High-content siRNA screen reveals global ENaC regulators and potential cystic fibrosis therapy targets.

Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKι, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na+ and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.

Niclosamide, but not ivermectin, inhibits anoctamin 1 and 6 and attenuates inflammation of the respiratory tract.

Inflammatory airway diseases like cystic fibrosis, asthma and COVID-19 are characterized by high levels of pulmonary cytokines. Two well-established antiparasitic drugs, niclosamide and ivermectin, are intensively discussed for the treatment of viral inflammatory airway infections. Here, we examined these repurposed drugs with respect to their anti-inflammatory effects in airways in vivo and in vitro. Niclosamide reduced mucus content, eosinophilic infiltration and cell death in asthmatic mouse lungs in vivo and inhibited release of interleukins in the two differentiated airway epithelial cell lines CFBE and BCi-NS1.1 in vitro. Cytokine release was also inhibited by the knockdown of the Ca2+-activated Cl- channel anoctamin 1 (ANO1, TMEM16A) and the phospholipid scramblase anoctamin 6 (ANO6, TMEM16F), which have previously been shown to affect intracellular Ca2+ levels near the plasma membrane and to facilitate exocytosis. At concentrations around 200 nM, niclosamide inhibited inflammation, lowered intracellular Ca2+, acidified cytosolic pH and blocked activation of ANO1 and ANO6. It is suggested that niclosamide brings about its anti-inflammatory effects at least in part by inhibiting ANO1 and ANO6, and by lowering intracellular Ca2+ levels. In contrast to niclosamide, 1 µM ivermectin did not exert any of the effects described for niclosamide. The present data suggest niclosamide as an effective anti-inflammatory treatment in CF, asthma, and COVID-19, in addition to its previously reported antiviral effects. It has an advantageous concentration-response relationship and is known to be well tolerated.

A TMEM16J variant leads to dysregulated cytosolic calcium which may lead to renal disease.

SIGIRR (single immunoglobulin IL-1 related receptor), PKP3 (plakophilin 3), and TMEM16J (anoctamin 9), a putative calcium-activated ion channel and phospholipid scramblase, control the immune response and the extent of inflammation. Variants of SIGIRR/PKP3/TMEM16J lead to severe inflammatory diseases such as pneumonia, enterocolitis, and kidney graft rejection. Meta-analysis of genome-wide association studies identified TMEM16J-T604A as a promotor for chronic kidney disease (CKD), but the disease mechanism and function of TMEM16J remain unknown. Here, we demonstrate TMEM16J as a calcium-activated calcium-permeable channel, which is expressed in the endoplasmic reticulum (ER). TMEM16J controls the intracellular distribution of calcium, and inhibits intracellular receptor-mediated Ca2+ signals and Ca2+ -dependent activation of ion channels, but augments transcription and release of pro-inflammatory cytokines. Renal epithelial cells expressing the variant TMEM16J-T604A show enhanced calcium signals when compared to cells expressing wt-TMEM16J, and demonstrate spontaneous transcription and release of cytokines. This study identifies TMEM16J as an important regulator of intracellular Ca2+ signals, ion channel activity, and cytokine release. TMEM16J may therefore affect immune response in renal tissue and immune cells.

Broadening the clinical spectrum: molecular mechanisms and new phenotypes of ANO3-dystonia.

