SARS-CoV-2 may hijack GPCR signaling pathways to dysregulate lung ion and fluid transport.

The tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, toward the host cells is determined, at least in part, by the expression and distribution of its cell surface receptor, angiotensin-converting enzyme 2 (ACE2). The virus further exploits the host cellular machinery to gain access into the cells; its spike protein is cleaved by a host cell surface transmembrane serine protease 2 (TMPRSS2) shortly after binding ACE2, followed by its proteolytic activation at a furin cleavage site. The virus primarily targets the epithelium of the respiratory tract, which is covered by a tightly regulated airway surface liquid (ASL) layer that serves as a primary defense mechanism against respiratory pathogens. The volume and viscosity of this fluid layer is regulated and maintained by a coordinated function of different transport pathways in the respiratory epithelium. We argue that SARS-CoV-2 may potentially alter evolutionary conserved second-messenger signaling cascades via activation of G protein-coupled receptors (GPCRs) or by directly modulating G protein signaling. Such signaling may in turn adversely modulate transepithelial transport processes, especially those involving cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC), thereby shifting the delicate balance between anion secretion and sodium absorption, which controls homeostasis of this fluid layer. As a result, activation of the secretory pathways including CFTR-mediated Cl- transport may overwhelm the absorptive pathways, such as ENaC-dependent Na+ uptake, and initiate a pathophysiological cascade leading to lung edema, one of the most serious and potentially deadly clinical manifestations of COVID-19.

The variable clinical manifestations are characteristic of the ongoing global outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)(1). The majority of the infected patients are asymptomatic or report mild symptoms and recover completely. Approximately 2/3 of the patients with severe COVID-19, however, develop pneumonia, with ∼25% requiring mechanical ventilation, which in turn is associated with up to 80% of the mortality (2); demographically, these are usually older patients with comorbid conditions. Both the expression level of angiotensin-converting enzyme 2 (ACE2), the entry point of SARS-CoV-2 into cells, in the respiratory epithelia (3, 4) and the extent of the resulting inflammatory response (5) are suspected to determine the rapid deterioration of patients and the development of an acute respiratory distress syndrome (6). SARS-CoV-2 shows a gradient infectivity or replication efficiency from the proximal to distal airways that parallels ACE2 expression (7), with ciliated cells being the most ACE2-expressing cells. In upper airways, the ciliated cells dominate the nasal epithelium and show a high rate of infectivity whereas alveolar type II (ATII) epithelial cells are preferentially targeted by the virus in the distal airways (7). In line with competitive binding of SARS-CoV-2 to endogenous or exogenous ACE2, human recombinant soluble ACE2 reduced SARS-CoV-2 load in cell cultures and organoids (8), and the encouraging treatment of a patient with severe COVID-19 with intravenous infusion of soluble ACE2 was reported recently (9). Overall, ACE2 expression in airway epithelia is, however, not high (10) and is not only proposed to serve as an entry portal for the virus (3, 4) but also fulfills an important protective role against lung injury (11, 12). Furthermore, interventions which supposedly increase ACE2 expression in animal models (13), such as angiotensin receptor blockers (ARBs), have not been found to be associated with higher mortality in severe COVID-19 (14).

Taken together, the variable expression pattern of ACE2 in respiratory epithelia and its complex role in lung physiology and pathophysiology indicate that ACE2 (or TMPRSS2) expression levels and patterns may not fully reflect the susceptibility of cells to the virus but rather that other factor(s) may, at least in part, play a role in severe COVID-19 lung disease. Here, we consider the possibility that failure of the primary defense mechanism of the respiratory tract, the mucociliary clearance (MCC) and a disruption in alveolar liquid clearance (15, 16), may present one of the key factors in the pathophysiology of COVID-19 lung disease.

