signals serve as mucin secretagogues that contribute to mucus hypersecretion in asthma. Adenosine triphosphate (ATP), released from damaged epithelial cells or through mechanical stretching of airway smooth muscle, acts as a potent secretagogue for MUC5AC^121,122. Cysteinyl leukotrienes and reactive oxygen species (ROS) generated during inflammation can damage airway epithelial cells and lead to dysregulated mucin production and impaired mucociliary clearance^123–126. A mechanism for ROS-mediated mucus dysfunction may occur via the NLRP3 inflammasome¹²⁷.

Exocytic machinery involved in baseline and stimulated mucin secretion

The secretory pathway is responsible for the synthesis, folding, and delivery of mature mucin granules to the plasma membrane. Ultimately, these granules are secreted into the airway lumen through regulated exocytosis (see Figure 4). This final step has been thoroughly characterized through extensive research that has identified the components of the exocytic machinery and their distinct roles in the secretory process^128,129.

Mucin is secreted at a slow baseline rate and a high stimulated rate. Both secretory mechanisms are regulated and involve extracellular ligands and calcium dependent activation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Although some components of the exocytic machinery are shared among baseline and stimulated secretion, some are distinct¹³⁰.

Baseline secretion plays a crucial role in maintaining homeostatic levels of mucus in the airways, supporting hydration and mucociliary defense¹³¹. Key components involved in baseline secretion include Munc13–2, Munc18–1, and SNAP23¹³⁰. Deletion of these proteins in mice lead to an accumulation of mucin-packed secretory granules in airway epithelial cells^8,131–133.

Stimulated secretion leads to the rapid release and swelling of the mucin. Additional components such as Munc18–2, Syntaxin 3, Synaptotagmin 2 and VAMP8 come into play^{129,132–135}. Together, these proteins facilitate the rapid docking and fusion of numerous secretory granules with the apical plasma membrane, resulting in mucin release and hydration. However, if mucins are released too quickly, it can cause issues like mucus adhesion to epithelial surfaces, impaired ciliary transport, and airflow obstruction¹³⁶.

The descriptions above primarily relate to surface airway epithelial cells, which have been extensively studied in both mouse models and human tissue culture systems. Mice possess only a limited number of submucosal glands in the proximal trachea, and the exocytic mechanisms of these glands remain unexplored¹³⁷. However, it is likely that the exocytic mechanisms are highly conserved among apically secreting cells of the respiratory epithelium, given their close developmental relationships.

A deeper understanding of the molecular mechanisms driving mucin exocytosis could lead to novel strategies for enhancing mucin secretion to bolster host defense or for suppressing it to prevent airway occlusion. Recently, it was shown that disruption of the calcium triggered membrane fusion reduced stimulated mucin secretion and attenuated mucus occlusion in a mouse model of asthma¹²⁹. While considerable progress has been made in elucidating and targeting the later stages of secretion, there is a significant gap in knowledge regarding the earlier steps involved in mucin trafficking and synthesis. Mucous cells have developed specialized mechanisms, including unique ER-to-Golgi transport pathways^138–140 and the unconventional dispersion of Golgi structures^141–143, to efficiently manage the synthesis and transport of these large and complex cargoes. Exploring these processes further could provide valuable insights into how to regulate mucin secretion effectively.

IMMUNOREGULATION THROUGH A MUCIN SIGLEC-AXIS

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a family of receptors primarily expressed on immune cells that recognize sialic acid-bearing glycoconjugates on various surfaces, including mucins^144,145. These molecules play critical roles in modulating immune responses by initiating intracellular signaling cascades upon ligand binding. Siglec biology, particularly Siglec-mucin interactions, has significant implications in the pathogenesis and therapeutic targeting of asthma.

Siglecs are classified into two groups: evolutionarily conserved Siglecs (Siglec-1, −2, −4, and −15) and CD33-related Siglecs, which include Siglecs-3, 5–12, and 14–16 in humans and Siglec-E, Siglec-F, Siglec-G, and Siglec-H in mice¹⁴⁴. Despite differences in expression patterns between human and murine immune cells, Siglecs consistently bind to sialylated glycans, commonly present in mucins, through their extracellular carbohydrate recognition domains (CRDs). These CRDs exhibit specificity toward different glycosidic linkages and modifications of sialic acids, allowing Siglecs to selectively engage various ligands^144,146,147.

