Dusik Kim
email: dusikkim@gmail.com

Junwei Huang

Arnaud Billet

Asmahan Abu-Arish

Julie Goepp

Elizabeth Matthes

Marc A Tewfik

Saul Frenkiel

John W Hanrahan
email: john.hanrahan@mcgill.ca

Cftrc

Pendrin mediates bicarbonate secretion and enhances CFTR function in airway surface epithelia

Keywords: SLC26A4, anion exchanger, ABCC7, cystic fibrosis, COPD, asthma

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INTRODUCTION

Anion secretion by airway epithelia is required for efficient mucociliary clearance of inhaled particles and pathogens from the lungs. Electrogenic Cl -secretion generates a transepithelial potential that drives paracellular sodium flow, and the resulting salt transport generates an osmotic gradient that drives fluid secretion [START_REF] Frizzell | Physiology of chloride and fluid secretion[END_REF]. Bicarbonate is also secreted into the airway lumen and this transport is reduced in cystic fibrosis (CF) indicating an important role for CFTR [START_REF] Smith | Camp stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia[END_REF]. Despite the importance of HCO 3 -in CF and other respiratory diseases the mechanisms mediating apical HCO 3 - efflux remain uncertain.

Airway epithelia express apical cAMP-and Ca 2+ -activated channels that are permeable to both Cl - and HCO 3 - [START_REF] Gray | Anion selectivity and block of the small conductance chloride channel on pancreatic duct cells[END_REF][START_REF] Poulsen | Bicarbonate conductance and ph regulatory capability of cystic fibrosis transmembrane conductance regulator[END_REF][START_REF] Linsdell | Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions[END_REF][START_REF] Hanrahan | A secretory cl channel from epithelial cells studied in heterologous expression systems[END_REF]. Under bi-ionic conditions, the equilibrium permeability ratio for CFTR (P HCO3- /P Cl-) is 0.14 with HCO 3 -bathing the extracellular aspect [START_REF] Linsdell | Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions[END_REF][START_REF] Hanrahan | A secretory cl channel from epithelial cells studied in heterologous expression systems[END_REF] and somewhat higher (0.25) when HCO 3 -is present intracellularly [START_REF] Linsdell | Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions[END_REF]. The Calu-3 cell line expresses CFTR at high levels and generates a large forskolin-stimulated short-circuit current (I sc ) that is carried exclusively by HCO 3 -(7-10). Electrogenic HCO 3 -secretion is also observed across airway epithelia ex vivo during 4 cAMP stimulation. Early studies of canine trachea showed that a fraction of the cAMP-stimulated I sc is carried by HCO 3 -(11, 12), although electroneutral HCO 3 -secretion under particular conditions was not excluded, for example during inflammation when the expression of transporters such as pendrin is increased [START_REF] Nofziger | Pendrin function in airway epithelia[END_REF][START_REF] Pedemonte | Thiocyanate transport in resting and il-4-stimulated human bronchial epithelial cells: Role of pendrin and anion channels[END_REF]. Pendrin mediates Cl -/HCO 3 - [START_REF] Soleimani | Pendrin: An apical cl(-)/oh(-)/hco(3)(-) exchanger in the kidney cortex[END_REF][START_REF] Royaux | Pendrin, encoded by the pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion[END_REF], Cl -/formate - [START_REF] Scott | Human pendrin expressed in xenopus laevis oocytes mediates chloride/formate exchange[END_REF], and Cl -/I - [START_REF] Scott | The pendred syndrome gene encodes a chloride-iodide transport protein[END_REF] exchange and its expression is strongly induced by IL-4, IL-13 and IL-17A [START_REF] Danahay | Interleukin-13 induces a hypersecretory ion transport phenotype in human bronchial epithelial cells[END_REF][START_REF] Galietta | Il-4 is a potent modulator of ion transport in the human bronchial epithelium in vitro[END_REF][START_REF] Adams | Il-17a induces pendrin expression and chloride-bicarbonate exchange in human bronchial epithelial cells[END_REF][START_REF] Scanlon | Epithelial anion transporter pendrin contributes to inflammatory lung pathology in mouse models of bordetella pertussis infection[END_REF][START_REF] Nakao | Identification of pendrin as a common mediator for mucus production in bronchial asthma and chronic obstructive pulmonary disease[END_REF][START_REF] Nakagami | The epithelial anion transporter pendrin is induced by allergy and rhinovirus infection, regulates airway surface liquid, and increases airway reactivity and inflammation in an asthma model[END_REF], proinflammatory cytokines that are implicated in several airway diseases [START_REF] Wills-Karp | Interleukin-13: Central mediator of allergic asthma[END_REF]. Pendrin levels increase in animal models of bronchial asthma and chronic obstructive pulmonary disease (COPD) and are associated with mucous cell hyperplasia and mucus accumulation [START_REF] Nakao | Identification of pendrin as a common mediator for mucus production in bronchial asthma and chronic obstructive pulmonary disease[END_REF][START_REF] Kuperman | Dissecting asthma using focused transgenic modeling and functional genomics[END_REF]. Asthma and COPD symptoms are less severe in pendrin null mice and can be induced by ectopic pendrin expression, which implies a causal relationship whereby pendrin increases disease severity [START_REF] Nakao | Identification of pendrin as a common mediator for mucus production in bronchial asthma and chronic obstructive pulmonary disease[END_REF].

Although HCO 3 -transport by CFTR and pendrin clearly play important roles in airway pathophysiology, the interactions between these pathways and their relative contributions to HCO 3 -secretion are not well understood.

Exposing bronchial epithelial cell cultures to IL-4 increases cAMP-and Ca 2+ -stimulated I sc and elevates the expression of pendrin and many other transporters and channels [START_REF] Gorrieri | Goblet cell hyperplasia requires high bicarbonate transport to support mucin release[END_REF]. Since pendrin is an electroneutral anion exchanger, increased expression of apical CFTR and the basolateral Na + ,K + ,2Cl -cotransporter NKCC1 have been suggested as possible reasons for larger I sc responses to cAMP after IL-4 treatment [START_REF] Gorrieri | Goblet cell hyperplasia requires high bicarbonate transport to support mucin release[END_REF]. Alternatively, CFTR might be regulated by pendrin as occurs in mouse pancreatic duct [START_REF] Ko | A molecular mechanism for aberrant cftr-dependent hco 3transport in cystic fibrosis[END_REF] and with other SLC26A transporters notably SLC26A3, SLC26A6, and SLC26A9 [START_REF] Ko | A molecular mechanism for aberrant cftr-dependent hco 3transport in cystic fibrosis[END_REF][START_REF] Ko | Gating of cftr by the stas domain of slc26 transporters[END_REF][START_REF] Avella | Slc26a9 stimulates cftr expression and function in human bronchial cell lines[END_REF]. Conversely, CFTR can increase pendrin activity [START_REF] Ko | A molecular mechanism for aberrant cftr-dependent hco 3transport in cystic fibrosis[END_REF] through interaction of its R domain with the STAS domain of pendrin [START_REF] Ko | Gating of cftr by the stas domain of slc26 transporters[END_REF] although in the airways it remains unclear whether CFTR and pendrin are expressed in the same cells where they could physically interact.

The aim of this study was to clarify the roles of pendrin and CFTR in HCO 3 -secretion by nasal and bronchial surface epithelia. We began by localizing the transporters in bronchial tissue and well-differentiated primary cultures, then studied their expression and function in well differentiated primary nasal and bronchial epithelial cell cultures.

MATERIALS AND METHODS

Human airway tissue and cells

Lung and nasal samples were obtained with informed consent following protocols approved by the Institutional Review Boards of the IRCM and McGill University (#A08-M70-14B). Cell isolation and histological methods are detailed in SUPPLEMENTAL INFORMATION.

Immunostaining pendrin and CFTR in bronchial sections and primary cultures

Fresh tissue specimens were collected within 48 h of lung resection and fixed in 10 % neutral buffered formalin overnight at 4 °C for paraffin embedding and sectioning. Primary cells were cultured on collagen-coated Transwell™ inserts, incubated with PBS on the apical side for 15 min at 37 o C in a 5% CO 2 incubator, and rinsed three times to reduce mucus immediately before immunostaining. (see SUPPLEMENTAL INFORMATION for details).

RNA interference and qRT-PCR

Adenovirus particles (Biofocus DPI/Galapagos, Leiden, Netherlands) were used to express shRNAs that target pendrin transcripts (see SUPPLEMENTAL INFORMATION). Pendrin shRNA reduced pendrin mRNA levels by ~50% (Supplementary Figure 1)

Intracellular pH measurement

Intracellular pH was measured using an inverted fluorescence microscope (IX81, Olympus, Center Valley PA) and imaging system (Photon Technology International, Edison NJ) as described [START_REF] Huang | Most bicarbonate secretion by calu-3 cells is mediated by cftr and independent of pendrin[END_REF][START_REF] Kim | Role of anion exchangers in cl-and hco3-secretion by the human airway epithelial cell line calu-3[END_REF]. Cells were loaded with the ratiometric pH-sensitive dye BCECF-AM and studied at 37 o C with independent perfusion of the apical and basolateral sides as described in the SUPPLEMENTAL INFORMATION.

Short-circuit current measurements

Snapwell™ inserts were mounted in Ussing chambers to measure (I sc (μA cm -2 ).

Transepithelial voltage was clamped at 0 mV except during 2 s bipolar pulses to ±1 mV at 100 s intervals to monitor resistance. Basolateral solution contained (mmol l HPO 4 , and 10 glucose. After I sc had stabilized, amiloride was added apically to block Na + absorption and this was followed by sequential apical additions of forskolin (10 μM), CFTR inhibitor, CFTR inh -172 (10 μM) and ATP (100 μM).

Patch clamp

BHK cells stably expressing wild-type CFTR were seeded on plastic coverslips at low density and transfected the following day with the expression plasmid pReceiver-M29 containing the CMV promoter to drive expression of EGFP-SLC26A4 (EGFP fused to the N-terminus of pendrin; Genecopoeia, Germantown MD). Patch-clamp experiments were performed 1-2 days after transfection. Whole cell Cl -currents were measured after compensating for pipette capacitance electronically in the cell-attached mode. (see SUPPLEMENTAL INFORMATION).

Fluid transport

ASL height was measured using an LSM-780 confocal microscope (Zeiss, Jena, Germany) equipped with a multiline argon laser (488nm, 561nm) after labeling the surface liquid with BCECF-dextran (see SUPPLEMENTAL INFORMATION). Secretions were collected by aspiration and measured using a micropipet as described previously [START_REF] Huang | Basolateral chloride loading by ae2: Role in fluid secretion by the human airway epithelial cell line calu-3[END_REF] after 2 days exposure to basolateral 10 μM forskolin and apical 10 μM amiloride.

Statistics

Data are means ± standard error of mean (s.e.m.) of n observations except the histogram in Fig. 7C, which shows means ± standard deviation (s.d.). Data sets were compared using the Student's t-test (GraphPad Prism) with p<0.05 considered significant unless otherwise indicated.