Anoctamin 3 (ANO3) belongs to a family of transmembrane proteins that form phospholipid scramblases and ion channels. A large number of ANO3 variants were identified as the cause of craniocervical dystonia, but the underlying pathogenic mechanisms remain obscure. It was suggested that ANO3 variants may dysregulate intracellular Ca2+ signalling, as variants in other Ca2+ regulating proteins like hippocalcin were also identified as a cause of dystonia. In this study, we conducted a comprehensive evaluation of the clinical, radiological, and molecular characteristics of four individuals from four families who carried heterozygous variants in ANO3. The median age at follow-up was 6.6 years (ranging from 3.8 to 8.7 years). Three individuals presented with hypotonia and motor developmental delay. Two patients exhibited generalized progressive dystonia, while one patient presented with paroxysmal dystonia. Additionally, another patient exhibited early dyskinetic encephalopathy. One patient underwent bipallidal deep brain stimulation (DBS) and showed a mild but noteworthy response, while another patient is currently being considered for DBS treatment. Neuroimaging analysis of brain MRI studies did not reveal any specific abnormalities. The molecular spectrum included two novel ANO3 variants (V561L and S116L) and two previously reported ANO3 variants (A599D and S651N). As anoctamins are suggested to affect intracellular Ca2+ signals, we compared Ca2+ signalling and activation of ion channels in cells expressing wild type ANO3 and cells expressing ANO variants. Novel V561L and S116L variants were compared with previously reported A599D and S651N variants and with wtANO3 expressed in fibroblasts isolated from patients or when overexpressed in HEK293 cells. We identified ANO3 as a Ca2+-activated phospholipid scramblase that also conducts ions. Impaired Ca2+ signalling and compromised activation of Ca2+ dependent K+ channels were detected in cells expressing ANO3 variants. In the brain striatal cells of affected patients, impaired activation of KCa3.1 channels due to compromised Ca2+ signals may lead to depolarized membrane voltage and neuronal hyperexcitability and may also lead to reduced cellular viability, as shown in the present study. In conclusion, our study reveals the association between ANO3 variants and paroxysmal dystonia, representing the first reported link between these variants and this specific dystonic phenotype. We demonstrate that ANO3 functions as a Ca2+-activated phospholipid scramblase and ion channel; cells expressing ANO3 variants exhibit impaired Ca2+ signalling and compromised activation of Ca2+-dependent K+ channels. These findings provide a mechanism for the observed clinical manifestations and highlight the importance of ANO3 for neuronal excitability and cellular viability.

Functional Interdependence of Anoctamins May Influence Conclusions from Overexpression Studies.

Anoctamin 6 (ANO6, TMEM16F) is a phospholipid (PL) scramblase that moves PLs between both plasma membrane (PM) leaflets and operates as an ion channel. It plays a role in development and is essential for hemostasis, bone mineralization and immune defense. However, ANO6 has also been shown to regulate cellular Ca2+ signaling and PM compartments, thereby controlling the expression of ion channels such as CFTR. Given these pleiotropic effects, we investigated the functional interdependence of the ubiquitous ANO6 with other commonly co-expressed anoctamins. As most expression studies on anoctamins use HEK293 human embryonic kidney cells, we compared ion currents, PL scrambling and Ca2+ signals induced by the overexpression of anoctamins in HEK293 wild-type parental and ANO6-knockout cells. The data suggest that the endogenous expression of ANO6 significantly affects the results obtained from overexpressed anoctamins, particularly after increasing intracellular Ca2+. Thus, a significant interdependence of anoctamins may influence the interpretation of data obtained from the functional analysis of overexpressed anoctamins.

Lipopolysaccharide augments microglial GABA uptake by increasing GABA transporter-1 trafficking and bestrophin-1 expression.

Gamma-aminobutyric acid (GABA), the principal inhibitory neurotransmitter in the brain, affects numerous immune cell functions. Microglia, the brain's resident innate immune cells, regulate GABA signaling through GABA receptors and express the complete GABAergic machinery for GABA synthesis, uptake, and release. Here, the use of primary microglial cell cultures and ex vivo brain tissue sections allowed for demonstrating that treatment with lipopolysaccharide (LPS) increased microglial GABA uptake as well as GABA transporter (GAT)-1 trafficking. This effect was not entirely abolished by treatment with GAT inhibitors (GAT-Is). Notably, LPS also induced microglial upregulation of bestrophin-1 (BEST-1), a Ca2+ -activated Cl- channel permeable to GABA. Combined administration of GAT-Is and a BEST-1 inhibitor completely abolished LPS-induced microglial GABA uptake. Interestingly, increased microglial GAT-1 membrane turnover via syntaxin 1A was detected in LPS-treated cultures after BEST-1 blockade. Altogether, these findings provided evidence for a novel mechanism through which LPS may trigger the inflammatory response by directly altering microglial GABA clearance and identified the GAT-1/BEST-1 interplay as a potential novel mechanism involved in brain inflammation.

KCNE1 does not shift TMEM16A from a Ca2+ dependent to a voltage dependent Cl- channel and is not expressed in renal proximal tubule.

