ß1 Integrin regulates adult lung alveolar epithelial cell inflammation.

Integrins, the extracellular matrix receptors that facilitate cell adhesion and migration, are necessary for organ morphogenesis; however, their role in maintaining adult tissue homeostasis is poorly understood. To define the functional importance of ß1 integrin in adult mouse lung, we deleted it after completion of development in type 2 alveolar epithelial cells (AECs). Aged ß1 integrin-deficient mice exhibited chronic obstructive pulmonary disease-like (COPD-like) pathology characterized by emphysema, lymphoid aggregates, and increased macrophage infiltration. These histopathological abnormalities were preceded by ß1 integrin-deficient AEC dysfunction such as excessive ROS production and upregulation of NF-κB-dependent chemokines, including CCL2. Genetic deletion of the CCL2 receptor, Ccr2, in mice with ß1 integrin-deficient type 2 AECs impaired recruitment of monocyte-derived macrophages and resulted in accelerated inflammation and severe premature emphysematous destruction. The lungs exhibited reduced AEC efferocytosis and excessive numbers of inflamed type 2 AECs, demonstrating the requirement for recruited monocytes/macrophages in limiting lung injury and remodeling in the setting of a chronically inflamed epithelium. These studies support a critical role for ß1 integrin in alveolar homeostasis in the adult lung.

Gas6/MerTK signaling is negatively regulated by NF-κB and supports lung carcinogenesis.

Growth arrest-specific 6 (Gas6) has been implicated in carcinogenesis through activation of its receptors, particularly MerTK. To investigate whether Gas6 plays a role in resistance to NF-κB inhibitors, which have not proven to be effective agents for lung cancer therapy, we studied lung cancer models induced by urethane injection or expression of mutant Kras (KrasG12D). We found that Gas6 is primarily produced by macrophages during tumorigenesis and that Gas6 is negatively regulated by NF-κB. Since Gas6 is a vitamin K dependent protein, we used low-dose warfarin to block Gas6 production and showed that this treatment inhibited tumorigenesis in both the urethane and KrasG12D models, most prominently in mice with targeted deletion of IKKß in myeloid cells (IKKßΔMye mice). In addition, MerTK deficient mice had reduced urethane-induced tumorigenesis. Inhibition of the Gas6-MerTK pathway in all these models reduced macrophages and neutrophils in the lungs of tumor-bearing mice. Analysis of mouse lung tumors revealed MerTK staining on tumor cells and in vitro studies showed that Gas6 increased proliferation of human lung cancer cell lines. To assess the therapeutic potential for combination treatment targeting NF-κB and Gas6-MerTK, we injected Lewis Lung Carcinoma cells subcutaneously and treated mice with Bay 11-70852 (NF-κB inhibitor) and/or Foretinib (MerTK inhibitor). While individual treatments were ineffective, combination therapy markedly reduced tumor growth, blocked tumor cell proliferation, reduced tumor-associated macrophages, and increased CD4+ T cells. Together, our studies unmask a role for Gas6-MerTK signaling in lung carcinogenesis and indicate that up-regulation of Gas6 production in macrophages could be a major mechanism of resistance to NF-κB inhibitors.

Natriuretic peptide receptor C contributes to disproportionate right ventricular hypertrophy in a rodent model of obesity-induced heart failure with preserved ejection fraction with pulmonary hypertension.

Heart failure with preserved ejection fraction (HFpEF) currently has no therapies that improve mortality. Right ventricular dysfunction and pulmonary hypertension are common in HFpEF, and thought to be driven by obesity and metabolic syndrome. Thus, we hypothesized that an animal model of obesity-induced HFpEF with pulmonary hypertension would provide insight into the pathogenesis of right ventricular failure in HFpEF. Two strains of mice, one susceptible (AKR) and one resistant (C3H) to obesity-induced HFpEF, were fed high fat (60% fat) or control diet for 0, 2, or 20 weeks and evaluated by cardiac catheterization and echocardiography for development of right ventricular dysfunction, pulmonary hypertension, and HFpEF. AKR, but not C3H, mice developed right ventricular dysfunction, pulmonary hypertension, and HFpEF. NPRC, which antagonizes beneficial natriuretic peptide signaling, was found in RNA sequencing to be the most differentially upregulated gene in the right ventricle, but not left ventricle or lung, of AKR mice that developed pulmonary hypertension and HFpEF. Overexpression of NPRC in H9C2 cells increased basal cell size and increased expression of hypertrophic genes, MYH7 and NPPA. In conclusion, we have shown that NPRC contributes to right ventricular modeling in obesity-induced pulmonary hypertension-HFpEF by increasing cardiomyocyte hypertrophy. NPRC may represent a promising therapeutic target for right ventricular dysfunction in pulmonary hypertension-HFpEF.

