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Excessive inflammation in cystic fibrosis (CF) lung disease is a contributor to progressive pulmonary decline. Effective and well-tolerated anti-inflammatory therapy may preserve lung function, thereby improving quality and length of life. In this paper, we assess the anti-inflammatory effects of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-l,9(11)-dien-28-oic acid (CDDO) in preclinical models of CF airway inflammation. In our experiments, mice carrying the R117H Cftr mutation have significantly reduced airway inflammatory responses to both LPS and flagellin when treated with CDDO before inflammatory challenge. Anti-inflammatory effects observed include reduced airway neutrophilia, reduced concentrations of proinflammatory cytokines and chemokines, and reduced weight loss. Our findings with the synthetic triterpenoids in multiple cell culture models of CF human airway epithelia agree with effects previously described in other disease models (e.g., neoplastic cells). These include the ability to reduce NF-[kappa]B activation while increasing nuclear factor erythroid-related factor 2 (Nrf2) activity. As these two signaling pathways appear to be pivotal in regulating the net inflammatory response in the CF airway, these compounds are a promising potential anti-inflammatory therapy for CF lung disease. anti-inflammatory therapy; antioxidant; nuclear factor-[kappa]B; nuclear factor erythroid-related factor 2; synthetic triterpenoid doi: 10.1152/ajplung.00171.2009.

The pathophysiology of cystic fibrosis (CF) inflammatory lung disease is not well understood. CF airway epithelial cells respond to inflammatory stimuli with increased production of proinflammatory cytokines as a result of increased NF-[kappa]B activation. Peroxisome proliferator-activated receptor-[gamma] (PPA[R[gamma]) inhibits NF-[kappa]B activity and is reported to be reduced in CF. If PPAR[gamma] participates in regulatory dysfunction in the CF lung, perhaps PPAR[gamma] ligands might be useful therapeutically. Cell models of CF airway epithelium were used to evaluate PPAR[gamma] expression and binding to NF-[kappa]B at basal and under conditions of inflammatory stimulation by Pseudomonas aeruginosa or TNF[alpha]/IL-1[beta]. An animal model of CF was used to evaluate the potential of PPAR[gamma] agonists as therapeutic agents in vivo. In vitro, PPAR[gamma] agonists reduced IL-8 and MMP-9 release from airway epithelial cells in response to PAO1 or TNF[alpha]/IL-1[beta] stimulation. Less NF-[kappa]B bound to PPAR[gamma] in CF than normal cells, in two different assays; PPAR[gamma] agonists abrogated this reduction. PPAR[gamma] bound less to its target DNA sequence in CF cells. To test the importance of the reported PPAR[gamma] inactivation by phosphorylation, we observed that inhibitors of ERK, but not JNK, were synergistic with PPAR[gamma] agonists in reducing IL-8 secretion. In vivo, administration of PPAR[gamma] agonists reduced airway inflammation in response to acute infection with P. aeruginosa in CF, but not wildtype, mice. In summary, PPAR[gamma] inhibits the inflammatory response in CF, at least in part by interaction with NF-[kappa]B in airway epithelial cells. PPAR[gamma] agonists may be therapeutic in CF. nuclear factor-[kappa]B; cytokines; disease models; animal; in vitro; lung diseases; Pseudomonas aeruginosa

FXYD5, also known as dysadherin, belongs to a family of tissue-specific regulators of the [Na.sup.+]-[K.sup.+]-ATPase. We determined the kinetic effects of FXYD5 on [Na.sup.+]-[K.sup.+]-ATPase pump activity in stably transfected Madin-Darby canine kidney cells. FXYD5 significantly increased the apparent affinity for [Na.sup.+] twofold and decreased the apparent affinity for [K.sup.+] by 60% with a twofold increase in [V.sub.max] of [K.sup.+], a pattern that would increase activity and [Na.sup.+] removal from the cell. To test the effect of increased [Na.sup.+] uptake on FXYD5 expression, we analyzed MadinDarby canine kidney cells stably transfected with an inducible vector expressing all three subunits of the epithelial [Na.sup.+] channel (ENaC). [Na.sup.+]-[K.sup.+]-ATPase activity increased sixfold after 48-h ENaC induction, but FXYD5 expression decreased 75%. FXYD5 expression was also decreased in lung epithelia from mice that over-express ENaC, suggesting that chronic [Na.sup.+] absorption by itself downregulates epithelial FXYD5 expression. Patients with cystic fibrosis (CF) display ENaC-mediated hyperabsorption of [Na.sup.+] in the airways, accompanied by increased [Na.sup.+]-[K.sup.+]-ATPase activity. However, FXYD5 was significantly increased in the lungs and nasal epithelium of CF mice as assessed by RT-PCR, immunohistochemistry, and immunoblot analysis (P < 0.001). FXYD5 was also upregulated in nasal scrapings from human CF patients compared with controls (P < 0.02). Treatment of human tracheal epithelial cells with a CFTR inhibitor (I-172) confirmed that loss of CFTR function correlated with increased FXYD5 expression (P < 0.001), which was abrogated by an inhibitor of NF-[kappa]B. Thus FXYD5 is upregulated in CF epithelia, and this change may exacerbate the [Na.sup.+] hyperabsorption and surface liquid dehydration observed in CF airway epithelia. [Na.sup.+]-[K.sup.+]-ATPase; sodium transport