RESULTS

Pendrin co-localizes with CFTR in ciliated airway surface epithelial cells

Bronchial tissue sections were immunostained for both CFTR and pendrin. In non-CF bronchus (Figure 1A, top row of images), most CFTR was detected at the apical pole of ciliated cells that covered much of the airway surface (green fluorescence, epithelium outlined by white dotted lines).

A similar distribution was observed using anti-pendrin antibody (Figure 1A, PDS, red). Colocalization of CFTR and pendrin is indicated by yellow in the merged images (Figure 1A). CF bronchus had barely detectable CFTR immunostaining as expected and moderately increased pendrin staining compared to non-CF tissue (second row of images in Figure 1A). Specificity of CFTR immunostaining in surface epithelium was further confirmed by the absence of signal when primary anti-CFTR antibody was omitted (Figure 1A, WO 1 o ). Omitting the anti-pendrin primary antibody caused a similar decrease in the pendrin signal (data not shown). CFTR and pendrin immunostaining were less prominent in submucosal gland mucus (MC) and serous (SC) cells (Figure 1B). The glands (outlined using white dotted lines in the fluorescence images) were identified as mucous cells (MC) or serous cells (SC) based on their granularity and by comparison with sections that had been stained for mucins using Alcian-blue periodic acid Schiff stain (AB-PAS; data not shown). Although a diffuse signal was detected for both proteins in serous cells, it was weak and was not near the apical membrane (Figure 1B). These results indicate that pendrin is prominently expressed in surface epithelial cells in vivo but not in submucosal glands.

Pendrin expression was also examined en face in first passage primary nasal and bronchial epithelial cultures treated with vehicle (0.1 % DMSO) or IL-4 (10 ng/ml) for 2 days (Figure 1C).

Immunostaining was low in non-CF nasal inferior turbinate and bronchial cells under control conditions (Figure 1C, left panels) but increased after pretreatment with IL-4 (Figure 1C, right panels). Although pendrin immunostaining varied between patients, it was usually higher in CF bronchial cells compared to non-CF cells and was further increased by IL-4.

X-Z images constructed from image stacks revealed more prominent pendrin expression at the apical pole of both non-CF and CF bronchial cells after 2 days pretreatment with IL-4 (10 ng/ml) compared to vehicle (0.1% DMSO, Figure 1D). Cellular coexpression of pendrin and CFTR in vitro was assessed using non-CF bronchial cells that had been maintained at the air-liquid interface for ≥4 weeks and then treated with DMSO or IL-4 (10 ng/ml) for 2 days (Figure 1E). X-Y images revealed increased pendrin (red) immunostaining after IL-4 exposure and coexpression with CFTR in some cells (white arrowheads). When well-differentiated bronchial epithelial cells treated with DMSO or IL-4 were gently scraped from the culture supports, fixed and immunostained, CFTR (green) was clearly concentrated near the apical pole of the cell (Figure 1F, left panels) and its levels and distribution were not noticeably affected by IL-4 pretreatment. By contrast, pendrin immunostaining (red, center panels) was more intense after IL-4, forming a band at the apical pole (shown by the white arrowheads) that was colocalized with CFTR at the bases of cilia (yellow merged signal, bottom right panel).

Pendrin mRNA expression is elevated in CF cells and further increased by IL-4

We used qRT-PCR to quantify mRNA transcripts coding for CFTR, pendrin, and the pendrinrelated anion channel SLC26A9 [START_REF] Bertrand | Slc26a9 is a constitutively active, cftr-regulated anion conductance in human bronchial epithelia[END_REF] under the conditions used below for functional studies. In non-CF nasal and bronchial cells, CFTR mRNA levels normalized to GAPDH mRNA appeared 3-to 7-fold higher than those for pendrin under basal conditions (Figure 2A,B) and SLC26A9 transcripts were 3-to 5-fold more abundant than those for CFTR. These differences between genes are only approximate as they rely on different primer sets, nevertheless they confirm the low expression of pendrin under basal conditions. Pendrin transcript levels were much higher in CF than in non-CF bronchial cells (Figure 2C), consistent with the immunostaining shown in Figure 1A (native tissue) and Figure 1C (primary culture).

Exposure to IL-4 (10 ng/ml) for 2 days dramatically increased pendrin mRNA in non-CF nasal and bronchial cells (normalized to DMSO vehicle, Figure 2E,F). IL-4 also elevated pendrin expression in CF bronchial epithelial cells, although the relative increase (2-to 5-fold) was smaller than in non-CF cells due to the higher basal expression in CF (Fig. 2G). The Calu-3 cell line was also compared with primary surface epithelial cells (Figure 2D) and had higher levels of CFTR mRNA but low pendrin and SLC26A9 mRNA expression, consistent with previous studies [START_REF] Huang | Most bicarbonate secretion by calu-3 cells is mediated by cftr and independent of pendrin[END_REF][START_REF] Shan | Bicarbonatedependent chloride transport drives fluid secretion by the human airway epithelial cell line calu-3[END_REF][START_REF] Kim | Role of anion exchangers in cl-and hco3-secretion by the human airway epithelial cell line calu-3[END_REF]. Moreover, IL-4 did not increase pendrin mRNA expression in the Calu-3 (Figure 2H) consistent with our previous studies [START_REF] Huang | Most bicarbonate secretion by calu-3 cells is mediated by cftr and independent of pendrin[END_REF]. Figure 2I summarizes the relative expression of CFTR and pendrin transcripts in non-CF and CF cells in untreated (naïve) cells and after pretreatment with IL-4. All values were normalized to those for non-CF cells treated with vehicle. Consistent with immunostaining results, pendrin mRNA expression in CF bronchial cells was generally higher than in non-CF cells and was further increased several-fold by IL-4.

Pendrin mediates Cl -/HCO 3 -and Cl -/I -antiport and enhances forskolin-stimulated electrogenic anion transport

We studied apical bicarbonate transport in nasal and bronchial epithelial cells by measuring the increase in intracellular pH (ΔpH i ) when cells were briefly challenged with low-Cl -saline (gluconate replacement) on the apical side. ΔpH i in nasal cells was small under basal conditions (Figure 3A) but became much larger after the cells had been pretreated with IL-4 for 2 days (Figure 3B). The rate of re-acidification upon restoring extracellular Cl -(dpH i /dt, dashed red lines) was also increased by 3-to 4-fold, indicating faster Cl -/HCO 3 -antiport in IL-4-treated nasal cells (0.029 ± 0.013 vs 0.113 ± 0.004 pH units min -1 , n=4, Figure 3B). Iodide can be carried by pendrin [START_REF] Scott | The pendred syndrome gene encodes a chloride-iodide transport protein[END_REF] but not by most other anion exchangers and provides a useful tool for identifying pendrin-mediated transport [START_REF] Garnett | Novel role for pendrin in orchestrating bicarbonate secretion in cystic fibrosis transmembrane conductance regulator (cftr)expressing airway serous cells[END_REF]. Selectivity was assessed as the re-acidification rate after ΔpH i when low-Cl - solution was replaced with one containing I -(Figure 3C,F). This rate was similar when apical gluconate was replaced with either Cl -or I -(0.113 ± 0.004 vs 0.083 ± 0.059 pH units, n=4-5, not significant p=0.37) consistent with HCO 3 -/I -exchange and pendrin-mediated base efflux. Similar results were obtained when non-CF bronchial epithelial cells were pretreated with vehicle for 2 days before challenge with low-Cl -solution; there was little increase in pH i irrespective of forskolin stimulation (Figure 3D). By contrast, after treatment with IL-4 for 2 days acute challenge with low-Cl -extracellular solution caused abrupt intracellular alkalinization in bronchial cells that was further enhanced by forskolin stimulation (ΔpH i =0.046 ± 0.065 (basal) vs 0.288 ± 0.023 pH units (with IL-4 and forskolin, n=4; Figure 3E-G). A spontaneous decline in ΔpH i during pulses with apical 0 Cl -solution under these conditions suggests that basolateral Cl -entry may become rate-limiting for apical anion exchange during forskolin stimulation (Fig. 3E, second pulse). Taken together, these results suggest that pendrin expression induced by IL-4 increases apical HCO 3 -/Cl - antiport which is further enhanced by CFTR activation in nasal and bronchial epithelial cells.

Further evidence for the role of CFTR in apical anion exchange comes from the partial inhibition of ΔpH i by the CFTR inhibitor CFTR inh -172 (Supplementary Figure 2). Remarkably, forskolin alone did not enhance ΔpH i in primary cells despite the presence of functional CFTR channels, in contrast to previous results in Calu-3 cells where ΔpH i was also elicited by apical low-Cl -solution only during forskolin stimulation. This indicates that ΔpH i is not mediated by CFTR in primary cells even though forskolin enhances ΔpH i after IL-4 pretreatment. The results can be readily explained if electrically-coupled HCO 3 -influx is negligible in primary cells, pendrin mediates most apical anion exchange after IL-4 pretreatment, and forskolin activation of CFTR enhances the anion exchange via pendrin. IL-4 alters the expression of many ion transporters and channels [START_REF] Gorrieri | Goblet cell hyperplasia requires high bicarbonate transport to support mucin release[END_REF]. To assess the specific contribution of pendrin to ΔpH i we compared IL-4 effects when bronchial cells had been transduced with adenoviruses expressing scrambled or pendrin-specific shRNA. In naïve cells (i.e. that had not been treated with IL-4), intracellular alkalinization was not significant regardless of the adenovirus used; i.e. expressing scrambled (0.015 ± 0.018 pH units, n=4; Scr KD, Figure 4A) or pendrin-specific shRNA (0.047 ± 0.048 pH units, n=4; PDS KD, Figure 4B). As summarized in Figure 4E, forskolin (0.024 ± 0.015, Scrambled shRNA vs 0.036 ± 0.007 pH units, Pendrin shRNA, n=4) and CFTR inh -172 (0.017 ± 0.04, Scrambled shRNA vs 0.029 ± 0.023 pH units, Pendrin shRNA, n=4) also had no effect on ΔpH i under these conditions. By contrast, after pretreatment with IL-4 (10 ng/ml) for 2 days ΔpH i was greatly increased in control cells expressing scrambled shRNA when challenged with apical Cl --free solution (0.196 ± 0.014 pH units, n=4; Figure 4C). Importantly, these large ΔpH i responses were reduced >50% by adenoviral shRNA targeting pendrin (0.08 ± 0.025 pH units, n=4; Figure 4D). Further, ΔpH i was enhanced by 80% when cells were challenged with apical Cl --free solution during exposure to forskolin (0.353 ± 0.025 pH units, n=4), pendrin shRNA inhibited this forskolin-dependent stimulation of ΔpH i (0.201 ± 0.022 pH units, n=4), and CFTR inh -172 inhibited ΔpH i to pre-forskolin levels (0.115 ± 0.04 pH units, n=4). These results confirm that IL-4 enhances ΔpH i by increasing pendrinmediated antiport and further suggest that forskolin activation of CFTR enhances anion exchange (Figure 4F).

