Fine mapping of the major bleomycin-induced pulmonary fibrosis susceptibility locus in mice.

Susceptibility to fibrotic lung disease differs among people and among inbred strains of mice exposed to bleomycin where C57BL/6J mice are susceptible and C3H/HeJ mice are spared fibrotic disease. Genetic mapping studies completed in offspring derived from these inbred strains revealed the inheritance of C57BL/6J alleles at loci, including the major locus on chromosome 17, called Blmpf1 bleomycin-induced pulmonary fibrosis 1, to be linked to pulmonary fibrosis in treated mice. In the present study, to reduce the interval of Blmpf1, we bred and phenotyped a panel of subcongenic mice with C3H/HeJ alleles in a C57BL/6J background. Subcongenic mice received bleomycin via osmotic minipump and the fibrosis phenotype was measured histologically. Inheritance of C3H/HeJ alleles from 34.31 to 35.02 Mb was revealed to spare bleomycin-induced pulmonary fibrosis of C57BL/6J mice. From database analysis, 40 protein coding genes have been mapped to this reduced Blmpf1 interval, 18 of which contain C57BL/6J:C3H/HeJ sequence polymorphisms predicted to affect protein structure or to confer allele-dependent expression, and by RT-PCR analysis of lung tissue, we show 6 of these genes to differ in expression between C57BL/6J and C3H/HeJ mice. Genes known to regulate T cell numbers and activation (Btnl family, Notch4) are among the limited list of potential causal variants leading to lung disease in this model and the bronchoalveolar lavage of protected subcongenic mice had fewer lymphocytes, post bleomycin, than did C57BL/6J mice. We conclude that Blmpf1genes contributing to the susceptibility to bleomycin-induced pulmonary fibrosis could alter the adaptive immune response of C57BL/6J mice.

Positional cloning reveals strain-dependent expression of Trim16 to alter susceptibility to bleomycin-induced pulmonary fibrosis in mice.

Pulmonary fibrosis is a disease of significant morbidity, with no effective therapeutics and an as yet incompletely defined genetic basis. The chemotherapeutic agent bleomycin induces pulmonary fibrosis in susceptible C57BL/6J mice but not in mice of the C3H/HeJ strain, and this differential strain response has been used in prior studies to map bleomycin-induced pulmonary fibrosis susceptibility loci named Blmpf1 and Blmpf2. In this study we isolated the quantitative trait gene underlying Blmpf2 initially by histologically phenotyping the bleomycin-induced lung disease of sublines of congenic mice to reduce the linkage region to 13 genes. Of these genes, Trim16 was identified to have strain-dependent expression in the lung, which we determined was due to sequence variation in the promoter. Over-expression of Trim16 by plasmid injection increased pulmonary fibrosis, and bronchoalveolar lavage levels of both interleukin 12/23-p40 and neutrophils, in bleomycin treated B6.C3H-Blmpf2 subcongenic mice compared to subcongenic mice treated with bleomycin only, which follows the C57BL/6J versus C3H/HeJ strain difference in these traits. In summary we demonstrate that genetic variation in Trim16 leads to its strain-dependent expression, which alters susceptibility to bleomycin-induced pulmonary fibrosis in mice.

Cystic fibrosis mouse model-dependent intestinal structure and gut microbiome.

Mice with a null mutation in the cystic fibrosis transmembrane conductance regulator (Cftr) gene show intestinal structure alterations and bacterial overgrowth. To determine whether these changes are model-dependent and whether the intestinal microbiome is altered in cystic fibrosis (CF) mouse models, we characterized the ileal tissue and intestinal microbiome of mice with the clinically common ΔF508 Cftr mutation (FVB/N Cftr(tm1Eur)) and with Cftr null mutations (BALB/c Cftr(tm1UNC) and C57BL/6 Cftr(tm1UNC)). Intestinal disease in 12-week-old CF mice, relative to wild-type strain controls, was measured histologically. The microbiome was characterized by pyrosequencing of the V4-V6 region of the 16S rRNA gene and intestinal load was measured by RT-PCR of the 16S rRNA gene. The CF-associated increases in ileal crypt to villus axis distention, goblet cell hyperplasia, and muscularis externa thickness were more severe in the BALB/c and C57BL/6 Cftr(tm1UNC) mice than in the FVB/N Cftr(tm1Eur) mice. Intestinal bacterial load was significantly increased in all CF models, compared to levels in controls, and positively correlated with circular muscle thickness in CF, but not wild-type, mice. Microbiome profiling identified Bifidobacterium and groups of Lactobacillus to be of altered abundance in the CF mice but overall bacterial frequencies were not common to the three CF strains and were not correlative of major histological changes. In conclusion, intestinal structure alterations, bacterial overgrowth, and dysbiosis were each more severe in BALB/c and C57BL/6 Cftr(tm1UNC) mice than in the FVB/N Cftr(tm1Eur) mice. The intestinal microbiome differed among the three CF mouse models.

