Carla M. P. Ribeiro

Cystic fibrosis airway epithelial Ca2+ i signaling: the mechanism for the larger agonist-mediated Ca2+ i signals in human cystic fibrosis airway epithelia.

In cystic fibrosis (CF) airways, abnormal epithelial ion transport likely initiates mucus stasis, resulting in persistent airway infections and chronic inflammation. Mucus clearance is regulated, in part, by activation of apical membrane receptors coupled to intracellular calcium (Ca2+i) mobilization. We have shown that Ca2+i signals resulting from apical purinoceptor (P2Y2-R) activation are increased in CF compared with normal human airway epithelia. The present study addressed the mechanism for the larger apical P2Y2-R-dependent Ca2+i signals in CF human airway epithelia. We show that the increased Ca2+i mobilization in CF was not specific to P2Y2-Rs because it was mimicked by apical bradykinin receptor activation, and it did not result from a greater number of P2Y2-R or a more efficient coupling between P2Y2-Rs and phospholipase C-generated inositol 1,4,5-trisphosphate. Rather, the larger apical P2Y2-R activation-promoted Ca2+i signals in CF epithelia resulted from an increased density and Ca2+ storage capacity of apically confined endoplasmic reticulum (ER) Ca2+ stores. To address whether the ER up-regulation resulted from ER retention of misfolded ΔF508 CFTR or was an acquired response to chronic luminal airway infection/inflammation, three approaches were used. First, ER density was studied in normal and CF sweat duct human epithelia expressing high levels of ΔF508 CFTR, and it was found to be the same in normal and CF epithelia. Second, apical ER density was morphometrically analyzed in airway epithelia from normal subjects, ΔF508 homozygous CF patients, and a disease control, primary ciliary dyskinesia; it was found to be greater in both CF and primary ciliary dyskinesia. Third, apical ER density and P2Y2-R activation-mobilized Ca2+i, which were investigated in airway epithelia in a long term culture in the absence of luminal infection, were similar in normal and CF epithelia. To directly test whether luminal infection/inflammation triggers an up-regulation of the apically confined ER Ca2+ stores, normal airway epithelia were chronically exposed to supernatant from mucopurulent material from CF airways. Supernatant treatment expanded the apically confined ER, resulting in larger apical P2Y2-R activation-dependent Ca2+i responses, which reproduced the increased Ca2+i signals observed in CF epithelia. In conclusion, the mechanism for the larger Ca2+i signals elicited by apical P2Y2-R activation in CF airway epithelia is an expansion of the apical ER Ca2+ stores triggered by chronic luminal airway infection/inflammation. Greater ER-derived Ca2+i signals may provide a compensatory mechanism to restore, at least acutely, mucus clearance in CF airways.

Patterns of Epithelial Cell Invasion by Different Species of the Burkholderia cepacia Complex in Well-Differentiated Human Airway Epithelia

ABSTRACT Burkholderia cepacia has emerged as a serious respiratory pathogen in cystic fibrosis (CF) patients. The clinical course of B. cepacia infections is variable, but ∼20% of patients eventually succumb to the cepacia syndrome, which is characterized as a fatal necrotizing pneumonia with bacteremia. The mechanisms that permit B. cepacia to cause bacteremia are not yet known but probably involve sequential penetration of airway barriers. This study evaluated the abilities of different species of the B. cepacia complex, including a strain from the ET12 lineage (BC-7, genomovar III, cblA+), which is associated with most cepacia syndrome fatalities among CF populations, a genomovar IV strain (HI2258), and a genomovar II strain (J-1) to penetrate polarized, well-differentiated human airway epithelial cell cultures. As revealed by light and electron microscopy, all three B. cepacia strains tested circumvented the mechanical barriers of mucus and ciliary transport to penetrate the airway epithelium but they used different routes. The BC-7 strain (genomovar III) formed biofilms in close proximity to the apical cell surface, followed by invasion and destruction of epithelial cells. This process involved disruption of the glycocalyx and rearrangements of the actin cytoskeleton. The HI2258 strain (genomovar IV) did not form biofilms, and the majority of bacteria that penetrated the epithelium were located between epithelial cells, suggesting paracytosis. Strain J-1 penetrated the epithelium both by cell destruction and paracytosis. These studies suggest that the distinct invasion pathways employed by B. cepacia may account for differences in virulence between B. cepacia genomovars.