Pendrin expression increases cAMP-stimulated HCO 3 -and fluid secretion

To examine the role of pendrin in forskolin stimulated fluid and HCO 3 -secretion, primary nasal cells cultured at the air-liquid interface for at least 2 weeks were transduced with scrambled (Scr) or pendrin-specific (PDS) shRNA using adenovirus particles and exposed to amiloride and vehicle (0.1% DMSO) or IL-4 (10 ng/ml) for 48 h (see MATERIALS AND METHODS). BCECF-dextran was delivered onto the surface by evaporation from perfluorocarbon (FC-72) and confocal imaging was performed 1-2 h later. Image z-stacks were constructed to determine the ASL height under each condition. Cells were studied under control conditions, after forskolin (10 μM) alone, and after treatment with both forskolin and IL-4 (10 ng/ml) using cells from 4 different patients. Images were taken from the same location ~0.88 mm from the edge of the Transwell. Forskolin increased ASL height in both control (Scr) and pendrin knockdown cells (PDS), and the stimulation by forskolin was larger after treatment with IL-4. The effects of IL-4 and shRNA targeting PDS on ASL height during forskolin stimulation are summarized for 7 -9 monolayers in Fig. 5B. Forskolin stimulation was not affected significantly by shRNA against PDS alone, although compared to Scr, shRNA although there was a trend towards smaller responses. However pretreating cells with both IL-4 and Scr shRNA (Scr KD/IL-4) increased ASL height significantly during forskolin stimulation compared to pretreatment with Scr shRNA alone (Scr KD; p<0.01, n=7-9, Fig. 5B).

Moreover, this increase was diminished when cells were pretreated with both IL- μM cpt-cAMP and pH was measured while equilibrated with 5% CO 2 / 95% air (5 measurements using cells from 3 patients, n=3, * p<0.05, ** p<0.01). PDS knockdown had little effect on pH under basal conditions, however pretreatment with IL-4 caused a significant increase in pH (p<0.01) that was significantly blunted by shRNA targeting pendrin (Figure 5D). These results indicate that pendrin upregulation by IL-4 enhances cAMP-stimulated secretion of both fluid and HCO 3 -through pendrin-dependent mechanisms.

The IL-4-induced increase in CFTR activity is pendrin-dependent

If pendrin enhances fluid secretion by increasing cAMP stimulation of CFTR as indicated above, then IL-4 should also increase the short-circuit current (I sc ) response to forskolin and the increase should be sensitive to pendrin knockdown. To test those predictions, I sc was measured across highly differentiated primary cell cultures after exposure to vehicle (0.1% DMSO) or IL-4 for 2 days. IL-4 dramatically increased I sc stimulation by both forskolin (ΔI sc =4.663 ± 0.905 vs 14.06 ± 0.582 μA cm -2 , n=4, P<0.0001) and ATP (ΔI sc =5.595 ± 1.071 vs 14.26 ± 0.793 μA cm -2 , n=4, P=0.0003) in nasal cells, indicating that cAMP-and Ca 2+ -activated anion secretion were both enhanced by IL-4 (Supplementary Figure 3). Larger purinergic responses were expected as they are mediated by TMEM16A, a Ca 2+ -activated Cl -channel that is induced by IL-4 [START_REF] Galietta | Il-4 is a potent modulator of ion transport in the human bronchial epithelium in vitro[END_REF][START_REF] Caputo | Tmem16a, a membrane protein associated with calcium-dependent chloride channel activity[END_REF]. Similar results were obtained using well-differentiated bronchial epithelial cells (Supplementary Figure 3C,D). IL-4 induction resulted in 2-to 3-fold larger forskolin responses (ΔI sc =4.7 ± 0.379 vs 12.55 ± 1.146 μA cm -2 , n=4-5, P=0.0002) that were sensitive to CFTR inh -172, indicating an increase in CFTR activation (Supplementary Figure 3F). CFTR inh -172 nearly abolished the forskolin response in both nasal and bronchial cells, indicating that IL-4 enhances CFTR responses to forskolin without increasing CFTR mRNA expression (Figure 2, see also ref [START_REF] Gorrieri | Goblet cell hyperplasia requires high bicarbonate transport to support mucin release[END_REF]).

IL-4 increases the expression of many genes therefore we examined whether the enhanced forskolin response was due specifically to pendrin. Nasal and bronchial cells that had been pretreated with vehicle or IL-4 for 2 days were transduced with adenoviruses encoding scrambled or pendrin-specific shRNA. Incubating cells with IL-4 increased their responses to forskolin by 2to 3-fold compared to vehicle (non-CF nasal; ΔI sc =6.11 ± 0.624 vs 13.15 ± 0.692 μA cm -2 , non-CF bronchial; 4.24 ± 0.60 vs 12.07 ± 0.778 μA cm -2 , n=4, P=0.0002; Figure 6A,C). This IL-4induced increase was inhibited >70% by pendrin shRNA (non-CF nasal 8.348 ± 1.036 μA cm -2 , n=4, P=0.008; non-CF bronchial 7.953 ± 0.917 μA cm -2 , n=4, P=0.014, Figure 6B,D). These results suggest that inducing pendrin expression with IL-4 enhances the activation of CFTR by forskolin. Large ATP-stimulated currents were observed after IL-4 exposure and they were unaffected by pendrin shRNA (non-CF nasal; ΔI sc =11.6 ± 0.437 vs 11.05 ± 0.668 μA cm -2 , n=4, P=0.5212; non-CF bronchial; 13.2 ± 0.682 vs 14.35 ± 0.743 μA cm -2 , n=4, P=0.299), consistent with a distinct, pendrin-independent mechanism of action (i.e. increased expression of Ca 2+activated Cl -channels). We conclude from these I sc experiments that IL-4 elevates pendrin expression, which in turn enhances the activation of CFTR by cAMP. Such modulation of CFTR would also explain the ability of IL-4 to increase forskolin-stimulated fluid secretion (Figure 5).

Pendrin increases cAMP-stimulated CFTR current in transfected cells

To study the effect of pendrin on CFTR activity without cytokine induction of other genes we studied whole cell Cl -current in BHK cells stably expressing CFTR. Currents were measured by stepping from a holding potential of -40 mV to test potentials of -100 to +100 mV in 20 mV increments under basal conditions and after sequential exposure to forskolin (Fsk, 10 µM) and CFTR inhibitor (CFTR Inh -172, 10 µM). In cells stably expressing CFTR forskolin activated 80 pA/pF currents that reached a maximum after 6 -7 minutes. By contrast, forskolin stimulation was faster in cells co-expressing CFTR with EGFP-pendrin and the maximum current was almost doubled (Figure 7A). Current-voltage relations revealed a 5-fold increase in slope conductance in control cells during forskolin stimulation and a 9-fold increase in cells co-expressing pendrin (identified by their green fluorescence; Figure 7B). Forskolin-stimulated current was abolished by CFTRin-172 and was therefore mediated by CFTR (Figure 7A,B). The mean slope conductances measured in the absence and presence of pendrin coexpression are summarized in Figure 7C. These results indicate that pendrin expression enhances forskolin activation of CFTR.

DISCUSSION

The present studies indicate that pendrin and CFTR are uniformly co-expressed in ciliated cells of the airway surface epithelium in native tissue and have overlapping expression in welldifferentiated primary airway cell cultures. Most HCO 3 -secretion across nasal and bronchial epithelial primary cultures pretreated with IL-4 appears mediated by pendrin, which also enhances cAMP activation of CFTR. Pendrin expressed during inflammation would increase luminal pH through electroneutral, anion exchange-mediated bicarbonate transport while simultaneously increasing the volume of secreted fluid by enhancing cAMP-stimulated, electrogenic chloride flux through CFTR channels.

Pendrin expression and co-localization with CFTR

Colocalization of CFTR and pendrin at the apical pole of ciliated surface cells is consistent with the previous reports demonstrating CFTR mRNA in bronchial surface epithelium and lamina propria [START_REF] Trezise | In vivo cell-specific expression of the cystic fibrosis transmembrane conductance regulator[END_REF], CFTR immunofluorescence in ciliated cells of the airway surface [START_REF] Kreda | Characterization of wildtype and f508 cystic fibrosis transmembrane regulator in human respiratory epithelia[END_REF], and pendrin immunohistochemical staining in mouse airway surface epithelium [START_REF] Nakao | Identification of pendrin as a common mediator for mucus production in bronchial asthma and chronic obstructive pulmonary disease[END_REF]. CFTR and pendrin expression both appeared much lower in submucosal gland serous cells, and neither protein was detected in mucous cells. Low CFTR expression in the glands agrees with some [START_REF] Kreda | Characterization of wildtype and f508 cystic fibrosis transmembrane regulator in human respiratory epithelia[END_REF] but not all [START_REF] Engelhardt | Expression of the cystic fibrosis gene in adult human lung[END_REF] previous studies, and the variable results may simply reflect cellular heterogeneity of CFTR expression in native tissue [START_REF] Jiang | Cellular heterogeneity of cftr expression and function in the lung: Implications for gene therapy of cystic fibrosis[END_REF]. Impaction of CF submucosal glands in the fetus [START_REF] Ornoy | Pathological confirmation of cystic fibrosis in the fetus following prenatal diagnosis[END_REF] and extensive studies of secretion by single glands in vitro [START_REF] Widdicombe | Airway gland structure and function[END_REF][START_REF] Ballard | Liquid secretion properties of airway submucosal glands[END_REF] have established beyond doubt that CFTR is functional in glands, although weak immunostaining in glands compared to ciliated surface cells of the bronchus suggests its expression is relatively low.

Pendrin mRNA levels were elevated in primary human bronchial and nasal cell cultures pretreated with the proinflammatory cytokine IL-4, consistent with previous studies [START_REF] Pedemonte | Thiocyanate transport in resting and il-4-stimulated human bronchial epithelial cells: Role of pendrin and anion channels[END_REF][START_REF] Gorrieri | Goblet cell hyperplasia requires high bicarbonate transport to support mucin release[END_REF]. Under control conditions, pendrin mRNA expression (normalized to GAPDH) appeared 2-to 10-fold lower than CFTR and 10-to 30-fold lower than SLC26A9. Transcripts amplified using different primers cannot be compared quantitatively, nevertheless these large differences confirm that pendrin expression is low under basal conditions and dramatically increases (>100-fold) after IL-4.