Basal levels of glutathione peroxidase correlate with onset of radiation induced lung disease in inbred mouse strains.

Biomarkers predicting for the radiation-induced lung responses of pneumonitis or fibrosis are largely unknown. Herein we investigated whether markers of oxidative stress and intracellular antioxidants, measured within days of radiation exposure, are correlated with the lung tissue injury response occurring weeks later. Mice of the eight inbred strains differing in their susceptibility to radiation-induced pulmonary fibrosis, and in the duration of asymptomatic survival, received 18 Gy whole thorax irradiation and were killed 6 h, 24 h, or 7 days later. Control mice were not irradiated. Lung levels of antioxidants superoxide dismutase, catalase, glutathione peroxidase (GPx), and glutathione, and of oxidative damage [reactive oxygen species (ROS) and 8-hydroxydeoxyguanosine (8-OHdG)], were biochemically determined. GPx was additionally measured through gene expression and immunohistochemical assessment of lung tissue, and activity in serum. ROS and 8-OHdG were increased postirradiation and exhibited significant strain and time-dependent variability, but were not strongly predictive of radiation-induced lung diseases. Antioxidant measures were not dramatically changed postirradiation and varied significantly among the strains. Basal GPx activity (r = 0.73, P = 0.04) in the lung and the pulmonary expression of GPx2 (r = 0.94, P = 0.0003) correlated with postirradiation asymptomatic survival, whereas serum GPx activity was inversely correlated (r = -0.80, P = 0.01) with fibrosis development. In conclusion, pulmonary oxidative stress and antioxidant markers were more affected by inbred strain than radiation over 7 days posttreatment. Lung GPx activity, and GPx2 expression, predicted for survival from lethal pneumonitis, and serum GPx for fibrosis, in this panel of mice.

Strain-dependent airway hyperresponsiveness and a chromosome 7 locus of elevated lymphocyte numbers in cystic fibrosis transmembrane conductance regulator-deficient mice.

We previously observed the lungs of naive BALB/cJ Cftr(tm1UNC) mice to have greater numbers of lymphocytes, by immunohistochemical staining, than did BALB wild type littermates or C57BL/6J Cftr(tm1UNC) mice. In the present study, we initially investigated whether this mutation in Cftr alters the adaptive immunity phenotype by measuring the lymphocyte populations in the lungs and spleens by FACS and by evaluating CD3-stimulated cytokine secretion, proliferation, and apoptosis responses. Next, we assessed a potential influence of this lymphocyte phenotype on lung function through airway resistance measures. Finally, we mapped the phenotype of pulmonary lymphocyte counts in BALB × C57BL/6J F2 Cftr(tm1UNC) mice and reviewed positional candidate genes. By FACS analysis, both the lungs and spleens of BALB Cftr(tm1UNC) mice had more CD3(+) (both CD4(+) and CD8(+)) cells than did littermates or C57BL/6J Cftr(tm1UNC) mice. Cftr(tm1UNC) and littermate mice of either strain did not differ in anti-CD3-stimulated apoptosis or proliferation levels. Lymphocytes from BALB Cftr(tm1UNC) mice produced more IL-4 and IL-5 and reduced levels of IFN-γ than did littermates, whereas lymphocytes from C57BL/6J Cftr(tm1UNC) mice demonstrated increased Il-17 secretion. BALB Cftr(tm1UNC) mice presented an enhanced airway hyperresponsiveness to methacholine challenge compared with littermates and C57BL/6J Cftr(tm1UNC) mice. A chromosome 7 locus was identified to be linked to lymphocyte numbers, and genetic evaluation of the interval suggests Itgal and Il4ra as candidate genes for this trait. We conclude that the pulmonary phenotype of BALB Cftr(tm1UNC) mice includes airway hyperresponsiveness and increased lymphocyte numbers, with the latter trait being influenced by a chromosome 7 locus.

