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1.
Am J Respir Cell Mol Biol ; 44(6): 824-30, 2011 Jun.
Article in English | MEDLINE | ID: mdl-20693405

ABSTRACT

Chronic lung colonization with Pseudomonas aeruginosa is anticipated in cystic fibrosis (CF). Abnormal terminal glycosylation has been implicated as a candidate for this condition. We previously reported a down-regulation of mannose-6-phosphate isomerase (MPI) for core N-glycan production in the CFTR-defective human cell line (IB3). We found a 40% decrease in N-glycosylation of IB3 cells compared with CFTR-corrected human cell line (S9), along with a threefold-lower surface attachment of P. aeruginosa strain, PAO1. There was a twofold increase in intracellular bacteria in S9 cells compared with IB3 cells. After a 4-hour clearance period, intracellular bacteria in IB3 cells increased twofold. Comparatively, a twofold decrease in intracellular bacteria occurred in S9 cells. Gene augmentation in IB3 cells with hMPI or hCFTR reversed these IB3 deficiencies. Mannose-6-phosphate can be produced from external mannose independent of MPI, and correction in the IB3 clearance deficiencies was observed when cultured in mannose-rich medium. An in vivo model for P. aeruginosa colonization in the upper airways revealed an increased bacterial burden in the trachea and oropharynx of nontherapeutic CF mice compared with mice treated either with an intratracheal delivery adeno-associated viral vector 5 expressing murine MPI, or a hypermannose water diet. Finally, a modest lung inflammatory response was observed in CF mice, and was partially corrected by both treatments. Augmenting N-glycosylation to attenuate colonization of P. aeruginosa in CF airways reveals a new therapeutic avenue for a hallmark disease condition in CF.


Subject(s)
Cystic Fibrosis/metabolism , Cystic Fibrosis/microbiology , Epithelium/metabolism , Pseudomonas aeruginosa/metabolism , Animals , Cell Line , Cell Separation , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Flow Cytometry , Gene Expression Profiling , Genetic Therapy/methods , Glycosylation , Humans , Inflammation , Mannose/chemistry , Mannose-6-Phosphate Isomerase/metabolism , Mice
2.
Am J Respir Cell Mol Biol ; 44(6): 922-9, 2011 Jun.
Article in English | MEDLINE | ID: mdl-20724552

ABSTRACT

Cystic fibrosis (CF), the most common fatal monogenic disease in the United States, results from mutations in CF transmembrane conductance regulator (CFTR), a chloride channel. The mechanisms by which CFTR mutations cause lung disease in CF are not fully defined but may include altered ion and water transport across the airway epithelium and aberrant inflammatory and immune responses to pathogens within the airways. We have shown that Cftr(-/-) mice mount an exaggerated IgE response toward Aspergillus fumigatus, with higher levels of IL-13 and IL-4, mimicking both the T helper cell type 2-biased immune responses seen in patients with CF. Herein, we demonstrate that these aberrations are primarily due to Cftr deficiency in lymphocytes rather than in the epithelium. Adoptive transfer experiments with CF splenocytes confer a higher IgE response to Aspergillus fumigatus compared with hosts receiving wild-type splenocytes. The predilection of Cftr-deficient lymphocytes to mount T helper cell type 2 responses with high IL-13 and IL-4 was confirmed by in vitro antigen recall experiments. Conclusive data on this phenomenon were obtained with conditional Cftr knockout mice, where mice lacking Cftr in T cell lineages developed higher IgE than their wild-type control littermates. Further analysis of Cftr-deficient lymphocytes revealed an enhanced intracellular Ca(2+) flux in response to T cell receptor activation. This was accompanied by an increase in nuclear localization of the calcium-sensitive transcription factor, nuclear factor of activated T cell, which could drive the IL-13 response. In summary, our data identified that CFTR dysfunction in T cells can lead directly to aberrant immune responses. These findings implicate the lymphocyte population as a potentially important target for CF therapeutics.