The TMEM16A (ANO1) Cl- channel is activated by Ca2+ in a voltage-dependent manner. It is broadly expressed and was shown to be also present in renal proximal tubule (RPT). KCNQ1 is an entirely different K+ selective channel that forms the cardiac IKS potassium channel together with its ß-subunit KCNE1. Surprisingly, KCNE1 has been claimed to interact with TMEM16A, and to be required for activation of TMEM16A in mouse RPT. Interaction with KCNE1 was reported to switch TMEM16A from a Ca22+-dependent to a voltage-dependent ion channel. Here we demonstrate that KCNE1 is not expressed in mouse RPT. TMEM16A expressed in RPT is activated by angiotensin II and ATP in a KCNE1-independent manner. Coexpression of KCNE1 does not change TMEM16A to a voltage gated Cl- channel and Ca2+-dependent regulation of TMEM16A is fully maintained in the presence of KCNE1. While overexpressed KCNE1 slightly affects Ca2+-dependent regulation of TMEM16A, the data provide no evidence for KCNE1 being an auxiliary functional subunit for TMEM16A.

Pharmacological inhibitors of the cystic fibrosis transmembrane conductance regulator exert off-target effects on epithelial cation channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αßγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δßγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.

A polycystin-2 protein with modified channel properties leads to an increased diameter of renal tubules and to renal cysts.

Mutations in the PKD2 gene cause autosomal-dominant polycystic kidney disease but the physiological role of polycystin-2, the protein product of PKD2, remains elusive. Polycystin-2 belongs to the transient receptor potential (TRP) family of non-selective cation channels. To test the hypothesis that altered ion channel properties of polycystin-2 compromise its putative role in a control circuit controlling lumen formation of renal tubular structures, we generated a mouse model in which we exchanged the pore loop of polycystin-2 with that of the closely related cation channel polycystin-2L1 (encoded by PKD2L1), thereby creating the protein polycystin-2poreL1. Functional characterization of this mutant channel in Xenopus laevis oocytes demonstrated that its electrophysiological properties differed from those of polycystin-2 and instead resembled the properties of polycystin-2L1, in particular regarding its permeability for Ca2+ ions. Homology modeling of the ion translocation pathway of polycystin-2poreL1 argues for a wider pore in polycystin-2poreL1 than in polycystin-2. In Pkd2poreL1 knock-in mice in which the endogenous polycystin-2 protein was replaced by polycystin-2poreL1 the diameter of collecting ducts was increased and collecting duct cysts developed in a strain-dependent fashion.

The SLC26A9 inhibitor S9-A13 provides no evidence for a role of SLC26A9 in airway chloride secretion but suggests a contribution to regulation of ASL pH and gastric proton secretion.

The solute carrier 26 family member A9 (SLC26A9) is an epithelial anion transporter that is assumed to contribute to airway chloride secretion and surface hydration. Whether SLC26A9 or CFTR is responsible for airway Cl- transport under basal conditions is still unclear, due to the lack of a specific inhibitor for SLC26A9. In the present study, we report a novel potent and specific inhibitor for SLC26A9, identified by screening of a drug-like molecule library and subsequent chemical modifications. The most potent compound S9-A13 inhibited SLC26A9 with an IC50 of 90.9 ± 13.4 nM. S9-A13 did not inhibit other members of the SLC26 family and had no effects on Cl- channels such as CFTR, TMEM16A, or VRAC. S9-A13 inhibited SLC26A9 Cl- currents in cells that lack expression of CFTR. It also inhibited proton secretion by HGT-1 human gastric cells. In contrast, S9-A13 had minimal effects on ion transport in human airway epithelia and mouse trachea, despite clear expression of SLC26A9 in the apical membrane of ciliated cells. In both tissues, basal and stimulated Cl- secretion was due to CFTR, while acidification of airway surface liquid by S9-A13 suggests a role of SLC26A9 for airway bicarbonate secretion.

NCX1 represents an ionic Na+ sensing mechanism in macrophages.

Inflammation and infection can trigger local tissue Na+ accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (MΦs) and their antimicrobial activity. Enhanced Na+-driven MΦ function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na+ sensing in MΦs remained unclear. High extracellular Na+ levels (high salt [HS]) trigger a substantial Na+ influx and Ca2+ loss. Here, we show that the Na+/Ca2+ exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na+ influx, concomitant Ca2+ efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na+ and is required for amplifying inflammatory and antimicrobial MΦ responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate MΦ function.

Pathogenic Relationships in Cystic Fibrosis and Renal Diseases: CFTR, SLC26A9 and Anoctamins.

