Molecular mechanisms of dexamethasone actions in COVID-19: Ion channels and airway surface liquid dynamics

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Introduction
COVID-19, caused by the novel coronavirus SARS-CoV-2, primarily affects the respiratory system, leading to symptoms that range from mild upper respiratory symptoms to acute respiratory distress syndrome (ARDS).The COVID-19 pandemic has spurred extensive research into the mechanisms of SARS-CoV-2 infection, disease progression, and potential treatments.Among the factors under investigation, the role of corticosteroids in COVID-19 has garnered significant attention in light of the clinical trials showing their advantages in reducing mortality in severe COVID-19.The therapeutic actions of corticosteroids in severe COVID-19 have been attributed to their known anti-inflammatory actions in the airways [1,2].Severe cases of COVID-19 often involve ARDS characterized by a profound inflammatory 'cytokine storm' and a severely compromised lung function.Dexamethasone has emerged as a crucial therapeutic agent in the management of severe COVID-19 cases.Beyond its well-known anti-inflammatory effects, emerging evidence suggests that dexamethasone may also modulate ion channels to inhibit electrolyte and water secretion into the airways [3], potentially influencing airway surface liquid dynamics, pulmonary oedema and disease outcomes.Pulmonary oedema, characterized by an abnormal accumulation of fluid in the lungs, is a life-threatening condition in ARDS associated with severe COVID-19 [4].The pathophysiology of COVID-19-induced pulmonary oedema involves a complex interplay of airway surface liquid dynamics, inflammation, endothelial dysfunction, and immune response dysregulation.Dexamethasone, a synthetic pure Abbreviations: ASL, Airway surface liquid; ARDS, Acute respiratory distress syndrome; CaCC, Calcium activated chloride channel; CFTR, Cystic fibrosis transmembrane conductance regulator; ENaC, epithelial sodium channel; EVLW, Extravascular lung water; GPCR, G-protein coupled receptor; GR, Glucocorticoid receptor; GRE, Glucocorticoid response element; ICU, Intensive care unit; PCL, Periciliary layer; VEGF, Vascular endothelial growth factor.
E-mail address: bjpharvey@rcsi.ie.glucocorticoid with potent anti-inflammatory and immunosuppressive properties, has garnered attention for its potential to address these various aspects of airway inflammation and oedema in the regulation and management of COVID-19 [5].
In COVID-19, an exaggerated immune response contributes to lung inflammation, which can lead to pulmonary oedema.The utility of dexamethasone in managing pulmonary oedema varies depending on the underlying cause.It has been most extensively studied and proven effective in the context of ARDS and certain inflammatory lung diseases.Clinical studies have shown that dexamethasone can mitigate the inflammatory response associated with ARDS in severe COVID-19, thereby reducing the severity of pulmonary oedema.The ground-breaking revelation about dexamethasone efficacy in treating severe COVID-19 came from the RECOVERY (Randomized Evaluation of COVID-19 Therapy) trial [6].This large-scale study demonstrated that dexamethasone reduced the risk of death in critically ill COVID-19 patients requiring mechanical ventilation or supplemental oxygen.The results showed a significant 17 % reduction in mortality for patients on mechanical ventilation and an 8 % reduction for those on oxygen support.Since this study, dexamethasone has been used effectively to reduce ICU admissions and mortality in severe COVID-19.Studies investigating the anti-inflammatory and anti-secretory properties of dexamethasone further support its utility in managing COVID-19-induced lung pathology.By reducing inflammation and airway secretion, the glucocorticoid likely plays a pivotal role in preventing and alleviating pulmonary oedema.Recent reports of anti-secretory, pro-absorptive effects of dexamethasone via the regulation of ion channels point to an additional role for the glucocorticoid in modulating airway surface liquid volume.This brief review aims to explore the molecular mechanisms of dexamethasone action in resolving airway flooding by regulating the activity of airway epithelial ion channels together with anti-inflammatory responses, while acknowledging the complex and multifaceted nature of COVID-19 disease.