Upon ligand binding, Siglecs initiate intracellular signaling through immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or activating motifs (ITAMs), which upon phosphorylation, suppress or enhance immune responses, respectively^144,146,147. These signals contribute to the modulation of innate and adaptive immunity, making Siglecs vital players in immune homeostasis and inflammation. In asthma, Siglecs regulate critical immune cells, including eosinophils, neutrophils, and macrophages, all of which are integral to the inflammatory response and mucus production in the airways.

Siglec-Mucin Interaction in Asthma

MUC5AC and MUC5B, two predominant mucins in the airway, are heavily sialylated and serve as endogenous ligands for Siglecs^148–150. In asthma, where mucin overproduction contributes to airway obstruction, the interaction between Siglecs and mucins can likely influence both immune cell function and mucus regulation. For this review, we will highlight the inhibitory Siglecs linked to allergic inflammation in both humans and mice (Figure 6).

Figure 6. Mucin-Siglec signaling axis.

Airway mucins carry α2–3 linked Sia glycans with affinity for mouse Siglec-E and Siglec-F, as well as human Siglec-8 and Siglec-9. Ligand association stimulates phosphorylation of ITIM and ITIM-like domains via p38 Ras (not shown). Once activated these domains phosphorylate SHP phosphatases that can suppress downstream kinase-driven signaling. The Siglecs depicted here are expressed on numerous leukocytes including eosinophils, mast cells, neutrophils, monocytes, macrophages, and others.

Siglec-E engagement reduces excessive neutrophil activation and recruitment, limiting inflammation and mucus secretion^151,152. Its human paralog, Siglec-9, shows similar regulatory functions on neutrophils in viral and tumor models^153,154. Neutrophils, known to infiltrate asthmatic airways, can exacerbate inflammation and mucus production, as discussed above. Siglec-9 is also highly expressed on natural killer (NK) cells, a subset of innate lymphoid cells known for their antiviral activity; Siglec-9 acts as a negative regulator of NK cell cytotoxicity during respiratory viral infection¹⁵⁵. Understanding the roles of Siglec-E and Siglec-9 is critical, as NK cell activation status correlates with the severity of viral-induced asthma exacerbations¹⁵⁶.

Siglec-F, the functional paralog of human Siglec-8, is highly expressed on eosinophils, a key cell type involved in allergic inflammation¹⁵⁷. Siglec-8 negatively regulates eosinophil-mediated responses by inhibiting their activation and promoting cell death¹⁵⁸. In murine models of asthma, the deletion of Siglec-F impairs eosinophil apoptosis and results in enhanced eosinophilic inflammation and mucus hypersecretion^159,160. However, the downstream signaling mechanisms do not appear to fully depend on ITIM phosphorylation^161,162.

Beyond eosinophils, Siglec-F is also highly expressed in murine airspace macrophages (AMs)^163,164. Along with mucociliary clearance, AMs are crucial in maintaining lung homeostasis by serving as sentinels that contribute to host defense without causing overwhelming inflammation and tissue damage¹⁶⁵. In murine models of allergic inflammation, macrophages contribute to both inflammation and tissue repair, depending on the stimuli used and subsequent polarization state¹⁶⁶. Siglec-F engagement on AMs may promote the resolution of inflammation by suppressing proinflammatory signals, thereby reducing mucus production. The precise role of Siglec-8 on human macrophages in the context of asthma remains largely unexplored.

Determining roles played by Siglecs on innate cells is an active area of research for therapeutics. Beyond innate immune regulation, Siglecs also influence adaptive immunity, particularly B cell responses in type 2 inflammation. Orgel et al. targeted both CD22 (Siglec-2) and the B cell receptor recognizing peanut allergen with liposomes¹⁶⁷. As a result, IgE-mediated sensitization to peanuts was inhibited, although it is unclear if this effect was due to the inhibitory function of Siglec-2 or impaired B cell maturation into plasma cells.

THERAPEUTIC DIRECTIONS

Conventional therapies for asthma including bronchodilators, muscarinic agonists, and corticosteroids, treat the inflammation in the airways to control symptom burden and prevent exacerbations¹⁶⁸. The only FDA-approved inhaled mucolytic therapy currently in use is N-acetylcysteine (NAC). NAC can act as a reducing agent, but this activity is weak. High doses needed to achieve target activity are not well tolerated due to irritant effects that cause bronchospasm^169,170.

Currently, other mucolytic therapies are being developed that disrupt disulfide bonds or weaken non-covalent mucin interactions^{86,136,171–178}. Like NAC, these therapies attempt to non-selectively treat symptoms associated with pathologic mucin hypersecretion^88,174,179. One potential downside to this approach is that future symptom burden may not be addressed if medications that are cleared sputum do not change underlying mechanisms responsible for symptoms prior to being expectorated.

