Thromboxane-Prostanoid Receptor Signaling Drives Persistent Fibroblast Activation in Pulmonary Fibrosis.

Rationale: Although persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF), mechanisms regulating persistent fibroblast activation in the lungs have not been fully elucidated. Objectives: On the basis of our observation that lung fibroblasts express TBXA2R (thromboxane-prostanoid receptor) during fibrosis, we investigated the role of TBXA2R signaling in fibrotic remodeling. Methods: We identified TBXA2R expression in lungs of patients with IPF and mice and studied primary mouse and human lung fibroblasts to determine the impact of TBXA2R signaling on fibroblast activation. We used TBXA2R-deficient mice and small-molecule inhibitors to investigate TBXA2R signaling in preclinical lung fibrosis models. Measurements and Main Results: TBXA2R expression was upregulated in fibroblasts in the lungs of patients with IPF and in mouse lungs during experimental lung fibrosis. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, thereby suggesting that an alternative ligand activates profibrotic TBXA2R signaling. In contrast to thromboxane, F2-isoprostanes, which are nonenzymatic products of arachidonic acid induced by reactive oxygen species, were persistently elevated during fibrosis. F2-isoprostanes induced TBXA2R signaling in fibroblasts and mediated a myofibroblast activation profile due, at least in part, to potentiation of TGF-ß (transforming growth factor-ß) signaling. In vivo treatment with the TBXA2R antagonist ifetroban reduced profibrotic signaling in the lungs, protected mice from lung fibrosis in three preclinical models (bleomycin, Hermansky-Pudlak mice, and radiation-induced fibrosis), and markedly enhanced fibrotic resolution after bleomycin treatment. Conclusions: TBXA2R links oxidative stress to fibroblast activation during lung fibrosis. TBXA2R antagonists could have utility in treating pulmonary fibrosis.

STAT1 Represses Cytokine-Producing Group 2 and Group 3 Innate Lymphoid Cells during Viral Infection.

The appropriate orchestration of different arms of the immune response is critical during viral infection to promote efficient viral clearance while limiting immunopathology. However, the signals and mechanisms that guide this coordination are not fully understood. IFNs are produced at high levels during viral infection and have convergent signaling through STAT1. We hypothesized that STAT1 signaling during viral infection regulates the balance of innate lymphoid cells (ILC), a diverse class of lymphocytes that are poised to respond to environmental insults including viral infections with the potential for both antiviral or immunopathologic functions. During infection with respiratory syncytial virus (RSV), STAT1-deficient mice had reduced numbers of antiviral IFN-γ+ ILC1 and increased numbers of immunopathologic IL-5+ and IL-13+ ILC2 and IL-17A+ ILC3 compared with RSV-infected wild-type mice. Using bone marrow chimeric mice, we found that both ILC-intrinsic and ILC-extrinsic factors were responsible for this ILC dysregulation during viral infection in STAT1-deficient mice. Regarding ILC-extrinsic mechanisms, we found that STAT1-deficient mice had significantly increased expression of IL-33 and IL-23, cytokines that promote ILC2 and ILC3, respectively, compared with wild-type mice during RSV infection. Moreover, disruption of IL-33 or IL-23 signaling attenuated cytokine-producing ILC2 and ILC3 responses in STAT1-deficient mice during RSV infection. Collectively, these data demonstrate that STAT1 is a key orchestrator of cytokine-producing ILC responses during viral infection via ILC-extrinsic regulation of IL-33 and IL-23.

p52 Overexpression Increases Epithelial Apoptosis, Enhances Lung Injury, and Reduces Survival after Lipopolysaccharide Treatment.

Although numerous studies have demonstrated a critical role for canonical NF-κB signaling in inflammation and disease, the function of the noncanonical NF-κB pathway remains ill-defined. In lung tissue from patients with acute respiratory distress syndrome, we identified increased expression of the noncanonical pathway component p100/p52. To investigate the effects of p52 expression in vivo, we generated a novel transgenic mouse model with inducible expression of p52 in Clara cell secretory protein-expressing airway epithelial cells. Although p52 overexpression alone did not cause significant inflammation, p52 overexpression caused increased lung inflammation, injury, and mortality following intratracheal delivery of Escherichia coli LPS. No differences in cytokine/chemokine expression were measured between p52-overexpressing mice and controls, but increased apoptosis of Clara cell secretory protein-positive airway epithelial cells was observed in transgenic mice after LPS stimulation. In vitro studies in lung epithelial cells showed that p52 overexpression reduced cell survival and increased the expression of several proapoptotic genes during cellular stress. Collectively, these studies demonstrate a novel role for p52 in cell survival/apoptosis of airway epithelial cells and implicate noncanonical NF-κB signaling in the pathogenesis of acute respiratory distress syndrome.