Anti-inflammatory action of dexamethasone in COVID-19
Severe cases of COVID-19 are characterized by a 'cytokine storm', an excessive and dysregulated immune response, more often associated with over stimulation of inflammatory cytokines IL-6, IL-8, TNFα and IFN-γ [7].The anti-inflammatory actions of dexamethasone in COVID-19 have been reviewed extensively elsewhere [1,8] and are briefly summarised here so as to place these immune responses in the context of pulmonary oedema.Dexamethasone can reinforce immune responses in COVID-19 through the regulation of immune cells, as well as calming inflammatory responses via the inhibition of inflammatory transcription factors Nuclear factor-κB (NF-κB) and transcription factor complex Activator Protein-1 (AP-1).This leads to the suppression of proinflammatory cytokines (Il-1, IL-2, IL-6, IL-8) while blocking the transcriptional activation of TNFα and IFN-γ (Fig. 1) [9][10][11].Dexamethasone also promotes the expression of anti-inflammatory cytokines (IL-4, IL-10) and helper T-cells and B-cells [12].In addition, dexamethasone may accelerate the resolution phase in the late inflammatory response by stimulating the expression of pro-resolution lipid mediators such as resolvin D1 and lipoxin A4 in COVID-19 [13].Dexamethasone exerts its anti-inflammatory effects by binding to glucocorticoid receptors (GR), leading to a cascade of anti-inflammatory and immunosuppressive responses.These anti-inflammatory actions of dexamethasone are genomic responses, with a latency of hours to days, involving the binding of the glucocorticoid with the glucocorticoid receptor in the cytoplasm, followed by GR dimerization, recruitment of coactivators, nuclear translocation, chromatin binding and activation of glucocorticoid response elements to induce de novo synthesis of anti-inflammatory proteins or repress the transcription of inflammatory proteins (Fig. 1).Dexamethasone may also suppress pro-inflammatory cytokines indirectly through ion channels regulating Ca 2+ influx in T cells.The voltage-gated K + channel Kv1.3 controls Ca 2+ influx and IFNγ production in T cells which have been shown to be reduced by dexamethasone treatment in COVID-19 patients [14].The well-described anti-inflammatory action of dexamethasone certainly contributes to the resolution of lung inflammation in severe COVID-19.Several clinical observations, Fig. 1.Anti-Inflammatory actions of dexamethasone in the airways: Dexamethasone repression of pro-inflammatory cytokines and stimulated expression of antiinflammatory cytokines (IL-4, IL-10, lipocortin) and pro-resolution lipid mediators (resolvin-D1, lipoxin A4) are transduced by genomic signalling via nuclear glucocorticoid receptors (GR) interacting with glucocorticoid response elements (GRE).Dexamethasone inhibits the transcription factors NFkB and AP-1 in the cytosol where the ligand-activated GR acts through kinases, such as IKK kinase complex and MAPK, in the trans-repression of these transcription factors and regulation of the cytokine storm, working in concert with GRE-mediated expression of anti-inflammatory cytokines and pro-resolution lipids.In this way, dexamethasone represses the production of pro-inflammatory cytokines such as interleukin IL-1, IL-2, IL-6, IL-8, TNF, IFN-γ, and nflammatory mediators VEGF and prostaglandins.Importantly, these inflammatory cytokines and mediators are linked to SARS-CoV-2 severity.The anti-inflammatory and pro-resolution responses to dexamethasone may synergise to reduce the cytokine storm seen in severe COVID-19 airway disease.

B.J. Harvey
however, indicate additional mechanisms are involved in the therapeutic actions of dexamethasone in COVID-19.Firstly, significant beneficial effects of dexamethasone on the length of stay in ICU and mortality are only observed in patients with severe COVID-19 under active ventilation and supplemental oxygen and not in patients with mild to moderate disease [6], although airway inflammation is also present in the latter.Secondly, there was concern at the very start of the pandemic for flash-pulmonary oedema so fluid resuscitation was kept to a minimum.These observations point to additional beneficial effects of dexamethasone on airway secretion to reduce flooding of the airways.Possible targets are the ion channels which regulate airway surface liquid height and volume.

