Metabolic profiling of murine radiation-induced lung injury with Raman spectroscopy and comparative machine learning.

Radiation-induced lung injury (RILI) is a dose-limiting toxicity for cancer patients receiving thoracic radiotherapy. As such, it is important to characterize metabolic associations with the early and late stages of RILI, namely pneumonitis and pulmonary fibrosis. Recently, Raman spectroscopy has shown utility for the differentiation of pneumonitic and fibrotic tissue states in a mouse model; however, the specific metabolite-disease associations remain relatively unexplored from a Raman perspective. This work harnesses Raman spectroscopy and supervised machine learning to investigate metabolic associations with radiation pneumonitis and pulmonary fibrosis in a mouse model. To this end, Raman spectra were collected from lung tissues of irradiated/non-irradiated C3H/HeJ and C57BL/6J mice and labelled as normal, pneumonitis, or fibrosis, based on histological assessment. Spectra were decomposed into metabolic scores via group and basis restricted non-negative matrix factorization, classified with random forest (GBR-NMF-RF), and metabolites predictive of RILI were identified. To provide comparative context, spectra were decomposed and classified via principal component analysis with random forest (PCA-RF), and full spectra were classified with a convolutional neural network (CNN), as well as logistic regression (LR). Through leave-one-mouse-out cross-validation, we observed that GBR-NMF-RF was comparable to other methods by measure of accuracy and log-loss (p > 0.10 by Mann-Whitney U test), and no methodology was dominant across all classification tasks by measure of area under the receiver operating characteristic curve. Moreover, GBR-NMF-RF results were directly interpretable and identified collagen and specific collagen precursors as top fibrosis predictors, while metabolites with immune and inflammatory functions, such as serine and histidine, were top pneumonitis predictors. Further support for GBR-NMF-RF and the identified metabolite associations with RILI was found as CNN interpretation heatmaps revealed spectral regions consistent with these metabolites.

The effect of spatial resolution on deep learning classification of lung cancer histopathology.

Objective: The microscopic analysis of biopsied lung nodules represents the gold-standard for definitive diagnosis of lung cancer. Deep learning has achieved pathologist-level classification of non-small cell lung cancer histopathology images at high resolutions (0.5-2 µm/px), and recent studies have revealed tomography-histology relationships at lower spatial resolutions. Thus, we tested whether patterns for histological classification of lung cancer could be detected at spatial resolutions such as those offered by ultra-high-resolution CT. Methods: We investigated the performance of a deep convolutional neural network (inception-v3) to classify lung histopathology images at lower spatial resolutions than that of typical pathology. Models were trained on 2167 histopathology slides from The Cancer Genome Atlas to differentiate between lung cancer tissues (adenocarcinoma (LUAD) and squamous-cell carcinoma (LUSC)), and normal dense tissue. Slides were accessed at 2.5 × magnification (4 µm/px) and reduced resolutions of 8, 16, 32, 64, and 128 µm/px were simulated by applying digital low-pass filters. Results: The classifier achieved area under the curve ≥0.95 for all classes at spatial resolutions of 4-16 µm/px, and area under the curve ≥0.95 for differentiating normal tissue from the two cancer types at 128 µm/px. Conclusions: Features for tissue classification by deep learning exist at spatial resolutions below what is typically viewed by pathologists. Advances in knowledge: We demonstrated that a deep convolutional network could differentiate normal and cancerous lung tissue at spatial resolutions as low as 128 µm/px and LUAD, LUSC, and normal tissue as low as 16 µm/px. Our data, and results of tomography-histology studies, indicate that these patterns should also be detectable within tomographic data at these resolutions.

Radiomic analysis for early differentiation of lung cancer recurrence from fibrosis in patients treated with lung stereotactic ablative radiotherapy.