Upregulation was greater than reported previously (~30-fold ( 27)), perhaps due to differences in cell culture conditions. IL-4 only increased pendrin mRNA expression by ~2.5-fold in CF bronchial epithelial cells, where pendrin expression was already elevated. High basal pendrin expression implies that CF cells may be in an inflamed state, although that suggestion has been controversial [START_REF] Machen | Innate immune response in cf airway epithelia: Hyperinflammatory?[END_REF] and was not confirmed by measuring inflammatory cytokines and NFκB activity in well differentiated primary bronchial epithelial cells [START_REF] Becker | Cytokine secretion by cystic fibrosis airway epithelial cells[END_REF]. Further studies are needed to understand the mechanism responsible for high basal pendrin expression. Perhaps only some manifestations of inflammation are constitutively increased in CF epithelial cells.

Unlike tissue sections, CFTR and pendrin expression overlapped only partially in primary cultures;

i.e. some ciliated cells expressed both proteins while others expressed only CFTR or pendrin.

Further studies are needed to determine if ciliated cells are incompletely differentiated even under ALI conditions. Pendrin was reported exclusively in cells lacking cilia after pretreatment with IL-17A [START_REF] Adams | Il-17a induces pendrin expression and chloride-bicarbonate exchange in human bronchial epithelial cells[END_REF]. We observed apical co-localization of CFTR and pendrin in ciliated cells after IL-4 but cannot exclude the possibility that pendrin is also present in goblet and club cells.

Well-differentiated surface epithelium and Calu-3 cells secrete HCO 3 -through different mechanisms

Results obtained with nasal and bronchial primary cells contrast with those from the Calu-3 cell line. Pendrin mRNA levels (after normalization to GAPDH) were much lower (i.e. 20-fold) in Calu-3 under basal conditions and were not increased by IL-4, consistent with our recent findings [START_REF] Huang | Most bicarbonate secretion by calu-3 cells is mediated by cftr and independent of pendrin[END_REF]. Stable knockdown of pendrin in Calu-3 cells using lentiviral shRNA reduced pendrin expression measured by qRT-PCR and immunofluorescence staining as expected but did not affect rates of HCO 3 -or fluid secretion. HCO 3 -transport under pH-stat conditions and net HCO 3 - flux across basolaterally-permeabilized monolayers were also unaffected by pendrin knockdown.

In Calu-3 cells, low extracellular Cl -solution produced a large ΔpH i during forskolin activation of CFTR because it is a major determinant of the membrane potential and its activation leads to electrically-coupled Cl -efflux and HCO 3 -influx (45). This CFTR dependence of ΔpH i in Calu-3 was further suggested by changes in intracellular pH when apical Cl -and HCO 3 -concentrations were varied in control and pendrin knockdown cells, but not in CFTR deficient cells [START_REF] Huang | Most bicarbonate secretion by calu-3 cells is mediated by cftr and independent of pendrin[END_REF]. Finally, IL-4 had little effect on pendrin expression in Calu-3 and did not alter fluid or HCO 3 -secretion, unlike the primary airway surface cells used in the present study. We conclude that Calu-3 and surface airway epithelial cells are fundamentally different with respect to HCO 3 -transport and the role of pendrin. Calu-3 is an adenocarcinoma cell line that shares some characteristics with submucosal gland serous cells and is used as a model for them [START_REF] Haws | Calu-3, a model cftr expressing cell line: Novel features of cftr channel regulation[END_REF][START_REF] Shen | Calu-3: A human airway epithelial cell line that shows camp-dependent clsecretion[END_REF][START_REF] Haws | Cftr in calu-3 human airway cells: Channel properties and role in camp-activated clconductance[END_REF]. The negligible contribution of pendrin to bicarbonate secretion by Calu-3 cells may be appropriate for submucosal gland acinar cells, since we detected little pendrin in glands by immunostaining bronchial sections (Figure 1B).

Pendrin as a potential therapeutic target

Pendrin modulation of cAMP-stimulated fluid secretion by non-CF cells may contribute to the beneficial effects of beta-agonists used to treat allergic asthma. ASL height was increased on tracheal epithelial cell cultures from pendrin-deficient mice pretreated with IL-13, suggesting that inhibiting pendrin might improve mucus hydration and mucociliary clearance [START_REF] Nakagami | The epithelial anion transporter pendrin is induced by allergy and rhinovirus infection, regulates airway surface liquid, and increases airway reactivity and inflammation in an asthma model[END_REF][START_REF] Lee | Thick airway surface liquid volume and weak mucin expression in pendrin-deficient human airway epithelia[END_REF]. Increased ASL height has been observed during exposure to pendrin inhibitors [START_REF] Haggie | Inhibitors of pendrin anion exchange identified in a small molecule screen increase airway surface liquid volume in cystic fibrosis[END_REF] in the absence of cAMP stimulation. Since pendrin promotes cAMP-stimulated fluid secretion it may increase ASL height and mucociliary clearance during cAMP stimulation by endogenous agonists (adenosine, vasoactive intestinal peptide, epinephrine) and by therapeutics used to treat asthma such as betaagonists (51) and phosphodiesterase inhibitors [START_REF] Franciosi | Efficacy and safety of rpl554, a dual pde3 and pde4 inhibitor, in healthy volunteers and in patients with asthma or chronic obstructive pulmonary disease: Findings from four clinical trials[END_REF]. It will be important to exclude adverse effects of pendrin inhibitors on CFTR activation before pursuing them as therapeutics for CF, especially when used in combination with correctors and potentiators that only partially restore CFTR function.

Author disclosures are available with the text of this article at www.atsjournals.org.

FIGURES

Immunostaining pendrin and CFTR in bronchial sections and primary cultures

To image single isolated primary cells, ALI cultures were gently scraped into PBS and centrifuged onto a glass slide at using a Cytospin4 (450 rpm for 5 min, Thermo Scientific, Cheshire, UK).

After fixation in 10% neutral buffered formalin (15 min at RT), samples were rinsed with PBS (3 x 5 min), permeabilized using 1% Triton X-100, and blocked with 2% BSA in PBS for 1.5 h at RT Tissue sections (5 µm thick) were mounted on microscope slides, de-paraffinized in xylene baths 3 (3 x 5 min each), and rehydrated in alcohol baths (95%, 95%, 70%; 5 min each) followed by two 5 min rinses in ddH 2 O and equilibration in PBS. Sections were permeabilized using 0.5% Triton X-100 and blocked for 1 h with 2% bovine serum albumin (BSA) in PBS at room temperature (RT). Primary cells cultured on collagen-coated Transwell™ inserts were incubated with PBS on the apical side for 15 min at 37 o C in a 5% CO 2 incubator, rinsed three times to remove apical mucus secretions and then immunostained. To image single isolated primary cells, ALI cultures were washed three times with PBS at RT and gently scraped from the inserts into PBS and centrifuged onto a glass slide at using a Cytospin4 (450 rpm for 5 min, Thermo Scientific, Cheshire, UK). After fixation in 10% neutral buffered formalin (15 min at RT), samples were rinsed with PBS (3 x 5 min), permeabilized using 1% Triton X-100, and blocked with 2% BSA in PBS for 1.5 h at RT.

Intracellular pH measurements

Any mucus on the apical surface was removed by 5-10 min incubation with PBS containing Nacetyl-l-Cysteine (1 mg ml -1 ) at 37°C and the cells were rinsed three times with PBS at RT. Then confluent cell monolayers were washed for 30 min in HCO 3 --buffered solution and incubated with the pH-sensitive dye BCECF-AM (2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester) (5 μmol l -1 ) in HCO 3 --buffered 5% CO 2 solution at 37 o C for ≥45 min. Cells were then rinsed again and mounted in a chamber that allowed independent apical and basolateral perfusion. Experiments were carried out at 37 °C using a thermostatically controlled platform (FC-5, Live Cell Instr., Seoul Korea). Flow to both surfaces was maintained at 1.5 ml min -1 using a four-channel peristaltic pump (205S, Watson Marlow, Wilmington MA). The normal HCO 3 - solution contained (mmol l -1 ): 116 NaCl, 25 Na HCO Intracellular BCECF was excited alternately at 440 and 490 nm (1 s each wavelength) every 5 s.

The signal was not perfectly uniform therefore fluorescence intensities were measured in at least 10 regions of interest (ROIs) using Easy Ratio Pro software (Horiba Sci. Mississauga, ON).

Unbiased sampling of pH i was performed by measuring fluorescence in five ROIs per field; one at the outer edge of each of four quadrants and one near the center. These ROIs covered ∼2/3 of the field of view using a 40× objective. Fluorescence intensities in several ROIs were averaged to obtain one measurement from each experiment. Fluorescence ratios (F490/F440) were recorded and displayed continuously. To calculate pH i , the F490/F440 was calibrated using high-K +nigericin solution that contained (mmol l -1 ): 140 KCl, 1 CaCl 2 , 1 MgSO 4 , 20 HEPES and 20 μmol l -1 nigericin, and was adjusted to different pH values. Graphs and statistical analyses were performed using Microsoft Excel and Prism 5 software.

Measurement of ASL height

ASL height was measured using an LSM-780 confocal microscope (Zeiss, Jena, Germany) equipped with a multiline Argon laser (488 nm, 561 nm) after transduction with adenovirus particles directing expression of scrambled (Scr) or pendrin (PDS) shRNA. Cells were exposed to amiloride (10 μM) alone, or together with forskolin (10 μM) or forskolin + IL-4 (10 ng/ml) for 48 h before experiments. To visualize the ASL, BCECF-dextran was delivered onto the apical surface in perfluorocarbon FC-72 that was allowed to evaporate for 1-2 h before imaging the surface liquid.

xz scanning images (1.06 μm apart, 40 -100 images) were collected from 4 -6 different regions at ~0.88 mm from the edge of the Transwell to determine fluid height.

Patch clamp

Transfections were performed using Gene Juice (Millipore) according to the manufacturer's instructions. Cells were cultured at 37 °C in 5% CO 2 . Voltage-clamp signals were recorded with an analog/digital interface (Digidata 1440; Axon Instruments, Inc., Burlingame, CA) and analyzed using pCLAMP version 10 (Axon Instruments). The external bath solution contained (in mM):

observed when cells were bathed with nominally bicarbonate-free solution. Finally, activation of CFTR would lower intracellular [Cl -] and this may enhance CFTR-mediated HCO 3 -secretion through a mole fraction effect on permeation (57) or an increase in P HCO3-/P Cl-due to WNK1-OSR1/SPAK signaling, which is activated by low intracellular Cl -(58).

  4 and PDS shRNA (Scr shRNA/IL-4; p<0.01, n=9), indicating that the positive effect of IL-4 on forskolinstimulated fluid secretion was pendrin-dependent. The effects of IL-4 and PDS shRNA on ASL height are summarized in Fig. 5B. To measure the volume and pH of secretions, fluid was collected by aspiration after 2 days and fluid volumes were compared. Changes in ASL volume paralleled those in ASL height, with IL-4 increasing forskolin-stimulated fluid volume ~2-fold (p<0.005, n=8) and PDS shRNA blunting the response (p<0.05, n=6). Finally, in separate experiments secretions were collected after 48 h continuous stimulation by 10 μM forskolin + 200

Figure 1 .