Dexamethasone and pulmonary oedema in COVID-19
Pulmonary oedema is still one of the main causes of mortality in COVID-19 and severe COVID-19 is associated with lung hypersecretion / fluid accumulation [4].Increased extravascular lung water (EVLW), an index of flooding of the airways, reflects long ICU dependency and mortality in COVID-19 associated ARDS [15].It is likely that failure in alveolar and bronchiolar fluid clearance plays a major role in the pathogenesis of COVID-19 pulmonary oedema.The imbalance of airway fluid hypersecretion, increased pulmonary vascular permeability and massive fluid exudation driven by Starling forces into the lung, combined with low pulmonary fluid clearance and increased colloidal pressure (a protein-rich EVLW), are key factors in the acute exacerbation of pulmonary oedema in COVID-19 patients.In the context of pulmonary oedema, the principal mechanisms by which dexamethasone could operate include anti-inflammatory effects, endothelial barrier protection, and anti-secretory actions to lower the volume of the airway surface liquid.By suppressing the release of pro-inflammatory cytokines, dexamethasone may reduce the excessive inflammatory response that often contributes to the development and progression of pulmonary oedema.Dexamethasone can also enhance the integrity of the pulmonary endothelial barrier, reducing vascular permeability and preventing fluid leakage into the lung tissue [16,17].These parameters have been investigated thoroughly and here we will focus on the novel aspects of dexamethasone action to reduce airway fluid secretion and lower airway surface liquid volume.

Airway surface liquid dynamics and mucociliary clearance
The airway surface liquid (ASL) is a thin hydrated layer bathing the airway epithelial cell surfaces and is composed of the periciliary layer (PCL) and a mucus layer overlying the PCL.The ASL is a critical component of the respiratory system and serves several vital functions: It acts as a protective barrier, trapping and removing inhaled pathogens, particulate matter, and foreign substances.It facilitates the movement of cilia, tiny hair-like structures on the airway epithelial cells, which help clear mucus and debris from the airways.It contains anti-bacterial compounds to fight infection and maintains the proper hydration of the airway epithelium, ensuring efficient gas exchange [18].Here we define ASL dynamics as the components of the layer of fluid overlying the airway surfaces which determine mucociliary clearance: the ASL height, hydration and viscosity, ciliary beat frequency and mucus secretion.The ASL is maintained under steady state conditions at a height just covering the length of outstretched cilia.The periciliary liquid layer has an optimal height of 7-10 μm equivalent to 1 µL/cm 2 of mucosal surface [19].The ASL is generated by transepithelial ion transport (and mucus secretions) with passive water flux following the Fig. 2. Ion transport generates the airway surface liquid height: Sodium ion absorption and chloride ion secretion generate an osmotic gradient for water flux across the airway epithelium.The transepithelial transport of Na + and Cl − is balanced so as to generate an optimal ASL height between 8 and 10 μm corresponding to the length of outstretched cilia.Sodium absorption occurs as a two-step process with Na + entry into the cell via ENaC Na + channels at the apical membrane and Na + is then pumped out of the cells via Na/K-ATPase pumps in the basolateral membrane.The charge balance and electrochemical driving force for Na + absorption is generated by the activity of KATP (Kir6.1)potassium ion channels in the basolateral membrane.Inhibition of any one of these ion transporters (ENaC, Na/K-ATPase, KATP) will produce a decrease in sodium and water absorption and as a consequence will increase ASLh, whereas stimulation of the Na + transport pathways will decrease ASLh.Chloride ion secretion also occurs as a two-step process, with Cl − entering the airway epithelial cell across the basolateral membranes via the Na:K:2Cl cotransporter and then transported down an electrochemical gradient across the apical membrane into the ASL via CFTR Cl − channels which support basal and cAMP-stimulated Cl − secretion, or via CaCC Cl − channels which are the pathway for calcium-activated Cl − secretion.The charge balance for CFTR-mediated Cl − secretion is provided by cAMP activated KCNQ1:KCNE3 K + channels and for CaCC channels via calcium-activated KCNN4 K + channels (the 'K + channel battery').Inhibition of any one the chloride ion secretion pathways will decrease water flux into the ASL and reduce ASLh, whereas stimulation of chloride ion secretion transporters will increase ASLh.

B.J. Harvey
osmotic gradient.The molecular mechanisms which sense the optimal ASL height (ASLh) are still unknown, whether they be osmotic stimuli, cilia tension or hormonal/paracrine signalling [20][21][22][23][24].The ion transporter mechanisms which generate the ASL, however, are welldescribed and are summarised in schematic form in Fig. 2. The optimal ASLh is a balance between net Cl − secretion and net Na + absorption with water flux following an osmotic gradient.Transepithelial Cl − secretion occurs by a two-step process with Cl − entry from the blood side across the basolateral cell membranes via the Na:K:2Cl cotransporter (NKCC).Chloride ions can then exit the cell across the apical membranes via cAMP-sensitive CFTR and calcium-activated Cl − channels (CaCC) [25].The charge balance for Cl − exit via CFTR is provided by cAMP-sensitive K + channels (KCNQ1:KCNE3) whereas Cl − flux via CaCC is balanced by calcium-dependent K + channels (KCNN4) [26][27][28][29].
