Frank B. McMahon

Repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis

Single-dose intratracheal bleomycin has been instrumental for understanding fibrotic lung remodeling, but fails to recapitulate several features of idiopathic pulmonary fibrosis (IPF). Since IPF is thought to result from recurrent alveolar injury, we aimed to develop a repetitive bleomycin model that results in lung fibrosis with key characteristics of human disease, including alveolar epithelial cell (AEC) hyperplasia. Wild-type and cell fate reporter mice expressing β-galactosidase in cells of lung epithelial lineage were given intratracheal bleomycin after intubation, and lungs were harvested 2 wk after a single or eighth biweekly dose. Lungs were evaluated for fibrosis and collagen content. Bronchoalveolar lavage (BAL) was performed for cell counts. TUNEL staining and immunohistochemistry were performed for pro-surfactant protein C (pro-SP-C), Clara cell 10 (CC-10), β-galactosidase, S100A4, and α-smooth muscle actin. Lungs from repetitive bleomycin mice had marked fibrosis with prominent AEC hyperplasia, similar to usual interstitial pneumonia (UIP). Compared with single dosing, repetitive bleomycin mice had greater fibrosis by scoring, morphometry, and collagen content; increased TUNEL+ AECs; and reduced inflammatory cells in BAL. Sixty-four percent of pro-SP-C+ cells in areas of fibrosis expressed CC-10 in the repetitive model, suggesting expansion of a bronchoalveolar stem cell-like population. In reporter mice, 50% of S100A4+ lung fibroblasts were derived from epithelial mesenchymal transition compared with 33% in the single-dose model. With repetitive bleomycin, fibrotic remodeling persisted 10 wk after the eighth dose. Repetitive intratracheal bleomycin results in marked lung fibrosis with prominent AEC hyperplasia, features reminiscent of UIP.

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.

β-Catenin in the Alveolar Epithelium Protects from Lung Fibrosis after Intratracheal Bleomycin

RATIONALE Alveolar epithelial cells (AECs) play central roles in the response to lung injury and the pathogenesis of pulmonary fibrosis. OBJECTIVES We aimed to determine the role of β-catenin in alveolar epithelium during bleomycin-induced lung fibrosis. METHODS Genetically modified mice were developed to selectively delete β-catenin in AECs and were crossed to cell fate reporter mice that express β-galactosidase (βgal) in cells of AEC lineage. Mice were given intratracheal bleomycin (0.04 units) and assessed for AEC death, inflammation, lung injury, and fibrotic remodeling. Mouse lung epithelial cells (MLE12) with small interfering RNA knockdown of β-catenin underwent evaluation for wound closure, proliferation, and bleomycin-induced cytotoxicity. MEASUREMENTS AND MAIN RESULTS Increased β-catenin expression was noted in lung parenchyma after bleomycin. Mice with selective deletion of β-catenin in AECs had greater AEC death at 1 week after bleomycin, followed by increased numbers of fibroblasts and enhanced lung fibrosis as determined by semiquantitative histological scoring and total collagen content. However, no differences in lung inflammation or protein levels in bronchoalveolar lavage were noted. In vitro, β-catenin-deficient AECs showed increased bleomycin-induced cytotoxicity as well as reduced proliferation and impaired wound closure. Consistent with these findings, mice with AEC β-catenin deficiency showed delayed recovery after bleomycin. CONCLUSIONS β-Catenin in the alveolar epithelium protects against bleomycin-induced fibrosis. Our studies suggest that AEC survival and wound healing are enhanced through β-catenin-dependent mechanisms. Activation of the developmentally important β-catenin pathway in AECs appears to contribute to epithelial repair after epithelial injury.