The airway surface liquid (ASL) is a thin layer of fluid that covers the respiratory tract (17) which consists of two components (18). The first part, a mucous layer (ML), traps and clears airways of inhaled harmful agents and sits on top of the second component, the periciliary layer (PCL), which provides the appropriate setting for cilia beating. The appropriate volume and viscosity of the fluid layer, an important determinant of the proper functioning of the MCC (18), is achieved through coordinated processes of secretion and absorption that are orchestrated by a synchronized operation of several transepithelial transport pathways (17). Among the numerous epithelial transporters, cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC) are recognized as the key players that determine ASL balance. CFTR functions as an ATP and protein kinase A (PKA)-dependent Cl− channel (19–21) and serves as a passageway for Cl− secretion whereas ENaC fulfills a key role in Na+ reabsorption (22, 23), with a well-described reciprocal relationship between these two proteins (24–28). In the airways, they regulate the ASL and play an important role in MCC (18). In the alveolar space, CFTR and ENaC participate in the regulation of the volume of the hypophase (a thin layer of the alveolar lining fluid) to maintain a thin air-liquid interface while preventing alveolar edema formation. Impaired ENaC function in ATII cells, as seen in acute lung injury, causes a reversal of alveolar chloride and fluid flux from absorptive to secretory mode (29–31), resulting in alveolar edema. Hence, any process that increases Cl− secretion or decreases Na+ reabsorption will interfere with MCC and alveolar fluid clearance, thus potentially impairing primary defense in the airways while concomitantly causing flooding of the alveolar space (30, 31).

An elaborate set of adaptor proteins (20, 32, 33) connect CFTR and ENaC to each other and serve as cellular signaling hub connecting these channels to intracellular signaling networks that regulate cellular homeostasis. Central to the regulation of this signaling hub are G protein-coupled receptors (GPCRs), the largest superfamily of receptors in humans (34), which regulate almost all known physiological processes (35, 36). GPCRs primarily function through signal transduction/propagation cascades (34, 37). Located at the cell surface, ligand-bound GPCRs transduce exogenous signals that activate GTP-binding “G” proteins which in turn activate effector proteins (such as adenylyl cyclase and phospholipases) and second messengers (e.g., calcium or cAMP) (34, 37). CFTR activity is regulated via the cAMP/PKA pathway (38) which is typically induced via Gαs-coupled GPCRs that stimulate adenylyl cyclase (AC), thereby raising cAMP levels and stimulating PKA (39). These endogenous signaling pathways are, however, frequently exploited by invading pathogens (40). For example, cholera toxin (from Vibrio cholerae) causes ADP-ribosylation of the α subunit of the stimulatory G protein, which in turn activates adenylate cyclase with a subsequent increase in cAMP that stimulates CFTR and triggers the excessive chloride secretion and fluid loss in the gut that are characteristics of cholera (41).

Two specific GPCRs are mainly involved in CFTR regulation and robustly expressed in airways, namely, A2B adenosine receptors and the β2 adrenergic receptors (39, 42). Under physiological conditions, the adenosine-CFTR regulation system is critical for the protection of mucosal airway surfaces (43) and alveolar surface layer (ASL) regulation (44). Viruses, however, are well recognized for their ability not only to exploit GPCRs to enter the host cells but also to use their intracellular signaling pathways for survival and replication (40). Based on this general concept, it is conceivable that SARS-CoV-2 may also potentially compromise GPCRs signaling and this effect may, at least in part, contribute to the pathophysiology of pulmonary edema. A simple and tempting speculation would be that SARS-CoV-2 may hijack the same GPCR signaling pathway that is used by cholera toxin (41)—namely, ADP-ribosylation of Gαs—to activate CFTR (Fig. 1) and turn on Cl− secretion, ultimately leading to lung edema in patients with COVID-19 (48, 49). At this point, however, this hypothesis seems unlikely as several reports suggest that SARS-CoV-2 in fact reverses cellular ADP-ribosylation to allow the virus to evade immune detection (51–53). Whether or not G proteins are involved in this response remains to be determined.

Figure 1.

