Anna M. van Heeckeren

Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa.

In cystic fibrosis (CF), defective function of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells and submucosal glands results in chronic pulmonary infection with Pseudomonas aeruginosa. The pulmonary infection incites an intense host inflammatory response, causing progressive suppurative pulmonary disease. Mouse models of CF, however, fail to develop pulmonary disease spontaneously. We examined the effects of bronchopulmonary infection on mice homozygous for the S489X mutation of the CFTR gene using an animal model of chronic Pseudomonas endobronchial infection. Slurries of sterile agarose beads or beads containing a clinical isolate of mucoid P. aeruginosa were instilled in the right lung of normal or CF mice. The mortality of CF mice inoculated with Pseudomonas-laden beads was significantly higher than that of normal animals: 82% of infected CF mice, but only 23% of normal mice, died within 10 d of infection (P = 0.023). The concentration of inflammatory mediators, including TNF-alpha, murine macrophage inflammatory protein-2, and KC/N51, in bronchoalveolar lavage fluid in CF mice 3 d after infection and before any mortality, was markedly elevated compared with normal mice. This inflammatory response also correlated with weight loss observed in both CF and normal littermates after inoculation. Thus, this model may permit examination of the relationship of bacterial infections, inflammation, and the cellular and genetic defects in CF.

Role for cystic fibrosis transmembrane conductance regulator protein in a glutathione response to bronchopulmonary pseudomonas infection.

ABSTRACT The lung maintains an elevated level of glutathione (GSH) in epithelial lining fluid (ELF) compared to serum. The mechanism(s) by which the lung maintains high levels of ELF GSH and factors that modulate them are largely unexplored. We hypothesized that lung cystic fibrosis transmembrane conductance regulator protein (CFTR) modulates GSH efflux in response to extracellular stress, which occurs with lung infections. Mice were challenged intratracheally with Pseudomonas aeruginosa, and on the third day of infection bronchoalveolar lavage fluid was obtained and analyzed for cytokines and antioxidants. Lung tissue antioxidants and enzyme activities were also assessed. P. aeruginosa lung infection increased levels of inflammatory cytokines and neutrophils in the ELF. This corresponded with a marked threefold increase in GSH and a twofold increase in urate levels in the ELF of P. aeruginosa-infected wild-type mice. A twofold increase in urate levels was also observed among lung tissue antioxidants of P. aeruginosa-infected wild-type mice. There were no changes in markers of lung oxidative stress associated with the P. aeruginosa lung infection. In contrast with wild-type mice, the CFTR knockout mice lacked a significant increase in ELF GSH when challenged with P. aeruginosa, and this correlated with a decrease in the ratio of reduced to oxidized GSH in the ELF, a marker of oxidative stress. These data would suggest that the lung adapts to infectious agents with elevated ELF GSH and urate. Individuals with lung diseases associated with altered antioxidant transport, such as cystic fibrosis, might lack the ability to adapt to the infection and present with a more severe inflammatory response.

Peroxisome proliferator-activated receptor-γ in cystic fibrosis lung epithelium

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-kappaB activation. Peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibits NF-kappaB activity and is reported to be reduced in CF. If PPARgamma participates in regulatory dysfunction in the CF lung, perhaps PPARgamma ligands might be useful therapeutically. Cell models of CF airway epithelium were used to evaluate PPARgamma expression and binding to NF-kappaB at basal and under conditions of inflammatory stimulation by Pseudomonas aeruginosa or TNFalpha/IL-1beta. An animal model of CF was used to evaluate the potential of PPARgamma agonists as therapeutic agents in vivo. In vitro, PPARgamma agonists reduced IL-8 and MMP-9 release from airway epithelial cells in response to PAO1 or TNFalpha/IL-1beta stimulation. Less NF-kappaB bound to PPARgamma in CF than normal cells, in two different assays; PPARgamma agonists abrogated this reduction. PPARgamma bound less to its target DNA sequence in CF cells. To test the importance of the reported PPARgamma inactivation by phosphorylation, we observed that inhibitors of ERK, but not JNK, were synergistic with PPARgamma agonists in reducing IL-8 secretion. In vivo, administration of PPARgamma agonists reduced airway inflammation in response to acute infection with P. aeruginosa in CF, but not wild-type, mice. In summary, PPARgamma inhibits the inflammatory response in CF, at least in part by interaction with NF-kappaB in airway epithelial cells. PPARgamma agonists may be therapeutic in CF.

