Airway surface hyperviscosity and defective mucociliary transport by IL-17/TNF-α are corrected by β-adrenergic stimulus

The fluid covering the surface of airway epithelia represents a first barrier against pathogens. The chemical and physical properties of the airway surface fluid are controlled by the activity of ion channels and transporters. In cystic fibrosis (CF), loss of CFTR chloride channel function causes airway surface dehydration, bacterial infection, and inflammation. We investigated the effects of IL-17A plus TNF-α, 2 cytokines with relevant roles in CF and other chronic lung diseases. Transcriptome analysis revealed a profound change with upregulation of several genes involved in ion transport, antibacterial defense, and neutrophil recruitment. At the functional level, bronchial epithelia treated in vitro with the cytokine combination showed upregulation of ENaC channel, ATP12A proton pump, ADRB2 β-adrenergic receptor, and SLC26A4 anion exchanger. The overall result of IL-17A/TNF-α treatment was hyperviscosity of the airway surface, as demonstrated by fluorescence recovery after photobleaching (FRAP) experiments. Importantly, stimulation with a β-adrenergic agonist switched airway surface to a low-viscosity state in non-CF but not in CF epithelia. Our study suggests that CF lung disease is sustained by a vicious cycle in which epithelia cannot exit from the hyperviscous state, thus perpetuating the proinflammatory airway surface condition.


Introduction
The airway epithelium represents an important barrier toward pathogenic agents that enter the respiratory system with inhaled air (1). One of the intrinsic defense mechanisms adopted by the epithelium is mucociliary clearance (MCC), in which mucus acts as a trap for bacteria, fungi, and viruses, and the beating cilia propel mucus toward oro-pharynx (2,3). MCC also requires the presence of an airway surface liquid (ASL), whose low viscosity facilitates ciliary beating. The chemical and physical properties of ASL are controlled by the coordinated activity of ion channels and transporters that mediate the transepithelial absorption and secretion of Na + , Cl -, HCO 3 -, and H + , as well as those of other solutes (4). ASL also contains antimicrobial molecules such as defensins, lactoferrin, and lysozyme (5). The innate defense mechanisms in the airway epithelium are further modulated during infection and inflammation. ILs, IFNs, and lipids released by cells of the immune system can reshape the transcriptome and proteome of airway epithelial cells to potentiate their defense barrier function, although excessive response may lead to lung damage (6)(7)(8). IL-4 and IL-13, which are characteristic of the Th2 immune response, boost mucus production, particularly that of MUC5AC mucin (9,10), and enhance expression and function of proteins (SLC26A4, ANO1, CFTR, SLC12A2) that mediate anion transport (11,12). The enhanced anion secretion, particularly that of HCO 3 -, could be instrumental in supporting mucus release and expansion (13)(14)(15), as well as in supporting innate antimicrobial activity in the ASL (16).
The fluid covering the surface of airway epithelia represents a first barrier against pathogens. The chemical and physical properties of the airway surface fluid are controlled by the activity of ion channels and transporters. In cystic fibrosis (CF), loss of CFTR chloride channel function causes airway surface dehydration, bacterial infection, and inflammation. We investigated the effects of IL-17A plus TNF-α, 2 cytokines with relevant roles in CF and other chronic lung diseases. Transcriptome analysis revealed a profound change with upregulation of several genes involved in ion transport, antibacterial defense, and neutrophil recruitment. At the functional level, bronchial epithelia treated in vitro with the cytokine combination showed upregulation of ENaC channel, ATP12A proton pump, ADRB2 β-adrenergic receptor, and SLC26A4 anion exchanger. The overall result of IL-17A/TNF-α treatment was hyperviscosity of the airway surface, as demonstrated by fluorescence recovery after photobleaching (FRAP) experiments. Importantly, stimulation with a β-adrenergic agonist switched airway surface to a low-viscosity state in non-CF but not in CF epithelia. Our study suggests that CF lung disease is sustained by a vicious cycle in which epithelia cannot exit from the hyperviscous state, thus perpetuating the proinflammatory airway surface condition. The defense function of the airway epithelium can be compromised in genetic diseases such as cystic fibrosis (CF) and primary ciliary dyskinesia (17,18). In CF, loss of function of the CFTR channel impairs Clsecretion. The consequence is the dehydration of the ASL, which results in defective MCC (19,20). CFTR also contributes to HCO 3 transport, either directly and/or by secreting Clthat is then exchanged with HCO 3 by SLC26A4 (21,22). The impairment of HCO 3 secretion may disrupt the activity of antimicrobial molecules and alter the properties and the release of mucus (13)(14)(15)(16). The combination of defects caused by lack of CFTR function leads to chronic bacterial infections that, in turn, sustain a severe inflammatory process with mucus hypersecretion. The perturbation of MCC in CF may also be due to enhanced function of ENaC, the epithelial channel that is responsible for Na + and fluid absorption -a process that counteracts secretion by CFTR (23).
It is still not clear to what extent the functional abnormalities occurring in the lungs of CF patients are a direct consequence of CFTR loss of function or the result of subsequent infection and inflammation. In a previous study, we found that ATP12A, a proton pump expressed in the apical membrane (AM) of airway epithelial cells, is upregulated in the lungs of CF patients (24). The activity of ATP12A is considered to be pathogenic, since acidification of the airway surface may abolish antibacterial activity and make mucus secretions more viscous (13)(14)(15)(16)25). We also found that differential ATP12A expression observed in ex vivo samples disappears when CF and non-CF epithelia are examined under sterile culture conditions. ATP12A expression was markedly induced in both cell types following treatment with IL-4 (24). These findings suggest that ATP12A upregulation in vivo is a consequence of inflammation and not a direct effect of CFTR loss. The aim of the present study was to investigate the link between ATP12A and inflammation. By testing different proinflammatory stimuli, we discovered that the treatment of bronchial epithelia in vitro with IL-17A (IL-17) and TNF-α triggers a complex program that, besides upregulating ATP12A, alters the expression and function of multiple channels and transporters with alteration of the chemical and physical properties of airway surface.