Primary airway epithelial cells grown in air-liquid interface differentiate into cultures that resemble native epithelium morphologically, express ion transport similar to those in vivo, and secrete cytokines in response to stimuli. Comparisons of cultures derived from normal and cystic fibrosis (CF) individuals are difficult to interpret due to genetic differences besides CFTR. The recently discovered CFTR inhibitor, CFT[R.sub.inh]-172, was used to create a CF model with its own control to test if loss of CFTR-[Cl.sup.-] conductance alone was sufficient to initiate the CF inflammatory response. Continuous inhibition of CFTR-[Cl.sup.-] conductance for 3-5 days resulted in significant increase in IL-8 secretion at basal (P = 0.006) and in response to 109 Pseudomonas (P = 0.0001), a fourfold decrease in Smad3 expression (P = 0.02), a threefold increase in RhoA expression, and increased NF-[kappa]B nuclear translocation upon TNF-[alpha]/IL-1[beta] stimulation (P < 0.000001). CFTR inhibition by CFT[R.sub.inh]-172 over this period does not increase epithelial sodium channel activity, so lack of [Cl.sup.-] conductance alone can mimic the inflammatory CF phenotype. CFT[R.sub.inh]-172 does not affect IL-8, IL-6, or granulocyte/macrophage colony-stimulating factor secretion in two CF phenotype immortalized cell lines: 9/HTE[o.sup.-] pCEP-R and 16HBE14[o.sup.-] AS, or IL-8 secretion in primary CF cells, and inhibitor withdrawal abolishes the increased response, so CFT[R.sub.inh]-172 effects on cytokines are not direct. Five-day treatment with CFT[R.sub.inh]-172 does not affect cells deleteriously as evidenced by lactate dehydrogenase, trypan blue, ciliary activity, electron micrograph histology, and inhibition reversibility. Our results support the hypothesis that lack of CFTR activity is responsible for the onset of the inflammatory cascade in the CF lung. inflammation; Pseudomonas; cytokines; cystic fibrosis transmembrane conductance regulator; epithelial sodium channel

Role of Cftr genotype in the response to chronic Pseudomonas aeruginosa lung infection in mice. Am J Physiol Lung Cell Mol Physiol 287: L944-L952, 2004. First published July 9, 2004; doi: 10.1152/ajplung.00387.2003.--Patients with cystic fibrosis have a lesion in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which is associated with abnormal regulation of other ion channels, abnormal glycosylation of secreted and cell surface molecules, and vulnerability to bacterial infection and inflammation in the lung usually leading to the death of these patients. The exact mechanism(s) by which mutation in CFTR leads to lung infection and inflammation is not clear. Mice bearing different mutations in the murine homolog to CFTR (Cftr) (R117H, $489X, Y122X. and [DELTA]F508, all backcrossed to the C57BL/6J background) were compared with respect to growth and in their ability to respond to lung infection elicited with Pseudomonas aeruginosaladen agarose beads. Body weights of mice bearing mutations in Cftr were significantly smaller than wild-type mice at most ages. The inflammatory responses to P. aeruginosa-laden agarose beads were comparable in mice of all four Cftr mutant genotypes with respect to absolute and relative cell counts in bronchoalveolar lavage fluid, and cytokine levels (TNF-[alpha], IL-1[beta], IL-6, macrophage inflammatory protein-2, and keratinocyte chemoattractant) and eicosanoid levels ([PGE.sub.2] and [LTB.sub.4]) in epithelial lining fluid: the few small differences observed occurred only between cystic fibrosis mice bearing the $489X mutation and those bearing the knockout mutation Y122X. Thus we cannot implicate either misprocessing of CFTR or failure of CFTR to reach the plasma membrane in the genesis of the excess inflammatory response of CF mice. Therefore, it appears that any functional defect in CFTR produces comparable inflammatory responses to lung infections with P. aeruginosa. cystic fibrosis transmembrane conductance regulator gene; agarose beads; mutation; inflammation