Tissue Inhibitor of Metalloproteinase 1 Regulates Resistance to Infection

ABSTRACT Tissue inhibitor of metalloproteinase 1 (TIMP-1)-deficient mice are resistant to Pseudomonas aeruginosa corneal infections. Corneas healed completely in TIMP-1-deficient mice, and infections were cleared faster in TIMP-1-deficient mice than in wild-type littermates. Genetic suppression studies using matrix metalloproteinase (MMP)-deficient mice showed that MMP-9, MMP-3, and MMP-7 but not MMP-2 or MMP-12 are needed for resistance. Increased resistance was also seen during pulmonary infections. These results identify a novel pathway regulating infection resistance.

Nutritional Effects on Host Response to Lung Infections with Mucoid Pseudomonas aeruginosa in Mice

ABSTRACT In cystic fibrosis, a recessive genetic disease caused by defects in the cystic fibrosis conductance regulator (CFTR), the main cause of death is lung infection and inflammation. Nutritional deficits have been proposed to contribute to the excessive host inflammatory response in both humans and Cftr-knockout mice. Cftr-knockout mice and gut-corrected Cftr-knockout mice expressing human CFTR primarily in the gut were challenged with Pseudomonas aeruginosa-laden agarose beads; they responded similarly with respect to bronchoalveolar lavage cell counts and levels of the acute-phase cytokines tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-6. Wild-type mice fed the liquid diet used to prevent intestinal obstruction in Cftr-knockout mice had inflammatory responses to P. aeruginosa-laden agarose beads similar to those of wild-type mice fed an enriched solid diet, so dietary effects are unlikely to account for differences between wild-type mice and mice with cystic fibrosis. Finally, since cystic fibrosis patients and Cftr-knockout mice have an imbalance in fatty acids (significantly lower-than-normal levels of docosahexaenoic acid), the effects of specific supplementation with docosahexaenoic acid of wild-type and Cftr-knockout mice on their inflammatory responses to P. aeruginosa-laden agarose beads were tested. There were no significant differences (P = 0.35) in cumulative survival rates between Cftr-knockout mice and wild-type mice provided with either the liquid diet Peptamen or Peptamen containing docosahexaenoic acid. In conclusion, diet and docosahexaenoic acid imbalances alone are unlikely to explain the differences in the host response to lung infections with mucoid P. aeruginosa between mice with cystic fibrosis and their wild-type counterparts.

Monitoring infection and inflammation in murine models of cystic fibrosis with magnetic resonance imaging.

To evaluate magnetic resonance imaging (MRI) in assessing lung inflammation longitudinally in genetic mouse models of cystic fibrosis (CF). MRI is used to view soft tissues noninvasively, but the lung is challenging to image.

HE3286, an oral synthetic steroid, treats lung inflammation in mice without immune suppression

Background17α-Ethynyl-5-androsten-3β, 7β, 17β-triol (HE3286) is a synthetic derivative of an endogenous steroid androstenetriol (β-AET), a metabolite of the abundant adrenal steroid deyhdroepiandrosterone (DHEA), with broad anti-inflammatory activities. We tested the ability of this novel synthetic steroid with improved pharmacological properties to limit non-productive lung inflammation in rodents and attempted to gauge its immunological impact.Methods and ResultsIn mice, oral treatment with HE3286 (40 mg/kg) significantly (p < 0.05) decreased neutrophil counts and exudate volumes (~50%) in carrageenan-induced pleurisy, and myeloperoxidase in lipopolysaccharide-induced lung injury. HE3286 (40 mg/kg) was not found to be profoundly immune suppressive in any of the classical animal models of immune function, including those used to evaluate antigen specific immune responses in vivo (ovalbumin immunization). When mice treated for two weeks with HE3286 were challenged with K. pneumoniae, nearly identical survival kinetics were observed in vehicle-treated, HE3286-treated and untreated groups.ConclusionsHE3286 represents a novel, first-in-class anti-inflammatory agent that may translate certain benefits of β-AET observed in rodents into treatments for chronic inflammatory pulmonary disease.