Transepithelial Na + absorption is also a two-step process conforming to the Ussing model [30].Sodium ions enter the cell from the airway side via the epithelial Na + channel (ENaC) and exit the cell across the basolateral membranes via the Na/K-ATPase pump.Charge balance for sodium ion absorption is dependent on recycling of K + via inwardly rectifying ATP-sensitive K + channels (Kir6.1,KATP) [31].The activity of the basolateral K + channels and Na/K-ATPase pump are vital in maintaining the favourable electrochemical driving forces for all sodium absorptive and chloride secretory ion transport routes.Transepithelial water flux mainly takes a cellular route via aquaporin channels located in apical and basolateral membranes and water moves down its osmotic gradient generated by Na + absorption and Cl − secretion [32].Activation or inhibition of any one of the ion channels, transporters or pumps along the cellular transport routes will affect the absorption and secretion rates for Na + and Cl − and water and, by consequence, modulate the ASLh.The generation of the optimal ASLh is essential for an effective mucociliary clearance [33].
Mucociliary clearance is a key physiological component of airway health to clear the large and smaller airways of invasive pathogens, excess mucus and inspired inflammatory particles [34][35][36].This mucociliary 'escalator' is dependent on airway surface liquid dynamics involving the generation of an optimal ASLh and the beating of cilia lining the upper airways [37].During an active stroke the cilia make contact with the overlying mucus layer and propel mucus and trapped pathogens towards the mouth at a rate of ~3 mm min − 1 [38].In this way the airways are kept relatively sterile and clean of irritants.The generation of the ASLh and ciliary beating are under the control of ion channels which regulate, respectively, the electrical driving force for electrolyte and water transport across the airway epithelium and control calcium and protein kinase signalling essential for the 'whiplash' stroke of cilia [39][40][41].Dysfunction in these regulatory mechanisms of ASL dynamics and mucocilary clearance can contribute to pulmonary oedema in COVID-19.

ASL dynamics in COVID-19
Severe COVID-19 is characterised by hypersecretion of airway surface fluid and dysregulation of airway surface liquid dynamics leading to poor mucocciliary clearance.The SARS-2-CoV virus is thought to destabilise adherens junctions which can disrupt tight junctions in the airway epithelium [42][43][44].A leaky airway epithelium allows plasma transudation from the blood side into the airways.The increased subepithelial hydrostatic pressure and intraluminal colloid pressure as a result of widespread pulmonary inflammation and cytokine damage to the respiratory epithelium contribute to bulk flow of fluid into the lungs.This hypersecretion and plasma transudation become an important contributor to the degradation of airway surface liquid dynamics and flooding of the airways in severe COVID-19 [4].
While direct research on ASL height and ASL dynamics in COVID-19 is limited, there are several reasons why it could be of interest in explaining therapeutic effects of dexamethasone in COVID-19 (Fig. 3): Dexamethasone can lower ASL height by inhibiting the ion channels which produce chloride secretion [45,46] and activating the ion channels supporting sodium absorption [47], thus inducing water flow out of the airway surface into the blood and contributing to lowering the ASL height and volume to reduce flooding of the airways.Alterations in ASL height also impact the efficacy of cilia to clear mucus which is compromised in a flooded airway, thus potentially affecting the body's ability to clear pathogens and viral particles, including SARS-CoV-2.Dexamethasone regulation of ASL height and the state of the airway epithelial barrier may influence the cytokine storm response in the respiratory tract to further reducing inflammation.In the following section we will explore the molecular mechanisms underpinning the modulation of airway surface liquid dynamics by dexamethasoneregulated ion channels.

