Taylor P. Sherrill

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.

Duration and Intensity of NF-κB Activity Determine the Severity of Endotoxin-Induced Acute Lung Injury

Activation of innate immunity in the lungs can lead to a self-limited inflammatory response or progress to severe lung injury. We investigated whether specific parameters of NF-κB pathway activation determine the outcome of acute lung inflammation using a novel line of transgenic reporter mice. Following a single i.p. injection of Escherichia coli LPS, transient NF-κB activation was identified in a variety of lung cell types, and neutrophilic inflammation resolved without substantial tissue injury. However, administration of LPS over 24 h by osmotic pump (LPS pump) implanted into the peritoneum resulted in sustained, widespread NF-κB activation and neutrophilic inflammation that culminated in lung injury at 48 h. To determine whether intervention in the NF-κB pathway could prevent progression to lung injury in the LPS pump model, we administered a specific IκB kinase inhibitor (BMS-345541) to down-regulate NF-κB activation following the onset of inflammation. Treatment with BMS-345541 beginning at 20 h after osmotic pump implantation reduced lung NF-κB activation, concentration of KC and MIP-2 in lung lavage, neutrophil influx, and lung edema measured at 48 h. Therefore, sustained NF-κB activation correlates with severity of lung injury, and interdiction in the NF-κB pathway is beneficial even after the onset of lung inflammation.

Airway Epithelium Controls Lung Inflammation and Injury through the NF-κB Pathway

Although airway epithelial cells provide important barrier and host defense functions, a crucial role for these cells in development of acute lung inflammation and injury has not been elucidated. We investigated whether NF-κB pathway signaling in airway epithelium could decisively impact inflammatory phenotypes in the lungs by using a tetracycline-inducible system to achieve selective NF-κB activation or inhibition in vivo. In transgenic mice that express a constitutively active form of IκB kinase 2 under control of the epithelial-specific CC10 promoter, treatment with doxycycline induced NF-κB activation with consequent production of a variety of proinflammatory cytokines, high-protein pulmonary edema, and neutrophilic lung inflammation. Continued treatment with doxycycline caused progressive lung injury and hypoxemia with a high mortality rate. In contrast, inducible expression of a dominant inhibitor of NF-κB in airway epithelium prevented lung inflammation and injury resulting from expression of constitutively active form of IκB kinase 2 or Escherichia coli LPS delivered directly to the airways or systemically via an osmotic pump implanted in the peritoneal cavity. Our findings indicate that the NF-κB pathway in airway epithelial cells is critical for generation of lung inflammation and injury in response to local and systemic stimuli; therefore, targeting inflammatory pathways in airway epithelium could prove to be an effective therapeutic strategy for inflammatory lung diseases.

Epithelial NF-κB activation promotes urethane-induced lung carcinogenesis

Chronic inflammation is linked to carcinogenesis in several organ systems. In the lungs, NF-κB, 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-κB activation and inflammation in the lungs after urethane treatment. However, a resistant strain (C57B6) failed to activate NF-κB or induce lung inflammation. In FVB mice, we identified urethane-induced NF-κB 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-κB specifically in airway epithelial cells, we found that urethane-induced lung inflammation was blocked and tumor formation was reduced by >50%. Selective NF-κB 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-κB signaling in airway epithelium is integral to tumorigenesis in the urethane model and identify the NF-κB pathway as a potential target for chemoprevention of lung cancer.

Differential Pathogenesis of Respiratory Syncytial Virus Clinical Isolates in BALB/c Mice

ABSTRACT 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.

Targeted Immunomodulation of the NF-κB Pathway in Airway Epithelium Impacts Host Defense against Pseudomonas aeruginosa

We investigated the impact of inflammatory signaling in airway epithelial cells on host defense against Pseudomonas aeruginosa, a major cause of nosocomial pneumonia. In mice, airway instillation of P. aeruginosa resulted in NF-κB activation in the lungs that was primarily localized to the bronchial epithelium at 4 h, but was present in a variety of cell types by 24 h. We modulated NF-κB activity in airway epithelium by intratracheal delivery of adenoviral vectors expressing RelA (AdRelA) or a dominant inhibitor of NF-κB before P. aeruginosa infection. Bacterial clearance was enhanced by up-regulation of NF-κB activity following AdRelA administration and was impaired by treatment with a dominant inhibitor of NF-κB. The TNF-α concentration in lung lavage was increased by AdRelA treatment and beneficial effects of NF-κB up-regulation were abrogated in TNF-α-deficient mice. In contrast, NF-κB inhibition reduced MIP-2 expression and neutrophil influx following P. aeruginosa infection. Therefore, inflammatory signaling through the NF-κB pathway in airway epithelial cells critically regulates the innate immune response to P. aeruginosa.

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.

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.

Candida albicans Septin Mutants Are Defective for Invasive Growth and Virulence

ABSTRACT Hyphal growth of Candida albicans is implicated as an important virulence factor for this opportunistic human pathogen. Septin proteins, a family of cytoskeletal elements that regulate membrane events and are important for proper morphogenesis of C. albicans, were examined for their role in tissue invasion and virulence in the mouse model of systemic infection. In vitro, septin mutants are only mildly defective for hyphal growth in liquid culture but display pronounced defects for invasive growth into agar. In vivo, the septin mutants were found to exhibit attenuated virulence. However, mice infected with the mutants displayed high fungal burdens in their kidneys without obvious symptoms of disease. Histological examination of infected kidneys revealed defects in organ invasion for the cdc10Δ and cdc11Δ deletion mutants, which displayed both reduced tissue penetration and noninvasive fungal masses. Thus, the septin proteins are necessary for invasive growth, which appears to be more important to the successful pathogenesis of C. albicans than hyphal growth alone.

Tumor Necrosis Factor-α Promotes Malignant Pleural Effusion

Tumor necrosis factor (TNF)-α 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-α 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-α were assessed using combined experimentation with TNF-α gene–deficient mice and in vivo TNF-α neutralization. To expand the scope of preclinical data, TNF-α and vascular endothelial growth factor (VEGF) expression were determined in human cancer cell lines and human MPE. In the MPE model, TNF-α of host and tumor origin was present. TNF-α neutralization significantly limited tumor dissemination, effusion formation, vascular hyperpermeability, TNF-α and VEGF expression, and angiogenesis, thereby improving survival. In contrast, these variables were not different between TNF-α gene–sufficient and TNF-α gene–deficient mice. In mouse cancer cells, TNF-α functioned via nuclear factor-κB– and neutral sphingomyelinase–dependent pathways to induce TNF-α and VEGF, respectively. These results were recapitulated in human cancer cells, and a correlation was detected between TNF-α and VEGF content of human MPE. We conclude that tumor-derived TNF-α is important in the development of MPE in mice, and provide preclinical evidence supporting the efficacy of TNF-α blockade against malignant pleural disease. [Cancer Res 2007;67(20):9825–34]