Interleukin-17 Pathophysiology and Therapeutic Intervention in Cystic Fibrosis Lung Infection and Inflammation

ABSTRACT Cystic fibrosis (CF) is characterized by an excessive neutrophilic inflammatory response within the airway as a result of defective cystic fibrosis transmembrane receptor (CFTR) expression and function. Interleukin-17A induces airway neutrophilia and mucin production associated with Pseudomonas aeruginosa colonization, which is associated with the pathophysiology of cystic fibrosis. The objectives of this study were to use the preclinical murine model of cystic fibrosis lung infection and inflammation to investigate the role of IL-17 in CF lung pathophysiology and explore therapeutic intervention with a focus on IL-17. Cftr-deficient mice (CF mice) and wild-type mice (WT mice) infected with P. aeruginosa had robust IL-17 production early in the infection associated with a persistent elevated inflammatory response. Intratracheal administration of IL-17 provoked a neutrophilic response in the airways of WT and CF animals which was similar to that observed with P. aeruginosa infection. The neutralization of IL-17 prior to infection significantly improved the outcomes in the CF mice, suggesting that IL-17 may be a therapeutic target. We demonstrate in this report that the pathophysiological contribution of IL-17 may be due to the induction of chemokines from the epithelium which is augmented by a deficiency of Cftr and ongoing inflammation. These studies demonstrate the in vivo contribution of IL-17 in cystic fibrosis lung disease and the therapeutic validity of attenuating IL-17 activity in cystic fibrosis.

829. Preferential Transfection of Tissue Macrophages Following Intravenous Administration of sec-R Targeted hCFTR DNA Enhances Survival of CF Mice Inoculated with Pseudomonas aeruginosa Agar Beads

Previously, we have shown that modification of Poly K compacted DNA complexes with a peptide ligand (C105Y) that binds the serpin enzyme complex receptor (sec-R) targets them to lung, liver and spleen after intravenous (IV) administration and to airway epithelial cells after intranasal (IN) administration in CF mice. Predominantly, tissue macrophages were transfected following IV dosing. Since both epithelial cells and macrophages express CFTR, we took advantage of these predilection to test whether CFTR delivery to macrophages or to epithelial cells protected against the consequences of Pseudomonas aeruginosa infection. We tested the effect of different routes of administration of sec-R targeted hCFTR, IN versus IV, on alleviating the consequence of the inoculation of CF mice with Pseudomonas aeruginosa laden agar beads. This method of infection induces inflammation in CF mice that models advanced disease in CF patients. Three groups of S489X/FABP-hCFTR mice (n=14 per group) housed in sterile micro-isolator cages were studied. Group 1 received a 50 μl bolus of a 1.0 M NaCl solution containing 10 μg of sec-R targeted compacted hCFTR plasmid (codes for human CFTR driven by the elongation factor 1 promoter) via the tail vein injection and 50 μl 1.0 M NaCl alone IN. Group 2 received 50 μl sec-R targeted hCFTR IN and 50 μl 1.0 M NaCl alone IV. Control group animals received 50 μl 1.0 M NaCl IV and IN. One day following dosing the mice were inoculated with 5 × 104 cfu P. aeruginosa embedded in agar beads, and their weights recoded. We monitored the mice for 10 days following infection. Ten of fourteen mice that received IV administration of sec-R targeted hCFTR survived the course of the experiment, while 6/14 survived for the IN dosed group and 3/14 survived for the control group (p=0.03, ANOVA). Weight loss that occurs following infection was reversed one day earlier on day 4 for groups 1 and 2 versus day 5 for control animals. These results were reproduced in 2 other experiments. In a fourth experiment, analysis of bronchoalveolar lavage (BAL) from animals similarly dosed and infected, but sacrificed on day 3 following infection revealed no significant differences in cell counts or production of TNF-α, IL-1β, IL-6, or mip-2/KC (mouse IL-8 homologs). Based on previous observations, these data suggest that delivery of hCFTR to tissue macrophages rather than airway epithelial cells allows for better protection against and/or recovery from infection in CF mice. Subtle decreases in cytokine production may be sufficient for this, although other event involved in infection and inflammation not assayed for by us may have been impacted more significantly by hCFTR gene transfer to tissue macrophages.