Song Huang

CFTR in a lipid raft-TNFR1 complex modulates gap junctional intercellular communication and IL-8 secretion

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause a chronic inflammatory response in the lung of patients with Cystic Fibrosis (CF). We have showed that TNF-alpha signaling through the Src family tyrosine kinases (SFKs) was defective as determined by an inability of TNF-alpha to regulate gap junctional communication (GJIC) in CF cells. Here, we sought to elucidate the mechanisms linking TNF-alpha signaling to the functions of CFTR at the molecular level. In a MDCKI epithelial cell model expressing wild-type (WtCFTR) or mutant CFTR lacking its PDZ-interacting motif (CFTR-DeltaTRL), TNF-alpha increased the amount of WtCFTR but not CFTR-DeltaTRL in detergent-resistant membrane microdomains (DRMs). This recruitment was modulated by SFK activity and associated with DRM localization of TNFR1 and c-Src. Activation of TNFR1 signaling also decreased GJIC and markedly stimulated IL-8 production in WtCFTR cells. In contrast, the absence of CFTR in DRMs was associated with abnormal TNFR1 signaling as revealed by no recruitment of TNFR1 and c-Src to lipid rafts in CFTR-DeltaTRL cells and loss of regulation of GJIC and IL-8 secretion. These results suggest that localization of CFTR in lipid rafts in association with c-Src and TNFR1 provides a responsive signaling complex to regulate GJIC and cytokine signaling.

Interaction of connexins with protein partners in the control of channel turnover and gating

In addition to neuronal synapses (chemical or electrical), gap junctions constitute a second type of cell communication system. Gap junctions are structures that facilitate the coordination of physiology of individual cells in an organ. Therefore, gap junctional communication (GJC) provides a mechanism for regulating the biological function of a whole tissue or organ. Gap junction channels are now well characterized; they are complexes of transmembrane proteins, termed connexins that directly link the cytoplasm of adjacent cells. These intercellular channels act as molecular sieves, allowing the passage of ions, small metabolites and second messengers. Connexins are members of a large family of at least 20 proteins with shared membrane topology. A connexin exhibits four alpha-helical transmembrane domains (M1–M4), two extracellular loops (E1 and E2), a cytoplasmic loop (CL), and cytoplasmic NH2and COOH-termini (NT and CT). Connexins are classified into three/four groups, identified by a Greek letter, according to sequence homology. In comparison to most membrane proteins, gap junctions are dynamic structures with short half-lives. The life cycle of a connexin (Cx) molecule begins with the non-covalent oligomerization of six connexin monomers into annular structures called connexons. This process is thought to occur soon after co-translational folding during protein insertion into the endoplasmic reticulum (ER) (Falk et al., 1997; Diez et al., 1999) or during its transport to the cell surface (Musil and Goodenough, 1993; Yeager et al., 1998; Evans et al., 1999). After translocation of assembled connexons to the plasma membrane, two connexons on opposing cell surfaces pair to form intercellular channels, which then cluster into paracrystallin arrays commonly referred to as gap junction plaques (Goodenough et al., 1996; Kumar and Gilula, 1996). The retrieval of gap junction plaques from the cell surface has been proposed to entail the endocytosis of partial or complete junctional plaques as a double membrane annular junction that is subsequently degraded or possibly re-utilized although it is unlikely that this represents the sole mechanism for gap junction internalization and degradation.

Potential of in vitro reconstituted 3D human airway epithelia (MucilAir™) to assess respiratory sensitizers

Respiratory sensitizers are considered as substances of higher risk, at the same level as carcinogens, mutagens and toxic chemicals for reproduction. Presently, there is no validated assay for identifying the respiratory sensitizers. Based on a fully differentiated and functional in vitro cell model of the human airway epithelium, MucilAir™, we attempt to develop such assay. To this end, we invented a novel method, using Dextran as carrier, for applying the water insoluble chemicals to the apical surface of the airway epithelia. Using the Dextran carrier method, we successfully tested some reference chemical compounds known to cause respiratory sensitisation in human beings, including MDI, TMA and HCPt. Interestingly, these chemical sensitizers differentially up-regulated the releases of certain cytokines and chemokines involved in allergic responses. We believe that based on MucilAir™ an in vitro assay could be developed for identification and characterization of the respiratory sensitizers.

Growth and characterization of different human rhinovirus C types in three-dimensional human airway epithelia reconstituted in vitro.