Allergic airway inflammation decreases lung bacterial burden following acute Klebsiella pneumoniae infection in a neutrophil- and CCL8-dependent manner.

The Th17 cytokines interleukin-17A (IL-17A), IL-17F, and IL-22 are critical for the lung immune response to a variety of bacterial pathogens, including Klebsiella pneumoniae. Th2 cytokine expression in the airways is a characteristic feature of asthma and allergic airway inflammation. The Th2 cytokines IL-4 and IL-13 diminish ex vivo and in vivo IL-17A protein expression by Th17 cells. To determine the effect of IL-4 and IL-13 on IL-17-dependent lung immune responses to acute bacterial infection, we developed a combined model in which allergic airway inflammation and lung IL-4 and IL-13 expression were induced by ovalbumin sensitization and challenge prior to acute lung infection with K. pneumoniae. We hypothesized that preexisting allergic airway inflammation decreases lung IL-17A expression and airway neutrophil recruitment in response to acute K. pneumoniae infection and thereby increases the lung K. pneumoniae burden. As hypothesized, we found that allergic airway inflammation decreased the number of K. pneumoniae-induced airway neutrophils and lung IL-17A, IL-17F, and IL-22 expression. Despite the marked reduction in postinfection airway neutrophilia and lung expression of Th17 cytokines, allergic airway inflammation significantly decreased the lung K. pneumoniae burden and postinfection mortality. We showed that the decreased lung K. pneumoniae burden was independent of IL-4, IL-5, and IL-17A and partially dependent on IL-13 and STAT6. Additionally, we demonstrated that the decreased lung K. pneumoniae burden associated with allergic airway inflammation was both neutrophil and CCL8 dependent. These findings suggest a novel role for CCL8 in lung antibacterial immunity against K. pneumoniae and suggest new mechanisms of orchestrating lung antibacterial immunity.

Identification of myeloid cell subsets in murine lungs using flow cytometry.

Although the antibody-based recognition of cell-surface markers has been widely used for the identification of immune cells, overlap in the expression of markers by different cell types and the inconsistent use of antibody panels have resulted in a lack of clearly defined signatures for myeloid cell subsets. We developed a 10-fluorochrome flow cytometry panel for the identification and quantitation of myeloid cells in the lungs, including pulmonary monocytes, myeloid dendritic cells, alveolar and interstitial macrophages, and neutrophils. After the initial sorting of viable CD45(+) leukocytes, we detected three leukocyte subpopulations based on CD68 expression: CD68(-), CD68(low), and CD68(hi). Further characterization of the CD68(hi) population revealed CD45(+)/CD68(hi)/F4/80(+)/CD11b(-)/CD11c(+)/Gr1(-) alveolar macrophages and CD45(+)/CD68(hi)/F4/80(-)/CD11c(+)/Gr1(-)/CD103(+)/major histocompatibility complex (MHC) class II(hi) dendritic cells. The CD68(low) population contained primarily CD45(+)/CD68(low)/F4/80(+)/CD11b(+)/CD11c(+)/Gr1(-)/CD14(low) interstitial macrophages and CD45(+)/CD68(low)/F4/80(+)/CD11b(+)/CD11c(-)/Gr1(low)/CD14(hi) monocytes, whereas the CD68(-) population contained neutrophils (CD45(+)/CD68(-)/F4/80(-)/CD11b(+)/Gr1(hi)). The validity of cellular signatures was confirmed by a morphological analysis of FACS-sorted cells, functional studies, and the depletion of specific macrophage subpopulations using liposomal clodronate. We believe our approach provides an accurate and reproducible method for the isolation, quantification, and characterization of myeloid cell subsets in the lungs, which may be useful for studying the roles of myeloid cells during various pathological processes.

IL-17A induces signal transducers and activators of transcription-6-independent airway mucous cell metaplasia.