Subject(s)
CD3 Complex/biosynthesis , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cytokines/metabolism , Lymphocytes/metabolism , Mutation , Animals , Aspergillus/metabolism , Calcium/metabolism , Hypersensitivity/metabolism , Immune System , Immunoglobulin E/chemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , NFATC Transcription Factors/metabolism , Spleen/cytology
3.
Mol Ther ; 18(3): 511-8, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19935781

ABSTRACT

Asthma and allergic rhinitis are almost invariable accompanied by elevated levels of immunoglobin E (IgE), and more importantly a genetic link between IgE levels and airway hyper-responsiveness has been established. We hypothesized that expression of soluble receptors directed against interleukin (IL)-13 and IL-17e would prevent the cytokines from engaging the cell-bound receptors and therefore help to attenuate allergic responses in a Cftr(-/-)-dependent mouse model of exaggerated-IgE responses. Cftr(-/-) mice were injected with recombinant adeno-associated virus 1 (rAAV1) intramuscularly expressing soluble receptors to IL-17e (IL-17Rh1fc) or IL-13 (IL-13Ralpha2Fc). Total IgE levels, in mice receiving the IL-17Rh1fc and IL-13Ralpha2Fc therapy, were lower than in the control group. Interestingly Aspergillus fumigatus (Af)-specific IgE levels were undetectable in both the mice receiving the IL-17Rh1fc and IL-13Ralpha2Fc therapies. Further flow cytometry analysis of intracellular gene expression suggests that blocking IL-17e may be interfering with signaling upstream of CD4+ and CD11b+ cells and reducing IgE levels by affecting signaling on these cell populations. In contrast it appears that IL-13 blockade acts downstream to reduce IgE levels probably by directly affecting B-cell maturation. These studies demonstrate the feasibility of targeting T helper 2 (Th2) cytokines with rAAV-delivered fusion proteins as a means to treat aberrant immune responses.


Subject(s)
Gene Transfer Techniques , Genetic Therapy/methods , Hypersensitivity/genetics , Immunoglobulin E/metabolism , Interleukin-13/genetics , Interleukin-17/genetics , Animals , CD11b Antigen/biosynthesis , CD4 Antigens/biosynthesis , Enzyme-Linked Immunosorbent Assay/methods , Flow Cytometry/methods , Hypersensitivity/therapy , Immune System , Immunoglobulin E/genetics , Mice , Mice, Transgenic , Th2 Cells/metabolism
4.
J Gene Med ; 10(1): 51-60, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18023072

ABSTRACT

Recently, we have developed a model of airway inflammation in a CFTR knockout mouse utilizing Aspergillus fumigatus crude protein extract (Af-cpe) to mimic allergic bronchopulmonary aspergillosis (ABPA) 1, an unusual IgE-mediated hypersensitivity syndrome seen in up to 15% of cystic fibrosis (CF) patients and rarely elsewhere. We hypothesized that replacement of CFTR via targeted gene delivery to airway epithelium would correct aberrant epithelial cytokine signaling and ameliorate the ABPA phenotype in CFTR-deficient (CFTR 489X - /-, FABP-hCFTR + / +) mice. CFTR knockout mice underwent intra-tracheal (IT) delivery of recombinant adeno-associated virus serotype 5 (rAAV5Delta-264CFTR) or rAAV5-GFP at 2.58 x 10(12) viral genomes/mouse. All mice were then sensitized with two serial injections (200 microg) of crude Af antigen via the intra-peritoneal (IP) route. Untreated mice were sensitized without virus exposure. Challenges were performed 2 weeks after final sensitization, using a 0.25% solution containing Aspergillus fumigatus crude protein extract delivered by inhalation on three consecutive days. The rAAV5Delta-264CFTR-treated mice had lower total serum IgE levels (172513 ng/ml +/- 1312) than rAAV5-GFP controls (26 892 ng/ml +/- 3715) (p = 0.037) and non-treated, sensitized controls (24 816 +/- 4219 ng/ml). Serum IgG1 levels also were lower in mice receiving the CFTR vector. Interestingly, splenocytes from rAAV5Delta-264CFTR-treated mice secreted less IL-13, INFg, TNFa, RANTES and GM-CSF after ConA stimulation. Gene therapy with rAAV5Delta-264CFTR attenuated the hyper-IgE response in this reproducible CF mouse model of ABPA, with systemic effects also evident in the cytokine response of stimulated splenocytes.


