Development and Application of a High-Throughput Method for the Purification and Analysis of Surface Carbohydrates from Klebsiella pneumoniae.

Klebsiella pneumoniae (Kp) is a Gram-negative bacterium, and a leading cause of neonatal sepsis in low- and middle-income countries, often associated with anti-microbial resistance. Two types of polysaccharides are expressed on the Kp cell surface and have been proposed as key antigens for vaccine design: capsular polysaccharides (known as K-antigens, K-Ags) and O-antigens (O-Ags). Historically, Kp has been classified using capsule serotyping and although 186 distinct genotypes have been predicted so far based on sequence analysis, many structures are still unknown. In contrast, only 11 distinct OAg serotypes have been described. The characterization of emerging strains requires the development of a high-throughput purification method to obtain sufficient K- and O-Ag material to characterize the large collection of serotypes and gain insight on structural features and potential cross-reactivity that could allow vaccine simplification. Here, this was achieved by adapting our established method for the simple purification of O-Ags, using mild acetic acid hydrolysis performed directly on bacterial cells, followed by filtration and precipitation steps. The method was successfully applied to purify the surface carbohydrates from different Kp strains, thereby demonstrating the robustness and general applicability of the purification method developed. Further, antigen characterization showed that the purification method had no impact on the structural integrity of the polysaccharides and preserved labile substituents such as O-acetyl and pyruvyl groups. This method can be further optimized for scaling up and manufacturing to support the development of high-valency saccharide-based vaccines against Kp.

Comparison of Shigella GMMA and glycoconjugate four-component formulations in animals.

Shigellosis is leading bacterial cause of diarrhea with high prevalence in children younger than 5 years in low- and middle-income countries, and increasing number of reports of Shigella cases associated to anti-microbial resistance. No vaccines against Shigella are still licensed, but different candidates based on the O-antigen portion of lipopolysaccharides are in clinic. Generalized Modules for Membrane Antigens (GMMA) have been proposed as an alternative delivery system for the O-antigen, and a 4-component vaccine candidate (altSonflex1-2-3), containing GMMA from S. sonnei and S. flexneri 1b, 2a and 3a is being tested in a phase 1/2 clinical trial, with the aim to elicit broad protection against the most prevalent Shigella serotypes. Here, the 4-component GMMA vaccine candidate has been compared to a more traditional glycoconjugate formulation for the ability to induce functional antibodies in mice and rabbits. In mice, in the absence of Alhydrogel, GMMA induce higher IgG antibodies than glycoconjugates and stronger bactericidal titers against all Shigella serotypes. In the presence of Alhydrogel, GMMA induce O-antigen specific IgG levels similar to traditional glycoconjugates, but with a broader range of IgG subclasses, resulting in stronger bactericidal activity. In rabbits, GMMA elicit higher functional antibodies than glycoconjugates against S. sonnei, and similar responses to S. flexneri 1b, 2a and 3a, independently from the presence of Alhydrogel. Different O-antigen based vaccines against Shigella are now in clinical stage and it will be of particular interest to understand how the preclinical findings in the different animal models translate in humans.

Testing a Recombinant Form of Tetanus Toxoid as a Carrier Protein for Glycoconjugate Vaccines.

Glycoconjugate vaccines play a major role in the prevention of infectious diseases worldwide, with significant impact on global health, enabling the polysaccharides to induce immunogenicity in infants and immunological memory. Tetanus toxoid (TT), a chemically detoxified bacterial toxin, is among the few carrier proteins used in licensed glycoconjugate vaccines. The recombinant full-length 8MTT was engineered in E. coli with eight individual amino acid mutations to inactivate three toxin functions. Previous studies in mice showed that 8MTT elicits a strong IgG response, confers protection, and can be used as a carrier protein. Here, we compared 8MTT to traditional carrier proteins TT and cross-reactive material 197 (CRM197), using different polysaccharides as models: Group A Streptococcus cell-wall carbohydrate (GAC), Salmonella Typhi Vi, and Neisseria meningitidis serogroups A, C, W, and Y. The persistency of the antibodies induced, the ability of the glycoconjugates to elicit booster response after re-injection at a later time point, the eventual carrier-induced epitopic suppression, and immune interference in multicomponent formulations were also evaluated. Overall, immunogenicity responses obtained with 8MTT glycoconjugates were compared to those obtained with corresponding TT and, in some cases, were higher than those induced by CRM197 glycoconjugates. Our results support the use of 8MTT as a good alternative carrier protein for glycoconjugate vaccines, with advantages in terms of manufacturability compared to TT.