Extensive Phenotyping of Individuals at Risk for Familial Interstitial Pneumonia Reveals Clues to the Pathogenesis of Interstitial Lung Disease

RATIONALE Asymptomatic relatives of patients with familial interstitial pneumonia (FIP), the inherited form of idiopathic interstitial pneumonia, carry increased risk for developing interstitial lung disease. OBJECTIVES Studying these at-risk individuals provides a unique opportunity to investigate early stages of FIP pathogenesis and develop predictive models of disease onset. METHODS Seventy-five asymptomatic first-degree relatives of FIP patients (mean age, 50.8 yr) underwent blood sampling and high-resolution chest computed tomography (HRCT) scanning in an ongoing cohort study; 72 consented to bronchoscopy with bronchoalveolar lavage (BAL) and transbronchial biopsies. Twenty-seven healthy individuals were used as control subjects. MEASUREMENTS AND MAIN RESULTS Eleven of 75 at-risk subjects (14%) had evidence of interstitial changes by HRCT, whereas 35.2% had abnormalities on transbronchial biopsies. No differences were noted in inflammatory cells in BAL between at-risk individuals and control subjects. At-risk subjects had increased herpesvirus DNA in cell-free BAL and evidence of herpesvirus antigen expression in alveolar epithelial cells (AECs), which correlated with expression of endoplasmic reticulum stress markers in AECs. Peripheral blood mononuclear cell and AEC telomere length were shorter in at-risk individuals than healthy control subjects. The minor allele frequency of the Muc5B rs35705950 promoter polymorphism was increased in at-risk subjects. Levels of several plasma biomarkers differed between at-risk subjects and control subjects, and correlated with abnormal HRCT scans. CONCLUSIONS Evidence of lung parenchymal remodeling and epithelial dysfunction was identified in asymptomatic individuals at risk for FIP. Together, these findings offer new insights into the early pathogenesis of idiopathic interstitial pneumonia and provide an ongoing opportunity to characterize presymptomatic abnormalities that predict progression to clinical disease.

TGFβ 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-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-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 TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 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(+)/β-gal(+)) fibroblasts. Attenuation of TGFβ 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.

Telomerase deficiency does not alter bleomycin-induced fibrosis in mice

ABSTRACT Idiopathic pulmonary fibrosis (IPF) is characterized by interstitial lung infiltrates, dyspnea, and progressive respiratory failure. Reports linking telomerase mutations to familial interstitial pneumonia (FIP) suggest that telomerase activity and telomere length maintenance are important in disease pathogenesis. To investigate the role of telomerase in lung fibrotic remodeling, intratracheal bleomycin was administered to mice deficient in telomerase reverse transcriptase (TERT) or telomerase RNA component (TERC) and to wild-type controls. TERT-deficient and TERC-deficient mice were interbred to the F6 and F4 generation, respectively, when they developed skin manifestations and infertility. Fibrosis was scored using a semiquantitative scale and total lung collagen was measured using a hydroxyprolinemicroplate assay. Telomere lengths were measured in peripheral blood leukocytes and isolated type II alveolar epithelial cells (AECs). Telomerase activity in type II AECs was measured using a real-time polymerase chain reaction (PCR)-based system. Following bleomycin, TERT-deficient and TERC-deficient mice developed an equivalent inflammatory response and similar lung fibrosis (by scoring of lung sections and total lung collagen content) compared to controls, a pattern seen in both early (F1) and later (F6 TERT and F4 TERC) generations. Telomere lengths were reduced in peripheral blood leukocytes and isolated type II AECs from F6 TERT-deficient and F4 TERC-deficient mice compared to controls. Telomerase deficiency in a murine model leads to telomere shortening, but does not predispose to enhanced bleomycin-induced lung fibrosis. Additional genetic or environmental factors may be necessary for development of fibrosis in the presence of telomerase deficiency.

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-1β 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-1β, 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.