Mucous cell metaplasia is a hallmark of asthma, and may be mediated by signal transducers and activators of transcription (STAT)-6 signaling. IL-17A is increased in the bronchoalveolar lavage fluid of patients with severe asthma, and IL-17A also increases mucus production in airway epithelial cells. Asthma therapeutics are being developed that inhibit STAT6 signaling, but the role of IL-17A in inducing mucus production in the absence of STAT6 remains unknown. We hypothesized that IL-17A induces mucous cell metaplasia independent of STAT6, and we tested this hypothesis in two murine models in which increased IL-17A protein expression is evident. In the first model, ovalbumin (OVA)-specific D011.10 Th17 cells were adoptively transferred into wild-type (WT) or STAT6 knockout (KO) mice, and the mice were challenged with OVA or PBS. WT-OVA and STAT6 KO-OVA mice demonstrated increased airway IL-17A and IL-13 protein expression and mucous cell metaplasia, compared with WT-PBS or STAT6 KO-PBS mice. In the second model, WT, STAT1 KO, STAT1/STAT6 double KO (DKO), or STAT1/STAT6/IL-17 receptor A (RA) triple KO (TKO) mice were challenged with respiratory syncytial virus (RSV) or mock viral preparation, and the mucous cells were assessed. STAT1 KO-RSV mice demonstrated increased airway mucous cell metaplasia compared with WT-RSV mice. STAT1 KO-RSV and STAT1/STAT6 DKO-RSV mice also demonstrated increased mucous cell metaplasia, compared with STAT1/STAT6/IL17RA TKO-RSV mice. We also treated primary murine tracheal epithelial cells (mTECs) from WT and STAT6 KO mice. STAT6 KO mTECs showed increased periodic acid-Schiff staining with IL-17A but not with IL-13. Thus, asthma therapies targeting STAT6 may increase IL-17A protein expression, without preventing IL-17A-induced mucus production.

Deficiency of gp91phox inhibits allergic airway inflammation.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a multienzyme complex, is the major source for production of reactive oxygen species (ROS). ROS are increased in allergic diseases, such as asthma, but the role of ROS in disease pathogenesis remains uncertain. We hypothesized that mice unable to generate ROS via the NADPH oxidase pathway would have decreased allergic airway inflammation. To test this hypothesis, we studied gp91phox(-/-) mice in a model of allergic airway inflammation after sensitization and challenge with ovalbumin. Serum, bronchoalveolar lavage fluid, and lungs were then examined for evidence of allergic inflammation. We found that mice lacking a functional NADPH oxidase complex had significantly decreased ROS production and allergic airway inflammation, compared with wild-type (WT) control animals. To determine the mechanism by which allergic inflammation was inhibited by gp91phox deficiency, we cultured bone marrow-derived dendritic cells from WT and gp91phox(-/-) mice and activated them with LPS. IL-12 expression was significantly increased in the gp91phox(-/-) bone marrow-derived dendritic cells, suggesting that the cytokine profile produced in the absence of gp91phox enhanced the conditions leading to T helper (Th) type 1 differentiation, while inhibiting Th2 polarization. Splenocytes from sensitized gp91phox(-/-) animals produced significantly less IL-13 in response to ovalbumin challenge in vitro compared with splenocytes from sensitized WT mice, suggesting that NADPH oxidase promotes allergic sensitization. In contrast, inflammatory cytokines produced by T cells cultured from WT and gp91phox(-/-) mice under Th0, Th1, Th2, and Th17 conditions were not significantly different. This study demonstrates the importance of NADPH oxidase activity and ROS production in a murine model of asthma.

A critical role for macrophages in promotion of urethane-induced lung carcinogenesis.

Macrophages have established roles in tumor growth and metastasis, but information about their role in lung tumor promotion is limited. To assess the role of macrophages in lung tumorigenesis, we developed a method of minimally invasive, long-term macrophage depletion by repetitive intratracheal instillation of liposomal clodronate. Compared with controls treated with repetitive doses of PBS-containing liposomes, long-term macrophage depletion resulted in a marked reduction in tumor number and size at 4 mo after a single i.p. injection of the carcinogen urethane. After urethane treatment, lung macrophages developed increased M1 macrophage marker expression during the first 2-3 wk, followed by increased M2 marker expression by week 6. Using a strategy to reduce alveolar macrophages during tumor initiation and early promotion stages (weeks 1-2) or during late promotion and progression stages (weeks 4-16), we found significantly fewer and smaller lung tumors in both groups compared with controls. Late-stage macrophage depletion reduced VEGF expression and impaired vascular growth in tumors. In contrast, early-stage depletion of alveolar macrophages impaired urethane-induced NF-κB activation in the lungs and reduced the development of premalignant atypical adenomatous hyperplasia lesions at 6 wk after urethane injection. Together, these studies elucidate an important role for macrophages in lung tumor promotion and indicate that these cells have distinct roles during different stages of lung carcinogenesis.