 1 Figure 1. Pendrin expression in bronchial tissue and primary airway epithelial cells. (A)

Figure 2 .

 2 Figure 2. CFTR, pendrin and SLC26A9 mRNA expression in surface airway epithelia and

Figure 3 .

 3 Figure 3. Effect of IL4 on the intracellular pH response (ΔpH i ) to apical Cl -removal in nasal and bronchial epithelial cells. Intracellular pHi was measured while Cl -in HCO 3 -

Figure 5 .

 5 Figure 5. Role of pendrin in IL-4-induced, forskolin-stimulated secretion by nasal

Figure 6 .

 6 Figure 6. Effect of pendrin knockdown on IL-4 and forskolin-stimulated anion secretion

Figure 7 .

 7 Figure 7. Effect of co-expressing pendrin on CFTR current in BHK cells. EGFP-pendrin

Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .

 234567 Figure 2. CFTR, pendrin and SLC26A9 mRNA expression in surface airway epithelia and the effect of IL-4 on pendrin in each cell type. 215x276mm (300 x 300 DPI)

  -1 ): 115 NaCl, 25 NaHCO 3 , 1.2 MgCl 2 , 1.2 CaCl 2 , 2.4 KH 2 PO 4 , 1.24 K 2 HPO 4 , and 10 D-glucose. Apical solution contained (mmol l -1 ): 1.2 NaCl, 115 Na-gluconate, 25 NaHCO 3 , 1.2 MgCl 2 , 4 CaCl 2 , 2.4 KH 2 PO 4 , 1.24 K 2

  3 -, 5 KCl, 1 CaCl 2 , 1 MgSO 4 , 2.8 NaHEPES, 4 2.2 HEPES and 10 D-glucose and Cl --free solution contained 116 sodium gluconate, 25 Na HCO 3 -, 5 potassium gluconate, 4 calcium gluconate, 1 MgSO 4 , 2.8 NaHEPES, 2.2 HEPES and 10 Dglucose. All solutions were adjusted to pH 7.4 by bubbling with a 95% O 2 , 5% CO 2 mixture at 37

o C.

Acknowledgements: The authors thank the Goodman Cancer Research Centre Histology Facility, McGill University for technical assistance with bronchial tissue histology. This work was supported by CF Canada, Canadian Institutes of Health Research, and the Canada Foundation for Innovation.

restored on the apical side. (C) pH i when apical Cl -was substituted with gluconate followed by iodide. Note the re-acidification rate with extracellular I -is similar to that with Cl -in panel B. (D) Effect of apical Cl -replacement on pH i in well differentiated primary bronchial epithelial cells under control conditions and during forskolin stimulation. Cells were pretreated with vehicle (0.1% DMSO) for 2 days then perfused with HCO

Histology

For histological studies, primary cell cultures were, then washed with PBS, fixed in 10% neutral buffered formalin (pH 6.8 -7.2, EMD Chemicals Inc.) overnight at 4°C, embedded in paraffin and sectioned at 5 µm. Slides were rehydrated and stained with standard hematoxylin and eosin (H&E) and Alcian-blue periodic acid Schiff (AB-PAS) stain. Morphology was assessed by imaging five regions under each condition with an Axioskop (Zeiss) wide-field microscope and analyzing the images using AxioVision 4 software.

Supplementary Figure 1 Comparison of pendrin mRNA expression in non-CF bronchial epithelia after pendrin knockdown using shRNA adenovirus.

Supplementary Figure 2 CFTR-dependent intracellular alkalinization response (ΔpHi)

after upregulation of pendrin by IL-4.

Supplementary Figure 3 Upregulation of CFTR-dependent short-circuit current (I sc ) by IL-4 in well-differentiated primary nasal and bronchial epithelial cells. 1 Gene-specific primers used for real-time qPCR using Taqman Gene Expression assay DISCUSSION (SUPPLEMENTARY)

Supplementary Table

Regulation of pendrin and CFTR function

Reciprocal CFTR -pendrin regulation was observed in both nasal and bronchial primary cells.

Forskolin activation of CFTR increased the ΔpH i induced by apical Cl --free solution, but only after pendrin expression had been elevated by IL-4. Thus, electrical coupling between HCO 3 -and Cl - flows is not detectable during CFTR activation and the ΔpH i during challenge with apical Cl --free solution is mainly due to electroneutral HCO 3 -entry through pendrin. Our finding that purinoceptor-activation of anion conductance also did not induce or increase ΔpH i after IL-4 pretreatment further strengthens the argument against electrical coupling between anions. IL-4 induced pendrin expression and increased bicarbonate and fluid secretion across nasal cells, and both these effects were suppressed by pendrin shRNA, which reduced pendrin mRNA levels by ~50% (Supplementary Figure 1). This indicates that pendrin both mediates HCO 3 -flux and enhances Cl -secretion through CFTR, with the latter driving most fluid transport. Purinoceptorstimulation of Ca 2+ -activated Cl -channels was also increased by IL-4 but was unaffected by pendrin shRNA, as expected when TMEM16A expression is elevated [START_REF] Pedemonte | Thiocyanate transport in resting and il-4-stimulated human bronchial epithelial cells: Role of pendrin and anion channels[END_REF][START_REF] Galietta | Il-4 is a potent modulator of ion transport in the human bronchial epithelium in vitro[END_REF][START_REF] Gorrieri | Goblet cell hyperplasia requires high bicarbonate transport to support mucin release[END_REF][START_REF] Caputo | Tmem16a, a membrane protein associated with calcium-dependent chloride channel activity[END_REF]. Reciprocal pendrin-CFTR regulation may result from a direct protein-protein interaction resembling those proposed for SLC26A3, SLC26A6 and SLC26A9 [START_REF] Ko | A molecular mechanism for aberrant cftr-dependent hco 3transport in cystic fibrosis[END_REF][START_REF] Ko | Gating of cftr by the stas domain of slc26 transporters[END_REF][START_REF] Avella | Slc26a9 stimulates cftr expression and function in human bronchial cell lines[END_REF](54)(55)(56). The present work provides direct evidence for pendrin-mediated regulation of CFTR in primary HBE cells and in non-epithelial BHK cells that stably express heterologous CFTR. Forskolin stimulation of CFTR-mediated whole cell Cl -currents was strongly enhanced in BHK cells by the co-expression of pendrin, and this was Supplementary Figure 1 Comparison of pendrin mRNA expression in non-CF bronchial epithelia after pendrin knockdown using shRNA adenovirus.


Guillaume Tintori

Pierre Nabokoff

Dr Ruqaya Buhaibeh

Dr David Bergé-Lefranc

Dr Sébastien Redon

Prof. P Julie Broggi
email: julie.broggi@univ-amu.fr

Patrice Vanelle
email: patrice.vanelle@univ-amu.fr.α

Vanelle

Base-free generation of organic electron donors from air-stable precursors

Organic electron donors (OEDs) are powerful reducing agents recognized for their potential in the reduction of challenging substrates and in original applications. Nonetheless, their stability issues in atmospheric oxygen or over time complicate their manipulation and storage. To overcome these constraints and enhance OED's practicality, new airand moisture-stable aminopyridinium carboxylate and carbonate precursors were synthesized and thermally activated to in-situ generate the potent electron donor. Carboxylate adducts proved to be excellent OEDlatent systems allowing easy and efficient reduction of challenging substrates. Their reducing properties are correlated to their structural characteristics by thermogravimetric and spectrometric analysis.

Organic electron donors (OEDs) with exceptionally negative redox potentials showed their potency and chemoselectivity in the reduction of challenging substrates. 1 They promote the formation of radical or anionic intermediates by single-or double-electron transfers. 2 These strong reducing agents are now attracting more and more the interest for original applications in diverse domains (coupling partners, polymerization initiators, redox switches, greenhouse gas reduction). 3 Their structures usually consist of two aza-cycles, such as imidazole or pyridine rings, connected at their center by an electron-rich double bond (Scheme 1). Typically, the enamine is prepared by addition of a strong base (NaH, KHMDS) on the cyclic iminium precursor salt. Some enamines (such as OED-2, Scheme 2) are stable under inert atmosphere and can be stored and manipulated in a glove box. 4 In other cases, the dimeric form is elusive (OED-1 has a half-life time of few minutes) and is rapidly converted into the corresponding carbene (Cb-1). 5 Thanks to the great resonance stabilization of their oxidized forms, pyridine and imidazole derivatives are powerful reducing agents with E1/2 (OED-2) = -1.27 V and E1/2 (OED-1) ≈ -1.20 V vs. SCE. To deal with stability issues, one solution is to in-situ generate the electron donor from the iminium salt in the presence of the substrate to be reduced. However, the concomitant use of excess amount of strong base is problematic as it presents limited tolerance to various functionalities and often gives complex mixtures.

To overcome these constraints and enhance OED's practicality, new strategies based on the activation of air-and moisture-stable masked OED-systems are crucial for the easy and efficient generation of organic reducers (Scheme 1). Our choice went to the use of carboxylate and carbonate adducts, well-known carbene precursors. 6 The advantage of these adducts is that any types of structures are virtually accessible by reaction of free N-heterocyclic carbenes (NHC) with CO2 atmosphere or by anion metathesis of azolium halides. Their thermolysis in-situ regenerate the free NHCs by decarboxylation, and CO2 is the only byproduct. This study proves that organic electron donors can also be formed during the process of thermal activation of carboxylate and carbonate adducts.

Scheme 1.

In-situ generation of OEDs.

Synthesis and characterization of OED-precursors

All new carboxylate and carbonate precursors of pyridinylidenetype reducers (DMAP-series) were synthesized in good yields using similar strategies than the ones reported for imidazoliumbased carboxy adducts (I-series in Figure 1). 7,8 Functionalization of the dimethylamino-pyridine core (DMAP) was obtained by a two-step procedure involving the deprotonation of the pyridinium Scheme 2. Potential species generated upon thermal decarboxylation and electron transfers.

salt followed by trapping of the generated species with carbon dioxide (Scheme 3). Whereas deprotonation of the 1,3-dimethylimidazolium iodide IMe-I with KHMDS gave the carbene Cb-1, the dimeric OED-2 was the only product isolated upon deprotonation of the pyridinium DMAP-Me-I. This further confirms the stability of bispyridinylidenes, even N-unbridged, compared to the elusive nature of imidazole-derived tetraazafulvalene (Scheme 2).