Glucocorticoid regulation of airway secretion and absorption
Glucocorticoids have been reported to modify the activity of ion channels which regulate airway secretion and absorption in a way which can reduce ASL height and maintain proper airway function in COVID-19.Indeed, both hydrocortisone and dexamethasone in addition to their anti-inflammatory actions, have been shown to activate sodium absorption via increased ENaC activity [48,49] and to inhibit chloride secretion via inhibition of calcium-dependent chloride secretion pathways [3,46] in airway epithelia.These combined pro-absorptive, antisecretory responses to glucocorticoids can draw water out of the airways.This is a physiological function of corticosteroids at birth.In the foetal lung, the alveolar spaces are flooded to facilitate gas exchange with maternal blood.This flooding is achieved by secretion of chloride ions to produce a net water flux into the alveolar space [50,51].At birth, this process is reversed and fluid secretion is greatly decreased while sodium absorption is stimulated via activation of ENaC and Na/K-ATPase pumps in the alveolar epithelium, mainly triggered by a surge in catecholamines and corticosteroids [52][53][54].The net effect is to reabsorb fluid and prevent alveolar oedema [55,56], thus producing a thin layered alveolar surface fluid ideal for gaseous exchange and to prepare the lungs for air breathing.ENaC channel expression and activity in the airway epithelium are modulated by glucocorticoids acting via glucocorticoid receptors in the alveoli and larger airways.Unlike other ENaC expressing epithelia such as the distal renal nephron, airway epithelium ENaC channels are not regulated by aldosterone or the mineralocorticoid receptor, although aldosterone has been reported to modulate intracellular calcium signalling in human bronchial epithelium [57] which may exert indirect effects on ion channel regulation of ASL dynamics.

Dexamethasone regulation of ion channels and ASL dynamics with relevance to COVID-19
Emerging evidence suggests that dexamethasone can modulate the activity of ion channels in the airway epithelium which affect immune responses [14], mucus secretion [58] and the hydration of the airways [59].Recent reports have highlighted the action of dexamethasone to inhibit the secretion of chloride ions which can lead to increased fluid absorption from the ASL into the airway epithelial cells [45].This response to dexamethasone can help in thinning the ASL, which would be beneficial in conditions where hypersecretion and pulmonary fluid accumulation are present in COVID-19.The anti-secretory effect of dexamethasone was shown to be the result of inhibition of CFTR and CaCC Cl − secretory pathways.The reduction in cAMP -dependent Cl − secretion is an indirect effect of dexamethasone and caused by an inhibition of KCNQ1 basolateral K + channels [27,45] (Fig. 4A).Dexamethasone also produces a rapid and sustained inhibition of calciumactivated Cl − secretion which again is indirect but involving a different class of calcium-activated KCNN4 K + channels (Fig. 4B) [27,45].
Dexamethasone may reduce ASL height further by stimulating ENaC and sodium ion absorption.This effect has been demonstrated in rat alveolar airway epithelium in which dexamethasone stimulated ENaC channel activity and increased sodium and water absorption [38] (Fig. 5A).An interesting finding of these studies was the ability of dexamethasone to upregulate α, β, and γENaC mRNA expression and enhance ENaC stability in the apical membrane [48,[60][61][62].Dexamethasone could also reverse the TNFα inhibition of ENaC and produce an even greater stimulation of sodium and water absorption [47] (Fig. 5B).This effect is very relevant to COVID-19 where TNFα is a component of the cytokine storm and could potentially exacerbate pulmonary oedema through inhibition of ENaC and water reabsorption The pro-absorptive actions of dexamethasone on ENaC do not appear to be sex-specific.A synergistic effect of dexamethasone with estrogen on ENaC was not observed in foetal-derived lung epithelium [63] The contribution of ENaC to adult alveolar function in health and disease is well documented [64].An interesting finding in this regard is the activation of ENaC by increased ASL volume [49] implicating a negative feedback regulation of ASLh during expansion of lung fluid volume.The stimulation of ENaC by dexamethasone and the potential to reduce ASLh can be expected to have positive effects on lung function in disease which cause flooding of the airways, in particular in severe COVID-19.The positive effects of dexamethasone on sodium and fluid absorption points to a role for ENaC in resolving pulmonary oedema in ARDS associated with COVID-19.This hypothesis is strengthened by observations on the essential role of ENaC mediated sodium absorption across an intact epithelial barrier in resolving alveolar oedema and reducing mortality [65][66][67].