Epithelial Yap/Taz are required for functional alveolar regeneration following acute lung injury.

A hallmark of idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases is dysregulated repair of the alveolar epithelium. The Hippo pathway effector transcription factors YAP and TAZ are implicated as essential for type 1 and type 2 alveolar epithelial cell (AT1 and AT2) differentiation in the developing lung, yet aberrant activation of YAP/TAZ is a prominent feature of the dysregulated alveolar epithelium in IPF. In these studies, we sought to define the functional role of YAP/TAZ activity during alveolar regeneration. We demonstrated that Yap and Taz were normally activated in AT2 cells shortly after injury, and deletion of Yap/Taz in AT2 cells led to pathologic alveolar remodeling, failure of AT2-to-AT1 cell differentiation, increased collagen deposition, exaggerated neutrophilic inflammation, and increased mortality following injury induced by a single dose of bleomycin. Loss of Yap/Taz activity prior to an LPS injury prevented AT1 cell regeneration, led to intraalveolar collagen deposition, and resulted in persistent innate inflammation. These findings establish that AT2 cell Yap/Taz activity is essential for functional alveolar epithelial repair and prevention of fibrotic remodeling.

Opposing effects of bortezomib-induced nuclear factor-κB inhibition on chemical lung carcinogenesis.

Since recent evidence indicates a requirement for epithelial nuclear factor (NF)-κB signaling in lung tumorigenesis, we investigated the impact of the NF-κB inhibitor bortezomib on lung tumor promotion and growth. We used an experimental model in which wild-type mice or mice expressing an NF-κB reporter received intraperitoneal urethane (1 g/kg) followed by twice weekly bortezomib (1 mg/kg) during distinct periods of tumor initiation/progression. Mice were serially assessed for lung NF-κB activation, inflammation and carcinogenesis. Short-term proteasome inhibition with bortezomib did not impact tumor formation but retarded the growth of established lung tumors in mice via effects on cell proliferation. In contrast, long-term treatment with bortezomib resulted in significantly increased lung tumor number and size. This tumor-promoting effect of prolonged bortezomib treatment was associated with perpetuation of urethane-induced inflammation and chronic upregulation of interleukin-1ß and proinflammatory C-X-C motif chemokine ligands (CXCL) 1 and 2 in the lungs. In addition to airway epithelium, bortezomib inhibited NF-κB in pulmonary macrophages in vivo, presenting a possible mechanism of tumor amplification. In this regard, RAW264.7 macrophages exposed to bortezomib showed increased expression of interleukin-1ß, CXCL1 and CXCL2. In conclusion, although short-term bortezomib may exert some beneficial effects, prolonged NF-κB inhibition accelerates chemical lung carcinogenesis by perpetuating carcinogen-induced inflammation. Inhibition of NF-κB in pulmonary macrophages appears to play an important role in this adverse process.

Differential pathogenesis of respiratory syncytial virus clinical isolates in BALB/c mice.

Airway mucus is a hallmark of respiratory syncytial virus (RSV) lower respiratory tract illness. Laboratory RSV strains differentially induce airway mucus production in mice. Here, we tested the hypothesis that RSV strains differ in pathogenesis by screening six low-passage RSV clinical isolates for mucogenicity and virulence in BALB/cJ mice. The RSV clinical isolates induced variable disease severity, lung interleukin-13 (IL-13) levels, and gob-5 levels in BALB/cJ mice. We chose two of these clinical isolates for further study. Infection of BALB/cJ mice with RSV A2001/2-20 (2-20) resulted in greater disease severity, higher lung IL-13 levels, and higher lung gob-5 levels than infection with RSV strains A2, line 19, Long, and A2001/3-12 (3-12). Like the line 19 RSV strain, the 2-20 clinical isolate induced airway mucin expression in BALB/cJ mice. The 2-20 and 3-12 RSV clinical isolates had higher lung viral loads than laboratory RSV strains at 1 day postinfection (p.i.). This increased viral load correlated with higher viral antigen levels in the bronchiolar epithelium and greater histopathologic changes at 1 day p.i. The A2 RSV strain had the highest peak viral load at day 4 p.i. RSV 2-20 infection caused epithelial desquamation, bronchiolitis, airway hyperresponsiveness, and increased breathing effort in BALB/cJ mice. We found that RSV clinical isolates induce variable pathogenesis in mice, and we established a mouse model of clinical isolate strain-dependent RSV pathogenesis that recapitulates key features of RSV disease.