Association analysis reveals genetic variation altering bleomycin-induced pulmonary fibrosis in mice.

Pulmonary fibrosis is a disease of significant morbidity, with an incompletely defined genetic basis. Here, we combine linkage and association studies to identify genetic variations associated with pulmonary fibrosis in mice. Mice were treated with bleomycin by osmotic minipump, and pulmonary fibrosis was histologically assessed 6 weeks later. Fibrosis was mapped in C57BL6/J (fibrosis-susceptible) × A/J (fibrosis-resistant) F2 mice, and the major identified linkage intervals were evaluated in consomic mice. Genome-wide and linkage-interval genes were assessed for their association with fibrosis, using phenotypic data from 23 inbred strains and the murine single-nucleotide polymorphism map. Susceptibility to pulmonary fibrosis mapped to a locus on chromosome 17, which was verified with consomic mice, and to three additional suggestive loci that may interact with alleles on chromosome 17 to affect the trait in F2 mice. Two of the loci, including the region on chromosome 17, are homologous to previously mapped loci of human idiopathic fibrosis. Of the 23 phenotyped murine strains, four developed significant fibrosis, and the majority presented minimal disease. Genome-wide and linkage region-specific association studies revealed 11 pulmonary expressed genes (including the autophagy gene Cep55, and Masp2, which is a complement component) to contain polymorphisms significantly associated with bleomycin-induced fibrotic lung disease. In conclusion, genomic approaches were used to identify linkage intervals and specific genetic variations associated with pulmonary fibrosis in mice. The common loci and similarities in phenotype suggest these findings to be of relevance to clinical pulmonary fibrosis.

A selenocysteine derivative therapy affects radiation-induced pneumonitis in the mouse.

The mechanism leading to the radiation-induced lung response of pneumonitis is largely unknown. Here we investigated whether treatment with 3,3'-diselenodipropionic acid (DSePA), which reduces radiation-induced oxidative stress in acute response models, decreases the lung response to irradiation. Mice of the C3H/HeJ (alveolitis/pneumonitis-responding) strain received 18 Gy whole-thorax irradiation, and a subset of these mice was treated with DSePA (2 mg/kg) three times per week, beginning at 2 hours after radiation treatment, and continuing in the postirradiation period until death because of respiratory distress symptoms. DSePA treatment increased the postirradiation survival time of mice by an average of 32 days (P = 0.0002). Radiation-treated and DSePA-treated mice presented lower levels of lipid peroxidation and augmented glutathione peroxidase in the lungs, compared with those levels measured in mice receiving radiation only, when mice receiving radiation only were killed because of distress symptoms, whereas catalase and superoxide dismutase levels did not show consistent differences among treatment groups. DSePA treatment decreased pneumonitis and the numbers of mast cells, neutrophils, and lymphocytes in the lungs and bronchoalveolar lavage, respectively, of irradiated mice relative to mice exposed to radiation alone. DSePA treatment also decreased the radiation-induced increase in granulocyte colony-stimulating factor levels in the bronchoalveolar lavage and lung-tissue expression of intercellular adhesion molecule-1 and E-selectin, while increasing the expression of glutathione peroxidase-4. We conclude that DSePA treatment reduces radiation-induced pneumonitis in mice by delaying oxidative damage and the inflammatory cell influx.

MicroRNA profiling of cystic fibrosis intestinal disease in mice.