Hypothetical concept for the dysregulation of lung epithelial ion and fluid transport by SARS-CoV-2. SARS-CoV-2 binds to ACE2, is endocytosed into the cell, and uses the host cellular machinery for RNA replication and translation resulting in the production of viral proteins. These viral proteins may potentially interact with G proteins, triggering the classic GPCR pathway and activating CFTR. Activation of CFTR in turn can inhibit ENaC. Secondary mediators may in parallel or alternatively activate GPCRs from the outside (45, 46). For example, ATP may serve as secondary mediator and engage A1AR [(green arrow) which in turn stimulates CFTR; activation of CFTR in turn can inhibit ENaC]. UTP can engage P2Y6R (blue arrow) and inhibit amiloride sensitive ion transport via ENaC. In addition, UTP can lead to purinergic-mediated calcium mobilization from intracellular stores, which may combine with the influx of calcium via TRPV4 (acting as a mechano- and/or chemosensor) to increase intracellular calcium levels that can in turn activate CFTR; this activation of CFTR can cause chloride and fluid secretion, and inhibition of sodium reabsorption via ENaC (ENaC is not depicted in the left side of the figure). The potential link between TRPV4 and CFTR (47) could involve a Ca2+-dependent activation of adenylyl cyclase and the classical PKA pathway, whereas tyrosine phosphorylation can cause PKA-independent CFTR activation (47–49). NHERF1 anchors CFTR (via ezrin, not shown) to the actin cytoskeleton. Increased levels of cAMP allow PKA and EPAC1 to promote opening and stabilization of CFTR at the plasma membrane (50), again causing chloride and fluid secretion. ACE2, angiotensin converting enzyme 2; cAMP, cyclic AMP; CFTR, cystic fibrosis transmembrane conductance regulator; ENaC, epithelial Na+ channel; EPAC1, exchange protein directly activated by cAMP; ER, endoplasmic reticulum; GPCR, G protein coupled-receptor; NHERF1, Na+/H+-exchanger regulatory factor isoform 1; PDZ, postsynaptic density-95 discs large, zona occludens-1 (PDZ) domain-containing protein; PKA, protein kinase A; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TRPV4, transient receptor potential vanilloid 4.

In addition to the established cAMP/PKA activation pathway, a second regulatory pathway for transepithelial ion transport acts through EPAC1 (the exchange protein directly activated by cAMP) and stabilizes CFTR at the plasma membrane by inhibiting its endocytosis (50). This pathway is also cAMP-dependent, with EPAC1 acting as an alternative cAMP effector (to PKA) and interacting with CFTR via the Na+/H+-exchanger regulatory factor isoform 1 (NHERF1) (32, 50). Importantly, EPAC1 plays an important role in the regulation of Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV viral replication (54), suggesting that both viruses exploit a cAMP-dependent signaling pathway for infection of and replication in human host cells. Notably, this pathway also links CFTR and NHERF to lysophosphatidic acid receptor 2 (LPA2) (42). LPA2 is a GPCR (55) and its stimulation by LPA inhibits CFTR activity; accordingly, in vitro disruption of the association between LPA2 and NHERF leads to activation of CFTR (56). Interestingly, in mice, LPA markedly decreased CFTR-mediated intestinal fluid secretion induced by cholera toxin (56). At present, little is known about lipid signaling in general and lysophosphatidic acid specifically in the context of SARS-CoV-2 infection.

In a recent perspective, Kuebler et al. (57) argued that the calcium channel, the transient receptor potential vanilloid 4 (TRPV4), may play an important role in COVID-19 clinical progression from mild symptoms to severe disease. This hypothesis is based on the rationale that activation of TRPV4 results in impaired barrier function of the alveolocapillary membrane, which is the hallmark of acute respiratory distress syndrome (ARDS) (6, 57). This pathway may be equally relevant to transepithelial ion transport, as TRPV4 stimulation activates CFTR via elevated intracellular calcium levels (47). It thus seems possible that the activation of both channels may synergistically contribute to the development of pulmonary edema in SARS-CoV-2 infection via independent and/or interdependent pathways.

Indeed, several viruses that cause respiratory infections have been shown to dysregulate ion transport in the airspaces (45,46, 58–60). Specifically, influenza virus (61, 62), respiratory syncytial virus (RSV) (63), and SARS-CoV (64) inhibit ENaC function in a protein kinase C (PKC)-dependent manner. Furthermore, the influenza M2 protein, which functions as a proton- conducting transmembrane channel, decreases ENaC activity by targeting the channel for ubiquitin-mediated proteasomal degradation (65). These findings indicate that the influenza virus interferes with the absorptive functions of ENaC and may thus contribute to pulmonary edema formation (45). In addition to ENaC dysregulation, the influenza M2 protein also promotes CFTR ubiquitination (66) while impairing CFTR maturation; the latter effect is likely caused by M2 protein functioning as a proton channel and increasing vesicular acidification. It is conceivable that SARS-CoV-2 may similarly exploit the PKC-dependent pathway and inhibit both CFTR and ENaC functions; in this case, only ENaC inhibition would contribute to lung edema formation. The severity of lung disease in patients with COVID-19, however, suggests that SARS-CoV-2 rather engages the classic GPCR pathway, resulting in a combined CFTR activation and ENaC inhibition.