Results
In a previous study, we found strong ATP12A expression in the bronchial epithelium of CF patients undergoing lung transplants (24). We asked whether ATP12A upregulation was a consequence of the advanced stage of the disease. Therefore, we investigated ATP12A expression in the nasal mucosa of CF patients of various age and clinical conditions. For this purpose, we adopted a simple nasal brushing procedure that we recently developed (26). After brushing, detached cells were immediately fixed and processed for immunofluorescence. Figure 1A shows that cells from many CF patients have enhanced expression of ATP12A in the AM. Furthermore, many CF samples showed unusual ATP12A expression in ciliated cells ( Figure 1A, CF7 sample). By combining results from 14 non-CF control individuals and 35 CF patients, we found that ATP12A was significantly more expressed in CF, with some patients showing a percentage of ATP12A-expressing cells as high as 50% to 60% ( Figure 1B). Interestingly, ATP12A was also significantly upregulated in non-CF individuals with rhinitis at the time of brushing ( Figure 1B). Because of the broad range of ATP12A expression in CF samples, we plotted the percentage of ATP12A-expressing cells versus age or pulmonary function indicated by forced expiratory volume in the first second (FEV 1 ) (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.164944DS1). We found no correlation. There was high ATP12A expression irrespective of age, so that even very young patients (<10 years) had high expression.
ATP12A is typically expressed in nonciliated mucus-producing cells (24). However, it has been shown, by single-cell RNA-Seq (scRNA-Seq), that ATP12A expression can appear in ciliated cells as an early sign of inflammation and transdifferentiation to goblet cells (27). Interestingly, in nasal samples, we found a correlation between ATP12A expression in the global cell population and extent of localization in ciliated cells ( Figure 1C). This finding suggests that ATP12A expression is related to the status of mucosal inflammation.
We treated bronchial epithelia in vitro with a panel of proinflammatory cytokines to further investigate the link between ATP12A expression and inflammation. In particular, we chose IL-1β and IL-17 plus TNF-α, stimuli that are associated with mucus hypersecretion and neutrophilic infiltration, respectively (28,29). In parallel, we also tested IFN-α, IFN-γ, and IL-6 as representative of other types of proinflammatory stimuli. After treatment (72 hours), ATP12A expression was investigated by immunofluorescence. We found that the IL-17/TNF-α combination was particularly effective in increasing the percentage of cells expressing ATP12A with respect to control and all other treatments (Figure 2A). This effect was also investigated by immunoblot. The IL-17/TNF-α combination, as well as IL-4, significantly increased the ATP12A band in both CF and non-CF epithelia ( Figure 2B; see complete unedited blots in the supplemental material). We evaluated ATP12A function by measuring pH under bicarbonate-free conditions (large volume in situ pH assay). We added the pH-sensitive fluorescent probe SNARF-1 conjugated to high-molecular weight dextran, dissolved in a modified PBS (60 μL, pH 7.3) with low buffer capacity, to the apical side of epithelia. This solution also contained ouabain (ATP12A inhibitor), bafilomycin A1 (V-ATPase inhibitor), both compounds together, or vehicle. The basolateral culture medium was buffered with Hepes (pH 7.4). (A) Low-magnification (top) and high-magnification (bottom) representative confocal microscope images showing ATP12A protein (green) in freshly fixed nasal brushings derived from non-CF and CF patients. MUC5AC (red) and acetylated tubulin (i.e., cilia, magenta) were also detected. Rectangles in the low-magnification images indicate the magnified regions. Rectangles in the high-magnification images show areas that were considered for ATP12A expression analysis. Scale bar: 20 μm. (B) Scatter dot plot showing percentage of ATP12A + cells in non-CF healthy individuals, CF patients, and in subjects with rhinitis (**P < 0.01 and ***P < 0.001 versus healthy control group by Kruskal-Wallis followed by Dunn's post hoc test). (C) Percentage of ATP12A + ciliated cells versus percentage of ATP12A + cells in the total population. The straight line shows the best linear fit of the data (Pearson's R = 0.6743, P < 0.001) JCI Insight 2022;7(22):e164944 https://doi.org/10.1172/jci.insight.164944

JCI
Fluorescence was recorded and pH was calculated at the starting point and after 1 and 3 hours following probe addition. Figure 2C shows that non-CF and CF epithelia acidify the apical solution after 3 hours. Unexpectedly, acidification was more marked in non-CF epithelia, with pH values close to 6.5 ( Figure 2C, left). Inclusion of proton pump inhibitors also led to some unexpected results. In non-CF cells, bafilomycin A1 and ouabain were both effective in partially reducing the acidification, and combination of the 2 compounds had an additive effect. In contrast, CF cells were mostly sensitive to ouabain and not to bafilomycin A1 ( Figure 2C, right). Treatment of CF and non-CF epithelia with IL-17/TNF-α induced marked acidification that was exclusively sensitive to ouabain. In fact, the bafilomycin-sensitive component observed in non-CF epithelia under untreated conditions disappeared after cytokine treatment.