Structures of DMAP-Me-CO2, DMAPi Pr-CO2

and DMAP-Me-HCO3 were confirmed by single crystal X-ray crystallography and compared to the data reported for the imidazolium counterparts (Table 1 and2). 8,10 Louie demonstrated that imidazolium carboxylates had a great resonance stabilization when the CO2 lay in the same plane as the imidazolium ring. On the other hand, an increase in N-substituent size clearly induced longer C-CO2 bond and higher torsional angles, thus causing decreased stability of the NHC-CO2 and lower decarboxylation temperatures. In our case, the C-CO2 bond lengths of DMAP-Me-CO2 and DMAP-i Pr-CO2 (1.53 Å and 1.529 Å, respectively) were longer than in IMe-CO2 (1.523 Å) but shorter than in I t Bu-CO2 (1.551 Å). As well, the carboxylate torsional angles with respect to the iminium ring were significantly more pronounced in the DMAP derivatives (90.84° and 77.61°, respectively) than in IMe-CO2 (29.03°). Note that the coordinated CO2 moiety in DMAP-Me-CO2 is perpendicular to the sixmembered pyridinium ring plane. Such a high dihedral angle had only been observed in the highly N-hindered IPr-CO2 (Pr = 2,6diisopropylphenyl, 88.14°). 8 Carbonyl stretching frequencies of DMAP-Me-CO2 and DMAP-i Pr-CO2 followed the same trend, displaying an intermediate frequency of 1624 cm -1 , between IMe-CO2 and I t Bu-CO2. These encouraging structural factors pointed toward a straightforward decarboxylation process for our novel DMAP carboxylates, similar to that of N-hindered NHC-CO2.

Table 1. X-ray and structural features of NHC-CO2.

NHC-CO2

C-CO2 bond length (Å)

Dihedral angle N-C-C-O (°) νCOasym (cm -1
)

IMe-CO2 1.523 [a]
29.03 (10, 6 and 15% of mass loss, respectively). Surprisingly, DMAP-Me-CO2 presented a much higher decomposition temperature (181°C). Its crystalline structure displays an orthorhombic cell with a perfect alignment of the amino-pyridiniums compared to the monoclinic crystalline structures of DMAP-i Pr-CO2 and DMAP-Me-HCO3. 11 The well-organized arrangement of DMAP-Me-CO2 improves atom interactions and confers a particular rigidity to the solid that might explain its higher thermal resistance by TGA. The crystallographic program PLATON© confirmed the presence of twice hydrogen bond interactions in DMAP-Me-CO2 compared to DMAP-i Pr-CO2 and DMAP-Me-HCO3. 11 In solution, however, DMAP-Me-CO2 have a better reactivity at 120°C than DMAP-i Pr-CO2 (vide infra Table 4, entry 8 vs. 11). [START_REF] Denning | carboxylate adducts is strongly influenced by the solvent polarity[END_REF] Given the low steric bulk around the CO2 unit, the breakability of the C-CO2 bond in amino-pyridinium carboxylates seemed more related to the electronic influence of the aminopyridine ring rather than the steric effect of the N-substituent. As a matter of fact, introduction of the bulkier isopropyl group (DMAP-i Pr-CO2) had little impact on the structural features and the decarboxylation temperature. Unfortunately, preparation of a tert-butyl analog, for the observation of a possible combined electronic and steric effect, failed.

Comparative study for the reduction of 1

Different temperatures of decarboxylation were first compared using the readily accessible and well-documented 1,3-dimethylimidazolium-2-carboxylate (IMe-CO2) 7 as OEDprecursor (Table 3). Below 150°C, reduction of the 1-iodo-2-(3phenoxypropyl)benzene 1, a benchmark substrate in OEDpromoted reduction, was zero or close to it (Entries 1-2) even assisted by microwave irradiations (Entry 3). 13 At 150°C, a good conversion (74%) into the reduced product 2 was observed (Entry 4). This temperature threshold is of the same order as the decarboxylation temperature measured by TGA for IMe-CO2 (Table 2). The drastic fall in conversion rate (19%) in a sealed system also confirmed the necessity for the loss of the CO2 gas (Entry 5). For a complete reduction of 1, several options were possible (for details see Table S1 in supporting information): i) the reaction time could be lengthened to 48h; ii) the quantity of precursor could be increased to 6 equivalents; iii) the flask could be purged with vacuum/N2 cycles performed at 150°C at the beginning of the reaction. This vacuum effect, certainly leading to better removal of the CO2, was not sufficient to decrease the reaction temperature or the precursor equivalents; iv) the solvent concentration could be increased to 0.08 M (Table 3,entry 6). This last option had a greater impact, allowing the reduction of IMe-CO2 loading to 3 equivalents (Entry 7). Note that the use of 3 equivalents of carboxylate adduct imply the in-situ generation of only 1.5 equivalents of OED with respect to the substrate. Even tough preparation of these reaction mixtures was performed in a glove-box for a more accurate comparative study, a full conversion was also obtained when the reaction was setup in air and degassed under N2 before heating (Entry 9). Different carboxylate and carbonate adducts were then compared expecting that the easy decarboxylation profile of Nhindered imidazolium carboxylates, such as I t Bu-CO2, would allow the reduction of 1 at lower temperatures (Table 4). Unfortunately, a linear decrease in conversion rates was observed as the bulk of the N-substituents increased (Entries 2-6). Knowing that the decarboxylation of these imidazolium adducts is not a problem at these working temperatures, 8 the steric hindrance around the N2C-carbon center seems to be a serious impediment to the generation of the corresponding OED dimeric form. As predicted by our structural analysis, aminopyridinium adducts, DMAP-Me-CO2 and DMAP-i Pr-CO2, were better candidates: At 120°c, higher conversion rates were obtained (68 and 15%, respectively -Entries 8 and 11) than with IMe-CO2 (6% -Table 3, entry 2). Moreover, after 6h at 150°C, 90% conversion into 2 was reached with DMAP-Me-CO2 versus 36% for IMe-CO2 (Entries 1 and 9). 150 [e] 0.08 95

[a] 1 (1 equiv.), IMe-CO2 (2-5 equiv.), DMF (0.04 or 0.08 M), Ar, 24h. [b] 1 H NMR conversion (isolated yield). [c] Microwave heating, 16h. [d] Closed

schlenck. [e] Set-up in air and degassed under N2 before heating. [a] 1 (1 equiv.), precursor (3 equiv.), DMF (0.08 M), Ar, 24h. [b] 1 H NMR conversion (isolated yield). [c] Reaction time: 6h.

As expected from TGA, the impact of the bulkier N-isopropyl group on the decarboxylation and reactivity of DMAP-i Pr-CO2 (Entries 11-12) was not as evident as in imidazole series. Hydrogen carbonate adducts IMe-HCO3 and DMAP-Me-HCO3 could also be used as OED-precursors but they were less efficient than their carboxylate counterparts (Entries 7 and 13). DMAP-Me-MeCO3 gave a good percentage of product 2 (88% -Entry 15) but this carbonate salt was sensitive to water and rapidly decomposed into DMAP-Me-HCO3 upon exposure to air.

Evidence supporting the involvement of an organic donor

First, decarboxylation of the precursor is required as highlighted by the initiation of reactivity at high temperatures or inhibition of the reduction of 1 in closed systems (Table 3). This raises the question about the nature of the generated active species: carbene or enamine? Based on the preferential form adopted in the Wanzlick equilibrium upon deprotonation of the cyclic iminium salt, 4,5 in-situ decarboxylation of IMe-CO2 should mainly lead to the formation of the carbene Cb-1, while DMAP-Me-CO2 should give OED-2 (Scheme 2). In the literature, there is no report on reaction of free carbenes on iodoarenes, [START_REF] Emerson-King | Bond activation by NHC, leading to the nucleophilic aromatic substitution of highly activated perfluorinated arenes, is reported[END_REF] while their reduction with organic reducers such as OED-1 and OED-2 is reported. 4b,5a Indeed, almost no reaction occurred using Cb-1 at room temperature (Table 5, Entry 2), while reduction of 1 was complete using the powerful organic reducer OED-2 (Entry 1). Interestingly, at 150°C, partial reduction of 1 was observed in the presence of Cb-1 (Entry 3). Two mechanisms could be considered starting from IMe-CO2 (Scheme 2): 1) At this high temperature, according to Van't Hoff law, the proportion of dimer OED-1 will be even lower than at room temperature. However, the electron transfer from OED-1 will be kinetically increased and could explain the progressive reduction of 1. 5,[START_REF] Alder | Although formation of OED-1 is kinetically and thermodynamically unfavorable, evidences of the Wanzlick equilibrium in solution between enetetramine and carbene have been reported[END_REF] 2) Deprotonation of the DMF by Cb-1 leading to the formation of a formamide-derived electron donor could also be a competitive pathway for the electron transfer. [START_REF]For the formation of a DMF-derived organic donor upon deprotonation at 130°C with KO t Bu (pKa (KO t Bu in DMSO) = 29.4 versus predicted pKa (Cb-1 in DMSO) = 21)[END_REF] The unfavorable formation of OED-1 and the poor conversion (12%) obtained with IMe-CO2 in N,N-dimethylacetamide (DMA) [START_REF]DMA) does not allow the in-situ formation of OED upon its deprotonation[END_REF] (Table 5, entry 5) would point mainly toward the formation of a formamide-derived OED. DMAP-Me-CO2 3 150 99 5 [c] IMe-CO2 A different mechanism appears to operate with DMAP-Me-CO2. The highly stable dimeric OED-2 is probably the major generated form. Conservation of the reactivity in DMA (Table 5, entry 4) excludes the formation of a formamide-derived OED. Evidences supporting the electron transfer mechanism were obtained with the addition of commonly used inhibitors. The reduction of 1 decreased to 59% in the presence of the radical trap TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy), 18% with the competitive electron acceptor p-DNB (para-dinitrobenzene) and 38% when the reaction was carried out in air (Entries 6-8). Finally, analysis of the residual compounds at the end of the reaction confirmed the in-situ formation of the bispyridinylidene OED-2 (Scheme 2). The same dimethylamino-pyridinium salt DMAP-Me-X was recovered from the aqueous phase starting either from the active form OED-2 11 or the carboxylate precursor. This pyridinium salt DMAP-Me-X probably arise from the thermal decomposition of the oxidized form OED-2 2+ . From a kinetic point of view, it seems that the first step of decarboxylation is the limiting step with a thermodynamic equilibrium between the OED-2 and the carboxylate form DMAP-Me-CO2. CO2 remains in the DMF solution and competes with substrate 1 toward OED-2. 11

Scope of the reaction

To further investigate the versatility of our masked OED-system, the reduction of various substrates commonly used in OEDpromoted reactions was examined. 1,[START_REF] Schoenebeck | [END_REF] To our delight, we totally and cleanly reduced aryl halide, sulfone, or sulfonamide derivatives (Scheme 4). Beside the required precursor's activation temperature, the potency of OED-2 was maintained using the same reaction conditions that previously reported (reducer loading, time, conversion). DMAP-Me-CO2 allowed the generation of OED-2 in the presence of the substrate, contrary to pre-formulated strong base/azolium salt solutions. 5 In addition, the reaction could be conveniently set-up in air and degassed under N2 before heating. The few low isolated yields were attributed to the volatility of the reduced products. Iodonaphtol and its acetate counterpart were not reduced, but both led to the recovery of iodonaphtol. The competitive nucleophilic nature of OED-2 1,19 certainly prevented the reduction in favor of the deprotonation of the alcohol or the deprotection of the acetate. In conclusion, we presented here an original concept of base-free generation of organic electron donors from air-and moisture-stable precursors. Newly synthesized aminopyridinium carboxylate and carbonate adducts were fully characterized. They presented interesting structural and electronic properties compared to imidazolium series. These pyridinium carboxylate and carbonate adducts could also provide access to original pyridinylidene ligands in organometallic chemistry. In our case, the aminopyridinium carboxylates proved to be excellent OEDprecursors allowing the same order of reduction than the powerful organic reducers. These OED-masked systems will doubtless facilitate the handling of oxygen sensitive and elusive organic reducers and encourage their broader use. Work is now in progress to develop new OED-precursors with further enhanced functionality and practicality.