New molecular diagnostic tools have recently allowed the discovery of human rhinovirus species C (HRV-C) that may be overrepresented in children with lower respiratory tract complications. Unlike HRV-A and HRV-B, HRV-C cannot be propagated in conventional immortalized cell lines and their biological properties have been difficult to study. Recent studies have described the successful amplification of HRV-C15, HRV-C11, and HRV-C41 in sinus mucosal organ cultures and in fully differentiated human airway epithelial cells. Consistent with these studies, we report that a panel of clinical HRV-C specimens including HRV-C2, HRV-C7, HRV-C12, HRV-C15, and HRV-C29 types were all capable of mediating productive infection in reconstituted 3D human primary upper airway epithelial tissues and that the virions enter and exit preferentially through the apical surface. Similar to HRV-A and HRV-B, our data support the acid sensitivity of HRV-C. We observed also that the optimum temperature requirement during HRV-C growth may be type-dependent.

The Use of In Vitro 3D Cell Models in Drug Development for Respiratory Diseases

In a certain way, drug developers are like the blind men in the well-known tale of “THE BLIND MEN AND THE ELEPHANT”, who believe the elephant to be like a water spout, a fan a pillar and a throne since they can only feel a different part but only one part of the elephant’s body such as trunk, ear, leg, back. Impossible to test a drug lead on human beings, drug developers also are obliged to forge a whole picture of the “elephant” – how a drug candidate would behave in a whole human body, by using information from the “parts” models. The task of the drug developers are far more complex and challenging than the blind men, instead of touching only the surface, they have to go deep into the human bodies: organs, tissues, cells, genes, proteins, lipids, hormones ... Furthermore, a living human being is a dynamic and interacting system, in a certain sense, the situation of the drug developers are even worse than the blind men: the blind men touch and feel a real elephant, the information that they get is true; a drug developer, most of the time, works on models, animal models or in vitro cell models, which are far from representing a human beings as a whole. As consequences, the information that one gets sometimes could be misleading. For drug developers, the misleading information could have serious consequences in terms of costs and human lives. This blind men’s approach may explain why a drug candidate, even though successfully passed pre-clinical stages, eventually failed at clinical trials. The failure of Torcetrapib, a drug developed by Pfizer, gives an example of just how difficult to develop a drug. Torcetrapib, designed to prevent heart attacks and strokes, is a cholesteryl-ester transfer protein (CETP) inhibitor. Genetic studies of the Japanese populations revealed that people with a deficiency of CETP presented a favorable lipid profile compared with unaffected family members: namely more High Density Lipoproteins (HDL, good) and less Low Density Lipoproteins (LDL, bad) (Inazu et al., 1990; Koizumi et al., 1991). CETP naturally became a target of drug development. Preclinical studies of Torcetrapib on different animal models (mice, rabbits, etc) didn’t reveal any severe side effects (Tall et al., 2007). But, medication of Torcetrapib on human beings caused severe hypertension and an increased mortality; apparently there was no obvious beneficial effects on coronary heart disease. The development of Torcetrapib was halted at 2006. This example illustrates another difficulty in drug development: the genetic heterogeneity of the human populations. The knowledge obtained from one group of people may not be applicable to another group of people. Quite often, this truth has been neglected.

Src Signaling Links Mediators of Inflammation to Cx43 Gap Junction Channels in Primary and Transformed CFTR-Expressing Airway Cells

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with recurrent pulmonary infections and inflammation. We previously reported that tumor necrosis factor (TNF)-α decreases gap junction connectivity in cell lines derived from the airway epithelium of non-cystic fibrosis (non-CF) subjects, a mechanism that was defective in cells derived from CF patients, and identified the tyrosine kinase c-Src as a possible bridge between TNF-α and Cx43. To examine whether this modulation also takes place in primary epithelial cells, the functional expression of Cx43 was studied in non-CF and CF airway cells, obtained from surgical polypectomies and turbinectomies, which were grown either on culture dishes or permeable filters. Expression of Cx43 was detected by immunofluorescence on cells grown under both culture conditions. Non-CF and CF airway cells also showed intercellular diffusion of Lucifer Yellow. Dye coupling was rapidly abolished in non-CF cells in the presence of TNF-α, lipopolysaccharide and lysophosphatidic acid, and could be prevented by tyrphostin47, an inhibitor of Src tyrosine kinases. This down-regulation, however, was not detected in CF airway cells. These data indicate that CFTR dysfunction is associated with altered Src signaling, resulting in the persistence of gap junction connectivity in primary and transformed CF airway cells.