The Cl--transporting proteins CFTR, SLC26A9, and anoctamin (ANO1; ANO6) appear to have more in common than initially suspected, as they all participate in the pathogenic process and clinical outcomes of airway and renal diseases. In the present review, we will therefore concentrate on recent findings concerning electrolyte transport in the airways and kidneys, and the role of CFTR, SLC26A9, and the anoctamins ANO1 and ANO6. Special emphasis will be placed on cystic fibrosis and asthma, as well as renal alkalosis and polycystic kidney disease. In essence, we will summarize recent evidence indicating that CFTR is the only relevant secretory Cl- channel in airways under basal (nonstimulated) conditions and after stimulation by secretagogues. Information is provided on the expressions of ANO1 and ANO6, which are important for the correct expression and function of CFTR. In addition, there is evidence that the Cl- transporter SLC26A9 expressed in the airways may have a reabsorptive rather than a Cl--secretory function. In the renal collecting ducts, bicarbonate secretion occurs through a synergistic action of CFTR and the Cl-/HCO3- transporter SLC26A4 (pendrin), which is probably supported by ANO1. Finally, in autosomal dominant polycystic kidney disease (ADPKD), the secretory function of CFTR in renal cyst formation may have been overestimated, whereas ANO1 and ANO6 have now been shown to be crucial in ADPKD and therefore represent new pharmacological targets for the treatment of polycystic kidney disease.

The Anion Channel TMEM16a/Ano1 Modulates CFTR Activity, but Does Not Function as an Apical Anion Channel in Colonic Epithelium from Cystic Fibrosis Patients and Healthy Individuals.

Studies in human colonic cell lines and murine intestine suggest the presence of a Ca2+-activated anion channel, presumably TMEM16a. Is there a potential for fluid secretion in patients with severe cystic fibrosis transmembrane conductance regulator (CFTR) mutations by activating this alternative pathway? Two-dimensional nondifferentiated colonoid-myofibroblast cocultures resembling transit amplifying/progenitor (TA/PE) cells, as well as differentiated monolayer (DM) cultures resembling near-surface cells, were established from both healthy controls (HLs) and patients with severe functional defects in the CFTR gene (PwCF). F508del mutant and CFTR knockout (null) mice ileal and colonic mucosa was also studied. HL TA/PE monolayers displayed a robust short-circuit current response (ΔIeq) to UTP (100 µM), forskolin (Fsk, 10 µM) and carbachol (CCH, 100 µM), while ΔIeq was much smaller in differentiated monolayers. The selective TMEM16a inhibitor Ani9 (up to 30 µM) did not alter the response to luminal UTP, significantly decreased Fsk-induced ΔIeq, and significantly increased CCH-induced ΔIeq in HL TA/PE colonoid monolayers. The PwCF TA/PE and the PwCF differentiated monolayers displayed negligible agonist-induced ΔIeq, without a significant effect of Ani9. When TMEM16a was localized in intracellular structures, a staining in the apical membrane was not detected. TMEM16a is highly expressed in human colonoid monolayers resembling transit amplifying cells of the colonic cryptal neck zone, from both HL and PwCF. While it may play a role in modulating agonist-induced CFTR-mediated anion currents, it is not localized in the apical membrane, and it has no function as an apical anion channel in cystic fibrosis (CF) and healthy human colonic epithelium.

The chloride channel CFTR is not required for cyst growth in an ADPKD mouse model.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of bilateral renal cysts which enlarge continuously, leading to compression of adjacent intact nephrons. The growing cysts lead to a progressive decline in renal function. Cyst growth is driven by enhanced cell proliferation and chloride secretion into the cyst lumen. Chloride secretion is believed to occur mainly by the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR), with some contribution by the calcium-activated chloride channel TMEM16A. However, our previous work suggested TMEM16A as a major factor for renal cyst formation. The contribution of CFTR to cyst formation has never been demonstrated in an adult ADPKD mouse model. We used mice with an inducible tubule-specific Pkd1 knockout, which consistently develop polycystic kidneys upon deletion of Pkd1. Cellular properties, ion currents, and cyst development in these mice were compared with that of mice carrying a co-deletion of Pkd1 and Cftr. Knockout of Cftr did not reveal any significant impact on cyst formation in the ADPKD mouse model. Furthermore, knockout of Cftr did not attenuate the largely augmented cell proliferation observed in Pkd1 knockout kidneys. Patch clamp analysis on primary renal epithelial cells lacking expression of Pkd1 indicated an only marginal contribution of CFTR to whole cell Cl- currents, which were clearly dominated by calcium-activated TMEM16A currents. In conclusion, CFTR does not essentially contribute to renal cyst formation in mice caused by deletion of Pkd1. Enhanced cell proliferation and chloride secretion is caused primarily by upregulation of the calcium-activated chloride channel TMEM16A.