Cystic fibrosis (CF) intestinal disease is characterized by alterations in processes such as proliferation and apoptosis which are known to be regulated in part by microRNAs. Herein, we completed microRNA expression profiling of the intestinal tissue from the cystic fibrosis mouse model of cystic fibrosis transmembrane conductance regulator (Cftr) deficient mice (BALBc/J Cftr(tm1UNC)), relative to that of wildtype littermates, to determine whether changes in microRNA expression level are part of this phenotype. We identified 24 microRNAs to be significantly differentially expressed in tissue from CF mice compared to wildtype, with the higher expression in tissue from CF mice. These data were confirmed with real time PCR measurements. A comparison of the list of genes previously reported to have decreased expression in the BALB×C57BL/6J F2 CF intestine to that of genes putatively targeted by the 24 microRNAs, determined from target prediction software, revealed 155 of the 759 genes of the expression profile (20.4%) to overlap with predicted targets, which is significantly more than the 100 genes expected by chance (p=1×10(-8)). Pathway analysis identified these common genes to function in phosphatase and tensin homolog-, protein kinase A-, phosphoinositide-3 kinase/Akt- and peroxisome proliferator-activated receptor alpha/retinoid X receptor alpha signaling pathways, among others, and through real time PCR experiments genes of these pathways were demonstrated to have lower expression in the BALB CF intestine. We conclude that altered microRNA expression is a feature which putatively influences both metabolic abnormalities and the altered tissue homeostasis component of CF intestinal disease.

IGF binding protein-3 treatment alters intestinal cell proliferation but not body weight of adult cystic fibrosis transmembrane conductance regulator deficient mice.

The intestinal phenotype of cystic fibrosis (CF) transmembrane conductance regulator deficient mice includes altered cell homeostasis and a distended crypt-villus axis, which, in previous work, was inversely proportional to body weight. To investigate this correlation, herein, we treated CF mice with IGF binding protein-3 (IGFBP-3), a protein which, as it has potent effects on cell proliferation and apoptosis, we hypothesized would alter the intestinal cell homeostasis, and assessed body weight. Six-week-old C57BL/6JxBALB F2 CF and WT mice received recombinant human IGFBP-3 (rhIGFBP-3, 20 mg/kg) or vehicle treatment, and weight gain, serum protein levels, and intestinal histology were assessed. Administration of rhIGFBP-3 to CF mice significantly increased the number of Igfbp-3 positive cells in the intestine and partially reversed the hyperproliferative phenotype of intestinal crypts and muscularis externa, while not affecting apoptosis. Serum Igfbp-3 levels were increased, and Igf-I, albumin, and triglycerides measures were decreased in CF compared with WT mice. rhIGFBP-3 treatment significantly increased serum albumin and triglycerides but did not affect weight gain in CF mice. We have identified rhIGFBP-3 treatment to reduce intestinal cell proliferation, resulting in decreases in crypt depth and muscularis externa thickness in CF mice.

Acute Radiation-Induced Hematopoietic Depletion Does Not Alter the Onset or Severity of Pneumonitis in Mice.

Survival from partial-body irradiation (PBI) may be limited by the development of the late lung injury response of pneumonitis. Herein we investigated the hypothesis that acute hematopoietic depletion alters the onset and severity of lung disease in a mouse model. To establish depletion, C3H/HeJ mice received 8 Gy PBI with shielding of only the tibiae, ankles and feet. One week after irradiation, blood lymphocyte and neutrophil counts were each significantly reduced (P < 0.04) in these mice compared to levels in untreated controls or in mice receiving 16 Gy to the whole thorax only. All 8 Gy PBI mice survived to the experimental end point of 16 weeks postirradiation. To determine whether the hematopoietic depletion affects lung disease, groups of mice received 8 Gy PBI plus 8 Gy whole-thorax irradiation (total lung dose of 16 Gy) or 16 Gy whole-thorax irradiation only. The weight loss, survival to onset of respiratory distress (P = 0.17) and pneumonitis score (P = 0.96) of mice that received 8 Gy PBI plus 8 Gy whole-thorax irradiation were not significantly different from those of mice receiving 16 Gy whole-thorax irradiation only. Mice in respiratory distress from PBI plus whole-thorax irradiation had significantly reduced (P = 0.02) blood monocyte counts compared to levels in distressed, whole-thorax irradiated mice, and symptomatic pneumonitis was associated with increased blood neutrophil counts (P = 0.04) relative to measures from irradiated, non-distressed mice. In conclusion, survivable acute hematopoietic depletion by partial-body irradiation did not alter the onset or severity of lethal pneumonitis in the C3H/HeJ mouse model.