The ER stress transducer IRE1β is required for airway epithelial mucin production

Inflammation of human bronchial epithelia (HBE) activates the endoplasmic reticulum (ER) stress transducer inositol-requiring enzyme 1 (IRE1)α, resulting in IRE1α-mediated cytokine production. Previous studies demonstrated ubiquitous expression of IRE1α and gut-restricted expression of IRE1β. We found that IRE1β is also expressed in HBE, is absent in human alveolar cells, and is upregulated in cystic fibrosis and asthmatic HBE. Studies with Ire1β−/− mice and Calu-3 airway epithelia exhibiting IRE1β knockdown or overexpression revealed that IRE1β is expressed in airway mucous cells, is functionally required for airway mucin production, and this function is specific for IRE1β vs. IRE1α. IRE1β-dependent mucin production is mediated, at least in part, by activation of the transcription factor X-box binding protein-1 (XBP-1) and the resulting XBP-1-dependent transcription of anterior gradient homolog 2, a gene implicated in airway and intestinal epithelial mucin production. These novel findings suggest that IRE1β is a potential mucous cell-specific therapeutic target for airway diseases characterized by mucin overproduction.

Azithromycin treatment alters gene expression in inflammatory, lipid metabolism, and cell cycle pathways in well-differentiated human airway epithelia.

Prolonged macrolide antibiotic therapy at low doses improves clinical outcome in patients affected with diffuse panbronchiolitis and cystic fibrosis. Consensus is building that the therapeutic effects are due to anti-inflammatory, rather than anti-microbial activities, but the mode of action is likely complex. To gain insights into how the macrolide azithromycin (AZT) modulates inflammatory responses in airways, well-differentiated primary cultures of human airway epithelia were exposed to AZT alone, an inflammatory stimulus consisting of soluble factors from cystic fibrosis airways, or AZT followed by the inflammatory stimulus. RNA microarrays were conducted to identify global and specific gene expression changes. Analysis of gene expression changes revealed that the AZT treatment alone altered the gene profile of the cells, primarily by significantly increasing the expression of lipid/cholesterol genes and decreasing the expression of cell cycle/mitosis genes. The increase in cholesterol biosynthetic genes was confirmed by increased filipin staining, an index of free cholesterol, after AZT treatment. AZT also affected genes with inflammatory annotations, but the effect was variable (both up- and down-regulation) and gene specific. AZT pretreatment prevented the up-regulation of some genes, such as MUC5AC and MMP9, triggered by the inflammatory stimulus, but the up-regulation of other inflammatory genes, e.g., cytokines and chemokines, such as interleukin-8, was not affected. On the other hand, HLA genes were increased by AZT. Notably, secreted IL-8 protein levels did not reflect mRNA levels, and were, in fact, higher after AZT pretreatment in cultures exposed to the inflammatory stimulus, suggesting that AZT can affect inflammatory pathways other than by altering gene expression. These findings suggest that the specific effects of AZT on inflamed and non-inflamed airway epithelia are likely relevant to its clinical activity, and their apparent complexity may help explain the diverse immunomodulatory roles of macrolides.

Human alveolar type II cells secrete and absorb liquid in response to local nucleotide signaling.

A balance sheet describing the integrated homeostasis of secretion, absorption, and surface movement of liquids on pulmonary surfaces has remained elusive. It remains unclear whether the alveolus exhibits an intra-alveolar ion/liquid transport physiology or whether it secretes ions/liquid that may communicate with airway surfaces. Studies employing isolated human alveolar type II (AT2) cells were utilized to investigate this question. Human AT2 cells exhibited both epithelial Na+ channel-mediated Na+ absorption and cystic fibrosis transmembrane conductance regulator-mediated Cl− secretion, both significantly regulated by extracellular nucleotides. In addition, we observed in normal AT2 cells an absence of cystic fibrosis transmembrane conductance regulator regulation of epithelial Na+ channel activity and an absence of expression/activity of reported calcium-activated chloride channels (TMEM16A, Bestrophin-1, ClC2, and SLC26A9), both features strikingly different from normal airway epithelial cells. Measurements of alveolar surface liquid volume revealed that normal AT2 cells: 1) achieved an extracellular nucleotide concentration-dependent steady state alveolar surface liquid height of ∼4 μm in vitro; 2) absorbed liquid when the lumen was flooded; and 3) secreted liquid when treated with UTP or forskolin or subjected to cyclic compressive stresses mimicking tidal breathing. Collectively, our studies suggest that human AT2 cells in vitro have the capacity to absorb or secrete liquid in response to local alveolar conditions.