Enhanced Systemic Humoral Immune Response Induced in Mice by Generalized Modules for Membrane Antigens (GMMA) Is Associated with Affinity Maturation and Isotype Switching.

Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from Gram-negative bacteria that can be used to design affordable subunit vaccines. GMMA have been observed to induce a potent humoral immune response in preclinical and clinical studies. In addition, in preclinical studies, it has been found that GMMA can be exploited as optimal antigen carriers for both protein and saccharide antigens, as they are able to promote the enhancement of the antigen-specific humoral immune response when the antigen is overexpressed or chemically conjugated to GMMA. Here we investigated the mechanism of this GMMA carrier effect by immunizing mice and using factor H binding protein and GMMA of Neisseria meningitidis B as an antigen-GMMA model. We confirmed that the antigen displayed on the GMMA surface increased the antigen-specific IgG production and, above all, the antibody functionality measured by the serum bactericidal activity. We found that the enhancement of the bactericidal capacity induced by GMMA carrying the antigen on the surface was associated with the increase in antibody affinity to the antigen, and with the switching toward IgG subclasses with more bactericidal potential. Thus, we conclude that the potent carrier effect of GMMA is due to their ability to promote a better quality of humoral immunity.

Vi polysaccharide and conjugated vaccines afford similar early, IgM or IgG-independent control of infection but boosting with conjugated Vi vaccines sustains the efficacy of immune responses.

Introduction: Vaccination with Vi capsular polysaccharide (Vi-PS) or protein-Vi typhoid conjugate vaccine (TCV) can protect adults against Salmonella Typhi infections. TCVs offer better protection than Vi-PS in infants and may offer better protection in adults. Potential reasons for why TCV may be superior in adults are not fully understood. Methods and results: Here, we immunized wild-type (WT) mice and mice deficient in IgG or IgM with Vi-PS or TCVs (Vi conjugated to tetanus toxoid or CRM197) for up to seven months, with and without subsequent challenge with Vi-expressing Salmonella Typhimurium. Unexpectedly, IgM or IgG alone were similarly able to reduce bacterial burdens in tissues, and this was observed in response to conjugated or unconjugated Vi vaccines and was independent of antibody being of high affinity. Only in the longer-term after immunization (>5 months) were differences observed in tissue bacterial burdens of mice immunized with Vi-PS or TCV. These differences related to the maintenance of antibody responses at higher levels in mice boosted with TCV, with the rate of fall in IgG titres induced to Vi-PS being greater than for TCV. Discussion: Therefore, Vi-specific IgM or IgG are independently capable of protecting from infection and any superior protection from vaccination with TCV in adults may relate to responses being able to persist better rather than from differences in the antibody isotypes induced. These findings suggest that enhancing our understanding of how responses to vaccines are maintained may inform on how to maximize protection afforded by conjugate vaccines against encapsulated pathogens such as S. Typhi.

Elucidating the role of N-acetylglucosamine in Group A Carbohydrate for the development of an effective glycoconjugate vaccine against Group A Streptococcus.

Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate against Group A Streptococcus infections. Native GAC consists of a polyrhamnose (polyRha) backbone with N-acetylglucosamine (GlcNAc) at every second rhamnose residue. Both native GAC and the polyRha backbone have been proposed as vaccine components. Here, chemical synthesis and glycoengineering were used to generate a panel of different length GAC and polyrhamnose fragments. Biochemical analyses were performed confirming that the epitope motif of GAC is composed of GlcNAc in the context of the polyrhamnose backbone. Conjugates from GAC isolated and purified from a bacterial strain and polyRha genetically expressed in E. coli and with similar molecular size to GAC were compared in different animal models. The GAC conjugate elicited higher anti-GAC IgG levels with stronger binding capacity to Group A Streptococcus strains than the polyRha one, both in mice and in rabbits. This work contributes to the development of a vaccine against Group A Streptococcus suggesting GAC as preferable saccharide antigen to include in the vaccine.

GMMA as an Alternative Carrier for a Glycoconjugate Vaccine against Group A Streptococcus.

Group A Streptococcus (GAS) causes about 500,000 annual deaths globally, and no vaccines are currently available. The Group A Carbohydrate (GAC), conserved across all GAS serotypes, conjugated to an appropriate carrier protein, represents a promising vaccine candidate. Here, we explored the possibility to use Generalized Modules for Membrane Antigens (GMMA) as an alternative carrier system for GAC, exploiting their intrinsic adjuvant properties. Immunogenicity of GAC-GMMA conjugate was evaluated in different animal species in comparison to GAC-CRM197; and the two conjugates were also compared from a techno-economic point of view. GMMA proved to be a good alternative carrier for GAC, resulting in a higher immune response compared to CRM197 in different mice strains, as verified by ELISA and FACS analyses. Differently from CRM197, GMMA induced significant levels of anti-GAC IgG titers in mice also in the absence of Alhydrogel. In rabbits, a difference in the immune response could not be appreciated; however, antibodies from GAC-GMMA-immunized animals showed higher affinity toward purified GAC antigen compared to those elicited by GAC-CRM197. In addition, the GAC-GMMA production process proved to be more cost-effective, making this conjugate particularly attractive for low- and middle-income countries, where this pathogen has a huge burden.

Novel Simple Conjugation Chemistries for Decoration of GMMA with Heterologous Antigens.

Outer Membrane Vesicles (OMV) constitute a promising platform for the development of efficient vaccines. OMV can be decorated with heterologous antigens (proteins or polysaccharides), becoming attractive novel carriers for the development of multicomponent vaccines. Chemical conjugation represents a tool for linking antigens, also from phylogenetically distant pathogens, to OMV. Here we develop two simple and widely applicable conjugation chemistries targeting proteins or lipopolysaccharides on the surface of Generalized Modules for Membrane Antigens (GMMA), OMV spontaneously released from Gram-negative bacteria mutated to increase vesicle yield and reduce potential reactogenicity. A Design of Experiment approach was used to identify optimal conditions for GMMA activation before conjugation, resulting in consistent processes and ensuring conjugation efficiency. Conjugates produced by both chemistries induced strong humoral response against the heterologous antigen and GMMA. Additionally, the use of the two orthogonal chemistries allowed to control the linkage of two different antigens on the same GMMA particle. This work supports the further advancement of this novel platform with great potential for the design of effective vaccines.

Generalized Modules for Membrane Antigens as Carrier for Polysaccharides: Impact of Sugar Length, Density, and Attachment Site on the Immune Response Elicited in Animal Models.