We analyzed the effect of IL-17 plus TNF-α at the transcriptome level using the QuantSeq 3′ mRNA-Seq. For comparison, we also investigated the individual effects of IL-17, TNF-α, and IL-4. This analysis revealed a profound change elicited by the IL-17/TNF-α combination, with upregulation of many genes. Supplemental Figure 2 shows data for the 80 top upregulated genes, with SLC26A4 at the top of the list. The extent of upregulation ranged from approximately 60-fold (SLC26A4) to 4-fold (KCNE3). The list includes genes involved in transepithelial transport (SLC26A4, SLC5A8, SLC5A1, SLC6A20), cytokine/chemokine signaling (CSF3, IL-19, CCL20, CXCL6, CXCL5, CXCL1), and modulation of immune response and antimicrobial activity (IDO1, NOS2, DEFB4A, LTF, DEFB4B). A selection of genes, with relevance to the topic of our study -i.e., ion transport and defense function -is shown in Figure 3, A and B. In addition to SLC26A4, the IL-17/TNF-α combination also increased the expression of ADRB2 (the β2-adrenergic receptor), the kinase SGK1, the ENaC subunits SCNN1B and SCNN1A, mucin MUC5B, and ATP12A. The extent of gene expression increase was relatively modest (between 5-and 2-fold) compared with that of the most upregulated genes (Supplemental Figure 2). The effects of IL-17/TNF-α combination for these selected genes were more like those of IL-17 alone ( Figure 3A). For example, SLC26A4 was upregulated by IL-17 but not by TNF-α. It is important to note that the analysis of IL-4 treatment revealed important differences ( Figure 3B). For example, IL-4 increased the expression of MUC5AC and not of MUC5B, which was instead downregulated (Figure 3, A and B). Also, in agreement with our previous studies (11,12,30), IL-4 enhanced the expression of the ANO1 (TMEM16A) Ca 2+ -activated Clchannel, which was instead unaffected by IL-17/TNF-α. Furthermore, the upregulation of ATP12A was more marked in IL-4-treated epithelia. The transcriptomic changes elicited by IL-17/TNF-α were investigated with functional annotation tools to identify the specific biological processes involved. Enriched processes include the response to bacteria and to bacterial components, cytokine production, mucosal immune response, and TLR signaling pathway ( Figure 3C). The Venn diagram in Figure 3D shows the comparison of the 3 gene expression profiles of cells treated with the IL-17/TNF-α in combination and with the single cytokines. The heatmaps in Supplemental Figure 3 (data available in Supplemental Table 1) show that the gene expression changes in CF and non-CF epithelia elicited by IL-17/TNF-α were quite similar.
We applied scRNA-Seq to further analyze the effects of IL-17/TNF-α treatment. The images in Figure  4A summarize the gene expression profile under control conditions (left) and after treatment (right). Each dot represents a single cell. Cells with similar transcriptomes are positioned close to each other, forming clusters numbered from 0 to 10. Figure 4B reports the position of cells in the 2D uniform manifold approximation and projection (UMAP) space with expression of particular genes selected because they are either markers of specific cell types (e.g., KRT5 for basal cells, FOXJ1 for ciliated cells, FOXI1 for ionocytes) or because they are modulated by IL-17/TNF-α. The cytokine combination markedly changes gene expression in the epithelium, as highlighted by the nature of the clusters. For example, cluster 2, which corresponds to basal cells (high KRT5 expression), almost does not contain treated cells. Instead, cluster 1 is strongly enriched in treated cells and shows predominant expression of genes induced by IL-17/TNF-α: SLC26A4, MUC5B, ADRB2, and ATP12A ( Figure 4B). Also, clusters 4-6 -which, based on FOXJ1 expression, probably represent ciliated cells -show a change in their transcriptional state. Clusters 4 and 6 contain more cells in untreated epithelia, while cluster 5 is enriched in treated epithelia.
We carried out short-circuit current recordings on non-CF and CF epithelia to evaluate the effect of IL-17/ TNF-α on electrogenic ion transport ( Figure 5, A and B). During recordings, we sequentially added: (a) amiloride (10 μM), to block ENaC-dependent Na + absorption; (b) CPT-cAMP (100 μM) followed by CFTR inh -172 (10 μM) to activate and inhibit CFTR, respectively; and (c) UTP (100 μM) and Ani9 (10 μM) to induce and block Ca 2+ -activated Clsecretion through TMEM16A (ANO1). Treatment with IL-17/TNF-α induced a significant increase in ENaC activity (i.e., the amplitude of amiloride effect) in both CF and non-CF epithelia.  This effect was unexpected, since other cytokines -namely, IL-4, IL-13, and IL-1β -actually decrease ENaC activity (31)(32)(33). CFTR current was also increased by IL-17/TNF-α, as recently reported (34), and this effect could be even detected in CF epithelia, despite the very low level of CFTR function. Instead, the response to UTP was not increased, in agreement with unaltered TMEM16A gene expression.
It can be noticed in the representative traces shown in Figure 5, A and B, that epithelia treated with IL-17/TNF-α show higher levels of basal current that persist even after addition of amiloride and CFTR inh -172 ( Figure 5A, red arrowhead). This current was also resistant to Ani9, an effective blocker of TMEM16A ( Figure 5, A and B). We hypothesized that the baseline current elicited by the cytokine combination is due to another electrogenic transport, possibly Na + -dependent glucose uptake at the AM. Indeed, the mRNA for SLC5A1 (also referred to as SGLT1, the sodium/glucose cotransporter) was upregulated (8-fold; Supplemental Figure 2) by IL-17/TNF-α. Accordingly, we tested mizagliflozin, a potent and selective inhibitor of SLC5A1. This compound caused a large drop of the baseline current in cytokine treated epithelia ( Figure 5C). In fact, the currents remaining after mizagliflozin addition in control and cytokine-treated epithelia were comparable.