Localized hypoxia links ER stress to lung fibrosis through induction of C/EBP homologous protein.

ER stress in type II alveolar epithelial cells (AECs) is common in idiopathic pulmonary fibrosis (IPF), but the contribution of ER stress to lung fibrosis is poorly understood. We found that mice deficient in C/EBP homologous protein (CHOP), an ER stress-regulated transcription factor, were protected from lung fibrosis and AEC apoptosis in 3 separate models where substantial ER stress was identified. In mice treated with repetitive intratracheal bleomycin, we identified localized hypoxia in type II AECs as a potential mechanism explaining ER stress. To test the role of hypoxia in lung fibrosis, we treated mice with bleomycin, followed by exposure to 14% O2, which exacerbated ER stress and lung fibrosis. Under these experimental conditions, CHOP-/- mice, but not mice with epithelial HIF (HIF1/HIF2) deletion, were protected from AEC apoptosis and fibrosis. In vitro studies revealed that CHOP regulates hypoxia-induced apoptosis in AECs via the inositol-requiring enzyme 1α (IRE1α) and the PKR-like ER kinase (PERK) pathways. In human IPF lungs, CHOP and hypoxia markers were both upregulated in type II AECs, supporting a conclusion that localized hypoxia results in ER stress-induced CHOP expression, thereby augmenting type II AEC apoptosis and potentiating lung fibrosis.

p52 expression enhances lung cancer progression.

While many studies have demonstrated that canonical NF-κB signaling is a central pathway in lung tumorigenesis, the role of non-canonical NF-κB signaling in lung cancer remains undefined. We observed frequent nuclear accumulation of the non-canonical NF-κB component p100/p52 in human lung adenocarcinoma. To investigate the impact of non-canonical NF-κB signaling on lung carcinogenesis, we employed transgenic mice with doxycycline-inducible expression of p52 in airway epithelial cells. p52 over-expression led to increased tumor number and progression after injection of the carcinogen urethane. Gene expression analysis of lungs from transgenic mice combined with in vitro studies suggested that p52 promotes proliferation of lung epithelial cells through regulation of cell cycle-associated genes. Using gene expression and patient information from The Cancer Genome Atlas (TCGA) database, we found that expression of p52-associated genes was increased in lung adenocarcinomas and correlated with reduced survival, even in early stage disease. Analysis of p52-associated gene expression in additional human lung adenocarcinoma datasets corroborated these findings. Together, these studies implicate the non-canonical NF-κB component p52 in lung carcinogenesis and suggest modulation of p52 activity and/or downstream mediators as new therapeutic targets.

Successful Establishment of Primary Type II Alveolar Epithelium with 3D Organotypic Coculture.

Alveolar type II (AT2) epithelial cells are uniquely specialized to produce surfactant in the lung and act as progenitor cells in the process of repair after lung injury. AT2 cell injury has been implicated in several lung diseases, including idiopathic pulmonary fibrosis and bronchopulmonary dysplasia. The inability to maintain primary AT2 cells in culture has been a significant barrier in the investigation of pulmonary biology. We have addressed this knowledge gap by developing a three-dimensional (3D) organotypic coculture using primary human fetal AT2 cells and pulmonary fibroblasts. Grown on top of matrix-embedded fibroblasts, the primary human AT2 cells establish a monolayer and have direct contact with the underlying pulmonary fibroblasts. Unlike conventional two-dimensional (2D) culture, the structural and functional phenotype of the AT2 cells in our 3D organotypic culture was preserved over 7 days of culture, as evidenced by the presence of lamellar bodies and by production of surfactant proteins B and C. Importantly, the AT2 cells in 3D cocultures maintained the ability to replicate, with approximately 60% of AT2 cells staining positive for the proliferation marker Ki67, whereas no such proliferation is evident in 2D cultures of the same primary AT2 cells. This organotypic culture system enables interrogation of AT2 epithelial biology by providing a reductionist in vitro model in which to investigate the response of AT2 epithelial cells and AT2 cell-fibroblast interactions during lung injury and repair.