Nanoparticle systems are being explored for the display of carbohydrate antigens, characterized by multimeric presentation of glycan epitopes and special chemico-physical properties of nano-sized particles. Among them, outer membrane vesicles (OMVs) are receiving great attention, combining antigen presentation with the immunopotentiator effect of the Toll-like receptor agonists naturally present on these systems. In this context, we are testing Generalized Modules for Membrane Antigens (GMMA), OMVs naturally released from Gram-negative bacteria mutated to increase blebbing, as carrier for polysaccharides. Here, we investigated the impact of saccharide length, density, and attachment site on the immune response elicited by GMMA in animal models, using a variety of structurally diverse polysaccharides from different pathogens (i.e., Neisseria meningitidis serogroup A and C, Haemophilus influenzae type b, and streptococcus Group A Carbohydrate and Salmonella Typhi Vi). Anti-polysaccharide immune response was not affected by the number of saccharides per GMMA particle. However, lower saccharide loading can better preserve the immunogenicity of GMMA as antigen. In contrast, saccharide length needs to be optimized for each specific antigen. Interestingly, GMMA conjugates induced strong functional immune response even when the polysaccharides were linked to sugars on GMMA. We also verified that GMMA conjugates elicit a T-dependent humoral immune response to polysaccharides that is strictly dependent on the nature of the polysaccharide. The results obtained are important to design novel glycoconjugate vaccines using GMMA as carrier and support the development of multicomponent glycoconjugate vaccines where GMMA can play the dual role of carrier and antigen. In addition, this work provides significant insights into the mechanism of action of glycoconjugates.

Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease.

Activation of the Ca2+ activated Cl- channel TMEM16A is proposed as a treatment in inflammatory airway disease. It is assumed that activation of TMEM16A will induce electrolyte secretion, and thus reduce airway mucus plugging and improve mucociliary clearance. A benefit of activation of TMEM16A was shown in vitro and in studies in sheep, but others reported an increase in mucus production and airway contraction by activation of TMEM16A. We analyzed expression of TMEM16A in healthy and inflamed human and mouse airways and examined the consequences of activation or inhibition of TMEM16A in asthmatic mice. TMEM16A was found to be upregulated in the lungs of patients with asthma or cystic fibrosis, as well as in the airways of asthmatic mice. Activation or potentiation of TMEM16A by the compounds Eact or brevenal, respectively, induced acute mucus release from airway goblet cells and induced bronchoconstriction in mice in vivo. In contrast, niclosamide, an inhibitor of TMEM16A, blocked mucus production and mucus secretion in vivo and in vitro. Treatment of airway epithelial cells with niclosamide strongly inhibited expression of the essential transcription factor of Th2-dependent inflammation and goblet cell differentiation, SAM pointed domain-containing ETS-like factor (SPDEF). Activation of TMEM16A in people with inflammatory airway diseases is likely to induce mucus secretion along with airway constriction. In contrast, inhibitors of TMEM16A may suppress pulmonary Th2 inflammation, goblet cell metaplasia, mucus production, and bronchoconstriction, partially by inhibiting expression of SPDEF.

Neisseria meningitidis Factor H Binding Protein Surface Exposure on Salmonella Typhimurium GMMA Is Critical to Induce an Effective Immune Response against Both Diseases.

GMMA, outer membrane vesicles resulting from hyperblebbing mutated bacterial strains, are a versatile vaccine platform for displaying both homologous and heterologous antigens. Periplasmic expression is a popular technique for protein expression in the lumen of the blebs. However, the ability of internalized antigens to induce antibody responses has not been extensively investigated. Herein, the Neisseria meningitidis factor H binding protein (fHbp) was heterologously expressed in the lumen of O-antigen positive (OAg+) and O-antigen negative (OAg-) Salmonella Typhimurium GMMA. Only the OAg- GMMA induced an anti-fHbp IgG response in mice if formulated on Alum, although it was weak and much lower compared to the recombinant fHbp. The OAg- GMMA on Alum showed partial instability, with possible exposure of fHbp to the immune system. When we chemically conjugated fHbp to the surface of both OAg+ and OAg- GMMA, these constructs induced a stronger functional response compared to the fHbp immunization alone. Moreover, the OAg+ GMMA construct elicited a strong response against both the target antigens (fHbp and OAg), with no immune interference observed. This result suggests that antigen localization on GMMA surface can play a critical role in the induction of an effective immune response and can encourage the development of GMMA based vaccines delivering key protective antigens on their surface.

O-Antigen Extraction, Purification, and Chemical Conjugation to a Carrier Protein.