TGFß signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment.

The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-ß (TGFß) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFß receptor 2 (TGFßR2) in lung epithelium were generated and crossed to cell fate reporter mice that express ß-galactosidase (ß-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFßR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFßR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4(+)/ß-gal(+)) fibroblasts. Attenuation of TGFß signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine.

NF-kappaB activation within macrophages leads to an anti-tumor phenotype in a mammary tumor lung metastasis model.

INTRODUCTION: Metastasis from primary tumor to the lungs is a major cause of the mortality associated with breast cancer. Both immune and inflammatory responses impact whether circulating mammary tumor cells successfully colonize the lungs leading to established metastases. Nuclear factor -kappaB (NF-κB) transcription factors regulate both immune and inflammatory responses mediated in part by the activities of macrophages. Therefore, NF-κB activity specifically within macrophages may be a critical determinant of whether circulating tumor cells successfully colonize the lungs. METHODS: To investigate NF-κB signaling within macrophages during metastasis, we developed novel inducible transgenic models which target expression of the reverse tetracycline transactivator (rtTA) to macrophages using the cfms promoter in combination with inducible transgenics that express either an activator (cIKK2) or an inhibitor (IκBα-DN). Doxycyline treatment led to activation or inhibition of NF-κB within macrophages. We used a tail vein metastasis model with mammary tumor cell lines established from MMTV-Polyoma Middle T-Antigen-derived tumors to investigate the effects of modulating NF-κB in macrophages during different temporal windows of the metastatic process. RESULTS: We found that activation of NF-κB in macrophages during seeding leads to a reduction in lung metastases. The mechanism involved expression of inflammatory cytokines and reactive oxygen species, leading to apoptosis of tumor cells and preventing seeding in the lung. Activation of NF-κB within macrophages after the seeding phase has no significant impact on establishment of metastases. CONCLUSIONS: Our results have identified a brief, defined window in which activation of NF-κB has significant anti-metastatic effects and inhibition of NF-κB results in a worse outcome.

Host-derived interleukin-5 promotes adenocarcinoma-induced malignant pleural effusion.

RATIONALE: IL-5 is a T helper 2 cytokine important in the trafficking and survival of eosinophils. Because eosinophils can be found in malignant pleural effusions (MPE) from mice and humans, we asked whether IL-5 is involved in the pathogenesis of MPE. OBJECTIVES: To determine the role of IL-5 in MPE formation. METHODS: The effects of IL-5 on experimental MPE induced in C57BL/6 mice by intrapleural injection of syngeneic lung (Lewis lung cancer [LLC]) or colon (MC38) adenocarcinoma cells were determined using wild-type (il5(+/+)) and IL-5-deficient (il5⁻(/)⁻) mice, exogenous administration of recombinant mouse (rm) IL-5, and in vivo antibody-mediated neutralization of endogenous IL-5. The direct effects of rmIL-5 on LLC cell proliferation and gene expression in vitro were determined by substrate reduction and microarray. MEASUREMENTS AND MAIN RESULTS: Eosinophils and IL-5 were present in human and mouse MPE, but the cytokine was not detected in mouse (LLC) or human (A549) lung and mouse colon (MC38) adenocarcinoma-conditioned medium, suggesting production by host cells in MPE. Compared with il5(+/+) mice, il5⁻(/)⁻ mice showed markedly diminished MPE formation in response to both LLC and MC38 cells. Exogenous IL-5 promoted MPE formation in il5(+/+) and il5⁻(/)⁻ mice, whereas anti-IL-5 antibody treatment limited experimental MPE in il5(+/+) mice. Exogenous IL-5 had no effects on LLC cell proliferation and gene expression; however, IL-5 was found to be responsible for recruitment of eosinophils and tumor-promoting myeloid suppressor cells to MPE in vivo. CONCLUSIONS: Host-derived IL-5 promotes experimental MPE and may be involved in the pathogenesis of human MPE.

