Stéphane Gagnon

A novel developmentally regulated gene in lung mesenchyme: homology to a tumor-derived trypsin inhibitor.

We used differential display-PCR (DD-PCR) to identify glucocorticoid-inducible genes that regulate lung development in late gestation. DD-PCR, a method to screen for differentially expressed genes, is based on a comparison of mRNAs isolated from a subset of two or more cell populations by analysis of RT-PCR products on DNA-sequencing gels. We isolated cDNA probes representing mRNAs expressed in primary cultures of rat lung fibroblasts, but not in epithelial cells, on fetal day 20. A day 20 glucocorticoid-treated fibroblast cDNA library was screened with a single probe to isolate the 3.1-kb cDNA late-gestation lung 1( LGL1; GenBank accession no. AF109674 ) encoding a deduced polypeptide of 188 amino acids. Northern analysis confirmed that LGL1is expressed in human, rat, and mouse fetal lungs, induced by glucocorticoid, developmentally regulated in fibroblasts but not detectable in epithelium. In situ hybridization confirmed LGL1 expression in the mesenchyme, but not in the epithelium, of fetal rat lung, kidney, and gut. The predicted LGL1 gene product (lgl1) showed 81% homology to P25TI, a polypeptide trypsin inhibitor recently identified in human glioblastoma and neuroblastoma cells but not detected in normal human tissues. Both lgl1 and P25TI belong to the CRISP family of cysteine-rich extracellular proteins. Trypsin is produced by both normal bronchial epithelial and lung adenocarcinoma cells. Although additional studies will be necessary to clearly establish a functional role for lgl1, we propose that lgl1 has a role in normal lung development that is likely to be via regulation of extracellular matrix degradation.

Glucocorticoid receptor mRNA and protein in fetal rat lung in vivo: modulation by glucocorticoid and androgen

Pulmonary glucocorticoid receptor (GR) is essential to timely preparation for the onset of breathing air at birth. We have previously used primary culture of late-gestation fetal rat lung cells to demonstrate differential regulation of GR by glucocorticoid depending on cell type. In this study, we hypothesized that the action of glucocorticoid on GR mRNA expression and protein elaboration in lung cells might be modulated by interactions present in vivo but not in primary culture. Given that male sex hormone (androgen) has an inhibitory effect on antenatal lung development, we also postulated that androgen would decrease antenatal lung GR. We report that antenatal maternal injection of the glucocorticoid dexamethasone (1 mg/kg) enhanced fetal lung cellular levels of GR mRNA and protein as assessed by in situ hybridization and immunocytochemistry (ICC), respectively. ICC was performed using polyclonal rabbit anti-human antibody that reacts with rat GR whether bound to ligand or not and does not interfere with GR binding to DNA. Levels of GR mRNA and protein were enhanced in cells throughout all areas of the lung tissue, suggesting that interactions occurring in intact tissue may override the previously reported direct inhibition by glucocorticoid of GR protein elaboration in isolated fetal rat lung epithelial cells. Furthermore, antenatal administration of the androgen 5alpha-dihydrotestosterone (0.2 mg/kg) reduced tissue levels of GR mRNA and protein, consistent with androgenic inhibition of antenatal lung development by decreasing GR. We conclude that glucocorticoids and androgens exert opposite effects on fetal lung GR.Pulmonary glucocorticoid receptor (GR) is essential to timely preparation for the onset of breathing air at birth. We have previously used primary culture of late-gestation fetal rat lung cells to demonstrate differential regulation of GR by glucocorticoid depending on cell type. In this study, we hypothesized that the action of glucocorticoid on GR mRNA expression and protein elaboration in lung cells might be modulated by interactions present in vivo but not in primary culture. Given that male sex hormone (androgen) has an inhibitory effect on antenatal lung development, we also postulated that androgen would decrease antenatal lung GR. We report that antenatal maternal injection of the glucocorticoid dexamethasone (1 mg/kg) enhanced fetal lung cellular levels of GR mRNA and protein as assessed by in situ hybridization and immunocytochemistry (ICC), respectively. ICC was performed using polyclonal rabbit anti-human antibody that reacts with rat GR whether bound to ligand or not and does not interfere with GR binding to DNA. Levels of GR mRNA and protein were enhanced in cells throughout all areas of the lung tissue, suggesting that interactions occurring in intact tissue may override the previously reported direct inhibition by glucocorticoid of GR protein elaboration in isolated fetal rat lung epithelial cells. Furthermore, antenatal administration of the androgen 5α-dihydrotestosterone (0.2 mg/kg) reduced tissue levels of GR mRNA and protein, consistent with androgenic inhibition of antenatal lung development by decreasing GR. We conclude that glucocorticoids and androgens exert opposite effects on fetal lung GR.