Cx26 regulates proliferation of repairing basal airway epithelial cells

The recovery of an intact epithelium following injury is critical for restoration of lung homeostasis, a process that may be altered in cystic fibrosis (CF). In response to injury, progenitor cells in the undamaged areas migrate, proliferate and re-differentiate to regenerate an intact airway epithelium. The mechanisms regulating this regenerative response are, however, not well understood. In a model of circular wound injury of well-differentiated human airway epithelial cell (HAEC) cultures, we identified the gap junction protein Cx26 as an important regulator of cell proliferation. We report that induction of Cx26 in repairing HAECs is associated with cell proliferation. We also show that Cx26 is expressed in a population of CK14-positive basal-like cells. Cx26 silencing in immortalized cell lines using siRNA and in primary HAECs using lentiviral-transduced shRNA enhanced Ki67-labeling index and Ki67 mRNA, indicating that Cx26 acts a negative regulator of HAEC proliferation. Cx26 silencing also markedly decreased the transcription of KLF4 in immortalized HAECs. We further show that CF HAECs exhibited deregulated expression of KLF4, Ki67 and Cx26 as well enhanced rate of wound closure in the early response to injury. These results point to an altered repair process of CF HAECs characterized by rapid but desynchronized initiation of HAEC activation and proliferation.

Establishment and characterization of an in vitro human small airway model (SmallAir

Graphical abstract Figure. No caption available. ABSTRACT We report here the establishment and characterization of an in vitro human small airway model (SmallAir™). The epithelial cells were isolated from the distal lungs by enzymatic digestion. After amplification, the cells were seeded on the microporous membrane of Transwell inserts. Once confluent, the cultures were switched to air‐liquid interface. After 3 weeks of culture, the epithelium became fully differentiated, with morphology of columnar epithelium, and a thickness of 10–15 &mgr;m. Most significantly, CC‐10, a specific marker of Club cells, was highly expressed in SmallAir™. CC‐10 was detected by both immune‐cytochemistry and Western Blot. As expected, SmallAir™ contained few Muc5‐Ac positive cells (goblet cells). In contrast, CC‐10 was not detected in MucilAir™, an in vitro model of the human nasal and bronchial epithelial model. Instead, Muc5‐Ac was highly expressed in MucilAir™. However, both MucilAir™ and SmallAir™ contain basal cells and ciliated cells, showing cilia beating and mucociliary clearance. Clearly, MucilAir™ and SmallAir™ are two distinct airway epithelial models.

Approaches to Study Differentiation and Repair of Human Airway Epithelial Cells

One of the main functions of the airway mucosa is to maintain a mechanical barrier at the air-surface interface and to protect the respiratory tract from external injuries. Differentiation of human airway epithelial cells (hAECs) to polarized airway mucosa can be reproduced in vitro by culturing the cells on microporous membrane at the air-liquid interface. Here, we describe approaches to study differentiation as well as repair of the hAECs by using a commercially available airway cell culture model called MucilAir™.

Propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures

Background: The leading cause of acute illnesses, respiratory viruses, typically cause self‐limited diseases, although severe complications can occur in fragile patients. Rhinoviruses (RVs), respiratory enteroviruses (EVs), influenza virus, respiratory syncytial viruses (RSVs), and coronaviruses are highly prevalent respiratory pathogens, but because of the lack of reliable animal models, their differential pathogenesis remains poorly characterized. Objective: We sought to compare infections by respiratory viruses isolated from clinical specimens using reconstituted human airway epithelia. Methods: Tissues were infected with RV‐A55, RV‐A49, RV‐B48, RV‐C8, and RV‐C15; respiratory EV‐D68; influenza virus H3N2; RSV‐B; and human coronavirus (HCoV)–OC43. Replication kinetics, cell tropism, effect on tissue integrity, and cytokine secretion were compared. Viral adaptation and tissue response were assessed through RNA sequencing. Results: RVs, RSV‐B, and HCoV‐OC43 infected ciliated cells and caused no major cell death, whereas H3N2 and EV‐D68 induced ciliated cell loss and tissue integrity disruption. H3N2 was also detected in rare goblet and basal cells. All viruses, except RV‐B48 and HCoV‐OC43, altered cilia beating and mucociliary clearance. H3N2 was the strongest cytokine inducer, and HCoV‐OC43 was the weakest. Persistent infection was observed in all cases. RNA sequencing highlighted perturbation of tissue metabolism and induction of a transient but important immune response at 4 days after infection. No majority mutations emerged in the viral population. Conclusion: Our results highlight the differential in vitro pathogenesis of respiratory viruses during the acute infection phase and their ability to persist under immune tolerance. These data help to appreciate the range of disease severity observed in vivo and the occurrence of chronic respiratory tract infections in immunocompromised hosts.