van Heeckeren, Anna M., Abraham Scaria, Mark D. Schluchter, Thomas W. Ferkol, Samuel Wadsworth, and Pamela B. Davis. Delivery of CFTR by adenoviral vector to cystic fibrosis mouse lung in a model of chronic Pseudomonas aeruginosa lung infection. Am J Physiol Lung Cell Mol Physiol 286: L717-L726, 2004. First published September 26, 2003; 10.1152/ajplung.00227.2003.--In cystic fibrosis (CF) there is an excessive inflammatory response to lung infections with Pseudomonas aeruginosa, which causes significant morbidity and mortality. Mice deficient in the cystic fibrosis conductance transmembrane regulator homolog (Cftr) have exaggerated production of proinflammatory cytokines in epithelial lining fluid and increased mortality in response to chronic bronchopulmonary infection with mucoid P. aeruginosa, compared with infected wild-type littermates. Whether delivery of CFTR to CF airways by an adenoviral vector (Ad2/CFTR-16) decreases cytokine production and mortality in response to chronic bronchopulmonary infection with mucoid P. aeruginosa was tested. CF mice [stock Cft[r.sup.tm1Unc_] TgN(FABPCFTR)#Jaw] were anesthetized with isoflurane and inoculated intranasally with either Ad2/CFTR-16, diluent (sucrose), or empty rector (Ad2/EV). Two weeks later, mice were anesthetized with 2.5% Avertin and inoculated transtracheally with P. aeruginosa-laden agarose beads (PA M57-15). The cumulative 10-day survival of mice pretreated with Ad2/CFTR-16 was significantly higher compared with mice pretreated with sucrose but not significantly higher than mice pretreated with Ad2/EV. After adjusting for differences in experiment, we found weight loss at 3 days for mice treated with Ad2/CFTR-16 to be significantly less than for the sucrose- or Ad2/ EV-treated groups. However, cytokine responses were similar in all groups 3 days after infection. In conclusion, the observed survival advantage of adenoviral delivery of CFTR to the CF lung may be due either to CFTR expression or possibly to proinflammatory effects of the adenoviral vector, of both. animal model; gene therapy; inflammation; lung infection; host response; cystic fibrosis; cystic fibrosis transmembrane conductance regulator

Research was conducted to study the hypothesis that foreign DNA condensed by poly-L-lysine coupled to a synthetic peptide ligand for the serpin enzyme complex receptor (SECR) can be targeted to and expressed in cells bearing the receptor in vitro. A molecular conjugate consisting of a synthetic peptide based on the SECR binding motif of human alpha1-AT covalently coupled to poly-L-lysine was used to introduce reporter genes into hepatoma cell lines in culture. Results indicate that SECR-mediated gene transfer gives efficient, specific uptake and expression of three reporter genes.

The regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator is suggested to act as a regulatory region to mediate the effects of protein kinase A (PKA) and adenosine 3',5'-cyclic monophosphate (cAMP). To test this hypothesis, the R domain was overexpressed in 9/HTEo- cells, a human epithelial cell line which exhibits cAMP-regulated CFTR. The results revealed reduction in Cl- conductance and inhibited its activity. The cells showed no changes in cAMP production and PKA activity, suggesting that the R domain only exerts its action on Cl- secretion.

A study was conducted to test the hypothesis that a low-residue liquid diet reduces the mortality rate in mice during the week after weaning. Mice were fed with Peptamen, a complete elemental, low-residue formula, and housed on corn cob bedding at 10 days of age. The results revealed that 88% of the mice survived until 50 days or maturity. The diet had no effect on the growth rate and size of the mice, suggesting that a combination of diet and bedding could improve survival and growth of mice.

Chronic hypoxia detected in lung homogenates positively regulates high and low-affinity beta-adrenergic (AR) receptors in the rat lung. Selective binding of low levels of agonist to beta-ARs marked with an antagonist radioligand facilitates the selective detection of beta-ARs in the agonist high-affinity state in the hypoxic and control slide-mounted portions of rat lung. Autoradiography was employed to identify high- and low-affinity beta-AR locations in lung parenchyma, specifically in the alveolar walls and walls of airways and blood vessels.