Objective. The development of radiation-induced fibrosis after stereotactic ablative radiotherapy (SABR) can obscure follow-up images and delay detection of a local recurrence in early-stage lung cancer patients. The objective of this study was to develop a radiomics model for computer-assisted detection of local recurrence and fibrosis for an earlier timepoint (<1 year) after the SABR treatment.Approach. This retrospective clinical study included CT images (n= 107) of 66 patients treated with SABR. A z-score normalization technique was used for radiomic feature standardization across scanner protocols. The training set for the radiomics model consisted of CT images (66 patients; 22 recurrences and 44 fibrosis) obtained at 24 months (median) follow-up. The test set included CT-images of 41 patients acquired at 5-12 months follow-up. Combinations of four widely used machine learning techniques (support vector machines, gradient boosting, random forests (RF), and logistic regression) and feature selection methods (Relief feature scoring, maximum relevance minimum redundancy, mutual information maximization, forward feature selection, and LASSO) were investigated. Pyradiomics was used to extract 106 radiomic features from the CT-images for feature selection and classification.Main results. An RF + LASSO model scored the highest in terms of AUC (0.87) and obtained a sensitivity of 75% and a specificity of 88% in identifying a local recurrence in the test set. In the training set, 86% accuracy was achieved using five-fold cross-validation. Delong's test indicated that AUC achieved by the RF+LASSO is significantly better than 11 other machine learning models presented here. The top three radiomic features: interquartile range (first order), Cluster Prominence (GLCM), and Autocorrelation (GLCM), were revealed as differentiating a recurrence from fibrosis with this model.Significance. The radiomics model selected, out of multiple machine learning and feature selection algorithms, was able to differentiate a recurrence from fibrosis in earlier follow-up CT-images with a high specificity rate and satisfactory sensitivity performance.

Raman microspectroscopy and machine learning for use in identifying radiation-induced lung toxicity.

OBJECTIVE: In this work, we explore and develop a method that uses Raman spectroscopy to measure and differentiate radiation induced toxicity in murine lungs with the goal of setting the foundation for a predictive disease model. METHODS: Analysis of Raman tissue data is achieved through a combination of techniques. We first distinguish between tissue measurements and air pockets in the lung by using group and basis restricted non-negative matrix factorization. We then analyze the tissue spectra using sparse multinomial logistic regression to discriminate between fibrotic gradings. Model validation is achieved by splitting the data into a training set containing 70% of the data and a test set with the remaining 30%; classification accuracy is used as the performance metric. We also explore several other potential classification tasks wherein the response considered is the grade of pneumonitis and fibrosis sickness. RESULTS: A classification accuracy of 91.6% is achieved on the test set of fibrotic gradings, illustrating the ability of Raman measurements to detect differing levels of fibrotic disease among the murine lungs. It is also shown via further modeling that coarser consideration of fibrotic grading via binning (ie. 'Low', 'Medium', 'High') does not degrade performance. Finally, we consider preliminary models for pneumonitis discrimination using the same methodologies.

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.

Human antigen R promotes lung fibroblast differentiation to myofibroblasts and increases extracellular matrix production.

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive scarring caused by excessive extracellular matrix (ECM) deposition and activation of α-SMA-expressing myofibroblasts. Human antigen R (HuR) is an RNA binding protein that promotes protein translation. Upon translocation from the nucleus to the cytoplasm, HuR functions to stabilize messenger RNA (mRNA) to increase protein levels. However, the role of HuR in promoting ECM production, myofibroblast differentiation, and lung fibrosis is unknown. Human lung fibroblasts (HLFs) treated with transforming growth factor ß1 (TGF-ß1) showed a significant increase in translocation of HuR from the nucleus to the cytoplasm. TGF-ß-treated HLFs that were transfected with HuR small interfering RNA had a significant reduction in α-SMA protein as well as the ECM proteins COL1A1, COL3A, and FN1. HuR was also bound to mRNA for ACTA2, COL1A1, COL3A1, and FN. HuR knockdown affected the mRNA stability of ACTA2 but not that of the ECM genes COL1A1, COL3A1, or FN. In mouse models of pulmonary fibrosis, there was higher cytoplasmic HuR in lung structural cells compared to control mice. In human IPF lungs, there was also more cytoplasmic HuR. This study is the first to show that HuR in lung fibroblasts controls their differentiation to myofibroblasts and consequent ECM production. Further research on HuR could assist in establishing the basis for the development of new target therapy for fibrotic diseases, such as IPF.

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.

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.

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.

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.

Acute adaptive immune response correlates with late radiation-induced pulmonary fibrosis in mice.