Figure 1 .

 1 Figure 1. OED-precursors used in this study (new structures in red).

  Data excerpted from reference 8 and 10.

Scheme 4 .

 4 Scheme 4. Scope of the reaction.

DMAP-Me-HCO3 could not be directly obtained from the reaction of the carboxylate DMAP-Me-CO2 with

	Scheme 3. Preparation of DMAP-derived carboxylate
	and carbonate precursors.
	4,5 Addition of CO2 on OED-2
	afforded DMAP-Me-CO2. Note that carboxylation of an
	aminoethylene functional group and formation of pyridinium
	carboxylates was not previously reported in the literature.
	Contrary to N-alkyl imidazolium carbonate, the N-methyl
	pyridinium carbonate water. Such lack of reactivity had been previously observed
	for N-aryl imidazolium carboxylates, where H2O was suspected
	of being only hydrogen-bonded to the CO2 moiety. 8 DMAP-Me-
	HCO3 was obtained by initial methylation of the DMAP with
	dimethyl carbonate ((MeO)2CO) followed by hydrolysis of the
	methyl carbonate anion of DMAP-Me-MeCO3. Acid-base
	reaction between the methyl carbonate anion and the pyridinium
	cation to form DMAP-Me-CO2 was not observed. Carbonate
	adducts usually present higher moisture stability and are thus
	more convenient to store. 9

Table 2 .

 2 Decarboxylation temperatures of carboxylate and carbonate precursors.

	IRR' adduct	-CO2 temp (°C) DMAP-R adduct	-CO2 temp (°C)
	IMe-CO2	140	DMAP-Me-CO2	181
	IMe-HCO3	40, 130	DMAP-Me-HCO3	55, 117
	IMe t Bu-CO2	113	DMAP-Me-MeCO3	34, 110

DMAP-Me-CO2 DMAP- i Pr-CO2 DMAP-Me-HCO3 I

	t Bu-CO2	63	DMAP-	i Pr-CO2	103
	Thermogravimetric analysis (TGA) further confirmed these
	conclusions showing intermediate decarboxylation temperatures
	for pyridinium derivatives (DMAP-Me-HCO3: 117°C, DMAP-Me-
	MeCO3: 110°C, DMAP-i Pr-CO2: 103°C, Table 2 and Figure S1-
	S2). 11 The first phenomena observed at 40, 55 and 34°C for
	IMe-HCO3,	DMAP-Me-HCO3	and	DMAP-Me-MeCO3
	corresponded to the prior loss of a molecule of H2O or MeOH

Table 3 .

 3 Optimization of the reduction of 1.

	[a]

Table 4 .

 4 Comparison of different carboxylate and carbonate precursors.

	[a]

Table 5 .

 5 Mechanistic study.

Acknowledgements

Aix-Marseille Université (AMU) and the Centre National de la Recherche Scientifique (CNRS) are gratefully acknowledged for financial support. G. T. thanks the Ministère de l'enseignement supérieur et de la recherche for his Ph.D. grant and P. N. thanks the Institut de Chimie Radicalaire for his master internship grant. We warmly thank Dr. Vincent Remusat for NMR analyses and the Spectropole (Fédération des Sciences Chimiques de Marseille) for MS, elemental analyses and X-ray determination. Our thanks to Michel Giorgi for valuable discussions on X-ray analysis.

Keywords: Electron transfer • organic electron donor • reduction reaction • carboxylate adduct • decarboxylation

Layout 2:

COMMUNICATION

To enhance the practicality of organic electron donors (OED), new air-and moisture-stable carboxylate and carbonate precursors were synthesized and thermally activated to in-situ generate the potent electron donor. Carboxylate adducts proved to be excellent OED-latent systems, allowing the easy and efficient reduction of challenging substrates. Base-free generation of organic electron donors from air-stable precursors

Guillaume


We describe a new approach to making plasmonic metamolecules with well-controlled resonances at optical wavelengths. Metamolecules are highly symmetric, subwavelength-scale clusters of metal and dielectric. They are of interest for metafluids, isotropic optical materials with applications in imaging and optical communications. For such applications, the morphology must be precisely controlled: the optical response is sensitive to nanometerscale variations in the thickness of metal coatings and the distances between metal surfaces. To achieve this precision, we use a multistep colloidal synthesis approach. Starting from highly monodisperse seeds, we grow octahedral clusters of polymer spheres using seededgrowth polymerization. We then overgrow the silica and remove the polystyrene to create a dimpled template. Finally, we attach six silica satellites to the template and coat them with gold. Using singlecluster spectroscopy, we show that the plasmonic resonances are reproducible from cluster to cluster. By comparing the spectra to theory, we show that the multi-step synthesis approach can control the distances between metallic surfaces to nanometer-scale precision. More broadly, our approach shows how metamolecules can be produced in bulk by combining different, high-yield colloidal synthesis steps, analogous to how small molecules are produced by multi-step chemical reactions.

Fabricating plasmonic building blocks (or "metamolecules") for optical metamaterials [1][2][3] requires precise positioning of metal and dielectric. The optical response is sensitive to nanometer-scale variations in both the thickness of metal coatings and the distances between metal surfaces. To date, two general approaches have been developed to create such structures: lithography, in which metal and dielectric are patterned and deposited stepwise [4][5][6][7][8] , and self-assembly, in which nanoparticles form nanoclusters, driven by DNA-mediated 9,10 or capillary interactions 11,12 . Lithography offers precise control over morphology but limited throughput. Self-assembly driven only by electrostatic, DNA-mediated, or capillary interactions offers high precision but limited control over yield.

Here we demonstrate a third approach that is based on neither lithography nor self-assembly: multi-step colloidal synthesis. We focus on the creation of nanoclusters with octahedral symmetry because their high symmetry may prove useful in metafluids [13][14][15] . We first synthesize a silica template with octahedrally coordinated "dimples" 16,17 , as shown in steps 1-4 of Fig. 1. This method takes advantage of several high-yield synthetic steps: a multi-stage silicaparticle synthesis process 18 that results in seed particles with a polydispersity index of only 1.002 (see ESI sec. 1.1); the templated synthesis of polystyrene colloidal molecules 19 , which results in octahedral clusters with a yield as high as 80% 18,20 ; the overgrowth of the silica core followed by removal of the polystyrene spheres 16 ; and the regioselective functionalization of the resulting concave dimples in the silica.

The challenge is to attach metal to the resulting functionalized, dimpled, octahedrally symmetric silica particles such that the distance between metal surfaces is controlled to nanometer-scale precision. In self-assembly approaches, this precision can be achieved by functionalizing gold nanoshells with a self-assembled monolayer and clustering them with capillary forces 11,12 , but this method yields clusters with many different morphologies. Another self-assembly approach involves attaching DNA-functionalized gold nanoparticles to DNA origami templates 10 . This method also leads to precise control J o u r n a l N a m e over the separation distance, but it produces clusters with different numbers of particles and morphologies. By contrast, the dimpled templates offer a highly consistent morphology, but until now no approach has led to precise control over the separation distance. In previous work, gold satellites were grown directly on the dimpled templates 21 . This approach requires iterative growth and oxidative etching of the gold, which results in the satellites varying in size and shape. Hence the interparticle distances were not well controlled. In the present study, we first attempted to lock presynthesized, highly monodisperse, ultra-smooth gold spheres 22 to the dimples, but we obtained a low yield of clusters with six particles attached, perhaps because of the large density difference between silica and gold. Therefore, instead of attaching gold directly to the dimples, we pursue a multi-step approach, as shown in steps 5-7 of Fig. 1. We first attach silica spheres to the dimples and then grow a gold layer on top of them. We lock highly monodisperse silica particles (polydispersity index 1.001) onto the dimples, building on a previously developed recipe 23 . This recipe takes advantage of the PS molecules that are grafted to the silica particles in step 1 and that remain after dissolution of the PS spheres in step 3. The chains are aminated and then coupled to carboxylated silica particles through a peptide reaction in dimethyl formamide. The solvent allows the aminated PS chains to extend, facilitating the capture of the particles by the dimples. To fill all the dimples, we use a large excess of silica particles (400:1). Then we use a seededgrowth method 24 to grow a gold film of controlled thickness on the silica. Growth occurs only on the silica satellites and not the central particle because the satellites are functionalized with carboxyl groups, which we can selectively convert to amine groups prior to gold plating. Gold seeds do not attach to the central particle because it is hydrophobic (ESI sec. 1.2.1). Thus, we control the separation distance by taking advantage of the very low polydispersity of the silica synthesis reaction 18,25 and the precision of the seeded-growth gold plating technique. Furthermore, because the silica core size and gold shell thickness can be changed independently, this approach provides independent control over the two geometrical features that control the frequency of the plasmon resonances: the separation distance and the separation-to-diameter ratio 26 . Transmission electron microscopy (TEM) shows that the synthesis is successful. As shown in Fig. 2a, we control the thickness of the gold shell by changing the amount of gold precursor in the synthesis step. We find that for a shell thickness of about 25 nm, the geometry remains consistent from cluster to cluster (Fig. 2b). Elemental mapping using energy-dispersive X-ray analysis (EDX) shows that the gold layer is specifically deposited on each of the six silica satellites, while the central silica particle remains gold-free (Fig. 2c).

Because the gold shells are separated by only a few nanometers, too small to measure precisely with TEM, we infer the separation distances and their variation using single-cluster spectroscopy and simulation. The plasmon resonances provide the most stringent test of the precision of our technique because they are sensitive to small changes in the separation 13 . We first locate isolated octahedral clusters on a marked grid using TEM, then transfer the TEM grid to a microscope stage. We set the illumination angle such that the objective collects the scattered light only. After finding the same nanoclusters in this dark-field setup, we measure the spectrum of light scattered from each individual cluster using a spectrometer attached to the microscope. The setup and representative images are shown in Fig. 3a.