Airway Epithelial Inflammation-induced Endoplasmic Reticulum Ca2+ Store Expansion Is Mediated by X-box Binding Protein-1

Inflamed cystic fibrosis (CF) human bronchial epithelia (HBE), or normal HBE exposed to supernatant from mucopurulent material (SMM) from CF airways, exhibit endoplasmic reticulum (ER)/Ca2+ store expansion and amplified Ca2+-mediated inflammation. HBE inflammation triggers an unfolded protein response (UPR) coupled to mRNA splicing of X-box binding protein-1 (XBP-1). Because spliced XBP-1 (XBP-1s) promotes ER expansion in other cellular models, we hypothesized that XBP-1s is responsible for the ER/Ca2+ store expansion in inflamed HBE. XBP-1s was increased in freshly isolated infected/inflamed CF in comparison with normal HBE. The link between airway epithelial inflammation, XBP-1s, and ER/Ca2+ store expansion was then addressed in murine airways challenged with phosphate-buffered saline or Pseudomonas aeruginosa. P. aeruginosa-challenged mice exhibited airway epithelial ER/Ca2+ store expansion, which correlated with airway inflammation. P. aeruginosa-induced airway inflammation triggered XBP-1s in ER stress-activated indicator (ERAI) mice. To evaluate the functional role of XBP-1s in airway inflammation linked to ER/Ca2+ store expansion, control, XBP-1s, or dominant negative XBP-1 (DN-XBP-1) stably expressing 16HBE14o- cell lines were used. Studies with cells transfected with an unfolded protein response element (UPRE) luciferase reporter plasmid confirmed that the UPRE was activated or inhibited by expression of XBP-1s or DN-XBP-1, respectively. Expression of XBP-1s induced ER/Ca2+ store expansion and potentiated bradykinin-increased interleukin (IL)-8 secretion, whereas expression of DN-XBP-1 inhibited bradykinin-dependent IL-8 secretion. In addition, expression of DN-XBP-1 blunted SMM-induced ER/Ca2+ store expansion and SMM-induced IL-8 secretion. These findings suggest that, in inflamed HBE, XBP-1s is responsible for the ER/Ca2+ store expansion that confers amplification of Ca2+-dependent inflammatory responses.

Role of Endoplasmic Reticulum Stress in Cystic Fibrosis–Related Airway Inflammatory Responses

Chronic airway infection and inflammation are hallmarks of cystic fibrosis (CF) pulmonary disease. The altered airway environment resulting from infection and inflammation can affect the innate defense of the airway epithelia. Luminal bacterial and inflammatory stimuli trigger an adaptation in human airway epithelia, characterized by a hyperinflammatory response to inflammatory mediators, which is mediated by an expansion of the endoplasmic reticulum (ER) and its Ca(2+) stores. Recent studies demonstrated that a form of ER stress, the unfolded protein response (UPR), is activated in airway epithelia by bacterial infection-induced airway inflammation. UPR-dependent signaling is responsible for the ER Ca(2+) store expansion-mediated amplification of airway inflammatory responses. These studies highlight the functional importance of the UPR in airway inflammation and suggest that targeting the UPR may be a therapeutic strategy for airway diseases typified by chronic inflammation. This article reviews the contribution of airway epithelia to airway inflammatory responses, discusses how expansion of the ER Ca(2+) stores in inflamed airway epithelia contributes to airway inflammation, describes the functional role of the UPR in these processes, and discusses how UPR activation might be relevant for CF airways inflammatory disease.

Anti-inflammatory effect of miglustat in bronchial epithelial cells ☆

The role of CFTR deficiency in promoting inflammation remains unclear. Perez et al. [A. Perez, A.C. Issler, C.U. Cotton, T.J. Kelley, A.S. Verkman and P.B. Davis, CFTR inhibition mimics the cystic fibrosis inflammatory profile. Am J Physiol Lung Cell Mol Physiol 2007; 292:L383-L395.] recently demonstrated that the inhibition of function of w/t CFTR produces an inflammatory profile that resembles that observed in CF patients, whereas we found that correction of F508del-CFTR function with MPB-07 down-modulates the inflammatory response to P. aeruginosa in CF bronchial cells [M.C. Dechecchi, E. Nicolis, V. Bezzerri, A. Vella, M. Colombatti, B.M. Assael, et al., MPB-07 reduces the inflammatory response to Pseudomonas aeruginosa in cystic fibrosis bronchial cells. Am J Respir Cell Mol Biol 2007; 36, 615-624.]. Since both evidence support a link between CFTR function and inflammation, we extended our investigation to other F508del-CFTR correctors, such as miglustat (Norez, 2006), an approved drug for Gaucher disease, in comparison with the galactose analogue NB-DGJ. We report here that miglustat but not NB-DGJ restores F508del-CFTR function in CF bronchial epithelial IB3-1 and CuFi-1 cells. Miglustat and NB-DGJ reduce the inflammatory response to P. aeruginosa in both CF and non-CF bronchial cells, indicating that the anti-inflammatory effect is independent of the correction of F508del-CFTR function. Miglustat also inhibits the inflammatory response induced by the supernatant of mucopurulent material obtained from the lower airway tract of cystic fibrosis patients with chronic bacterial colonization (Ribeiro, 2005). Both compounds do not interfere with the adherence of P. aeruginosa to the cells and reduce the expression of IL-8 not only after challenge with P. aeruginosa but also after exposure to TNF alpha or IL-1 beta, suggesting an effect on transduction proteins downstream and in common with different receptors for pathogens. Finally, miglustat has no major effects on overall binding activity of transcription factors NF-kappaBNF-kB and AP-1. Since miglustat is an approved drug, it could be investigated as a novel anti-inflammatory molecule to ameliorate lung inflammation in CF patients.