Specific effects of bortezomib against experimental malignant pleural effusion: a preclinical study.

BACKGROUND: We have previously shown that nuclear factor (NF)-kappaB activation of mouse Lewis lung carcinoma (LLC) specifically promotes the induction of malignant pleural effusions (MPE) by these cells. In the present studies we hypothesized that treatment of immunocompetent mice with bortezomib tailored to inhibit cancer cell NF-kappaB activation and not proliferation specifically inhibits MPE formation by LLC cells. RESULTS: Treatment of LLC cells with low concentrations of bortezomib (100 ng/ml) inhibited NF-kappaB activation and NF-kappaB-dependent transcription, but not cellular proliferation. Bortezomib treatment of immunocompetent C57BL/6 mice bearing LLC-induced subcutaneous tumors and MPEs significantly blocked tumor-specific NF-kappaB activation. However, bortezomib treatment did not impair subcutaneous LLC tumor growth, but was effective in limiting LLC-induced MPE. This specific effect was evidenced by significant reductions in effusion accumulation and the associated mortality and was observed with both preventive (beginning before MPE formation) and therapeutic (beginning after MPE establishment) bortezomib treatment. The favorable impact of bortezomib on MPE was associated with suppression of cardinal MPE-associated phenomena, such as inflammation, vascular hyperpermeability, and angiogenesis. In this regard, therapeutic bortezomib treatment had identical favorable results on MPE compared with preventive treatment, indicating that the drug specifically counteracts effusion formation. CONCLUSIONS: These studies indicate that proteasome inhibition tailored to block NF-kappaB activation of lung adenocarcinoma specifically targets the effusion-inducing phenotype of this tumor. Although the drug has limited activity against advanced solid lung cancer, it may prove beneficial for patients with MPE.

Contribution of epithelial-derived fibroblasts to bleomycin-induced lung fibrosis.

RATIONALE: Lung fibroblasts are key mediators of fibrosis resulting in accumulation of excessive interstitial collagen and extracellular matrix, but their origins are not well defined. OBJECTIVES: We aimed to elucidate the contribution of lung epithelium-derived fibroblasts via epithelial-mesenchymal transition (EMT) in the intratracheal bleomycin model. METHODS: Primary type II alveolar epithelial cells were cultured from Immortomice and exposed to transforming growth factor-beta(1) and epidermal growth factor. Cell fate reporter mice that permanently mark cells of lung epithelial lineage with beta-galactosidase were developed to study EMT, and bone marrow chimeras expressing green fluorescent protein under the control of the fibroblast-associated S100A4 promoter were generated to examine bone marrow-derived fibroblasts. Mice were given intratracheal bleomycin (0.08 unit). Immunostaining was performed for S100A4, beta-galactosidase, green fluorescent protein, and alpha-smooth muscle actin. MEASUREMENTS AND MAIN RESULTS: In vitro, primary type II alveolar epithelial cells undergo phenotypic changes of EMT when exposed to transforming growth factor-beta(1) and epidermal growth factor with loss of prosurfactant protein C and E-cadherin and gain of S100A4 and type I procollagen. In vivo, using cell fate reporter mice, approximately one-third of S100A4-positive fibroblasts were derived from lung epithelium 2 weeks after bleomycin administration. From bone marrow chimera studies, one-fifth of S100A4-positive fibroblasts were derived from bone marrow at this same time point. Myofibroblasts rarely derived from EMT or bone marrow progenitors. CONCLUSIONS: Both EMT and bone marrow progenitors contribute to S100A4-positive fibroblasts in bleomycin-induced lung fibrosis. However, neither origin is a principal contributor to lung myofibroblasts.

Epithelial NF-kappaB activation promotes urethane-induced lung carcinogenesis.