A Shared Pattern of ß-Catenin Activation in Bronchopulmonary Dysplasia and Idiopathic Pulmonary Fibrosis.

Wnt/ß-catenin signaling is necessary for normal lung development, and abnormal Wnt signaling contributes to the pathogenesis of both bronchopulmonary dysplasia (BPD) and idiopathic pulmonary fibrosis (IPF), fibrotic lung diseases that occur during infancy and aging, respectively. Using a library of human normal and diseased human lung samples, we identified a distinct signature of nuclear accumulation of ß-catenin phosphorylated at tyrosine 489 and epithelial cell cytosolic localization of ß-catenin phosphorylated at tyrosine 654 in early normal lung development and fibrotic lung diseases BPD and IPF. Furthermore, this signature was recapitulated in murine models of BPD and IPF. Image analysis of immunofluorescence colocalization demonstrated a consistent pattern of elevated nuclear phosphorylated ß-catenin in the lung epithelium and surrounding mesenchyme in BPD and IPF, closely resembling the pattern observed in 18-week fetal lung. Nuclear ß-catenin phosphorylated at tyrosine 489 associated with an increased expression of Wnt target gene AXIN2, suggesting that the observed ß-catenin signature is of functional significance during normal development and injury repair. The association of specific modifications of ß-catenin during normal lung development and again in response to lung injury supports the widely held concept that repair of lung injury involves the recapitulation of developmental programs. Furthermore, these observations suggest that ß-catenin phosphorylation has potential as a therapeutic target for the treatment and prevention of both BPD and IPF.

Bacterial-derived Neutrophilic Inflammation Drives Lung Remodeling in a Mouse Model of Chronic Obstructive Pulmonary Disease.

Loss of secretory IgA is common in the small airways of patients with chronic obstructive pulmonary disease and may contribute to disease pathogenesis. Using mice that lack secretory IgA in the airways due to genetic deficiency of polymeric Ig receptor (pIgR-/- mice), we investigated the role of neutrophils in driving the fibrotic small airway wall remodeling and emphysema that develops spontaneously in these mice. By flow cytometry, we found an increase in the percentage of neutrophils among CD45+ cells in the lungs, as well as an increase in total neutrophils, in pIgR-/- mice compared with wild-type controls. This increase in neutrophils in pIgR-/- mice was associated with elastin degradation in the alveolar compartment and around small airways, along with increased collagen deposition in small airway walls. Neutrophil depletion using anti-Ly6G antibodies or treatment with broad-spectrum antibiotics inhibited development of both emphysema and small airway remodeling, suggesting that airway bacteria provide the stimulus for deleterious neutrophilic inflammation in this model. Exogenous bacterial challenge using lysates prepared from pathogenic and nonpathogenic bacteria worsened neutrophilic inflammation and lung remodeling in pIgR-/- mice. This phenotype was abrogated by antiinflammatory therapy with roflumilast. Together, these studies support the concept that disruption of the mucosal immune barrier in small airways contributes to chronic obstructive pulmonary disease progression by allowing bacteria to stimulate chronic neutrophilic inflammation, which, in turn, drives progressive airway wall fibrosis and emphysematous changes in the lung parenchyma.

Epithelial NF-κB signaling promotes EGFR-driven lung carcinogenesis via macrophage recruitment.

Several studies have demonstrated that NF-κB activation is common in lung cancer; however, the mechanistic links between NF-κB signaling and tumorigenesis remain to be fully elucidated. We investigated the function of NF-κB signaling in epidermal growth factor receptor (EGFR)-mutant lung tumors using a transgenic mouse model with doxycycline (dox)-inducible expression of oncogenic EGFR in the lung epithelium with or without a dominant inhibitor of NF-κB signaling. NF-κB inhibition resulted in a significant reduction in tumor burden in both EGFR tyrosine kinase inhibitor (TKI)-sensitive and resistant tumors. However, NF-κB inhibition did not alter epithelial cell survival in vitro or in vivo, and no changes were detected in activation of EGFR downstream signaling pathways. Instead, we observed an influx of inflammatory cells (macrophages and neutrophils) in the lungs of mice with oncogenic EGFR expression that was blocked in the setting of NF-κB inhibition. To investigate whether inflammatory cells play a role in promoting EGFR-mutant lung tumors, we depleted macrophages and neutrophils during tumorigenesis and found that neutrophil depletion had no effect on tumor formation, but macrophage depletion caused a significant reduction in tumor burden. Together, these data suggest that epithelial NF-κB signaling supports carcinogenesis in a non-cell autonomous manner in EGFR-mutant tumors through recruitment of pro-tumorigenic macrophages.