We were intrigued by the marked upregulation of ENaC function elicited by IL-17/TNF-α. Since the increase in SCNN1A and SCNN1B transcripts was small ( Figure 3A), we considered the possibility of a posttranscriptional mechanism. ENaC activity is known to be regulated by proteolytic activation (35,36). Therefore, we tested the effect of elastase ( Figure 6A, top traces). Elastase was ineffective on epithelia kept under control conditions or treated with IL-17/TNF-α, indicating that ENaC is fully cleaved in both conditions and that the effect of the cytokine combination is not mediated by an increase in ENaC proteolysis. In parallel experiments, we incubated epithelia for 18 hours with the protease inhibitor camostat, which is effective on the proteases that regulate ENaC (37). As expected, camostat treatment reduced the size of ENaC currents and subsequent addition of elastase to camostat-treated epithelia resulted in rapid activation of currents (Figure 6A, bottom traces). The activating effect of elastase demonstrates that camostat reduces ENaC activity by preventing its proteolysis. Interestingly, we noticed a difference in the fraction of the total current that was sensitive to elastase treatment. This parameter was smaller in epithelia treated with IL-17/TNF-α ( Figure 6A).
Since the ENaC turnover at the plasma membrane -dependent on channel insertion, proteolytic activation, and internalization -is quite fast (38,39), we also added camostat during recordings of epithelia previously treated with/without IL-17/TNF-α ( Figure 6B). Camostat caused a rapid reduction in transepithelial current. We added amiloride during this decay phase. There was a sharp drop of the current that then reached a stable level. This behavior indicates that the current rundown caused by camostat is based on ENaC inhibition; otherwise, it would have continued in the presence of amiloride. Importantly, we noticed a difference in the time course of the decay between control-and IL-17/TNF-α-treated cells. The time required to reach half of the initial amplitude in control-treated epithelia was 10-14 minutes, a value comparable with what was previously reported: 11-17 minutes (38,39). With IL-17/TNF-α, the decay was instead significantly slower, with a mean half-time of ~28 minutes in both CF and non-CF epithelia ( Figure 6C). It is also interesting to note that the trend of the camostat-induced decay is different between control-and IL-17/TNF-α-treated epithelia. In the control condition, the decay appears to follow a single exponential fashion. With the cytokine combination, the decay appears biphasic, with an initial small and relatively rapid decay followed by a large and slower linear phase. The representative immunofluorescence images in Figure 6D show that SCNN1A protein is indeed increased on the apical side of cells treated with IL-17/TNF-α.
Internalization and degradation of ENaC is blocked by SGK1-dependent phosphorylation (40), which prevents ENaC ubiquitination by NEDD4L. Since SGK1 was one of the genes upregulated by IL-17/TNF-α ( Figure 3A), we used a SGK1 inhibitor to assess the effect on ENaC activity ( Figure 6E). GSK650394 caused a rapid rundown of ENaC currents. The rundown was not significantly different between control-and IL-17/TNF-α-treated CF epithelia and only modestly different in non-CF epithelia. Furthermore, elastase was ineffective when added during the GSK650394-induced current rundown (Supplemental Figure 4), indicating that the inhibitor is acting downstream from the proteolytic step. We also inhibited mTORC2, since this regulatory complex acts upstream from SGK1 to control ENaC function (41,42). The mTORC2 inhibitor PP242 also caused ENaC rundown, and the kinetics between controland cytokine-treated epithelia were not significantly different ( Figure 6F). These results support SGK1 upregulation as the mechanism responsible for the enhanced ENaC function in epithelia exposed to IL-17/ TNF-α. Indeed, SGK1 inhibition cancels the difference between control and treated epithelia.  We were also interested in investigating the consequences of ADRB2 transcript upregulation by IL-17/TNF-α ( Figure 3A). ADRB2 activation results in intracellular cAMP elevation, which should, in turn, activate CFTR. We carried out short-circuit current recordings, during which the ADRB2 agonist isoproterenol was added on the basolateral side of epithelia. Addition of 10 and 100 nM of isoproterenol elicited rapid activation of currents that could be similarly blocked by CFTR inh -172 and by the ADRB2 antagonist propranolol ( Figure 7A). Epithelia treated with IL-17/TNF-α showed a more than 2-fold increase in isoproterenol-induced currents.
Given the alteration by IL-17/TNF-α of multiple mechanisms involved in controlling airway surface chemical/physical properties and MCC (ENaC-dependent Na + absorption, CFTR-dependent anion secretion, ATP12A-dependent proton secretion), we carried out a series of functional assays to define the overall effect. First, we evaluated the transport of microbeads on the apical surface. Black microbeads dispersed in a small volume of saline solution were deposited on epithelial surface, and MCC was determined by timelapse microscopy. Under control conditions, microbeads visibly moved with a velocity of approximately 10 μm/second in both CF and non-CF epithelia (Supplemental Figure 5). Instead, epithelia treated for 72 hours with IL-17/TNF-α showed a near arrest of microbead transport. Importantly, mucociliary transport was resumed when non-CF epithelia treated with the cytokine combination were stimulated for 3 hours with basolateral isoproterenol. In contrast, despite the addition of the β-adrenergic agonist, microbeads remained static in CF epithelia (Supplemental Figure 5).
To clarify the reason for the arrest of microbead transport, we investigated the properties of the apical surface with the Fluorescence Recovery After Photobleaching (FRAP) technique. To apply FRAP, we needed a short distance between the objective and the epithelium, which was impeded by the narrow cup-like shape of the porous support (Snapwell) used to generate the epithelia. To overcome this limitation, we seeded epithelial cells on the opposite side of the porous membrane. To obtain the air-liquid interface (ALI) condition, the medium was removed from the bottom part. Control short-circuit recordings showed that, under this upsidedown configuration, cells are still able to form tight epithelia with normal ion transport properties. We added FITC-dextran dissolved in a small volume (5 μL) of saline solution and waited for 3 hours. Representative images in Figure 7, B and C, show that photobleaching was followed by a fast recovery in CF and non-CF epithelia kept under untreated conditions. After 72 hours of treatment with IL-17/TNF-α, we observed a dramatic slowing down of fluorescence recovery. In most cases, there was nearly no recovery at all. Importantly, when we added isoproterenol to the basolateral (top) side, we observed in non-CF epithelia a significant restoration of fluorescence recovery ( Figure 7B). In contrast, CF epithelia did not respond to isoproterenol ( Figure 7C).