Toll-like receptor-4 genotype influences the survival of cystic fibrosis mice.

Toll-like receptor (Tlr) 4 is a lipopolysaccharide (LPS) receptor that contributes to the regulation of intestinal cell homeostasis, a condition that is altered in the intestines of cystic fibrosis mice. Herein, we assessed whether Tlr4 genotype influences cystic fibrosis intestinal disease by producing and phenotyping 12-wk (adult)- and 4-day (neonate)-old mice derived from BALB cystic fibrosis transmembrane conductance regulator, Cftr(+/tm1Unc) and C.C3-Tlr4(Lps-d)/J (Tlr4(-/-)), progenitors. Intestinal disease was assayed through mouse survival, crypt-villus axis (CVA) length, cell proliferation, bacterial load, bacterial classification, inflammatory cell infiltrate, and mucus content measures. Of the 77 Cftr(-/-) (CF) mice produced, only one Cftr/Tlr4 double-mutant mouse lived to the age of 12 wk while the majority of the remainder succumbed at approximately 4 days of age. The survival of CF Tlr4(+/-) mice exceeded that of both CF Tlr4(+/+) and Cftr/Tlr4 double-mutant mice. Adult CF mice presented increased Tlr4 expression, CVA length, crypt cell proliferation, and bacterial load relative to non-CF mice, but no differences were detected in Tlr4(+/-) compared with Tlr4(+/+) CF mice. The double-mutant neonates did not differ from Tlr4(+/+) or Tlr4(+/-) CF mice by intestinal CVA length or bacterial load, but fewer Tlr4(+/-) CF neonates presented with luminal mucus obstruction in the distal ileum, and the intestinal mast cell increase of CF mice was not evident in double-mutant neonates. We conclude that Tlr4 deficiency reduces the survival, but does not alter the intestinal phenotypes, of extended CVA or increased bacterial load in BALB CF mice.

Persistent osteopenia in adult cystic fibrosis transmembrane conductance regulator-deficient mice.

RATIONALE: A loss of function mutation in the cystic fibrosis transmembrane conductance regulator gene is believed to be an independent risk factor for bone disease in patients with cystic fibrosis. OBJECTIVES: The objective of this work was to use congenic mice as a preclinical model to examine the bone phenotype of Cftr(-/-) mice and control littermates at 8, 12, and 28 weeks of age. METHODS: The bone phenotype of control and Cftr(-/-) mice was evaluated by quantitative imaging, histologic and histomorphometric analyses, and serum levels of bone biomarkers. MEASUREMENTS AND MAIN RESULTS: At 12 weeks of age, Cftr(-/-) mice were smaller, had lower bone mineral density, cortical bone thinning, and altered trabecular architecture compared with Cftr(+/+) or Cftr(+/-) control mice. In skeletally mature 28-week-old mice, there were persistent deficits in cortical and trabecular bone structure in Cftr(-/-) mice despite significant, quantifiable improvements. Cftr(-/-) mice also had lower serum insulin-like growth factor-I levels at 12 weeks of age than did control mice, whereas parathyroid hormone and 25-hydroxyvitamin D levels were not significantly different. CONCLUSIONS: Persistent osteopenia and structural abnormalities in adult Cftr(-/-) mice, in the absence of overt respiratory and gastrointestinal disease, suggest that loss of Cftr function has a direct impact on bone metabolism in Cftr(-/-) mice that is not sex specific or subject to haplotype insufficiency.

Radiation-induced lung response of AcB/BcA recombinant congenic mice.