Integration of genomic and non-genomic actions of dexamethasone in COVID-19
Dexamethasone modulates airway surface liquid dynamics by regulating the activity of ion channels and transporters via both latent nuclear-initiated genomic and rapid membrane-initiated non-genomic cell signalling mechanisms (Fig. 6).Non-genomic corticosteroid responses are characterised by a rapid onset of signal transduction usually  Dexamethasone interacts with a pertussis toxin sensitive G-protein coupled receptor (GPCR) possibly coupled to a membrane glucocorticoid receptor (mGR) to activate adenylyl cyclase IV, cAMP, PKA, PKCδ to phosphorylate KCNQ1:KCNE3 causing the uncoupling of the K + channel complex and collapsing the electrical driving force for CFTR-mediated Cl − secretion [27,45,46].Dexamethasone also activates PKC signalling pathways to phosphorylate and inhibit KCNN4 channels and thus reduce the electrical driving force for CaCC -mediated Cl − secretion.Although the anti-secretory responses are rapid in onset they are also sustained, most likely as a result of prolonged kinase phosphorylation of the channels and delayed genomic activation of mRNA expression of ion channel proteins.Since dexamethasone targets oestrogen-regulated KCNQ1 channels, this may confer sexual dimorphism on the anti-secretory response to the glucocorticoid.Genomic Signalling: Dexamethasone stimulates Na + absorption by increasing the transcription of ENaC Na + channel subunits α, β, γ in parallel with stimulating the expression of the Na/K-ATPase pump subunits α1, β1 [48,62,71].The combined effect of anti-secretion and pro-absorption responses to dexamethasone results in transcellular water absorption from the airway surface to the blood side.The anti-inflammatory response will also enhance the maintenance of the epithelial barrier to decrease fluid exudation into the airways.Genomic and non-genomic dexamethasone signalling synergise to reduce airway surface liquid volume and help to resolve pulmonary oedema.

B.J. Harvey
within minutes and are differentiated from latent genomic transcriptional responses requiring hours to days for activation [68].A membrane-associated glucocorticoid receptor (mGR) has been postulated to transduce such rapid responses for glucocorticoids [69], including a plasmamembrane-tethered form of the canonical GR [70] or by direct ligand activation of a G-protein coupled receptor (GPCR), [71].The molecular identity of the putative membrane GR remains elusive and may work in tandem with a GPCR [72].The rapid actions of dexamethasone on airway ion channels and sensitivity to pertussis toxin and adenylyl cyclase inhibitors, suggest an association of the membrane glucocorticoid receptor with a GPCR [73] which transduces downstream signaling via adenylyl cyclase (identified as AC isoform IV) and cAMP/ PKA modulation of ion channel gating [45].
Under steady state conditions or in the presence of cAMP-activating agonists (such as adrenergic agonists), chloride secretion occurs via CFTR.Dexamethasone reduces CFTR-mediated Cl − secretion as a result of inhibition of the basolateral membrane KCNQ1 K + channel which exists as a heterodimer with a KCNE3 regulatory subunit (which confers cAMP sensitivity to the KCNQ1 channel).Dexamethasone rapidly induces a PKCδ phosphorylation of the KCNQ1:KCNE3 channel complex resulting in its dissociation and collapse of the channel conductance.The closure of the KCNQ1 channel causes membrane electrical depolarization which reduces the electrical driving force for Cl − exit via CFTR.This is very similar to the molecular mechanism for the anti-secretory response to oestrogen involving oestradiol inhibition of KCNQ1 channels.Since oestrogen regulates the mRNA and protein expression of KCNQ1 and PKCδ [74], this may confer a sexual dimorphism upon dexamethasone anti-secretory actions in airway epithelia.It is worth noting that clinically proven sex differences exist with a female advantage in COVID-19 severity and mortality, underpinned by oestrogen actions on immune responses and airway biology [75,76].
In the absence of CFTR or when Cl − secretion is activated by calciummobilizing agonists (muscarinic receptor agonists such as acetylcholine), chloride secretion occurs principally through CaCC channels.Dexamethasone indirectly inhibits Cl − exit via CaCC channels by causing a rapid PKC -dependent phosphorylation and closure of basolateral KCNN4 channels which reduces the electrical driving force for calcium-stimulated Cl − secretion (Fig. 6).Thus dexamethasone can elicit a very potent anti-secretory response in airway epithelium by inhibiting both basal and cAMP-sensitive Cl − transport (60 % reduction) as well as Ca 2+ activated Cl − secretion (70 % reduction) [27,45].The antisecretory responses to dexamethasone are rapid, occurring within minutes, whereas dexamethasone stimulation of sodium absorption involves latent genomic signalling transduced by nuclear GR to increase the transcription of ENaC channel α, β,γ subunits [48,62] together with parallel stimulation of α1, β1 subunits of the Na/K-ATPase pump (Fig. 6) [77].Latent transcriptional effects of glucocorticoids have also been demonstrated in regulating the expression of ENaC and CFTR during the development of a well-differentiated human bronchial epithelium [78].It is quite interesting that dexamethasone (and other glucocorticoids such as hydrocortisone) exert both rapid and latent effects on ion transporters in the airways.These actions most likely synergise to produce a rapid and sustained response to meet the demands of maintaining an acute onset and chronic regulation of airway surface liquid volume essential for effective mucociliary clearance.