We investigated the effect of IL-17/TNF-α and isoproterenol on apical pH level using conditions that included HCO 3 -. In one set of experiments, we added a fixed volume (200 μL) of saline solution to the apical side of epithelia (large volume ex situ pH assay). We also added mineral oil to minimize exchange of CO 2 between the solution and the atmosphere. After 6 hours, the aqueous apical solution was recovered to measure pH with SNARF-1 dextran conjugate. We found that the solution was essentially acidic, with a pH value close to 6.3 in both CF and non-CF epithelia ( Figure 7D). The pH only slightly changed by IL-17/ TNF-α treatment. However, in agreement with ATP12A upregulation, epithelia treated with the cytokine combination showed a large alkalinization (approximately 0.9-1.0 pH units) when exposed to ouabain (Figure 7D). Epithelia treated with IL-17/TNF-α also responded to the SLC26A4 inhibitor PDS inh -A01 (43), with a small (approximately 0.2-0.3 pH units) but significant acidic shift in pH ( Figure 7D). These results indicate that both ATP12A and SLC26A4 are involved in setting the apical pH, possibly with a larger contribution by ATP12A. Surprisingly, stimulation of epithelia with isoproterenol elicited a significant alkalinization (approximately 0.6 pH units) in both CF and non-CF epithelia previously treated with IL-17/TNF-α ( Figure 7E). The alkalinization by isoproterenol was significantly reduced by PDS inh -A01 in CF epithelia and showed no additivity with ouabain ( Figure 7E). We tested YS-01 as a second type of SLC26A4 inhibitor (44). We compared side-by-side PDS inh -A01 and YS-01 on SLC26A4 transport with a functional assay in FRT cells (Supplemental Figure 6, A and B). Both compounds were effective, although YS-01 appeared more potent, with near total SLC26A4 inhibition at 5 μM (Supplemental Figure 6, A and B). We tested the effect of YS-01 on the apical pH of epithelia treated with IL-17/TNF-α (Supplemental Figure 6, C and D). YS-01 generated a significant acidic shift in both CF and non-CF epithelia (Supplemental Figure 6, C and D). A similar effect was also seen in CF but not in non-CF epithelia after stimulation with isoproterenol.
We asked whether the alkalinization by isoproterenol is due to SLC26A4 upregulation or ATP12A inhibition. We reasoned that, if isoproterenol increases the activity of SLC26A4, we should have seen a significant change in pH in the presence of ouabain. However, this was not the case, since the pH values with ouabain plus/minus isoproterenol were essentially identical (Supplemental Figure 7, A and B). Furthermore, isoproterenol also caused a significant alkalinization under HCO 3 --free conditions, which excludes SLC26A4 contribution (Supplemental Figure 7, C and D). Such results indicate that the effect of isoproterenol on pH is due to inhibition of ATP12A.
In a second set of experiments, to measure apical pH in a more physiological context, we "stained" the apical surface with a small volume (5 μL) of saline solution containing SNARF-1 dextran (small volume in situ pH assay). With this technique, we could confirm that isoproterenol caused significant alkalinization of the apical surface in CF and non-CF epithelia ( Figure 7F). While carrying out these experiments, we noticed a peculiar behavior of SNARF-1 dextran whose distribution, as a colored pattern, could be easily detected by the naked eye and with a low magnification stereomicroscope. In epithelia kept under control conditions, the probe appeared to diffuse on a large area of the epithelium ( Figure 7F). In epithelia treated with IL-17/TNF-α, the probe remained confined in a smaller circular area characterized by very sharp borders. In non-CF epithelia, stimulation with isoproterenol reversed this situation, allowing rapid diffusion of the probe over the epithelium ( Figure 7F). In CF epithelia, isoproterenol was ineffective.
We asked how HCO 3 transport affects the properties of airway surface in epithelia treated with IL-17/ TNF-α. Therefore, we carried out, in parallel, FRAP experiments in the presence and absence of HCO 3 in the basolateral medium, with 5% CO 2 and pure air in the atmosphere, respectively. Surprisingly, the apical surface of epithelia treated with the cytokine combination remained fluid under HCO 3 --free conditions, whereas epithelia in the presence of HCO 3 developed the expected viscous state ( Figure 8A). We also carried out experiments with YS-01 to block SLC26A4 function. YS-01 decreased viscosity in both non-CF and CF epithelia treated with IL-17/TNF-α, indicating that SLC26A4 has an important role in establishing the viscous state by the cytokine combination ( Figure 8B). Finally, we asked whether mutant CFTR rescue in CF epithelia with pharmacological correctors of F508del mutation (17) is effective in decreasing apical viscosity. We treated epithelia for 72 hours with/without IL-17/TNF-α. In the last 24 hours, epithelia were treated with the corrector combination VX-809/VX-445 or with vehicle. Then, all epithelia were stimulated with isoproterenol. CF epithelia treated with IL-17/TNF-α and vehicle showed the expected slow fluorescence recovery. However, epithelia that were pretreated with correctors showed a significant improvement in airway surface properties, as indicated by faster fluorescence recovery ( Figure 8C).

Discussion
The initial aim of our study was to elucidate the conditions that lead to ATP12A upregulation in the airways of CF patients (24). By analyzing nasal samples, we confirmed ATP12A upregulation in CF patients, even in individuals of very young age. We found a broad range of values that could be explained with different levels of nasal mucosa inflammation at the time of cell collection. Indeed, there was a positive correlation between ATP12A expression and ATP12A localization in ciliated cells (27).