Neutrophil-Derived IL-1ß Impairs the Efficacy of NF-κB Inhibitors against Lung Cancer.

Although epithelial NF-κB signaling is important for lung carcinogenesis, NF-κB inhibitors are ineffective for cancer treatment. To explain this paradox, we studied mice with genetic deletion of IKKß in myeloid cells and found enhanced tumorigenesis in Kras(G12D) and urethane models of lung cancer. Myeloid-specific inhibition of NF-κB augmented pro-IL-1ß processing by cathepsin G in neutrophils, leading to increased IL-1ß and enhanced epithelial cell proliferation. Combined treatment with bortezomib, a proteasome inhibitor that blocks NF-κB activation, and IL-1 receptor antagonist reduced tumor formation and growth in vivo. In lung cancer patients, plasma IL-1ß levels correlated with poor prognosis, and IL-1ß increased following bortezomib treatment. Together, our studies elucidate an important role for neutrophils and IL-1ß in lung carcinogenesis and resistance to NF-κB inhibitors.

Epithelial-Derived Inflammation Disrupts Elastin Assembly and Alters Saccular Stage Lung Development.

The highly orchestrated interactions between the epithelium and mesenchyme required for normal lung development can be disrupted by perinatal inflammation in preterm infants, although the mechanisms are incompletely understood. We used transgenic (inhibitory κB kinase ß transactivated) mice that conditionally express an activator of the NF-κB pathway in airway epithelium to investigate the impact of epithelial-derived inflammation during lung development. Epithelial NF-κB activation selectively impaired saccular stage lung development, with a phenotype comprising rapidly progressive distal airspace dilation, impaired gas exchange, and perinatal lethality. Epithelial-derived inflammation resulted in disrupted elastic fiber organization and down-regulation of elastin assembly components, including fibulins 4 and 5, lysyl oxidase like-1, and fibrillin-1. Fibulin-5 expression by saccular stage lung fibroblasts was consistently inhibited by treatment with bronchoalveolar lavage fluid from inhibitory κB kinase ß transactivated mice, Escherichia coli lipopolysaccharide, or tracheal aspirates from preterm infants exposed to chorioamnionitis. Expression of a dominant NF-κB inhibitor in fibroblasts restored fibulin-5 expression after lipopolysaccharide treatment, whereas reconstitution of fibulin-5 rescued extracellular elastin assembly by saccular stage lung fibroblasts. Elastin organization was disrupted in saccular stage lungs of preterm infants exposed to systemic inflammation. Our study reveals a critical window for elastin assembly during the saccular stage that is disrupted by inflammatory signaling and could be amenable to interventions that restore elastic fiber assembly in the developing lung.

Airway bacteria drive a progressive COPD-like phenotype in mice with polymeric immunoglobulin receptor deficiency.

Mechanisms driving persistent airway inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. As secretory immunoglobulin A (SIgA) deficiency in small airways has been reported in COPD patients, we hypothesized that immunobarrier dysfunction resulting from reduced SIgA contributes to chronic airway inflammation and disease progression. Here we show that polymeric immunoglobulin receptor-deficient (pIgR(-/-)) mice, which lack SIgA, spontaneously develop COPD-like pathology as they age. Progressive airway wall remodelling and emphysema in pIgR(-/-) mice are associated with an altered lung microbiome, bacterial invasion of the airway epithelium, NF-κB activation, leukocyte infiltration and increased expression of matrix metalloproteinase-12 and neutrophil elastase. Re-derivation of pIgR(-/-) mice in germ-free conditions or treatment with the anti-inflammatory phosphodiesterase-4 inhibitor roflumilast prevents COPD-like lung inflammation and remodelling. These findings show that pIgR/SIgA deficiency in the airways leads to persistent activation of innate immune responses to resident lung microbiota, driving progressive small airway remodelling and emphysema.

Chronic NF-κB activation links COPD and lung cancer through generation of an immunosuppressive microenvironment in the lungs.