Subject(s)
Aspergillosis, Allergic Bronchopulmonary/therapy , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/therapeutic use , Dependovirus/metabolism , Genetic Therapy , Mutation/genetics , Transduction, Genetic , Animals , Aspergillosis, Allergic Bronchopulmonary/chemically induced , Aspergillosis, Allergic Bronchopulmonary/genetics , Aspergillus fumigatus , Cell Proliferation/drug effects , Complex Mixtures , Concanavalin A/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/deficiency , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cytokines/metabolism , Disease Models, Animal , Fluorescein-5-isothiocyanate/metabolism , Gene Expression Regulation/drug effects , Green Fluorescent Proteins/metabolism , Humans , Immunoglobulin E/blood , Immunologic Factors/metabolism , Mice , Mice, Inbred CFTR , Mutant Proteins/genetics , Mutant Proteins/metabolism , Spleen/cytology , Spleen/drug effects , Transgenes
5.
Lab Invest ; 86(2): 130-40, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16424881

ABSTRACT

To gain insight into aberrant cytokine regulation in cystic fibrosis (CF), we compared the phenotypic manifestations of allergen challenge in gut-corrected CFTR-deficient mice with background-matched C57Bl6 (B6) mice. Aspergillus fumigatus (Af) antigen was used to mimic allergic bronchopulmonary aspergillosis, a peculiar hyper-IgE syndrome with a high prevalence in CF patients. CFTR-/-, C57BL/6 and FVB/NJ mice were sensitized with Af antigen by serial intraperitoneal injections. Control mice were mock sensitized with PBS. Challenges were performed by inhalation of Af antigen aerosol. After Af antigen challenge, histologic analysis showed goblet cell hyperplasia and lymphocytic infiltration in both strains. However, total serum IgE levels were markedly elevated in CF mice. Sensitized CF mice showed a five-fold greater IgE response to sensitization as compared with B6- and FVB-sensitized controls. Additional littermate controls to fully normalize for B6-FVB admixture in the strain background confirmed the role of CFTR mutation in the hyper-IgE syndrome. Cytokine mRNA levels of IL-5 and GM-CSF in the bronchoalveolar lavage (BAL) fluid, and BAL cell differentials indicated that CFTR mutation caused a shift from an IL-5-predominant to an IL-4-predominant cytokine profile. This system models a very specific type of airway inflammation in CF and could provide insights into pathogenesis and treatment of the disease.


Subject(s)
Aspergillus fumigatus/immunology , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Hypersensitivity/immunology , Immunoglobulin E/biosynthesis , Allergens/immunology , Animals , Bronchoalveolar Lavage Fluid , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Enzyme-Linked Immunosorbent Assay , Immunoglobulin E/immunology , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction
6.
Hum Gene Ther ; 15(9): 832-41, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15353038

ABSTRACT

Despite extensive experience with recombinant adeno-associated virus (rAAV) 2 vectors in the lung, gene expression has been low in the context of cystic fibrosis (CF) gene therapy, where the large size of the cystic fibrosis transmembrane conductance regulator (CFTR) coding sequence has prompted the use of compact endogenous promoter elements. We evaluated the possibility that gene expression from recombinant adeno-associated virus (rAAV) could be improved by using alternate AAV capsid serotypes that target different cell-surface receptors (i.e., rAAV5) and/or using stronger promoters. The relative activities of the cytomegalovirus (CMV) Rous sarcoma virus (RSV) promoter, the CMV enhancer/beta-actin (CB) promoter combination, and the CMV enhancer/RSV promoter hybrid were assessed in vitro in a CF bronchial cell line. The CB promoter was the most efficient. AAV capsid serotypes, rAAV2 and rAAV5, were also compared, and rAAV5 was found to be significantly more efficient. Based on these studies a rAAV5-CB-promoter-driven CFTR minigene vector was then used to correct the CF chloride transport defect in vitro, as well as the hyperinflammatory lung phenotype in Pseudomonas-agarose bead challenged CF mouse lungs in vivo. These studies provide functional characterization of a new version of rAAV-CFTR vectors.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/therapy , Dependovirus/genetics , Genetic Vectors , Actins/genetics , Animals , Chloride Channels/drug effects , Cloning, Molecular , Cystic Fibrosis/genetics , DNA, Recombinant/therapeutic use , Dependovirus/metabolism , Gene Expression , Genetic Therapy , Luciferases/analysis , Luciferases/genetics , Mice , Pneumonia/microbiology , Pneumonia/pathology , Promoter Regions, Genetic , Pseudomonas aeruginosa/pathogenicity , Weight Loss
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