The genetic factors that influence the development of radiotherapy-induced lung disease are largely unknown. Herein we identified a strain difference in lung response to radiation wherein A/J mice developed alveolitis with increased levels of pulmonary mast cells and cells in bronchoalveolar lavage while the phenotype in C57BL/6J mice was fibrosis with fewer inflammatory cells. To identify genomic loci that may influence these phenotypes, we assessed recombinant congenic (RC) mice derived from the A/J and C57BL/6J strains for their propensity to develop alveolitis or fibrosis after 18 Gy whole-thorax irradiation. Mouse survival, lung histopathology and bronchoalveolar lavage cell types were recorded. Informative strains for each of mast cell influx, bronchoalveolar cell numbers, alveolitis and fibrosis were identified. In mice with the A/J strain background, the severity of alveolitis correlated with increased mast cell numbers while in C57BL/6J background strain mice fibrosis was correlated with the percentage of neutrophils in lavage. The data for RC mice support the association of specific inflammatory cells with the development of radiation-induced lung disease and provide informative strains with which to dissect the genetic basis of these complex traits.

A Chromosome 6, not Natural Killer Cell, Contribution to Radiation- and Bleomycin-Induced Lung Disease in Mice.

Inbred strains of mice differ in susceptibility to both radiation-induced and bleomycin-induced pulmonary fibrosis and these traits have been mapped to a common locus on chromosome 6 which harbors genes of natural killer cell function. To investigate this putative locus of fibrosis susceptibility we assessed the fibrotic response of chromosome-6 consomic mice (B6.6A), and of mice deficient for natural killer cells, C57BL/6J Ly49A transgenic mice, after each of thoracic irradiation and bleomycin treatment via osmotic minipump. Thoracic irradiation resulted in less than 15% survival at 26 weeks in parental strain C57BL/6J and A/J mice, due to the development of pneumonitis with fibrosis in C57BL/6J (B6) mice, and pneumonitis in A/J mice. One hundred percent of consomic B6.6A mice survived at 26 weeks after thoracic irradiation, and developed a fibrosis level similar to that of fibrosis-resistant A/J mice, after irradiation ( P = 0.38) or bleomycin challenge ( P = 0.32). C57BL/6J Ly49A transgenic mice were confirmed through flow cytometric analysis to be deficient in NK cells, but the post-irradiation survival of these mice was not significantly different from that of wild-type littermate mice ( P = 0.64). Extent of pulmonary fibrosis by histological examination did not differ between C57BL/6J Ly49A transgenic mice and wild-type littermate mice in response to either irradiation ( P = 0.14) or bleomycin treatment ( P = 0.62). We conclude that chromosome 6 genes, but not NK cells, contribute to the susceptibility to both radiation-induced and bleomycin-induced pulmonary fibrosis of C57BL/6J mice.

X chromosome transmission ratio distortion in Cftr +/- intercross-derived mice.

BACKGROUND: Cystic fibrosis (CF) mice, created with a genetically engineered mutation in the Cystic fibrosis transmembrane conductance regulator (Cftr) gene, may develop intestinal plugs which limit their survival past weaning. In a studied population of genetically mixed CF mice differences in allelic ratios at particular loci, between surviving CF mice and mice with the lethal intestinal defect, were used to map cystic fibrosis modifier gene one, Cfm1. Using this approach, we previously identified an X chromosome locus which may influence the survival to weaning of C57BL/6J x BALB/cJ F2 CF mice. We also detected two regions of transmission ratio distortion, independent of Cftr genotype, in a limited dataset. To investigate these findings, in this study we have genotyped 1208 three-week old F2 mice, and 186 day E15.5 embryos, derived from a congenic (C57BL/6J x BALB/cJ) F1 Cftr +/- intercross, for the putative distortion regions. RESULTS: An excess of homozygous BALB genotypes, compared to Mendelian expectations, was detected on chromosomes 5 (p = 5.7 x 10-15) and X (p = 3.0 x 10-35) in three-week old female mice but transmission ratio distortion was not evident in the tested region of chromosome 3 (p = 0.39). Significant pre-weaning lethality of CF mice occurred as 11.3% (137/1208) of the three-week old offspring were identified as CF mice. X chromosome genotypes were not, however, distorted in the female CF mice (p = 0.62), thus the significant non-Mendelian inheritance of this locus was dependent on CF status. The survival of CF embryos to day E15.5 was consistent with Mendelian expectations (42/186 = 23%), demonstrating the loss of CF mice to have occurred between E15.5 and three weeks of age. The excess of X chromosome homozygous BALB genotypes was recorded in female embryos (p = 0.0048), including CF embryos, indicating the distortion to be evident at this age. CONCLUSION: Two of three previously suggested loci of transmission ratio distortion were replicated as distorted in this mouse cross. The non-Mendelian inheritance of X chromosome genotypes implicates this region in the survival to weaning of non-CF mice.