Nuclear Factor (NF)-κB is positioned to provide the interface between COPD and carcinogenesis through regulation of chronic inflammation in the lungs. Using a tetracycline-inducible transgenic mouse model that conditionally expresses activated IκB kinase ß (IKKß) in airway epithelium (IKTA), we found that sustained NF-κB signaling results in chronic inflammation and emphysema by 4 months. By 11 months of transgene activation, IKTA mice develop lung adenomas. Investigation of lung inflammation in IKTA mice revealed a substantial increase in M2-polarized macrophages and CD4+/CD25+/FoxP3+ regulatory T lymphocytes (Tregs). Depletion of alveolar macrophages in IKTA mice reduced Tregs, increased lung CD8+ lymphocytes, and reduced tumor numbers following treatment with the carcinogen urethane. Alveolar macrophages from IKTA mice supported increased generation of inducible Foxp3+ Tregs ex vivo through expression of TGFß and IL-10. Targeting of TGFß and IL-10 reduced the ability of alveolar macrophages from IKTA mice to induce Foxp3 expression on T cells. These studies indicate that sustained activation of NF-κB pathway links COPD and lung cancer through generation and maintenance of a pro-tumorigenic inflammatory environment consisting of alternatively activated macrophages and regulatory T cells.

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.

Endothelial HIF signaling regulates pulmonary fibrosis-associated pulmonary hypertension.

Pulmonary hypertension (PH) complicating chronic parenchymal lung disease, such as idiopathic pulmonary fibrosis, results in significant morbidity and mortality. Since the hypoxia-inducible factor (HIF) signaling pathway is important for development of pulmonary hypertension in chronic hypoxia, we investigated whether HIF signaling in vascular endothelium regulates development of PH related to pulmonary fibrosis. We generated a transgenic model in which HIF is deleted within vascular endothelial cells and then exposed these mice to chronic intraperitoneal bleomycin to induce PH associated with lung fibrosis. Although no differences in the degree of fibrotic remodeling were observed, we found that endothelial HIF-deficient mice were protected against development of PH, including right ventricle and pulmonary vessel remodeling. Similarly, endothelial HIF-deficient mice were protected from PH after a 4-wk exposure to normobaric hypoxia. In vitro studies of pulmonary vascular endothelial cells isolated from the HIF-targeted mice and controls revealed that endothelial HIF signaling increases endothelial cell expression of connective tissue growth factor, enhances vascular permeability, and promotes pulmonary artery smooth muscle cell proliferation and wound healing ability, all of which have the potential to impact the development of PH in vivo. Taken together, these studies demonstrate that vascular endothelial cell HIF signaling is necessary for development of hypoxia and pulmonary fibrosis associated PH. As such, HIF and HIF-regulated targets represent a therapeutic target in these conditions.

Expression of mutant bone morphogenetic protein receptor II worsens pulmonary hypertension secondary to pulmonary fibrosis.

Pulmonary fibrosis is often complicated by pulmonary hypertension (PH), and previous studies have shown a potential link between bone morphogenetic protein receptor II (BMPR2) and PH secondary to pulmonary fibrosis. We exposed transgenic mice expressing mutant BMPR2 and control mice to repetitive intraperitoneal injections of bleomycin for 4 weeks. The duration of transgene activation was too short for mutant BMPR2 mice to develop spontaneous PH. Mutant BMPR2 mice had increased right ventricular systolic pressure compared to control mice, without differences in pulmonary fibrosis. We found increased hypoxia-inducible factor (HIF)1-α stabilization in lungs of mutant-BMPR2-expressing mice compared to controls following bleomycin treatment. In addition, expression of the hypoxia response element protein connective tissue growth factor was increased in transgenic mice as well as in a human pulmonary microvascular endothelial cell line expressing mutant BMPR2. In mouse pulmonary vascular endothelial cells, mutant BMPR2 expression resulted in increased HIF1-α and reactive oxygen species production following exposure to hypoxia, both of which were attenuated with the antioxidant TEMPOL. These data suggest that expression of mutant BMPR2 worsens secondary PH through increased HIF activity in vascular endothelium. This pathway could be therapeutically targeted in patients with PH secondary to pulmonary fibrosis.

Aberrant activation of NF-κB signaling in mammary epithelium leads to abnormal growth and ductal carcinoma in situ.