Abstract 2873: Epithelial NF-κB signaling is required for different functions in Kras- and EGFR-mediated lung tumorigenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The transcription factor nuclear factor-κB (NF-κB) is a key mediator of the inflammatory response, and its activation correlates with poor prognosis in non-small cell lung cancer patients. Oncogenic KRAS and epidermal growth factor receptor (EGFR) mutations occur frequently in lung adenocarcinoma and are associated with increased NF-κB activation in lung tumors. Recent papers have shown that inhibition of epithelial NF-κB signaling in a mouse model of Kras-mediated lung tumorigenesis decreases tumor formation. However, the mechanisms linking NF-κB signaling to tumor formation remain unclear. We hypothesize that epithelial NF-κB signaling is a common requirement for lung tumorigenesis. For our studies, we used mice that inducibly express oncogenic KrasG12D or EGFRL858R+T790M as well as a dominant negative inhibitor of κB signaling (DN-IκB) in Clara Cell Specific Protein (CCSP)-expressing lung epithelium (designated KrasDN or EGFRDN mice). EGFRDN mice developed smaller and fewer tumors after two months of doxycycline administration compared to mice expressing EGFRL858R+T790M alone, while KrasDN mice did not develop any tumors. In KrasDN mice, loss of Kras transgene expression and increased cell death were observed after one week of doxycycline, suggesting that NF-κB signaling may be required for survival of Kras mutant cells in vivo. In EGFRDN mice, NF-κB inhibition led to a significant reduction in the percentage of BAL neutrophils when compared to mice expressing EGFRL858R+T790M alone. In addition, NF-κB inhibition led to reduced expression of CXCR2 ligands CXCL1 and CXCL2, which are known NF-κB-regulated neutrophil chemoattractants. In vitro inhibition of CXCR2 did not affect proliferation of EGFR-mutant cells, suggesting that the critical function of CXCL1 and CXCL2 may be inflammatory cell recruitment/activation. Future studies will investigate the effects of CXCR2 inhibition and neutrophil depletion on EGFR-mediated tumor formation in vivo. Together, these results suggest that NF-κB signaling is required for both Kras- and EGFR-mediated lung tumorigenesis, but NF-κBs function in a tumor may depend on the driver mutation. From these studies, we hope to identify NF-κB-regulated genes critical for tumor formation as potential targets for the development of better therapeutic strategies for lung cancer patients. Citation Format: Jamie A. Ausborn, Taylor Sherrill, Rinat Zaynagetdinov, Vasiliy Polosukhin, Frank McMahon, Fiona Yull, Timothy Blackwell. Epithelial NF-κB signaling is required for different functions in Kras- and EGFR-mediated lung tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2873. doi:10.1158/1538-7445.AM2013-2873

Abstract 313: K-ras- and EGFR-mediated lung tumor formation require epithelial NF-κB signaling to modulate the inflammatory microenvironment

The transcription factor nuclear factor-κB (NF-κB) is a key mediator of the inflammatory response, and its activation correlates with poor prognosis in non-small cell lung cancer patients. Oncogenic K-ras and epidermal growth factor receptor (EGFR) mutations occur frequently in lung adenocarcinoma and are associated with an inflammatory response in lung tumors. Recent papers have shown that inhibition of epithelial NF-κB signaling in a mouse model of K-ras-mediated lung tumorigenesis decreased tumor formation. However, the mechanisms linking NF-κB signaling to tumor formation remain unclear. We hypothesize that epithelial NF-κB signaling is a common requirement for lung tumorigenesis, causing activation and/or recruitment of inflammatory cells that promote lung tumor formation. For our studies, we used mice that inducibly express oncogenic K-rasG12D or EGFRT790M+L858R as well as a dominant negative inhibitor of κB signaling (DN-IκB) specifically in the lung epithelium. Mice expressing DN-IκB together with K-rasG12D or EGFRT790M+L858R developed significantly fewer tumors than mice expressing K-rasG12D or EGFRT790M+L858R alone after two months of doxycycline administration. However, two months of K-rasG12D or EGFRT790M+L858R expression did not significantly alter total inflammatory cell numbers in bronchoalveolar lavage fluid (BAL). EGFRT790M+L858R expression led to an increase in BAL neutrophils that was lost when NF-κB signaling was inhibited, while no changes in BAL macrophage, lymphocyte, or neutrophil subpopulations were observed in K-rasG12D-expressing mice with or without NF-κB signaling. At later time points, both K-rasG12D- and EGFRT790M+L858R-expressing mice demonstrated a profound inflammatory response, which was associated with an increased tumor burden. Using a model of long term alveolar macrophage depletion by intratracheal administration of liposomal clodronate, we found that depletion of alveolar macrophages decreased tumor burden in K-rasG12D-expressing mice. Together, these results suggest that, rather than causing a profound inflammatory influx, epithelial NF-κB signaling may be important for activating resident inflammatory cell populations that aid in K-ras- and EGFR-mediated tumor formation. From these studies, we hope to broaden understanding of the role of inflammatory signaling pathways in regulation of lung carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 313. doi:1538-7445.AM2012-313