The Th1/Th17 balance dictates the fibrosis response in murine radiation-induced lung disease.

Radiotherapy can result in lung diseases pneumonitis or fibrosis dependent on patient susceptibility. Herein we used inbred and genetically altered mice to investigate whether the tissue adaptive immune response to radiation injury influences the development of radiation-induced lung disease. Six inbred mouse strains were exposed to 18 Gy whole thorax irradiation and upon respiratory distress strains prone to pneumonitis with fibrosis presented an increased pulmonary frequency of Thelper (Th)17 cells which was not evident in strains prone solely to pneumonitis. The contribution of Th17 cells to fibrosis development was supported as the known enhanced fibrosis of toll-like receptor 2&4 deficient mice, compared to C57BL/6J mice, occurred with earlier onset neutrophilia, and with increased levels of pulmonary Th17, but not Th1, cells following irradiation. Irradiated Il17-/- mice lacked Th17 cells, and were spared both fibrosis and pneumonitis, as they survived to the end of the experiment with a significantly increased pulmonary Th1 cell frequency, only. Interferon-γ-/- mice, deficient in Th1 cells, developed a significantly enhanced fibrosis response compared to that of C57BL/6J mice. The tissue adaptive immune response influences the pulmonary disease response to radiotherapy, as an increased Th17 cell frequency enhanced and a Th1 response spared, fibrosis in mice.

Strain-dependent pulmonary gene expression profiles of a cystic fibrosis mouse model.

Cystic fibrosis (CF) lung disease severity is influenced by unknown genetic factors apart from the disease causative gene, cystic fibrosis transmembrane conductance regulator (CFTR). Previous studies have shown the C57BL/6J congenic Cftr(-/-) (B6 CF) mouse to develop a fibrotic lung disease compared with both CF mice of the BALB/c background and wild-type animals. In this report, gene expression profiling with microarrays was used to identify genes differentially expressed in the lungs of B6 and BALB CF mice compared with non-CF littermates. Seven hundred two genes or expressed sequence tags (ESTs) were identified to be differentially expressed between the B6 CF and non-CF control lungs (P < 0.05), and, by Gene Ontology classification, the B6 CF response included the cell proliferation categories of DNA metabolism and mitosis. In the response of BALB mice to nonfunctional Cftr, 943 genes/ESTs were differentially expressed compared with controls. The biological processes of apoptosis and T and B cell proliferation were prominent in the gene list of the BALB CF strain. In support of this strain difference, increased T lymphocyte infiltration was evident in the lungs of BALB CF mice, through immunohistochemical staining, compared with the lungs from both B6 CF and non-CF control mice. Four hundred forty-four genes/ESTs were differentially expressed between B6 CF and BALB CF mice (P < 0.05, fold > 2), including 56 that map to previously identified linkage intervals. These results suggest that the variable severity of CF lung disease in this mouse model is controlled by multiple genetic factors, including those of an immune response.

Universal and radiation-specific loci influence murine susceptibility to radiation-induced pulmonary fibrosis.