TMEM16A deficiency: a potentially fatal neonatal disease resulting from impaired chloride currents.

INTRODUCTION: TMEM16A is a calcium-activated chloride channel expressed in various secretory epithelia. Two siblings presented in early infancy with reduced intestinal peristalsis and recurrent episodes of haemorrhagic diarrhoea. In one of them, the episodes were characterised by hepatic pneumatosis with gas bubbles in the portal vein similar to necrotising enterocolitis of the newborn. METHODS: Exome sequencing identified a homozygous truncating pathogenic variant in ANO1. Expression analysis was performed using reverse transcription PCR, western blot and immunohistochemistry. Electrophysiological and cell biological studies were employed to characterise the effects on ion transport both in patient respiratory epithelial cells and in transfected HEK293 cells. RESULTS: The identified variant led to TMEM16A dysfunction, which resulted in abolished calcium-activated Cl- currents. Secondarily, CFTR function is affected due to the close interplay between both channels without inducing cystic fibrosis (CF). CONCLUSION: TMEM16A deficiency is a potentially fatal disorder caused by abolished calcium-activated Cl- currents in secretory epithelia. Secondary impairment of CFTR function did not cause a CF phenotyp, which may have implications for CF treatment.

Attenuation of near-ultraviolet, visible and near-infrared light in sound and carious human enamel and dentin.

OBJECTIVES: This in vitro study aimed to investigate the optical attenuation of light at 405, 660 and 780 nm sent through sound and carious human enamel and dentin, including respective individual caries zones, as well as microscopically sound-appearing tissue close to a carious lesion. MATERIALS AND METHODS: Collimated light transmission through sections of 1000-125-µm thickness was measured and used to calculate the attenuation coefficient (AC). The data were statistically analysed with a MANOVA and Tukey's HSD. Precise definition of measurement points enabled separate analysis within the microstructure of lesions: the outer and inner halves of enamel (D1, D2), the translucent zone (TZ) within dentin lesions and its adjacent layers, the enamel side of the translucent zone (ESTZ) and the pulpal side of the translucent zone (PSTZ). RESULTS: The TZ could be distinguished from its adjacent layers and from caries-free dentin at 125 µm. Sound-appearing dentin close to caries lesions significantly differed from caries-free dentin at 125 µm. While sound and carious enamel exhibited a significant difference (p < 0.05), this result was not found for D1 and D2 enamel lesions (p > 0.05). At 405 nm, no difference was found between sound and carious dentin (p > 0.05). CONCLUSIONS: Light optical means enable the distinction between sound and carious tissue and to identify the microstructure of dentin caries partially as well as the presence of tertiary dentin formation. Information on sample thickness is indispensable when interpreting the AC. CLINICAL RELEVANCE: Non-ionising light sources may be suitable to detect lesion progression and tertiary dentin.

Diagnostic validity of early proximal caries detection using near-infrared imaging technology on 3D range data of posterior teeth.

OBJECTIVES: This in vitro study analysed potential of early proximal caries detection using 3D range data of teeth consisting of near-infrared reflection images at 850 nm (NIRR). MATERIALS AND METHODS: Two hundred fifty healthy and carious permanent human teeth were arranged pairwise, examined with bitewing radiography (BWR) and NIRR and validated with micro-computed tomography. NIRR findings were evaluated from buccal, lingual and occlusal (trilateral) views according to yes/no decisions about presence of caries. Reliability assessments included kappa statistics and revealed high agreement for both methods. Statistical analysis included cross tabulation and calculation of sensitivity, specificity and AUC. RESULTS: Underestimation of caries was 24.8% for NIRR and 26.4% for BWR. Overestimation was 10.4% for occlusal NIRR and 0% for BWR. Trilateral NIRR had overall accuracy of 64.8%, overestimation of 15.6% and underestimation of 19.6%. NIRR and BWR showed high specificity and low sensitivity for proximal caries detection. CONCLUSIONS: NIRR achieved diagnostic results comparable to BWR. Trilateral NIRR assessments overestimated presence of proximal caries, revealing stronger sensitivity for initial caries detection than BWR. CLINICAL RELEVANCE: NIRR provided valid complement to BWR as diagnostic instrument. Investigation from multiple angles did not substantially improve proximal caries detection with NIRR.