Given the common ion channel targets of dexamethasone in modulating inflammation and airway secretion responses, it is interesting to speculate that both genomic and non-genomic actions of glucocorticoids could synergise to alleviate pulmonary oedema in COVID-19.Indeed, as summarised in Fig. 6, dexamethasone affects many common resolution targets of pulmonary oedema in the airway epithelium, from reducing inflammation by repressing inflammatory cytokines to modulating electrolyte and water transport to reduce airway flooding.The actions of dexamethasone can be viewed as early (non-genomic) and late (genomic) although many studies have now shown that a strict separation of these actions is most likely out-of-date as cross-talk between non-genomic (membrane-initiated signaling) and genomic (nuclear-initiated signaling) exists for all steroid receptors.The early effects of the steroid can be permissive for genomic events to occur and provide an additional mechanism for the regulation of gene transcription.Similiarly, nuclear signaling may influence the responsiveness of non-genomic responses through expression of receptors and protein kinases [79].

Conclusions
Dexamethasone, with its anti-inflammatory and immunosuppressive properties, has emerged as a valuable tool in the management of pulmonary oedema, particularly in the context of ARDS associated with severe COVID-19.The actions of dexamethasone to reduce airway fluid secretion combined with suppression of pro-inflammatory cytokines may contribute to the advantage of corticosteroid treatment seen in severe COVID-19 and other forms of acute respiratory distress syndrome.Although the role of dexamethasone in regulating (reducing) ASL volume in COVID-19 is still unknown, there is a good deal of evidence that glucocorticoids regulate airway surface liquid volume in other airway diseases characterised by ARDS and during pre-term and post-partum lung clearance through actions on ion channels (ENaC, CFTR).This knowledge combined with the clinical observations that increased extra-vascular lung water volume is a hallmark of severe COVID-19 [15], points to a therapeutic role for dexamethasonemodulated ion channel effects on ASL volume to be more than a speculative theory but rather a testable hypothesis [80].
Ion channels play a fundamental role in the regulation of airway surface liquid volume which is essential for maintaining a healthy respiratory function.Dysregulation of ion channels leads to altered ASL electrolyte and water composition and volume, contributing to various respiratory diseases and conditions.The concentration of dexamethasone required to effect changes in ion channel activity and ASL dynamics lie within a 'physiological' range.A comparison of dosages used in the RECOVERY Trial [6] would have yielded a steady-state dexamethasone concentration in extracellular fluid volume (19L) of 16 nmol/L (dosage 6 mg/day for 10 days).For comparison, dexamethasone activation of Na + absorption and inhibition of Cl − secretion was reported at 1 nmol/ L. Thus, the dexamethasone concentration used to modulate ion transport and airway surface liquid dynamics is well-within the range of the maintenance dosages commonly used to treat airway inflammation (0.3-0.5 mg/day equivalent to 0.8 -1.3 nmol/L).Moreover, the effects of dexamethasone on airway ion transport and ASL volume are receptormediated and involving protein kinase signaling which would rule out indirect non-specific effects of the glucocorticoid on the ion channels.