BACKGROUND: Approximately 1 in 5 women diagnosed with breast cancer are considered to have in situ disease, most often termed ductal carcinoma in situ (DCIS). Though recognized as a risk factor for the development of more invasive cancer, it remains unclear what factors contribute to DCIS development. It has been shown that inflammation contributes to the progression of a variety of tumor types, and nuclear factor kappa B (NF-κB) is recognized as a master-regulator of inflammatory signaling. However, the contributions of NF-κB signaling to tumor initiation are less well understood. Aberrant up-regulation of NF-κB activity, either systemically or locally within the breast, could occur due to a variety of commonly experienced stimuli such as acute infection, obesity, or psychological stress. In this study, we seek to determine if activation of NF-κB in mammary epithelium could play a role in the formation of hyperplastic ductal lesions. METHODS: Our studies utilize a doxycycline-inducible transgenic mouse model in which constitutively active IKKß is expressed specifically in mammary epithelium. All previously published models of NF-κB modulation in the virgin mammary gland have been constitutive models, with transgene or knock-out present throughout the life and development of the animal. For the first time, we will induce activation at later time points after normal ducts have formed, thus being able to determine if NF-κB activation can promote pre-malignant changes in previously normal mammary epithelium. RESULTS: We found that even a short pulse of NF-κB activation could induce profound remodeling of mammary ductal structures. Short-term activation created hyperproliferative, enlarged ducts with filled lumens. Increased expression of inflammatory markers was concurrent with the down-regulation of hormone receptors and markers of epithelial differentiation. Furthermore, the oncoprotein mucin 1, known to be up-regulated in human and mouse DCIS, was over-expressed and mislocalized in the activated ductal tissue. CONCLUSIONS: These results indicate that aberrant NF-κB activation within mammary epithelium can lead to molecular and morphological changes consistent with the earliest stages of breast cancer. Thus, inhibition of NF-κB signaling following acute inflammation or the initial signs of hyperplastic ductal growth could represent an important opportunity for breast cancer prevention.

Interleukin-5 facilitates lung metastasis by modulating the immune microenvironment.

Although the lung is the most common metastatic site for cancer cells, biologic mechanisms regulating lung metastasis are not fully understood. Using heterotopic and intravenous injection models of lung metastasis in mice, we found that IL5, a cytokine involved in allergic and infectious diseases, facilitates metastatic colonization through recruitment of sentinel eosinophils and regulation of other inflammatory/immune cells in the microenvironment of the distal lung. Genetic IL5 deficiency offered marked protection of the lungs from metastasis of different types of tumor cells, including lung cancer, melanoma, and colon cancer. IL5 neutralization protected subjects from metastasis, whereas IL5 reconstitution or adoptive transfer of eosinophils into IL5-deficient mice exerted prometastatic effects. However, IL5 deficiency did not affect the growth of the primary tumor or the size of metastatic lesions. Mechanistic investigations revealed that eosinophils produce CCL22, which recruits regulatory T cells to the lungs. During early stages of metastasis, Treg created a protumorigenic microenvironment, potentially by suppressing IFNγ-producing natural killer cells and M1-polarized macrophages. Together, our results establish a network of allergic inflammatory circuitry that can be co-opted by metastatic cancer cells to facilitate lung colonization, suggesting interventions to target this pathway may offer therapeutic benefits to prevent or treat lung metastasis.

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.

Molecular imaging of folate receptor ß-positive macrophages during acute lung inflammation.

Characterization of markers that identify activated macrophages could advance understanding of inflammatory lung diseases and facilitate development of novel methodologies for monitoring disease activity. We investigated whether folate receptor ß (FRß) expression could be used to identify and quantify activated macrophages in the lungs during acute inflammation induced by Escherichia coli LPS. We found that FRß expression was markedly increased in lung macrophages at 48 hours after intratracheal LPS. In vivo molecular imaging with a fluorescent probe (cyanine 5 polyethylene glycol folate) showed that the fluorescence signal over the chest peaked at 48 hours after intratracheal LPS and was markedly attenuated after depletion of macrophages. Using flow cytometry, we identified the cells responsible for uptake of cyanine 5-conjugated folate as FRß(+) interstitial macrophages and pulmonary monocytes, which coexpressed markers associated with an M1 proinflammatory macrophage phenotype. These findings were confirmed using a second model of acute lung inflammation generated by inducible transgenic expression of an NF-κB activator in airway epithelium. Using CC chemokine receptor 2-deficient mice, we found that FRß(+) macrophage/monocyte recruitment was dependent on the monocyte chemotactic protein-1/CC chemokine receptor 2 pathway. Together, our results demonstrate that folate-based molecular imaging can be used as a noninvasive approach to detect classically activated monocytes/macrophages recruited to the lungs during acute inflammation.