Susceptibility to radiation-induced pulmonary fibrosis is a heritable trait in mice. In a prior study of C57BL/6J (susceptible), C3Hf/Kam (resistant), and F1 and F2 mice derived from these strains, we estimated that approximately 38% of the measured phenotypic variation could be attributed to effects from a few genetic factors. In addition, we identified one genetic factor on chromosome 17 in the MHC region. To identify any additional genetic loci that might influence interstrain variability, we conducted a genome-wide linkage scan using 214 markers and the phenotypically extreme 94 (of 268) F2 mice. In regions exceeding suggestive linkage (LOD = 2.8), we followed up with additional markers. This scan revealed evidence for quantitative trait locus (QTL) on chromosomes 17 (LOD = 4.2), 1 (LOD = 4.5), and 18 (LOD = 3.9), which influence susceptibility to radiation-induced pulmonary fibrosis. An additional region containing a QTL on chromosome 6, LOD = 4.6, showed linkage in female mice only. The evidence for linkage to chromosome 18 weakened when it was analyzed jointly with other markers. These four loci are estimated to account for 70% of the genetic contribution to this trait with chromosome 17 and 1 accounting for 28 and 24%, respectively. To confirm and better define the influence of the chromosome 17-linked QTL on radiation sensitivity, we conducted studies on congenic mice in which the linked region on chromosome 17 had been transferred onto a B6.AKR or a C3.SW background. The chromosome 17-linked QTL was confirmed to influence the phenotype as the fibrotic radiation response of B6.AKR-H2(k) mice was significantly less than that of B6 mice (P = 0.0001). The QTL on chromosome 17 for radiation-induced lung fibrosis is within the same region as QTLs identified for lung damage after other insults, including bleomycin, ozone, and particle exposure, as well as for asthma, suggesting that this region of chromosome 17 may harbor a "universal" lung injury gene.

Streptomycin treatment alters the intestinal microbiome, pulmonary T cell profile and airway hyperresponsiveness in a cystic fibrosis mouse model.

Cystic fibrosis transmembrane conductance regulator deficient mouse models develop phenotypes of relevance to clinical cystic fibrosis (CF) including airway hyperresponsiveness, small intestinal bacterial overgrowth and an altered intestinal microbiome. As dysbiosis of the intestinal microbiota has been recognized as an important contributor to many systemic diseases, herein we investigated whether altering the intestinal microbiome of BALB/c Cftr(tm1UNC) mice and wild-type littermates, through treatment with the antibiotic streptomycin, affects the CF lung, intestinal and bone disease. We demonstrate that streptomycin treatment reduced the intestinal bacterial overgrowth in Cftr(tm1UNC) mice and altered the intestinal microbiome similarly in Cftr(tm1UNC) and wild-type mice, principally by affecting Lactobacillus levels. Airway hyperresponsiveness of Cftr(tm1UNC) mice was ameliorated with streptomycin, and correlated with Lactobacillus abundance in the intestine. Additionally, streptomycin treated Cftr(tm1UNC) and wild-type mice displayed an increased percentage of pulmonary and mesenteric lymph node Th17, CD8 + IL-17+ and CD8 + IFNγ+ lymphocytes, while the CF-specific increase in respiratory IL-17 producing γδ T cells was decreased in streptomycin treated Cftr(tm1UNC) mice. Bone disease and intestinal phenotypes were not affected by streptomycin treatment. The airway hyperresponsiveness and lymphocyte profile of BALB/c Cftr(tm1UNC) mice were affected by streptomycin treatment, revealing a potential intestinal microbiome influence on lung response in BALB/c Cftr(tm1UNC) mice.

Bleomycin-induced pulmonary fibrosis susceptibility genes in AcB/BcA recombinant congenic mice.

The genetic basis of susceptibility to pulmonary fibrosis is largely unknown. Initially, in this study, loci regulating the response of bleomycin-induced pulmonary fibrosis were mapped using a set of recombinant congenic strains bred from pulmonary fibrosis-resistant A/J and susceptible C57BL/6J (B6) mice. Linkage was identified (logarithm of the odds score = 4.9) on chromosome 9, and other suggestive loci were detected. The putative loci included alleles from both the B6 and A/J strains as increasing the fibrosis response of congenic mice. Gene expression analysis with microarrays revealed 3,304 genes or expressed sequence tags to be differentially expressed (P < 0.01) in lung tissue between bleomycin-treated B6 and A/J mice, and 246 of these genes mapped to potential susceptibility loci. Pulmonary genes differentially expressed between bleomycin-treated B6 and A/J mice included those of heparin binding and extracellular matrix deposition pathways. A review of available genomic sequences revealed 809 (43% of total) genes in the linkage intervals to have variations predicted to alter the encoded proteins or their regulation, 68 (8.4%) of which were also differentially expressed. Genomic approaches were combined to produce a set of candidate genes that may influence susceptibility to bleomycin-induced pulmonary fibrosis in the A/J:B6 mouse model.