A variety of bacterial infections have been tackled by glycoconjugates over the recent years, and more vaccines are either under development at preclinical level or in clinical trials. So far, licensed glycoconjugate vaccines have made use of capsular polysaccharides or derived fragments. Today, many glycoconjugates are making use of other classes of sugars, in particular, the O-antigen portion of lipopolysaccharide molecules. Here, we report a simplified method for O-antigen extraction and purification that avoids the step of lipopolysaccharide isolation. Also, a selective chemistry for terminal linkage of O-antigen chains to a carrier protein is described, together with analytical methods for intermediates and final conjugate characterization.

TMEM16A Mediates Mucus Production in Human Airway Epithelial Cells.

TMEM16A is a Ca2+-activated chloride channel that was shown to enhance production and secretion of mucus in inflamed airways. It is, however, not clear whether TMEM16A directly supports mucus production, or whether mucin and TMEM16A are upregulated independently during inflammatory airway diseases such as asthma and cystic fibrosis (CF). We examined this question using BCi-NS1 cells, a human airway basal cell line that maintains multipotent differentiation capacity, and the two human airway epithelial cell lines, Calu-3 and CFBE. The data demonstrate that exposure of airway epithelial cells to IL-8 and IL-13, two cytokines known to be enhanced in CF and asthma, respectively, leads to an increase in mucus production. Expression of MUC5AC was fully dependent on expression of TMEM16A, as shown by siRNA knockdown of TMEM16A. In addition, different inhibitors of TMEM16A attenuated IL-13-induced mucus production. Interestingly, in CFBE cells expressing F508 delCFTR, IL-13 was unable to upregulate membrane expression of TMEM16A or Ca2+-activated whole cell currents. The regulator of TMEM16A, CLCA1, strongly augmented both Ca2+- and cAMP-activated Cl- currents in cells expressing wtCFTR but failed to augment membrane expression of TMEM16A in F508 delCFTR-expressing CFBE cells. The data confirm the functional relationship between CFTR and TMEM16A and suggest an impaired upregulation of TMEM16A by IL-13 or CLCA1 in cells expressing the most frequent CF-causing mutation F508 delCFTR.

Rational Design of a Glycoconjugate Vaccine against Group A Streptococcus.

No commercial vaccine is yet available against Group A Streptococcus (GAS), major cause of pharyngitis and impetigo, with a high frequency of serious sequelae in low- and middle-income countries. Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate. Here, we explored the possibility to use GAS Streptolysin O (SLO), SpyCEP and SpyAD protein antigens with dual role of antigen and carrier, to enhance the efficacy of the final vaccine and reduce its complexity. All protein antigens resulted good carrier for GAC, inducing similar anti-GAC IgG response to the more traditional CRM197 conjugate in mice. However, conjugation to the polysaccharide had a negative impact on the anti-protein responses, especially in terms of functionality as evaluated by an IL-8 cleavage assay for SpyCEP and a hemolysis assay for SLO. After selecting CRM197 as carrier, optimal conditions for its conjugation to GAC were identified through a Design of Experiment approach, improving process robustness and yield This work supports the development of a vaccine against GAS and shows how novel statistical tools and recent advancements in the field of conjugation can lead to improved design of glycoconjugate vaccines.

Short Vi-polysaccharide abrogates T-independent immune response and hyporesponsiveness elicited by long Vi-CRM197 conjugate vaccine.