BACKGROUND: The lung response to radiation exposure can involve an immediate or early reaction to the radiation challenge, including cell death and an initial immune reaction, and can be followed by a tissue injury response, of pneumonitis or fibrosis, to this acute reaction. Herein, we aimed to determine whether markers of the initial immune response, measured within days of radiation exposure, are correlated with the lung tissue injury responses occurring weeks later. METHODS: Inbred strains of mice known to be susceptible (KK/HIJ, C57BL/6J, 129S1/SvImJ) or resistant (C3H/HeJ, A/J, AKR/J) to radiation-induced pulmonary fibrosis and to vary in time to onset of respiratory distress post thoracic irradiation (from 10-23 weeks) were studied. Mice were untreated (controls) or received 18 Gy whole thorax irradiation and were euthanized at 6 h, 1d or 7 d after radiation treatment. Pulmonary CD4+ lymphocytes, bronchoalveolar cell profile & cytokine level, and serum cytokine levels were assayed. RESULTS: Thoracic irradiation and inbred strain background significantly affected the numbers of CD4+ cells in the lungs and the bronchoalveolar lavage cell differential of exposed mice. At the 7 day timepoint greater numbers of pulmonary Th1 and Th17 lymphocytes and reduced lavage interleukin17 and interferonγ levels were significant predictors of late stage fibrosis. Lavage levels of interleukin-10, measured at the 7 day timepoint, were inversely correlated with fibrosis score (R=-0.80, p=0.05), while serum levels of interleukin-17 in control mice significantly correlated with post irradiation survival time (R=0.81, p=0.04). Lavage macrophage, lymphocyte or neutrophil counts were not significantly correlated with either of fibrosis score or time to respiratory distress in the six mouse strains. CONCLUSION: Specific cytokine and lymphocyte levels, but not strain dependent lavage cell profiles, were predictive of later radiation-induced lung injury in this panel of inbred strains.

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.

DNA damage at respiratory distress, but not acute time-points, correlates with tissue fibrosis following thoracic radiation exposure in mice.

PURPOSE: Radiation exposure can result in DNA damage but whether the extent of DNA damage correlates with the radiation-induced tissue injury in the lung is not known. We aimed to determine whether numbers of γH2AX foci, representing histone H2AX phosphorylation a marker of DNA damage, measured within days of radiation exposure, correlated with known later lung injury responses in eight inbred mouse strains. MATERIALS AND METHODS: Mice received 18 Gy pulmonary irradiation and numbers of γH2AX positive nuclei in the lung were immunohistochemically determined. RESULTS: Numbers of γH2AX foci, assessed up to seven days post irradiation did not correlate with pulmonary fibrosis. γH2AX counts from mice in respiratory distress, however, significantly correlated with fibrosis and lungs from mice treated with a fibrosis-reducing antagonist had fewer γH2AX foci. CONCLUSIONS: Acute response measures of pulmonary DNA damage did not predict for pathology, but levels of this marker in distressed mice were correlative of fibrosis.

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.

NKT deficient mice are not spared lung disease after exposure to thoracic radiotherapy.

The specific pathways through which radiation produces the lung injuries of pneumonitis (alveolitis) and fibrosis are unknown but may involve an altered immune response. In this study, we investigated the hypothesis that the radiation-induced lung phenotype of Ja18(-/-) mice [which lack invariant natural killer T (iNKT) cells] is altered relative to that of C57BL/6J genetic background strain. After 18 Gy whole-thorax irradiation male C57BL/6J mice succumbed to respiratory distress at 28-30 weeks postirradiation and although confirmed by flow cytometric analysis to be deficient in iNKT cells, the postirradiation survival of Ja18(-/-) mice was not significantly different from that of C57BL/6J mice (P = 0.87). Histologically, the lungs of both C57BL/6J and Ja18(-/-) mice developed fibrosing alveolitis over a similar time course with the same severity (P = 0.15). Analysis of the bronchoalveolar lavage revealed that the C57BL/6J mice and female Ja18(-/-) mice succumbed to respiratory distress with neutrophil numbers exceeding those of the Ja18(-/-) male mice and untreated control mice. In conclusion, the radiation-induced lung disease of Ja18(-/-) mice did not significantly differ from that of C57BL/6J mice.

Airway hyperresponsiveness in FVB/N delta F508 cystic fibrosis transmembrane conductance regulator mice.