The mitochondrial barriers segregate agonist-induced calcium-dependent functions in human airway epithelia.

In airway epithelia, purinergic receptor (P2Y2-R) stimulation of intracellular calcium (Ca2+ i)–regulated ion transport is restricted to the membrane domain ipsilateral to receptor activation, implying compartmentalization of Ca2+ i signaling. Because mitochondria can spatially restrict cellular Ca2+ i signals, immunocytochemical, electron microscopic, and fluorescent studies of mitochondria localization were performed in human airway epithelia. Although concentrated at the apical domain, mitochondria were found distributed at both the apical and the basolateral poles and in close association with the endoplasmic reticulum. The role of mitochondria in locally restricting P2Y2-R–induced Ca2+ i signals was investigated by measuring changes in mitochondrial Ca2+ (Ca2+ m) in human airway epithelial monolayers. P2Y2-R activation induced Ca2+ m accumulation in mitochondria confined to the domain ipsilateral to P2Y2-R stimulation, which was blocked by mitochondrial uncoupling with 1 μM CCCP and 2.5 μg/ml oligomycin. The role of mitochondria in restricting the cellular cross-talk between basolateral P2Y2-R–dependent Ca2+ i mobilization and apical membrane Ca2+-activated Cl− secretion was investigated in studies simultaneously measuring Ca2+ i and Cl− secretion in cystic fibrosis human airway epithelial monolayers. Activation of basolateral P2Y2-Rs produced similar increases in Ca2+ i in monolayers without and with pretreatment with uncouplers, whereas Ca2+ i-activated Cl− secretion was only efficiently triggered in mitochondria-uncoupled conditions. We conclude that (a) mitochondria function as a Ca2+ i-buffering system in airway epithelia, compartmentalizing Ca2+ i-dependent functions to the membrane ipsilateral to receptor stimulation; and (b) the mitochondria provide structural barriers that protect the airway epithelia against nonspecific activation of Ca2+ i-modulated functions associated with Ca2+ i signals emanating from the apical or the basolateral membrane domains.

AAV Exploits Subcellular Stress Associated with Inflammation, Endoplasmic Reticulum Expansion, and Misfolded Proteins in Models of Cystic Fibrosis

Barriers to infection act at multiple levels to prevent viruses, bacteria, and parasites from commandeering host cells for their own purposes. An intriguing hypothesis is that if a cell experiences stress, such as that elicited by inflammation, endoplasmic reticulum (ER) expansion, or misfolded proteins, then subcellular barriers will be less effective at preventing viral infection. Here we have used models of cystic fibrosis (CF) to test whether subcellular stress increases susceptibility to adeno-associated virus (AAV) infection. In human airway epithelium cultured at an air/liquid interface, physiological conditions of subcellular stress and ER expansion were mimicked using supernatant from mucopurulent material derived from CF lungs. Using this inflammatory stimulus to recapitulate stress found in diseased airways, we demonstrated that AAV infection was significantly enhanced. Since over 90% of CF cases are associated with a misfolded variant of Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR), we then explored whether the presence of misfolded proteins could independently increase susceptibility to AAV infection. In these models, AAV was an order of magnitude more efficient at transducing cells expressing ΔF508-CFTR than in cells expressing wild-type CFTR. Rescue of misfolded ΔF508-CFTR under low temperature conditions restored viral transduction efficiency to that demonstrated in controls, suggesting effects related to protein misfolding were responsible for increasing susceptibility to infection. By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. The presence of misfolded proteins did not affect cell surface attachment of virus or influence expression levels from promoter transgene cassettes in plasmid transfection studies, indicating exploitation occurs at the level of virion trafficking or processing. Thus, we surmised that factors enlisted to process misfolded proteins such as ΔF508-CFTR in the secretory pathway also act to restrict viral infection. In line with this hypothesis, we found that AAV trafficked to the microtubule organizing center and localized near Golgi/ER transport proteins. Moreover, AAV infection efficiency could be modulated with siRNA-mediated knockdown of proteins involved in processing ΔF508-CFTR or sorting retrograde cargo from the Golgi and ER (calnexin, KDEL-R, β-COP, and PSMB3). In summary, our data support a model where AAV exploits a compromised secretory system and, importantly, underscore the gravity with which a stressed subcellular environment, under internal or external insults, can impact infection efficiency.