Background Interleukin (IL)-33 is implicated in the pathophysiology of asthma and allergic diseases. However, our knowledge is limited regarding how IL-33 release is controlled. The transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2) plays a key role in antioxidant response regulation. Objective The goal of this project was to investigate the role of cellular oxidative stress in controlling IL-33 release in airway epithelium. Methods Complementary approaches were used that included human bronchial epithelial cells and mouse models of airway type-2 immunity that were exposed to fungus Alternaria extract. The clinically available Nrf2 activator 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me) was used to evaluate the role of Nrf2-induced antioxidant molecules. Results Human bronchial epithelial cells produced reactive oxygen species (ROS) when they were exposed to Alternaria extract. ROS scavengers, such as glutathione (GSH) and N-acetyl cysteine, prevented extracellular secretion of ATP and increases in intracellular calcium concentrations that precede IL-33 release. Administration of CDDO-Me to mice enhanced expression of a number of antioxidant molecules in the lungs and elevated lung levels of endogenous GSH. Importantly, CDDO-Me treatment reduced allergen-induced ATP secretion and IL-33 release by airway epithelial cells in vitro and protected mice from IL-33 release and asthma-like pathological changes in the lungs. Conclusions The balance between oxidative stress and antioxidant responses plays a key role in controlling IL-33 release in airway epithelium. The therapeutic potential of Nrf2 activators needs to be considered for asthma and allergic airway diseases.

Four behavioral experiments conducted in both the laboratory and the field provide evidence that adult sea lamprey (Petromyzon marinus) select spawning rivers based on the odor of larvae that they contain and that bile acids released by the larvae are part of this pheromonal odor. First, when tested in a recirculating maze, migratory adult lamprey spent more time in water scented with larvae. However, when fully mature, adults lost their responsiveness to larvae and preferred instead the odor of mature individuals. Second, when tested in a flowing stream, migratory adults swam upstream more actively when the water was scented with larvae. Third, when migratory adults were tested in a laboratory maze containing still water, they exhibited enhanced swimming activity in the presence of a 0.1 nM concentration of the two unique bile acids released by larvae and detected by adult lamprey. Fourth, when adults were exposed to this bile acid mixture within flowing waters, they actively swam into it. Taken together, these data suggest that adult lamprey use a bile acid based larval pheromone to help them locate spawning rivers and that responsiveness to this cue is influenced by current flow, maturity, and time of day. Although the precise identity and function of the larval pheromone remain to be fully elucidated, we believe that this cue will ultimately prove useful as an attractant in sea lamprey control.

The role of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in airway epithelial wound repair was investigated using normal human bronchial epithelial (NHBE) cells and a human airway epithelial cell line (Calu-3) of serous gland origin. Measurements of wound repair were performed using continuous impedance sensing to determine the time course for wound closure. Control experiments showed that the increase in impedance corresponding to cell migration into the wound was blocked by treatment with the actin polymerization inhibitor, cytochalasin D. Time lapse imaging revealed that NHBE and Calu-3 cell wound closure was dependent on cell migration, and that movement occurred as a collective sheet of cells. Selective inhibition of CFTR activity with CFTRinh-172 or short hairpin RNA silencing of CFTR expression produced a significant delay in wound repair. The CF cell line UNCCF1T also exhibited significantly slower migration than comparable normal airway epithelial cells. Inhibition of CFTR-dependent anion transport by treatment with CFTRinh-172 slowed wound closure to the same extent as silencing CFTR protein expression, indicating that ion transport by CFTR plays a critical role in migration. Moreover, morphologic analysis of migrating cells revealed that CFTR inhibition or silencing significantly reduced lamellipodia protrusion. These findings support the conclusion that CFTR participates in airway epithelial wound repair by a mechanism involving anion transport that is coupled to the regulation of lamellipodia protrusion at the leading edge of the cell.

Key points• Exposure of human bronchial epithelial (HBE) cells to fungal aeroallergens derived from Alternaria alternata stimulates Ca 2+ -dependent and Ca 2+ -independent ATP release across the apical membrane.• The Ca 2+ -dependent component was blocked by inhibitors of both ATP uptake and transport of exocytotic vesicles to the plasma membrane.• Treatment with inhibitors that target cysteine proteases significantly blocked Ca 2+ -dependent ATP release evoked by Alternaria in normal HBE cells, but not in cells derived from asthmatic patients.• The magnitude of ATP release and associated intracellular Ca 2+ mobilization was significantly greater in bronchial epithelial cells obtained from patients with asthma.• These findings establish a novel role for ATP release as a mechanism underlying Alternaria aeroallergen activation of airway mucosal immunity and that cells derived from patients with asthma exhibit greater responsiveness to these allergens.Abstract Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) that was dependent on the coordinated activation of specific purinergic receptor (P2Y 2 and P2X 7 ) subtypes. In the present study, pretreatment with a cell-permeable Ca 2+ -chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca 2+ ] i . Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC 50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca 2+ ] i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca 2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation.

The role of phosphodiesterase (PDE) isoforms in regulation of transepithelial Cl secretion was investigated using cultured monolayers of T84 cells grown on membrane filters. Identification of the major PDE isoforms present in these cells was determined using ion exchange chromatography in combination with biochemical assays for cGMP and cAMP hydrolysis. The most abundant PDE isoform in these cells was PDE4 accounting for 70-80% of the total cAMP hydrolysis within the cytosolic and membrane fractions from these cells. The PDE3 isoform was also identified in both cytosolic and membrane fractions accounting for 20% of the total cAMP hydrolysis in the cytosolic fraction and 15-30% of the total cAMP hydrolysis observed in the membrane fraction. A large portion of the total cGMP hydrolysis detected in cytosolic and membrane fractions of T84 cells was mediated by PDE5 (50-75%). Treatment of confluent monolayers of T84 cells with various PDE inhibitors produced significant increases in short-circuit current (Isc). The PDE3-selective inhibitors terqinsin, milrinone and cilostamide produced increases in Isc with EC50 values of 0.6 nM, 8.0 nM and 0.5 microM respectively. These values were in close agreement with the IC50 values for cAMP hydrolysis. The effects of the PDE1-(8-MM-IBMX) and PDE4-(RP-73401) selective inhibitors on Isc were significantly less potent than PDE3 inhibitors with EC50 values of >7 microM and >50 microM respectively. However, the effects of 8-MM-IBMX and terqinsin on Cl secretion were additive, suggesting that inhibition of PDE1 also increases Cl secretion. The effect of PDE inhibitors on Isc were significantly blocked by apical treatment with glibenclamide (an inhibitor of the CFTR Cl channel) and by basolateral bumetanide, an inhibitor of Na-K-2Cl cotransport activity. These results indicate that inhibition of PDE activity in T84 cells stimulates transepithelial Cl secretion and that PDE1 and PDE3 are involved in regulating the rate of secretion.