Polysaccharide-protein conjugates have been developed to overcome the T-independent response, hyporesponsiveness to repeated vaccination, and poor immunogenicity in infants of polysaccharides. To address the impact of polysaccharide length, typhoid conjugates made with short- and long-chain fractions of Vi polysaccharide with average sizes of 9.5, 22.8, 42.7, 82.0, and 165 kDa were compared. Long-chain-conjugated Vi (165 kDa) induced a response in both wild-type and T cell-deficient mice, suggesting that it maintains a T-independent response. In marked contrast, short-chain Vi (9.5 to 42.7 kDa) conjugates induced a response in wild-type mice but not in T cell-deficient mice, suggesting that the response is dependent on T cell help. Mechanistically, this was explained in neonatal mice, in which long-chain, but not short-chain, Vi conjugate induced late apoptosis of Vi-specific B cells in spleen and early depletion of Vi-specific B cells in bone marrow, resulting in hyporesponsiveness and lack of long-term persistence of Vi-specific IgG in serum and IgG+ antibody-secreting cells in bone marrow. We conclude that while conjugation of long-chain Vi generates T-dependent antigens, the conjugates also retain T-independent properties, leading to detrimental effects on immune responses. The data reported here may explain some inconsistencies observed in clinical trials and help guide the design of effective conjugate vaccines.

GMMA Is a Versatile Platform to Design Effective Multivalent Combination Vaccines.

Technology platforms are an important strategy to facilitate the design, development and implementation of vaccines to combat high-burden diseases that are still a threat for human populations, especially in low- and middle-income countries, and to address the increasing number and global distribution of pathogens resistant to antimicrobial drugs. Generalized Modules for Membrane Antigens (GMMA), outer membrane vesicles derived from engineered Gram-negative bacteria, represent an attractive technology to design affordable vaccines. Here, we show that GMMA, decorated with heterologous polysaccharide or protein antigens, leads to a strong and effective antigen-specific humoral immune response in mice. Importantly, GMMA promote enhanced immunogenicity compared to traditional formulations (e.g., recombinant proteins and glycoconjugate vaccines), without negative impact to the anti-GMMA immune response. Our findings support the use of GMMA as a "plug and play" technology for the development of effective combination vaccines targeting different bugs at the same time.

Transport properties in CFTR-/- knockout piglets suggest normal airway surface liquid pH and enhanced amiloride-sensitive Na+ absorption.

Previous analysis of CFTR-knockout (CFTR-/-) in piglets has provided important insights into the pathology of cystic fibrosis. However, controversies exist as to the true contribution of CFTR to the pH balance in airways and intestine. We therefore compared ion transport properties in newborn wild-type (CFTR+/+) and CFTR-knockout (CFTR-/- piglets). Tracheas of CFTR-/- piglets demonstrated typical cartilage malformations and muscle cell bundles. CFTR-/- airway epithelial cells showed enhanced lipid peroxidation, suggesting inflammation early in life. CFTR was mainly expressed in airway submucosal glands and was absent in lungs of CFTR-/- piglets, while expression of TMEM16A was uncompromised. mRNA levels for TMEM16A, TMEM16F, and αßγENaC were unchanged in CFTR-/- airways, while mRNA for SLC26A9 appeared reduced. CFTR was undetectable in epithelial cells of CFTR-/- airways and intestine. Small intestinal epithelium of CFTR-/- piglets showed mucus accumulation. Secretion of both electrolytes and mucus was activated by stimulation with prostaglandin E2 and ATP in the intestine of CFTR+/+, but not of CFTR-/- animals. pH was measured inside small bronchi using a pH microelectrode and revealed no difference between CFTR+/+ and CFTR-/- piglets. Intracellular pH in porcine airway epithelial cells revealed only a small contribution of CFTR to bicarbonate secretion, which was absent in cells from CFTR-/- piglets. In contrast to earlier reports, our data suggest a minor impact of CFTR on ASL pH. In contrast, enhanced amiloride-sensitive Na+ absorption may contribute to lung pathology in CFTR-/- piglets, along with a compromised CFTR- and TMEM16A-dependent Cl- transport.

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl- Channel TMEM16A.