Reduced Viability of Mice with Lung Epithelial-Specific Knockout of Glucocorticoid Receptor

Glucocorticoid (GC)-responsive epithelial-mesenchymal interactions regulate lung development. The GC receptor (GR) mediates GC signaling. Mice lacking GR in all tissues die at birth of respiratory failure. To determine the specific need for epithelial GR in lung development, we bred triple transgenic mice that carry SPC/rtTA, tet-O-Cre, and floxed, but not wild-type, GR genes. When exposed to doxycycline in utero, triple transgenic (GRepi⁻) mice exhibit a Cre-mediated recombination event that inactivates the floxed GR gene in airway epithelial cells. Immunofluorescence confirmed the elimination of GR in Cre-positive airway epithelial cells of late gestation GRepi⁻ mice. Embryonic Day 18.5 pups had a relatively immature appearance with increased lung cellularity and increased pools of glycogen in the epithelium. Postnatal Day 0.5 pups had decreased viability. We used quantitative RT-PCR to demonstrate that specific elimination of epithelial immunoreactive GR in GRepi⁻ mice is associated with reduced mRNA expression for surfactant proteins (SPs) A, B, C, and D; β- and γ-ENaC; T1α; the 10-kD Clara cell protein (CCSP); and aquaporin 5 (AQP5). Western blots confirmed reduced levels of AQP5 protein. No reduction in the levels of the GR transport protein importin (IPO)-13 was observed. Our findings demonstrate a requirement for lung epithelial cell GR in normal lung development. We speculate that impaired epithelial differentiation, leading to decreased SPs, transepithelial Na, and liquid absorption at birth, may contribute to the reduced survival of newborn mice with suppressed lung epithelial GR.

Intrinsic predisposition of naïve cystic fibrosis T cells to differentiate towards a Th17 phenotype

BackgroundCystic fibrosis (CF) is a complex, multi-system, life-shortening, autosomal recessive disease most common among Caucasians. Pulmonary pathology, the major cause of morbidity and mortality in CF, is characterized by dysregulation of cytokines and a vicious cycle of infection and inflammation. This cycle causes a progressive decline in lung function, eventually resulting in respiratory failure and death. The Th17 immune response plays an active role in the pathogenesis of CF pulmonary pathology, but it is not known whether the pathophysiology of CF disease contributes to a heightened Th17 response or whether CF naïve CD4+ T lymphocytes (Th0 cells) intrinsically have a heightened predisposition to Th17 differentiation.MethodsTo address this question, Th0 cells were isolated from the peripheral blood of CF mice, human CF subjects and corresponding controls. Murine Th0 cells were isolated from single spleen cell suspensions using fluorescence-activated cell sorting. Lymphocytes from human buffy coats were isolated by gradient centrifugation and Th0 cells were further isolated using a human naïve T cell isolation kit. Th0 cells were then assessed for their capacity to differentiate along Th17, Th1 or Treg lineages in response to corresponding cytokine stimulation. The T cell responses of human peripheral blood cells were also assessed ex vivo using flow cytometry.ResultsHere we identify in both mouse and human CF an intrinsically enhanced predisposition of Th0 cells to differentiate towards a Th17 phenotype, while having a normal propensity for differentiation into Th1 and Treg lineages. Furthermore, we identify an active Th17 response in the peripheral blood of human CF subjects.ConclusionsWe propose that these novel observations offer an explanation, at least in part, for the known increased Th17-associated inflammation of CF and the early signs of inflammation in CF lungs before any evidence of infection. Moreover, these findings point towards direct modulation of T cell responses as a novel potential therapeutic strategy for combating excessive inflammation in CF.