Each of the nine clusters we measure shows two major peaks, one at 791.5±6.5nm, another at 886.2±5.9nm (Fig. 3b). We identify the peak positions by searching for the local maxima in the spectra, which can be located precisely, despite the width of the peaks. The uncertainties represent one standard deviation about the mean of the peak positions of the nine spectra. Whereas the microscope images confirm that each nanocluster has octahedral symmetry and that the number and approximate positions of the core-shell particles are consistent from cluster to cluster, the small uncertainty in peak positions suggests that the separation distance is also consistent. To make a quantitative statement about how precisely this distance is controlled, we first use simulation to determine the origins of the resonances, then explore how changing the separation distance affects the resonances.

To understand the origin of the scattering peaks, we use finitedifference time-domain (FDTD) simulations. Our simulations account for the experimental setup (Figs. 4a andS1)-including the substrate and the numerical aperture of the lens-and the geometry of the nanoclusters (Figs. 4b andS2)-including the fact that the shells terminate at the surface of the central particle. We first estimate the positions of the particles and the interparticle separations from TEM images, then refine these values to obtain agreement between simulation and experiment. We find that the simulation reproduces the peaks in the experimental spectra when the interparticle separation is 2 nm (Fig. 4c).

By mapping the charge density in the simulations (Fig. 4c) and calculating the far-field radiation pattern at different wavelengths (Fig. S4), we find that the peak near 900 nm originates from an electric dipolar resonance, and the peak near 800 nm has an electric quadrupolar component. The simulation does not explain all the features of the spectra. The peak close to 700 nm-which appears in three of the nine spectra and corresponds to the electric quadrupolar resonance (Fig. S4)-and the variation in the heights of the main two peaks likely arise from roughness in the gold shells. Because this roughness is comparable to the gap between the shells, we expect the relative magnitudes of the dipolar and higher-order resonances to depend sensitively on the details of the topography in the gap [27][28][29] .

With this understanding of the peak origins, we examine how changing the separation distance affects the locations of the resonances. Previous theoretical studies have shown that the dipolar resonances in a tetrahedral cluster are sensitive to the ratio of the inter-particle separation distance to particle diameter 13 . When this ratio changes from 0.02 to 0.04, the electric dipolar resonance shifts by about 100 nm. From the separation estimated with TEM (2-5 nm) and the overall diameter of the core-shell geometry, we estimate that the separation-to-diameter ratio of our octahedral cluster is between 0.011 and 0.027. Within this range, we expect the resonance peaks to be at least as sensitive as those in the previous study 13 .

Wavelength (nm)

Fig. 4 Finite-difference time-domain (FDTD) simulations reproduce the locations of the measured peaks. a, Illustration of simulation geometry, showing 3D renderings of a scale model of a cluster, with perspective, top, and front views. The simulation accounts for the incidence angle, collection angle, and substrate in the experiments (ESI sec. 2). To mimic an unpolarized light source, we add the scattered intensity from two orthogonal polarizations, shown as s and p. b, Schematic of cross-section of an octahedral cluster, showing length scales used in the simulation. "Gap" refers to the separation distance between the gold shells. c, Spectra from simulation (gray curves) and experiment (red curve, corresponding to cluster 9 of Fig. 3b). The spectrum simulated at a gap size of 2 nm (top gray curve) reproduces the locations of the peaks in the measured spectrum. Increasing the gap by 1 nm or more in the simulation shifts the two peaks to wavelengths that are outside the range of the observations. Charge density maps at 808.7 and 892.5 nm show different resonant modes at the two peaks. See Fig. S3 for polarized spectra. We use our simulation to confirm this sensitivity. By changing the inter-particle separation from 2 nm (shell thickness 23 nm, diameter 137+46=183nm, separation-to-diameter ratio 0.011) to 5 nm (shell thickness 21.5 nm, diameter 137+43=180nm, separation-to-diameter ratio 0.022), the dipolar peak blue-shifts about 90 nm (Fig. 4c) and the higher-order peak blue-shifts about 50 nm. The uncertainty in peak positions from experimental measurements is an order of magnitude smaller, 5.9 nm for the dipolar peak and 6.5 nm for the higher-order peak. By assuming a linear relationship between peak positions and interparticle separations, we obtain a conservative estimate for the uncertainty in interparticle separation: 0.2-0.4 nm. In other words, if the gap varied by more than this amount from cluster to cluster, the simulated spectra would not match the data. From this result, we conclude that the multi-step synthesis approach can control the interparticle separation to nanometer precision.

To be clear, "nanometer precision" refers to the average distance between the gold layers on the silica satellites. As can be seen in Fig. 2a, the gold layers have a roughness that is larger than a nanometer. This roughness might contribute to the observed variation in amplitude of the peaks from cluster to cluster. However, the consistency of the peak positions indicates that the average separation distance is very well controlled. In previous measurements on individual nanoclusters 30,31 , the spectra varied from cluster to cluster. This variation can lead to spectral broadening in bulk measurements 32,33 , which might be avoided by using clusters made with our approach.

We estimate the morphology yield to be 60%, based on analysis of electron microscopy images at different steps along the synthesis pathway shown in Fig. 1. The yield of seed silica particles with octahedrally-coordinated polystyrene satellites (step 1) is 62% (total clusters counted N≈100), with the side products being silica cores with three, four, and five polystyrene satellites (Table S1). After overgrowth of the silica core (step 2) and removal of the polystyrene spheres (step 3), the yield of silica cores with octahedrally coordinated dimples is 60% (N≈100, Table S2). After functionalizing the dimples (step 4) and adding silica satellites in large excess (step 5), we observe that nearly all of the dimples are filled. Finally, after functionalizing the silica satellites (step 6) and growing gold shells on them (step 7), we find that 60% (N=25) of the resulting clusters have six gold shells attached to a central silica particle (Table S3). These results show that the step that limits the yield is likely the synthesis of octahedral silica/polystyrene colloidal molecules (step 1). Previous work 18,20 has shown that the yield of this step can be increased to more than 80%. We note also that because each step of the synthesis is done in bulk, the procedure can be scaled up to produce clusters at gram scales or more.

Future work might extend this approach to produce other plasmonic metamolecules 13 . To this end, one can control the morphology by changing the dimple geometry of the overgrown silica; by tuning the core size, it is possible to make tetrahedral and dodecahedral geometries 16,34 . Also, because the core size, shell thickness, and interparticle separation can be controlled independently of one another, there are many ways to tune the positions of the plasmon resonances.

More generally, our method illustrates the degree to which colloidal morphology can now be controlled through chemistry. The precision of our method stems from the versatility of seeded-growth reactions. These are used to create the highly monodisperse silica particles for the core and satellites, to control the thickness of the gold films (step 7 of Fig. 1), to overgrow the silica core (step 2), and to make the octahedral cluster of sacrificial polymer particles that coordinate the central silica core (step 1). The reaction in step 1 relies on a geometrical effect 35 that results in a narrow distribution of numbers of particles attached to a central core, even when the initial seeds of those particles are randomly distributed on the core surface 36 . A similar process in which spheres adsorb onto a central core-rather than grow on it-is the basis of a recent synthesis method for making patchy colloidal particles with controlled morphology 37 . Like our method, this method uses multiple high-yield synthesis steps to produce large quantities of specific clusters. Such schemes, which resemble the multi-step synthetic schemes used to produce complex molecules from organic reagents, represent a new and emerging paradigm in colloidal materials. They have clear advantages over selfassembly and lithography: they can produce bulk quantities of complex three-dimensional structures in bulk with no annealing required. Our results demonstrate that the multistep approach can meet even the most stringent requirements for precision over the assembly.

  Polyhedral plasmonic nanoclusters through multi-step colloidal chemistry † Nabila Tanjeem, ‡,1 Cyril Chomette, ‡,b Nicholas B. Schade, c Serge Ravaine, d Etienne Duguet, * ,b Mona Tréguer-Delapierre, * ,b and Vinothan N. Manoharan * ,a,c

2

 2

Fig. 1

 1 Fig. 1 Our synthesis scheme for plasmonic clusters consists of seven steps: (1) Seeded-growth emulsion polymerization of styrene on spherical silica particles with a diameter of 86 nm; (2) overgrowing the silica seed and (3) dissolving polystyrene, which results in structures with six concave dimples; (4) aminating the residual polystyrene at the bottom of the dimples to complete the template; (5) locking carboxylated silica nanospheres with a diameter of 137 nm onto the aminated dimples; (6) converting the carboxylate groups on the silica satellites into amine groups; (7) growing a thin gold shell on the spheres by the site-specific adsorption of gold nuclei and subsequent regrowth. See ESI sec. 1.1-1.4 for details on the synthesis.

Fig. 2

 2 Fig. 2 Electron microscopy shows the geometry of the nanoclusters. a, TEM images of silica/gold clusters with varying gold shell thicknesses, obtained with different amounts of gold precursor, from 5-35 mL. b, TEM image of clusters obtained for the regrowth condition of 25 mL gold precursor. Octahedral clusters are circled in red. Other particles are unattached gold nanoshells, which are formed when the excess silica particles added in step 4 of Fig. 1 are functionalized and coated with gold. Drying the sample for TEM imaging can cause these particles to aggregate. c, STEM-EDX elemental mapping of an octahedral cluster: bright field TEM image (top) and silicon, oxygen, gold, and superimposed EDX maps evidence the core-shell morphology of the six satellites. See ESI sec. 1.5 for details on the characterization methods.

Fig. 3

 3 Fig. 3 Single-cluster spectroscopy shows that individual clusters have two consistent peaks. a, Illustration of setup for measuring scattering spectra of individual nanoclusters (ESI sec. 1.5). An unpolarized halogen lamp illuminates the sample, and an objective (NA = 0.5) collects the scattered light from a a spot 1 µm in diameter, set by the aperture. TEM images show a cluster near marker "P" on the finder grid at low and high magnification. Dark-field micrograph, obtained from the setup, shows the same cluster. b, Measured spectra of nine individual octahedral clusters. Each spectrum is accompanied by a TEM image of the measured cluster and a 3D rendering showing its approximate orientation. Upward ticks on the wavelength axis show the peak positions. Gray regions denote the range of these positions. Cluster 3 is also shown in the micrographs of a.

Acknowledgments

We thank Arthur McClelland for help with the dark-field spectroscopy setup, Carolyn Marks and Adam Graham for help with TEM, and Sarah Kostinski for helpful discussions about the simulation. Some of the TEM experiments were performed at the Bordeaux Imaging Center and Plateforme de Caractérisation des Matériaux of the University of Bordeaux. We gratefully acknowledge Jérôme Majimel for his assistance in mapping analysis. This work was supported by the LabEx AMADEus (ANR-10-LABX-42) and IdEx Bordeaux (ANR-10-IDEX-03-02), that is, the Investissements d'Avenir programme of the French government managed by the Agence Nationale de la Recherche. The research was partially supported by the National Science Foundation through the Harvard University Materials Research Science and Engineering Center under grant number DMR-2011754. This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Coordinated Infrastructure Network (NNCI), which is supported by the National Science Foundation under NSF award no. 1541959. CNS is part of Harvard University.

Conflicts of interest

There are no conflicts to declare.