P2 receptors and purinergic signaling play an important role in alveolar homeostasis and contribute to several lung pathologies, including ARDS and ventilator-induced lung injury (67–70). P2 receptor activation primarily depends on ATP (and most likely UTP) that is present in the alveolar hypophase at very low concentrations (71) but increases in mechanically induced lung injury (70) and some viral infections (45, 72). Binding of extracellular nucleotides activates ionotropic (P2X) and metabotropic (P2Y) purinoreceptors, which—depending on the receptor involved—in turn trigger second messenger responses and signaling cascades (67, 70). These responses can be complex and diverse, as a considerable variety of P2X and P2Y receptors are expressed on both resident and nonresident populations of lung cells including alveolar type I and type II cells, endothelial cells, macrophages, and neutrophils (70). Several studies point to the possibility that some viruses including influenza virus A and RSV can regulate CFTR and ENaC- mediated transport pathways via secondary viral mediators (46). Specifically, ATP and UTP, probably released into the alveolar space via volume-regulated anion channels (73–77), pannexin 1 channel (78–80), or other ATP release mechanisms (67, 70, 81), can serve as secondary mediators of viruses and engage A1AR and P2Y6R, two GPCRs that are abundantly expressed on respiratory and/or alveolar epithelial cells (46). Increased UTP levels inhibit amiloride-sensitive ion transport via P2Y6R, whereas elevated levels of ATP cause increased CFTR-mediated Cl− secretion via the A1AR receptor; in combination, these differential effects will synergize to cause extensive alveolar edema (46). It is conceivable that SARS-CoV-2, replication of which requires high levels of ATP (82), similarly uses such secondary mediators which in turn will regulate epithelial ion and fluid transport in an auto- or paracrine fashion (Fig. 1). This signaling cascade may act in parallel to the classic intracellular pathways. A combination of both effects may create a vicious cycle that keeps ENaC consistently inhibited and CFTR active, thus accounting for the development of severe ARDS in COVID-19. The picture is further complicated by the fact that the disturbed balance of pro- and anti-inflammatory cytokines (83–86) caused by SARS-CoV-2 (5) may further dysregulate ion-transporting pathways and contribute to lung edema in COVID-19 (85).

At present, these discussions are by nature hypothetical. In the absence of appropriate tools to measure transepithelial ion and fluid transport in patients with COVID-19 or adequate animal models, the functional consequences of SARS-CoV-2 infection on the homeostasis of the epithelial lining fluid in the airways and alveolar space remain speculative. That notwithstanding, the clinical presentation of severe COVID-19 and the precedent of other respiratory viruses—including SARS-CoV and MERS—hijacking GPCR signaling pathways and dysregulating ENaC and CFTR point to a very real possibility for a similar scenario in SARS-CoV-2 infection. The resulting imbalance between absorptive and secretory pathways in the airspace may relevantly contribute to the formation (and/or lack of resolution) of alveolar edema, thus, mimicking the effects of cholera toxin in intestinal epithelial cells (41). The functional significance of these arguments and their implications in the pathophysiology of COVID-19 require further study (87) but may be critical (88) in managing the respiratory pathology that drives severe and ultimately, fatal COVID-19.

GRANTS

This work was supported, in whole or in part, by MBRU-COM Internal Grant Awards MBRU-CM-RG2018-04 and MBRU-CM-RG2018-05 (to M.U. and B.K.B.), Sandoq Al Watan Research & Development Grant SWARD-F2018-002 (to M.U. and B.K.B.), AlMahmeed Collaborative Research Award 2018 (to M.U. and B.K.B.), and Al Jalila Foundation Grant AJF201763 (to M.U.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

B.K.B. conceived and designed research; R.A.H. and M.U. prepared figure; R.A.H., W.M.K., M.U., and B.K.B. drafted manuscript; R.A.H., E.C-B., W.M.K., M.U., and B.K.B. edited and revised manuscript; R.A.H., E.C-B., W.M.K., M.U., and B.K.B. approved final version of manuscript.