T cell unresponsiveness in a pediatric cystic fibrosis patient: a case report

A girl was diagnosed with cystic fibrosis (CF) at birth, with repeatedly positive sweat tests and homozygous F508del mutations of her CF transmembrane conductance regulator (CFTR) gene. From an early age, her lung disease was more severe than her birth cohort peers despite aggressive treatment. At the age of 16 she was listed for lung transplantation, but prior to transplant was not on systemic corticosteroids or other immunosuppressive agents. In response to ex vivo stimulation, her pre-transplant peripheral blood T cells unexpectedly failed to produce detectable levels of IFN-γ, unlike cells from healthy controls or from another girl with CF and lung disease of comparable severity. Furthermore, naïve T cells freshly isolated from her peripheral blood showed a complete block of T cell differentiation into Th1, Th17 and Treg lineages, even in the presence of cytokines known to promote differentiation into the respective lineages. Her serology has been remarkably devoid of evidence of exposure to viruses that have been associated with T cell exhaustion. However, her freshly isolated naïve T cells showed sustained expression of markers of T cell exhaustion, which were further induced upon ex vivo stimulation, pointing to T cell exhaustion as the cause of the failure of naïve T cells to undergo differentiation in response to cytokine stimulation. Although excessive inflammation in CF lung can be both ineffective at clearing certain pathogens as well as destructive to the lung tissue itself, adequate inflammation is a component of an effective overall immune response to microbial pathogens. Our present findings suggest that intrinsic impairment of T cell differentiation may have contributed to the greater severity and more rapid progression of her CF lung disease than of the lung disease of most of her peers.

A Novel Gene Expressed in Lung Mesenchymal Cells Encodes a Hormonally and Developmentally Regulated Protein Homologous to a Tumour-Derived Trypsin Inhibitor

A Novel Gene Expressed in Lung Mesenchymal Cells Encodes a Hormonally and Developmentally Regulated Protein Homologous to a Tumour-Derived Trypsin Inhibitor

A Novel Developmental Gene Expressed in Late Gestation Lung Identified Using Representational Difference Analysis

A Novel Developmental Gene Expressed in Late Gestation Lung Identified Using Representational Difference Analysis

IDENTIFICATION OF GENES INVOLVED IN THE ADAPTATION OF THE LUNG TO THE AIR ENVIRONMENT. |[utrif]| 354

We are using the technique of differential display RT-PCR in order to identify genes important in lung development. We are particularly interested in genes whose products are essential to mesenchymal-epithelial interactions in late gestation lung. We isolated 38 cDNA probes representing mRNAs whose expression is modulated by glucocorticoid (GC) and dihydro-testosterone (DHT) in days 19-20 fibroblasts (fib) or epithelial cells (epi) of fetal rat lung. Among 18 fib-specific sequences, 16 represented mRNAs whose expression is increased in the presence of GC and decreased in the presence of DHT. This pattern of expression is expected for FPF, an activity generated by fibs which augments epi surfactant production. Twenty sequences were epi-specific and represent mRNAs meeting criteria for GC receptor (GR) stimulating factor (an activity produced by epi which stimulates fib GR). We sequenced 20 (15 epi, 5 fib) subclones (18 novel). Nine showed interesting homologies to sequences in Genbank. The predicted protein product of one epi-clone had 66-70% homology(over 60 residues) to 70 bacterial flagellins but had no identified homologs in eukaryotes. This suggests a novel protein, perhaps a precursor of a lung ciliary protein. The predicted protein products of 3 epi-clones had primary sequence homology or protein structural motifs suggestive of transcriptional regulators. One clone had homology to rat/mouse progesterone receptor (a stage and tissue-specific transcriptional regulator). Another had homology to yeast HPR1 protein (a positive transcriptional regulator). A third clone had a region with a leucine-zipper motif, a motif common to transcriptional activators. Transcriptional regulatory sequences are of interest as regulation of transcription is likely a key mechanism in the modulation of gene expression in late gestation lung development. These findings open new areas for the study of regulation of fetal lung maturation.

GLUCOCORTICOID ENHANCES AND ANDROGEN DIMINISHES EXPRESSION OF GLUCOCORTICOID RECEPTOR mRNA AND PROTEIN IN FETAL RAT LUNG TISSUE IN SITU . ▴ 399

GLUCOCORTICOID ENHANCES AND ANDROGEN DIMINISHES EXPRESSION OF GLUCOCORTICOID RECEPTOR mRNA AND PROTEIN IN FETAL RAT LUNG TISSUE IN SITU . ▴ 399