TMEM16A is a Ca2+ activated Cl- channel with important functions in airways, intestine, and other epithelial organs. Activation of TMEM16A is proposed as a therapy in cystic fibrosis (CF) to reinstall airway Cl- secretion and to enhance airway surface liquid (ASL). This CFTR-agnostic approach is thought to improve mucociliary clearance and lung function in CF. This could indeed improve ASL, however, mucus release and airway contraction may also be induced by activators of TMEM16A, particularly in inflamed airways of patients with asthma, COPD, or CF. Currently, both activators and inhibitors of TMEM16A are developed and examined in different types of tissues. Here we compare activation and inhibition of endogenous and overexpressed TMEM16A and analyze potential off-target effects. The three well-known blockers benzbromarone, niclosamide, and Ani9 inhibited both TMEM16A and ATP-induced Ca2+ increase by variable degrees, depending on the cell type. Niclosamide, while blocking Ca2+ activated TMEM16A, also induced a subtle but significant Ca2+ store release and inhibited store-operated Ca2+ influx. Niclosamide, benzbromarone and Ani9 also affected TMEM16F whole cell currents, indicating limited specificity for these inhibitors. The compounds Eact, cinnamaldehyde, and melittin, as well as the phosphatidylinositol diC8-PIP2 are the reported activators of TMEM16A. However, the compounds were unable to activate endogenous TMEM16A in HT29 colonic epithelial cells. In contrast, TMEM16A overexpressed in HEK293 cells was potently stimulated by these activators. We speculate that overexpressed TMEM16A might have a better accessibility to intracellular Ca2+, which causes spontaneous activity even at basal intracellular Ca2+ concentrations. Small molecules may therefore potentiate pre-stimulated TMEM16A currents, but may otherwise fail to activate silent endogenous TMEM16A.

Control of Ion Transport by Tmem16a Expressed in Murine Intestine.

Cl- secretion by the human and murine intestinal epithelium occurs through the cystic fibrosis transmembrane conductance regulator (cftr). However, the Ca2+ activated Cl- channel Tmem16a was shown to contribute to Cl- secretion, mainly, but not exclusively, as a basolaterally located Cl- channel that controls basolateral Ca2+ signaling, and thus activation of basolateral Ca2+ dependent Sk4 K+ channels. In intestinal goblet cells, Tmem16a was shown to regulated Ca2+ signals required for exocytosis of mucus. Because a recent report denied the existence and functional role of Tmem16a in murine intestine, we reexamined in detail expression of mRNA and protein for Tmem16a in mouse colon. In experiments using short-circuited Ussing chamber and whole cell patch-clamp techniques, we further compared ion transport in wild type (WT) colon with that in mice with intestinal epithelial specific knockout of Tmem16a. As reported earlier we fully confirm expression of Tmem16a in colonic epithelial cells and the role of Tmem16a for both Ca2+-dependent and cAMP-regulated ion secretion.

Niclosamide repurposed for the treatment of inflammatory airway disease.

Inflammatory airway diseases, such as asthma, cystic fibrosis (CF), and chronic obstructive pulmonary disease (COPD), are characterized by mucus hypersecretion and airway plugging. In both CF and asthma, enhanced expression of the Ca2+-activated Cl- channel TMEM16A is detected in mucus-producing club/goblet cells and airway smooth muscle. TMEM16A contributes to mucus hypersecretion and bronchoconstriction, which are both inhibited by blockers of TMEM16A, such as niflumic acid. Here we demonstrate that the FDA-approved drug niclosamide, a potent inhibitor of TMEM16A identified by high-throughput screening, is an inhibitor of both TMEM16A and TMEM16F. In asthmatic mice, niclosamide reduced mucus production and secretion, as well as bronchoconstriction, and showed additional antiinflammatory effects. Using transgenic asthmatic mice, we found evidence that TMEM16A and TMEM16F are required for normal mucus production/secretion, which may be due to their effects on intracellular Ca2+ signaling. TMEM16A and TMEM16F support exocytic release of mucus and inflammatory mediators, both of which are blocked by niclosamide. Thus, inhibition of mucus and cytokine release, bronchorelaxation, and reported antibacterial effects make niclosamide a potentially suitable drug for the treatment of inflammatory airway diseases, such as CF, asthma, and COPD.