Expression of SLC26A9 in Airways and Its Potential Role in Asthma.

SLC26A9 is an epithelial anion transporter with a poorly defined function in airways. It is assumed to contribute to airway chloride secretion and airway surface hydration. However, immunohistochemistry showing precise localization of SLC26A9 in airways is missing. Some studies report localization near tight junctions, which is difficult to reconcile with a chloride secretory function of SLC26A9. We therefore performed immunocytochemistry of SLC26A9 in sections of human and porcine lungs. Obvious apical localization of SLC26A9 was detected in human and porcine superficial airway epithelia, whereas submucosal glands did not express SLC26A9. The anion transporter was located exclusively in ciliated epithelial cells. Highly differentiated BCi-NS1 human airway epithelial cells grown on permeable supports also expressed SLC26A9 in the apical membrane of ciliated epithelial cells. BCi-NS1 cells expressed the major Cl- transporting proteins CFTR, TMEM16A and SLC26A9 in about equal proportions and produced short-circuit currents activated by increases in intracellular cAMP or Ca2+. Both CFTR and SLC26A9 contribute to basal chloride currents in non-stimulated BCi-NS1 airway epithelia, with CFTR being the dominating Cl- conductance. In wtCFTR-expressing CFBE human airway epithelial cells, SLC26A9 was partially located in the plasma membrane, whereas CFBE cells expressing F508del-CFTR showed exclusive cytosolic localization of SLC26A9. Membrane localization of SLC26A9 and basal chloride currents were augmented by interleukin 13 in wild-type CFTR-expressing cells, but not in cells expressing the most common disease-causing mutant F508del-CFTR. The data suggest an upregulation of SLC26A9-dependent chloride secretion in asthma, but not in the presence of F508del-CFTR.

Airway Delivery of Hydrogel-Encapsulated Niclosamide for the Treatment of Inflammatory Airway Disease.

Repurposing of the anthelminthic drug niclosamide was proposed as an effective treatment for inflammatory airway diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Niclosamide may also be effective for the treatment of viral respiratory infections, such as SARS-CoV-2, respiratory syncytial virus, and influenza. While systemic application of niclosamide may lead to unwanted side effects, local administration via aerosol may circumvent these problems, particularly when the drug is encapsulated into small polyethylene glycol (PEG) hydrospheres. In the present study, we examined whether PEG-encapsulated niclosamide inhibits the production of mucus and affects the pro-inflammatory mediator CLCA1 in mouse airways in vivo, while effects on mucociliary clearance were assessed in excised mouse tracheas. The potential of encapsulated niclosamide to inhibit TMEM16A whole-cell Cl- currents and intracellular Ca2+ signalling was assessed in airway epithelial cells in vitro. We achieved encapsulation of niclosamide in PEG-microspheres and PEG-nanospheres (Niclo-spheres). When applied to asthmatic mice via intratracheal instillation, Niclo-spheres strongly attenuated overproduction of mucus, inhibited secretion of the major proinflammatory mediator CLCA1, and improved mucociliary clearance in tracheas ex vivo. These effects were comparable for niclosamide encapsulated in PEG-nanospheres and PEG-microspheres. Niclo-spheres inhibited the Ca2+ activated Cl- channel TMEM16A and attenuated mucus production in CFBE and Calu-3 human airway epithelial cells. Both inhibitory effects were explained by a pronounced inhibition of intracellular Ca2+ signals. The data indicate that poorly dissolvable compounds such as niclosamide can be encapsulated in PEG-microspheres/nanospheres and deposited locally on the airway epithelium as encapsulated drugs, which may be advantageous over systemic application.

Drug Repurposing for Cystic Fibrosis: Identification of Drugs That Induce CFTR-Independent Fluid Secretion in Nasal Organoids.

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion. From a ~1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including ß2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in the Ussing chamber, and fluid secretion was not affected by TMEM16A, as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for medium-scale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.