We tested various types of stimuli in vitro in order to mimic the inflammation occurring in CF airways. We found that IL-17/TNF-α, a stimulus that is associated with bacterial infection and neutrophilic infiltration (29), is particularly effective in enhancing ATP12A expression and function. Our pH measurements in the apical compartment under HCO 3 --free conditions confirmed upregulation of ATP12A at the functional level, as indicated by strong enhancement of the ouabain-sensitive component. It has been shown  A and B) Representative traces (top) and summary of data (bottom) from short-circuit current recordings done on non-CF (A) and CF (B) bronchial epithelia, treated with/without IL-17/TNF-α combination. During recordings, epithelia were sequentially exposed to: amiloride (10 μM), CPT-cAMP (100 μM), CFTR inh -172 (I-172, 20 μM), UTP (100 μM), and Ani9 (5 μM). Red arrowhead: higher residual current in cytokine treated epithelia. The scatter dot plots report, for control and IL-17/TNF-α-treated(I+T-treated) epithelia, the amplitude of amiloride, CFTR inh -172, and UTP effects, which are representative of ENaC, CFTR, and TMEM16A function, respectively (*P < 0.05; **P < 0.01; and ***P < 0.001 versus control; Mann-Whitney U test). (C) Representative short-circuit current recordings (left) and summary of data (right) showing effect of mizagliflozin (10 μM) on control and I+T-treated epithelia. Mizagliflozin was added in the presence of amiloride, CFTR inh -172, and Ani9. (***P < 0.001 versus control; Mann-Whitney U test). that airway epithelia, particularly of distal bronchi, display proton secretion mediated by the electrogenic V-ATPase (45). This activity could be dependent on CFTR that, by transporting Cl -, provides the required counter ion required for electroneutrality. In agreement with this notion, we found that a bafilomycin-sensitive component was present in non-CF but not in CF epithelia. This finding probably explains why pH was actually more acidic in non-CF epithelia under resting conditions. Interestingly, the bafilomycin-sensitive proton secretion disappeared in non-CF epithelia after treatment with IL-17/TNF-α.

JCI
Surprisingly, IL-17/TNF-α treatment also affected other transepithelial transport mechanisms, including ENaC-dependent Na + absorption. ENaC function is regulated at the posttranscriptional level by a complex process involving trafficking to the plasma membrane, channel activation by proteolysis, and fast internalization/degradation by a ubiquitin-dependent mechanism (35,36,38,39,46,47). ENaC ubiquitination and internalization is prevented by SGK1-dependent phosphorylation (40). During short-circuit current recordings, we added camostat to inhibit endogenous proteases involved in ENaC activation. Camostat addition caused a rapid rundown of ENaC currents. We interpret this rundown as caused by a lack of compensation for the rapid removal of ENaC from the cell surface by proteolytic activation of new channels delivered to the plasma membrane. Importantly, ENaC function rundown was significantly slower in epithelia treated with IL-17/TNF-α. This finding indicates that ENaC upregulation elicited by the cytokine treatment may depend on a slower rate of internalization. In this respect, one of the genes induced by IL-17/TNF-α is SGK1. In our experiments, an SGK1 inhibitor elicited a rapid rundown of ENaC currents. Furthermore, inhibition of mTORC2, a complex that regulates ENaC through SGK1 (41,42), also caused rundown of ENaC currents. Such results suggest that SGK1 upregulation may be responsible, at least in part, for the higher ENaC function in epithelia treated with IL-17/TNF-α. To our knowledge, our findings are the first to show upregulation of ENaC by a proinflammatory stimulus. With other cytokines, including IL-4, IL-13, and IL-1β, the effect was actually inhibitory (31,33). The enhancement of ENaC function by IL-17/TNF-α is expected to worsen the impairment of MCC caused in CF by CFTR loss of function. Enhanced activity of ENaC in CF airways, with the consequent hyperabsorption of Na + and fluid, has been a debated topic (48)(49)(50)(51)(52). Our findings reveal a mechanism to explain ENaC upregulation in CF, not directly linked to CFTR defect but to the associated inflammation, and provide a further rationale for the use of ENaC inhibitors to treat CF patients (23).
As previously reported, treatment with IL-17 or IL-17/TNF-α strongly enhances the expression of the SLC26A4 Cl -/HCO 3 exchanger (21,22,53). Our work shows that IL-17/TNF-α also affects ATP12A. We carried out pH measurements in the presence of HCO 3 to assess the role of ATP12A and SLC26A4. In agreement with ATP12A and SLC26A4 upregulation, epithelia treated with IL-17/TNF-α showed alkalinization and acidification with ouabain and SLC26A4 inhibitors, respectively. These results indicate that both transporters contribute to setting apical pH. Interestingly, isoproterenol caused a marked alkalinization, which was of similar amplitude in both CF and non-CF epithelia and which, in part, resembled that produced by ouabain. We reasoned that the increase in pH by isoproterenol could either be caused by SLC26A4 activation or ATP12A inhibition. Our results are in favor of the second type of mechanism, since the effects of isoproterenol and ouabain on pH were not additive. The alkalinization by isoproterenol should then derive from downregulation of ATP12A activity that would uncover SLC26A4-dependent HCO 3 export. In this respect, we found that the SLC26A4 inhibitor indeed lowered pH in CF epithelia stimulated with isoproterenol. This effect was much lower in non-CF epithelia, possibly suggesting that another mechanism, perhaps CFTR, is contributing to HCO 3 secretion when SLC26A4 is inhibited. We asked how the different processes altered by IL-17/TNF-α affect the properties of the airway surface. Measurements of mucociliary transport with microbeads revealed a profound inhibition induced by the cytokine treatment. Importantly, the transport of microbeads was resumed in non-CF epithelia stimulated with a β-adrenergic agonist. Such changes were in agreement with the properties of the apical surface measured with the FRAP technique. Indeed, the diffusion of the high-molecular weight fluorescent dextran was nearly abolished in epithelia treated with IL-17/TNF-α, but it was recovered, only in non-CF epithelia, by isoproterenol stimulation. Such results suggest that impairment of mucociliary transport in epithelia treated with the cytokine combination is caused by hyperviscosity and, possibly, dehydration of airway surface. This condition could result from the combination of enhanced Na + absorption and apical surface acidification due to ENaC and ATP12A upregulation, respectively. In this regard, it should be noted that ATP12A upregulation by IL-13 was previously found to be associated with airway surface hyperviscosity (54). However, in our experiments, isoproterenol elicited alkalinization of apical fluid also in CF epithelia, but it did not affect viscosity. Therefore, the increase in pH is not able per se to make airway surface less viscous. In fact, the recovery of mucociliary transport elicited by isoproterenol only in non-CF epithelia implies that CFTR is endowed with a key function that controls the overall effect of cytokine treatment.