Dexamethasone effects on ASL dynamics reveal the hormone's influence beyond anti-inflammatory functions.Here we have highlighted these novel aspects of dexamethasone regulation of ion channels controlling airway secretion and absorption which can play and important role in reducing lung water volume and resolve airway flooding.Modulation of ENaC, CFTR, CaCC, KCNQ1 and KCNN4 ion channels by dexamethasone can reduce excessive airway hydration and improve mucociliary clearance.A synergistic action of genomic and non-genomic cell signalling may confer additional benefits for therapeutic actions dexamethasone in resolving ARDS in COVID-19.It is essential to acknowledge that dexamethasone is not without limitations in COVID-19 [72].Prolonged or high-dose glucocorticoid use can lead to a range of side effects, including immunosuppression, hyperglycaemia, and secondary infections.With respect to dysglycaemia adverse reactions, of the 19,355 patients who were eligible to receive dexamethasone in the RECOVERY COVID TRIAL [6], only 2 patients were found to present with hyperglycaemia associated with the dexamethasone treatment.Thus in this cohort of severe COVID-19 patients, the benefits of reduced mortality greatly outweighed the risks of transient hyperglycaemia.The question is raised whether such complications are found in milder cases.A recent clinical study showed that half of 'mild' (non-ICU) COVID-19 patients treated with dexamethasone presented with hyperglycaemia.B.J. Harvey [81].Again, the dysglycaemia was transient and manageable with systematic glucose monitoring and addition of rapid acting insulin with meals for optimal glycaemic control.
The timing of dexamethasone administration in the course of COVID-19 infection is crucial; it is typically recommended for use in severe cases, where an exaggerated immune response is more likely to be harmful.The beneficial actions of dexamethasone to mitigate the cytokine storm and prevent further inflammation in the lungs has proven to be a game-changer.However, it is to be noted that dexamethasone has not demonstrated the same benefit in mild or asymptomatic COVID-19 cases.In fact, it may be counterproductive in such cases by suppressing the body's natural defence mechanisms [82][83][84].
In the fight against the COVID-19 pandemic, dexamethasone has emerged as a valuable tool in the therapeutic arsenal, offering hope for patients with severe respiratory complications.Understanding the effects of glucocorticoids on ion channels in airway diseases holds significant therapeutic potential.Targeting specific ion channels affected by dexamethasone could offer novel therapeutic avenues for the management of airway diseases, potentially leading to more effective and personalized treatments [85].Further investigation into the precise molecular mechanisms underlying dexamethasone-ion channel interactions is warranted to unlock the full therapeutic potential of targeting corticosteroid-related pathways for the management of airway diseases.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 3 .
Fig. 3. ASL dynamics in airway diseases: In COVID-19, the SARS-CoV-2 virus causes severe lung inflammatory responses and fluid exudation which can cause flooding of the alveoli and central airways.The hugely increased ASLh renders mucociliary clearance difficult leading to increased viral load and a vicious cycle of inflammation and airway flooding.Dexamethasone can reduce ASL hydration, especially in the alveoli by stimulating Na + absorption (which is inhibited by cytokines and TNFα) and contribute to better survival in severe COVID-19 patients.

Fig. 4 .Fig. 5 .
Fig. 4. Dexamethasone inhibition of Cl − secretion: Dexamethasone inhibits (A) CFTR -mediated and (B) CaCC -mediated chloride ion secretion by inhibiting the activity of KCNQ1 and KCNN4 channels, respectively.Cl − secretion activated by cAMP agonists such as forskolin or adrenergic stimulation can be rapidly inhibited by dexamethasone acting on KCNQ1 channels, whereas dexamethasone targets KCNN4 channels to inhibit calcium-activated Cl − secretion stimulated by purinergic (ATP) or muscarinergic (acetylcholine) agonists.The diverse nature of the anti-secretory response indicates that dexamethasone can reduce ASL height irrespective of the cellular transport pathways and agonists of chloride ion secretion.Adapted from[45]

Fig. 6 .
Fig. 6.Genomic and Non-Genomic cell signalling mechanisms for anti-secretory and anti-inflammatory responses to dexamethasone: Membrane-initiated Signalling:Dexamethasone interacts with a pertussis toxin sensitive G-protein coupled receptor (GPCR) possibly coupled to a membrane glucocorticoid receptor (mGR) to activate adenylyl cyclase IV, cAMP, PKA, PKCδ to phosphorylate KCNQ1:KCNE3 causing the uncoupling of the K + channel complex and collapsing the electrical driving force for CFTR-mediated Cl − secretion[27,45,46].Dexamethasone also activates PKC signalling pathways to phosphorylate and inhibit KCNN4 channels and thus reduce the electrical driving force for CaCC -mediated Cl − secretion.Although the anti-secretory responses are rapid in onset they are also sustained, most likely as a result of prolonged kinase phosphorylation of the channels and delayed genomic activation of mRNA expression of ion channel proteins.Since dexamethasone targets oestrogen-regulated KCNQ1 channels, this may confer sexual dimorphism on the anti-secretory response to the glucocorticoid.Genomic Signalling: Dexa-