On the other hand, an agent that clears pathologic mucus and is then eliminated could allow for replacement with a healthy protective mucus layer. This may also be a critical point to consider in the context of mucus that is heterogenous. Reducing disulfides in regions of healthy mucus (∼3–6 mg/ml mucin content) could cause viscoelasticity to fall below levels needed for effective mucociliary clearance, even if regions of abnormal mucus with higher mucin concentrations are benefited⁸⁷. This dichotomy can be addressed by determining the extents of mucin disulfide reduction needed, the rates of mucus replacement, or the abilities of agents to target specific areas of abnormal mucus.

While there is in vitro evidence that corticosteroids inhibit goblet cell metaplasia and burden of Muc5ac¹⁸⁰, it is not clear that effects on mucous cell differentiation in vitro are direct or are related to anti-inflammatory responses in vivo. Along these lines, biologic therapies targeting allergic inflammation such as Dupilumab (IL-4/IL-13 inhibition) and Tezepelumab (TSLP inhibition), significantly improve symptoms in patients with asthma phenotypes that were refractory to traditional therapies¹⁸¹. In addition to inflammation, targeting these pathways has also been shown to improve mucus plugging, lung function, and other clinical indices^182–184, suggesting that the effects of biologic therapies on pathologic mucus and mucus plugging should be investigated further.

Although biologics currently in use for asthma mainly target type 2 inflammation, non-type 2 disease remains a challenge. Biologics targeting IL-17 or IL-1β are used clinically. However, IL-17 inhibition failed to improve asthma in a randomized trial, and two recent clinical trials designed to assess IL-1β inhibition with anakinra were withdrawn due to the COVID-19 pandemic and risks associated with allergen exposure and drug treatment^185,186. The diversity of signaling molecules and the costs of using biologics as treatments further highlight the need to consider molecules that directly target mucus since it is present across inflammatory spectra and patient endotypes.

Efficacy derived from targeting specific inflammatory responses also highlights the need to better understand systemic immunity. There have been numerous studies assessing the influence of the gut microbiome on asthma disease incidence, symptom control, and progression. Antibiotic administration in the first year of life in humans shows an increased risk of developing asthma via effects exerted on the gut/lung microbiome¹⁸⁷. In animal models of allergic asthma, some bacterial species in the gut have been associated with protection from asthma development. Mice supplemented with Lactobacillus johnsonii showed decreased bronchial responsiveness and decreased production of Th2 cytokines including IL-13¹⁸⁸, thus likely affecting mucus production/mucus plugging.

Accordingly, efforts to curtail inappropriate antibiotic use in children could potentially reduce asthma burden later in life. Lastly, studies have assessed the effects of dietary supplementation of probiotics, fiber, and short chain fatty acids in humans and mice as a way to modify the gut microbiome.¹⁸⁹. Further work is needed to determine whether these strategies can reduce mucosal inflammation and perhaps prevent or even treat asthma.

In addition to targeting the burden of extracellular mucus or the inflammatory processes that lead to mucin overproduction, controlling mucin production directly at mucosal surfaces remains an attractive approach. This could be achieved by reducing numbers of mucous cells via Notch pathway inhibition^59,190, by controlling transcription factors that regulate mucin expression¹⁹¹, by targeting MUC5AC and MUC5B expression at the mRNA or protein levels during synthesis, or by limiting their acute secretion by inhibiting exocytosis¹⁹².

Another approach would be to target enzymes involved in translation or post-translational modifications that are specific to pathogenic mucins. AGR2, for example, has relative specificity of expression within mucous cells. However, lack of AGR2 signaling could lead to protein mis-folding and ER stress which could be detrimental to the cells¹⁹³. The enzymes involved in protein glycosylation are also attractive targets for therapeutics as glycosylation states of leukocytes, antibodies, and epithelial cells have been shown to affect multiple lung diseases, including asthma¹⁹⁴. Fucosyltransferase 2 (FUT2) is a potential glycosyltransferase enzyme that could be targeted for treatment of asthma^108,109,174.

SUMMARY

Although crucial for host defense, mucus hypersecretion is a problem in numerous lung diseases, including asthma. Current therapies target bronchoconstriction and inflammation, with tremendous steps being made to improve responses in people with severe or difficult to treat disease. Steps towards improving mucus treatments will be made as we continue to better understand the basic cell and molecular biology of mucous cells, MUC5AC, and MUC5B. Importantly, the most effective interventions will need to be able to target pathogenic mucus while preserving the protective effects of mucus needed for host defense^{2,4,5,136,195}.