Our experiments that were initially guided to understand the basis of ATP12A upregulation in CF airways have revealed that IL-17/TNF-α is a potent stimulus that, besides affecting ATP12A protein expression and function, modifies other processes with high relevance to airway epithelium barrier function. Importantly, IL-17 is a cytokine whose role in CF lung disease is becoming increasingly apparent. In particular, it has been recently proposed that release of IL-17 from innate lymphoid cells is an important factor responsible for neutrophilic infiltration, inflammation, and lung damage in CF patients (55).
As an attempt to explain the overall output of the complex changes induced by IL-17/TNF-α, we propose the model depicted in Figure 8D, which is based on previous knowledge and results obtained in our study. This model integrates the coordinated activity of various ion channels and transporters (ENaC, ATP12A, SLC26A4) with a key role of CFTR and β-adrenergic stimulus in switching the epithelium between an absorptive and a secretory state. As found in the distal nephron, we propose that ENaC and JCI Insight 2022;7(22):e164944 https://doi.org/10.1172/jci.insight.164944 SLC26A4 work in parallel to generate a net absorption of NaCl (56). Indeed, the absorption of Na + through ENaC is followed by the uptake of Clmediated by SLC26A4. In this respect, Haggie and colleagues found that pharmacological inhibition of SLC26A4 results in decreased fluid absorption in airway epithelia (43). In agreement with this study, we found that SLC26A4 inhibition decreased apical viscosity in FRAP experiments. The activity of SLC26A4 implies that HCO 3 is released on the apical side. Excessive accumulation of HCO 3 -, which could actually reverse the direction of transported anions by SLC26A4, should be prevented to support NaCl absorption. Accordingly, the upregulation of ATP12A-dependent proton pumping could be a way to neutralize extruded HCO 3 -. The combined secretion of H + and HCO 3 would push the production of CO 2 that leaves the apical surface to airway lumen. It is also possible that ATP12A proton pumping, by preventing excessive alkalinization, sustains the activity of ENaC that is favored by acidic pH (57). In summary, the upregulation of ENaC, SLC26A4, and ATP12A by IL-17/TNF-α would be instrumental to promote NaCl/fluid absorption. In this respect, the presence of HCO 3 appears to be crucial for fluid absorption, since in its absence, we found that the apical surface remains fluid despite the treatment with the cytokine combination. The requirement of HCO 3 to generate the viscous state is unexpected and prompts reconsideration of the role of this anion in the homeostasis of airway surface properties.
Importantly, we found that the absorptive state induced by IL-17/TNF-α is significantly changed by the β-adrenergic stimulus. Isoproterenol decreased the viscosity of apical surface in non-CF epithelia treated with IL-17/TNF-α. The lack of effect of isoproterenol in CF epithelia highlights the key role of CFTR in this process. Also, we obtained results suggesting that the β-adrenergic stimulus inhibits ATP12A activity. To integrate these results, we postulate that isoproterenol switches the epithelium state from absorptive to secretory. Indeed, the opening of CFTR channel would allow Clefflux ( Figure 8D). Therefore, Clentering the cell by SLC26A4 would be recycled back to the apical surface. The net effect of CFTR and SLC26A4 working in parallel would be the enhanced secretion of HCO 3 -. We cannot exclude that part of HCO 3 secretion is also directly mediated by CFTR. Coupled to ATP12A inhibition, this would result in accumulation of HCO 3 -. This is expected to improve the fluidity of the apical surface and to promote antimicrobial activity (13)(14)(15)(16)25). To further support this model, additional evidence will need to be provided in future experiments. In particular, other pharmacological inhibitors of ATP12A, with improved selectivity with respect to ouabain, need to be found. Also, methods to measure local changes in HCO 3 concentration will need to be developed. In conclusion, we found that IL-17/TNF-α treatment induces a complex program that involves profound changes in ion transport mechanisms with alteration of airway surface properties. Such alterations may also involve changes in solid content and Ca 2+ concentration, parameters that did not evaluate in our study. IL-17/TNF-α is associated with bacterial infection and neutrophilic infiltration (29). The increased viscosity of the apical surface by IL-17/TNF-α treatment appears to be detrimental, since mucociliary transport is believed to be beneficial as an antibacterial defense mechanism. However, it can be speculated that transient and local immobilization of bacteria, which would result from increased viscosity, could be useful to avoid dispersion of bacteria over large surfaces and to promote killing by localized epitheliumand neutrophil-dependent mechanisms. In this respect, we found that IL-17/TNF-α enhances the expression of potential antimicrobial genes (SLC5A1, defensins, NOS2, LTF, IDO1). When needed, the absorptive state induced by IL-17/TNF-α would be turned off by the β-adrenergic stimulus to restore mucociliary transport. This switch would be disrupted in CF airways, thus triggering a cycle of inflammation and mucociliary transport impairment. Importantly, our experiments with CFTR correctors show that rescue  of F508del-CFTR function allows CF epithelia to acquire, at least in part, the low-viscosity state associated with β-adrenergic stimulus, potentially breaking the damaging cycle. It would be important to find out how the persistent hyperviscous state can be corrected in CF patients with undruggable CFTR mutations. Our results indicate that SLC26A4 inhibition could be an effective approach that could improve MCC in such patients. Our findings are also of high relevance to clarify the pathogenic mechanisms and to find possible therapeutic interventions for other, nongenetic, chronic obstructive respiratory diseases.