Chronic inflammation is linked to carcinogenesis in several organ systems. In the lungs, NF-kappaB, a central effector of inflammatory responses, is frequently activated in non-small-cell lung cancer, but its role in tumor promotion has not been studied. Several lines of evidence indicate that ethyl carbamate (urethane)-induced lung tumor formation, a prototypical mouse model of multistage lung carcinogenesis, is potentiated by inflammation. We found that mouse strains susceptible to lung tumor formation (FVB, BALB/c) exhibited early NF-kappaB activation and inflammation in the lungs after urethane treatment. However, a resistant strain (C57B6) failed to activate NF-kappaB or induce lung inflammation. In FVB mice, we identified urethane-induced NF-kappaB activation in airway epithelium, as well as type II alveolar epithelial cells and macrophages. Using an inducible transgenic mouse model (FVB strain) to express a dominant inhibitor of NF-kappaB specifically in airway epithelial cells, we found that urethane-induced lung inflammation was blocked and tumor formation was reduced by >50%. Selective NF-kappaB inhibition resulted in increased apoptosis of airway epithelial cells at 2 weeks after urethane treatment in association with a marked reduction of Bcl-2 expression. These studies indicate that NF-kappaB signaling in airway epithelium is integral to tumorigenesis in the urethane model and identify the NF-kappaB pathway as a potential target for chemoprevention of lung cancer.

Perfusion and Inflation of the Mouse Lung for Tumor Histology.

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform necropsies rapidly and efficiently from studies without sacrificing the quality of the tissues procured. The goal of this protocol is to present a method to rapidly perfuse, inflate, and fix mouse lungs for downstream histological analysis. This method does not standardize lung inflation; thus, it does not require any special procedures or equipment and instead simply instills fixative directly through the trachea following perfusion through the heart. This allows for sufficient estimation of tumor size, histology, and scoring. This also allows for the collection of frozen tissue prior to lung tissue fixation. This method is limited in that it does not allow for later morphometric quantification of the lung; however, it is more than sufficient for lung tumor analysis from genetically engineered mouse models (GEMMs), syngeneic models, as well as xenograft tumor and metastasis studies.

Host nuclear factor-kappaB activation potentiates lung cancer metastasis.

Epidemiologic and experimental evidence suggests that a link exists between inflammation and cancer, although this relationship has only recently begun to be elucidated for lung cancer, the most frequently fatal human tumor. Nuclear factor-kappaB (NF-kappaB), a transcription factor that controls innate immune responses in the lungs, has been implicated as an important determinant of cancer cell proliferative and metastatic potential; however, its role in lung tumorigenesis is uncertain. Here, we specifically examine the role of NF-kappaB-induced airway inflammation in lung cancer metastasis using a model of intravenous injection of Lewis lung carcinoma cells into immunocompetent C57Bl/6 mice. Induction of lung inflammation by direct and specific NF-kappaB activation in airway epithelial cells potentiates lung adenocarcinoma metastasis. Moreover, we identify resident lung macrophages as crucial effectors of lung susceptibility to metastatic cancer growth. We conclude that NF-kappaB activity in host tissue is a significant factor in the development of lung metastasis.

Tumor necrosis factor-alpha promotes malignant pleural effusion.

Tumor necrosis factor (TNF)-alpha is present in the microenvironment of human tumors, including malignant pleural effusion (MPE). Although the cytokine is produced in the pleural cavity by both tumor and host cells, its effects on MPE formation are unknown. In these studies, we sought to determine the role of TNF-alpha in the pathogenesis of MPE and to assess the therapeutic effects of its neutralization in a preclinical model. For this, MPEs were generated in immunocompetent mice using intrapleural injection of mouse lung adenocarcinoma cells. The roles of tumor- and host-derived TNF-alpha were assessed using combined experimentation with TNF-alpha gene-deficient mice and in vivo TNF-alpha neutralization. To expand the scope of preclinical data, TNF-alpha and vascular endothelial growth factor (VEGF) expression were determined in human cancer cell lines and human MPE. In the MPE model, TNF-alpha of host and tumor origin was present. TNF-alpha neutralization significantly limited tumor dissemination, effusion formation, vascular hyperpermeability, TNF-alpha and VEGF expression, and angiogenesis, thereby improving survival. In contrast, these variables were not different between TNF-alpha gene-sufficient and TNF-alpha gene-deficient mice. In mouse cancer cells, TNF-alpha functioned via nuclear factor-kappaB- and neutral sphingomyelinase-dependent pathways to induce TNF-alpha and VEGF, respectively. These results were recapitulated in human cancer cells, and a correlation was detected between TNF-alpha and VEGF content of human MPE. We conclude that tumor-derived TNF-alpha is important in the development of MPE in mice, and provide preclinical evidence supporting the efficacy of TNF-alpha blockade against malignant pleural disease.