BACKGROUND: Airway hyperresponsiveness is a feature of clinical CF lung disease. In this study, we investigated whether the FVB/N ΔF508 CFTR mouse model has altered airway mechanics. METHODS: Mechanics were measured in 12-14week old FVB/N Cftr(tm1Eur) (ΔF508) mice and wildtype littermates using the FlexiVent small animal ventilator. Lung disease was assayed by immunohistochemistry, histology and bronchoalveolar lavage analysis. RESULTS: Cftr(tm1Eur) mice presented with increased airway resistance, compared to wildtype littermates, in response to methacholine challenge. No differences in bronchoalveolar cell number or differential, or in tissue lymphocyte, goblet cell or smooth muscle actin levels were evident in mice grouped by Cftr genotype. The bronchoalveolar lavage of Cftr(tm1Eur) mice included significantly increased levels of interleukin 12(p40) and CXCL1 compared to controls. CONCLUSION: We conclude that the pulmonary phenotype of Cftr(tm1Eur) mice includes airway hyperresponsiveness in the absence of overt lung inflammation or airway remodeling.

MicroRNA profiling implicates the insulin-like growth factor pathway in bleomycin-induced pulmonary fibrosis in mice.

BACKGROUND: Idiopathic pulmonary fibrosis is a disease characterized by alveolar epithelial cell injury, inflammatory cell infiltration and deposition of extracellular matrix in lung tissue. As mouse models of bleomycin-induced pulmonary fibrosis display many of the same phenotypes observed in patients with idiopathic pulmonary fibrosis, they have been used to study various aspects of the disease, including altered expression of microRNAs. RESULTS: In this work, microRNA expression profiling of the lungs from treated C57BL/6J mice, relative to that of untreated controls, was undertaken to determine which alterations in microRNAs could in part regulate the fibrosis phenotype induced by bleomycin delivered through mini-osmotic pumps. We identified 11 microRNAs, including miR-21 and miR-34a, to be significantly differentially expressed (P < 0.01) in lungs of bleomycin treated mice and confirmed these data with real time PCR measurements. In situ hybridization of both miR-21 and miR-34a indicated that they were expressed in alveolar macrophages. Using a previously reported gene expression profile, we identified 195 genes to be both predicted targets of the 11 microRNAs and of altered expression in bleomycin-induced lung disease of C57BL/6J mice. Pathway analysis with these 195 genes indicated that altered microRNA expression may be associated with hepatocyte growth factor signaling, cholecystokinin/gastrin-mediated signaling, and insulin-like growth factor (IGF-1) signaling, among others, in fibrotic lung disease. The relevance of the IGF-1 pathway in this model was then demonstrated by showing lung tissue of bleomycin treated C57BL/6J mice had increased expression of Igf1 and that increased numbers of Igf-1 positive cells, predominantly in macrophages, were detected in the lungs. CONCLUSIONS: We conclude that altered microRNA expression in macrophages is a feature which putatively influences the insulin-like growth factor signaling component of bleomycin-induced 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.

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.

CXC receptor 1 and 2 and neutrophil elastase inhibitors alter radiation-induced lung disease in the mouse.

PURPOSE: We previously reported increased numbers of neutrophils to be associated with the development of the radiation-induced lung responses of alveolitis (pneumonitis) and fibrosis in mice. In the present study we investigated whether CXC receptor 1 and 2 antagonism with DF2156A, a small molecule inhibitor of neutrophil chemotaxis, or the neutrophil elastase inhibitor sivelestat decreases the lung response to irradiation. METHODS AND MATERIALS: KK/HIJ mice received 14 Gy whole-thorax irradiation, and a subset of them received drug treatment 3 times per week from the day of irradiation until they were killed because of respiratory distress symptoms. RESULTS: Irradiated mice receiving sivelestat survived 18% longer than did mice receiving radiation alone (73 vs 60 days for female mice, 91 vs 79 days for male mice), whereas postirradiation survival times did not differ between the group of mice receiving DF2156A and the radiation-only group. The numbers of neutrophils in lung tissue and in bronchoalveolar lavage fluid did not differ among groups of irradiated mice, but they significantly exceeded the levels in unirradiated control mice. The extent of alveolitis, assessed histologically, did not differ between irradiated mice treated with either drug and those receiving radiation alone, when assessed at the end of the experiment, but it was significantly reduced, as were the neutrophil measures, in sivelestat-treated mice at the common kill time of 60 days after irradiation. Mice treated with radiation and DF2156A developed significantly less fibrosis than did mice receiving radiation alone, and this difference was associated with decreased expression of interleukin-13 in lung tissue. CONCLUSIONS: We conclude that neutrophil elastase inhibition affects alveolitis and prolongs survival, whereas CXCR1/2 antagonism reduces radiation-induced fibrotic lung disease in mice without affecting the onset of distress.