Kube, Dianne, Ute Sontich, David Fletcher, and Pamela B. Davis. Proinflammatory cytokine responses to P. aeruginosa infection in human airway epithelial cell lines. Am J Physiol Lung Cell Mol Physiol 280: L493-L502, 2001.--A tendency toward excessive inflammation in cystic fibrosis (CF) patients often accompanies lung infections with Pseudomonas aeruginosa. We tested the cytokine response to P. aeruginosa in two pairs of human airway epithelial cell lines matched except for CF transmembrane conductance regulator activity. The [9/HTEo.sup.-] CF-phenotypic cell line produced significantly more interleukin (IL)-8, IL-6, and granulocyte-macrophage colony-stimulating factor but not regulated on activation normal T cell expressed and secreted (RANTES) in response to Pseudomonas than the [9/HTEo.sup.-] control line, and the differences widened over time. Similarly, a 16HBE cell line lacking transmembrane conductance regulator activity showed enhanced IL-8 and IL-6 responses compared with the control cell line. The pharmacology of the cytokine response also differed because dexamethasone reduced cytokine production to similar levels in the matched cell lines. The protracted proinfiammatory cytokine response of the CF-phenotypic cell lines suggests that the limiting mechanisms of normal cells are absent or attenuated. These results are consistent with in vivo observations in patients with CF and suggest that our novel cell lines may be useful for further investigation of the proinfiammatory responses in CF airways. cystic fibrosis; interleukin-8; interleukin-6; granulocyte-macrophage colony-stimulating factor; inflammation

Kube, Dianne, Lynn Adams, Aura Perez, and Pamela B. Davis. Terminal sialylation is altered in airway cells with impaired CFTR-mediated chloride transport. Am J Physiol Lung Cell Mol Physiol 280: L482-L492, 2001.--Reduced terminal sialylation at the surface of airway epithelial cells from patients with cystic fibrosis may predispose them to bacterial infection. To determine whether a lack of chloride transport or misprocessing of mutant cystic fibrosis transmembrane conductance regulator (CFTR) is critical for the alterations in glycosylation, we studied a normal human tracheal epithelial cell line (9/HTE[o.sup.-]) transfected with the regulatory (R) domain of CFTR, which blocks CFTR-mediated chloride transport; [Delta]F508 CFTR, which is misprocessed, wild-type CFTR; or empty vector. Reduced cAMP-stimulated chloride transport is seen in the R domain and [Delta]F508 transfectants. These two cell lines had consistent, significantly reduced binding of elderberry bark lectin, which recognizes terminal sialic acid in the [Alpha]-2,6 configuration. Binding of other lectins, including Maakia amurensis lectin, which recognizes sialic acid in the [Alpha]-2,3 configuration, was comparable in all cell lines. Because the cell surface change occurred in R domain-transfected cells, which continue to express wild-type CFTR, it cannot be related entirely to misprocessed or overexpressed CFTR. It is associated most closely with reduced CFTR activity. cystic fibrosis; cystic fibrosis transmembrane conductance regulator; [Delta]F508; regulatory domain; lectins; fluorescent imaging; immunogold labeling; 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine

Miller TJ, Davis PB. FXYD5 modulates Na~ absorption and is increased in cystic fibrosis airway epithelia. Am J Physiol Lung Cell Mo! Physiol 294: L654-L664, 2008. First published February 3, 2008; doi: 10.1 152/ajplung.00430.2007.-FXYD5, also known as dys- adherin, belongs to a family of tissue-specific regulators of the Na~-K~-ATPase. We determined the kinetic effects of FXYD5 on Na~-K~-ATPase pump activity in stably transfected Madin-Darby canine kidney cells. FXYD5 significantly increased the apparent affinity for Na~ twofold and decreased the apparent affinity for K~ by 60% with a twofold increase in Vmax of K~, a pattern that would increase activity and Na~ removal from the cell. To test the effect of increased Na~ uptake on FXYD5 expression, we analyzed Madin- Darby canine kidney cells stably transfected with an inducible vector expressing all three subunits of the epithelial Na~ channel (ENaC). Na~-K~-ATPase activity increased sixfold after 48-h ENaC induc- tion, but FXYD5 expression decreased 75%. FXYD5 expression was also decreased in lung epithelia from mice that overexpress ENaC, suggesting that chronic Na~ absorption by itself downregulates epi- thelial FXYD5 expression. Patients with cystic fibrosis (CF) display ENaC-mediated hyperabsorption of Na~ in the airways, accompanied by increased Na~-K~-ATPase activity. However, FXYD5 was sig- nificantly increased in the lungs and nasal epithelium of CF mice as assessed by RT-PCR, immunohistochemistry, and immunoblot anal- ysis (P < 0.001). FXYD5 was also upregulated in nasal scrapings from human CF patients compared with controls (P < 0.02). Treat- ment of human tracheal epithelial cells with a CFTR inhibitor (1-172) confirmed that loss of CFTR function correlated with increased FXYD5 expression (P < 0.00 1), which was abrogated by an inhibitor of NF-KB. Thus FXYDS is upregulated in CF epithelia, and this change may exacerbate the Na~ hyperabsorption and surface liquid dehydration observed in CF airway epithelia. [ABSTRACT FROM AUTHOR]