Methods
Analysis of nasal epithelial cells. The collection of nasal epithelial cells by brushing was previously described (26). The brush was immediately placed in 10% neutral buffered formalin and shipped to the laboratory, where cells were detached from the brush and deposited on silanized glass slides. After attachment (2-3 hours) in a humidified chamber, cells were treated for antigen retrieval with 10 mM citrate buffer and then permeabilized with 0.3% Triton X-100 in PBS for 5 minutes, blocked with 1% BSA in PBS (2 hours), and incubated overnight at 4°C with primary antibodies diluted in PBS containing 1% BSA (A7030, Sigma-Aldrich). Primary antibodies and dilutions were: rabbit anti-ATP12A (HPA039526, Sigma-Aldrich), 1:400; mouse IgG1 anti-MUC5AC (MA5-12178; Thermo Fisher Scientific), 1:200; and mouse IgG2B anti-acetylated tubulin (MilliporeSigma), 1:300. Samples were washed 3 times in PBS and incubated for 1 hour in the dark with the following secondary antibodies diluted 1:200 in PBS containing 1% BSA: goat anti-rabbit Alexa Fluor 488 (catalog A11008), goat anti-mouse IgG1 Alexa Fluor 546 (catalog A2112), and goat anti-mouse IgG2B Alexa Fluor 633 (catalog A21146) antibodies (all from Thermo Fisher Scientific). After 3 further washes in PBS, slides were mounted using Fluoroshield with DAPI (MilliporeSigma) to stain cell nuclei.
Immunofluorescence images were acquired with a laser scanning confocal microscope (TCS SPE; Leica Microsystems). For each sample, 200-500 cells were analyzed. To quantify ATP12A expression in the AM of cells, 2 regions of interest (ROIs) were selected: one on the AM and another placed halfway between the AM and the nucleus -i.e., the cytosol (C). ROI positioning was done in merged fluorescence and bright-field images to easily detect the AM in cells with low ATP12A expression. The mean fluorescence intensity of each ROI was calculated with the ImageJ software (NIH), and only cells with AM/C ratio higher than 2 were considered positive for ATP12A.
FRAP assay. Epithelial cells were plated on the bottom part of "flipped" Snapwell inserts. After cell adhesion, Snapwell inserts were returned to their normal position and medium was added to the top part of the Snapwell (basolateral with respect to cells) and removed from the bottom part. Epithelia were allowed to differentiate (2-3 weeks). For experiments done in the presence of HCO 3 -, the basolateral medium was PneumaCult ALI. For experiments done under HCO 3 --free conditions, the basolateral medium was Coon's modification of F12 containing 20 mM HEPES (pH 7.4). The apical epithelial surface was stained with 5 μL PBS containing FITC-Dextran (70 kDa, 2 μg/mL, Thermo Fisher Scientific). After 3 hours, Snapwells with epithelia were mounted on the stage of a Nikon Eclipse Ti-E Spinning Disk inverted microscope within a chamber that allowed control of temperature (37°C) and atmosphere (humidified 5% CO 2 /95% air or pure air depending on the presence of bicarbonate in the basolateral medium). Images of the stained epithelial surface were sequentially acquired for 5 seconds before and 60 seconds after photobleaching of a preselected circular ROI (50 μm diameter). After normalization for the initial value, the recovery of fluorescence at 30 seconds from bleach pulse was measured.
MicroParticle transport (MPT) assay. Black micro beads (6 μm diameter; 24293-5 Polybead Black Dyed Microspheres, Polysciences) suspended in 2 μL PBS were added to the apical side of epithelia. Microbead movement was recorded with a Leica M205FA Stereo microscope. Microbeads velocity was calculated with ImageJ using Manual Tracking plug-in.
Large-volume in situ pH assay. This assay was used to measure net ATP12A activity in HCO 3 --free conditions. In total, 60 μL of a modified PBS solution with low buffer capacity containing the pH-sensitive SNARF-1 probe coupled to 70 kDa dextran (0.1 mg/mL, D3304, Thermo Fisher Scientific) was added on the apical side of epithelia formed on Transwell inserts. The modified PBS solution had the following composition (in mM): 145 NaCl, 2.7 KCl, 0.81 Na 2 HPO 4 , 0.15 KH 2 PO 4 , 1 CaCl 2 , 0.5 MgCl 2 (pH 7.35). The microplates carrying Transwell inserts were introduced in a FLUOstar Omega microplate reader (BMG LABTECH). Fluorescence was measured using single excitation (544 nm) and double emission (590 and 640 nm) at the beginning of treatment and every 60 minutes for 3 hours. The ratio of fluorescence emitted at 590 and 640 nm was converted to pH values using a calibration curve.
Large-volume ex situ pH assay. In total, 200 μL of PBS with/without compounds was added on the apical side of epithelia formed on Snapwell inserts. The saline solution was covered by 200 μL of mineral oil to avoid evaporation and CO 2 exchange with atmosphere. The basolateral medium was PneumaCult ALI for experiments done in the presence of bicarbonate or Coon's modification of F12 containing 20 mM HEPES (pH 7.4) for bicarbonate-free conditions. Epithelia were then incubated at 37°C in 5% CO 2 /95% air or pure air atmosphere. After 6 hours, the apical fluid was recovered, centrifuged (2,000 g, 1 minute,