Bernadette R. Gochuico

Bleomycin and IL-1β–mediated Pulmonary Fibrosis is IL-17A Dependent

Idiopathic pulmonary fibrosis (IPF) is a destructive inflammatory disease with limited therapeutic options. To better understand the inflammatory responses that precede and concur with collagen deposition, we used three models of pulmonary fibrosis and identify a critical mechanistic role for IL-17A. After exposure to bleomycin (BLM), but not Schistosoma mansoni eggs, IL-17A produced by CD4+ and γδ+ T cells induced significant neutrophilia and pulmonary fibrosis. Studies conducted with C57BL/6 il17a−/− mice confirmed an essential role for IL-17A. Mechanistically, using ifnγ−/−, il10−/−, il10−/−il12p40−/−, and il10−/−il17a−/− mice and TGF-β blockade, we demonstrate that IL-17A–driven fibrosis is suppressed by IL-10 and facilitated by IFN-γ and IL-12/23p40. BLM-induced IL-17A production was also TGF-β dependent, and recombinant IL-17A–mediated fibrosis required TGF-β, suggesting cooperative roles for IL-17A and TGF-β in the development of fibrosis. Finally, we show that fibrosis induced by IL-1β, which mimics BLM-induced fibrosis, is also highly dependent on IL-17A. IL-17A and IL-1β were also increased in the bronchoalveolar lavage fluid of patients with IPF. Together, these studies identify a critical role for IL-17A in fibrosis, illustrating the potential utility of targeting IL-17A in the treatment of drug and inflammation-induced fibrosis.

MMP1 and MMP7 as Potential Peripheral Blood Biomarkers in Idiopathic Pulmonary Fibrosis

Background Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease associated with substantial morbidity and mortality. The objective of this study was to determine whether there is a peripheral blood protein signature in IPF and whether components of this signature may serve as biomarkers for disease presence and progression. Methods and Findings We analyzed the concentrations of 49 proteins in the plasma of 74 patients with IPF and in the plasma of 53 control individuals. We identified a combinatorial signature of five proteins—MMP7, MMP1, MMP8, IGFBP1, and TNFRSF1A—that was sufficient to distinguish patients from controls with a sensitivity of 98.6% (95% confidence interval [CI] 92.7%–100%) and specificity of 98.1% (95% CI 89.9%–100%). Increases in MMP1 and MMP7 were also observed in lung tissue and bronchoalveolar lavage fluid obtained from IPF patients. MMP7 and MMP1 plasma concentrations were not increased in patients with chronic obstructive pulmonary disease or sarcoidosis and distinguished IPF compared to subacute/chronic hypersensitivity pneumonitis, a disease that may mimic IPF, with a sensitivity of 96.3% (95% CI 81.0%–100%) and specificity of 87.2% (95% CI 72.6%–95.7%). We verified our results in an independent validation cohort composed of patients with IPF, familial pulmonary fibrosis, subclinical interstitial lung disease (ILD), as well as with control individuals. MMP7 and MMP1 concentrations were significantly higher in IPF patients compared to controls in this cohort. Furthermore, MMP7 concentrations were elevated in patients with subclinical ILD and negatively correlated with percent predicted forced vital capacity (FVC%) and percent predicted carbon monoxide diffusing capacity (DLCO%). Conclusions Our experiments provide the first evidence for a peripheral blood protein signature in IPF to our knowledge. The two main components of this signature, MMP7 and MMP1, are overexpressed in the lung microenvironment and distinguish IPF from other chronic lung diseases. Additionally, increased MMP7 concentration may be indicative of asymptomatic ILD and reflect disease progression.

Progressive Preclinical Interstitial Lung Disease in Rheumatoid Arthritis

BACKGROUND Early detection and treatment for interstitial lung disease (ILD) in patients with rheumatoid arthritis (RA) may ameliorate disease progression. The objective of this study was to identify asymptomatic lung disease and potential therapeutic targets in patients having RA and preclinical ILD (RA-ILD). METHODS Sixty-four adults with RA and 10 adults with RA and pulmonary fibrosis (RAPF) were referred to the National Institutes of Health, Bethesda, Maryland, and underwent high-resolution computed tomography (HRCT) and pulmonary physiology testing. Proteins capable of modulating fibrosis were quantified in alveolar fluid. RESULTS Twenty-one of 64 patients (33%) having RA without dyspnea or cough had preclinical ILD identified by HRCT. Compared with patients without lung disease, patients with RA-ILD had statistically significantly longer histories of cigarette smoking (P< .001), increased frequencies of crackles (P= .02), higher alveolar-arterial oxygen gradients (P= .004), and higher HRCT scores (P< .001). The HRCT abnormalities progressed in 12 of 21 patients (57%) with RA-ILD. The alveolar concentrations of platelet-derived growth factor-AB and platelet-derived growth factor-BB were statistically significantly higher in patients having RA-ILD (mean [SE], 497.3 [78.6] and 1473 [264] pg/mL, respectively) than in patients having RA without ILD (mean [SE], 24.9 [42.4] and 792.7 [195.0] pg/mL, respectively) (P< .001 and P=.047, respectively). The concentrations of interferon gamma and transforming growth factor beta(2) were statistically significantly lower in patients having RAPF (mean [SE], 5.59 [1.11] pg/mL and 0.94 [0.46] ng/mL, respectively) than in patients having RA without ILD (mean [SE], 14.1 [1.9] pg/mL and 2.30 [0.39] ng/mL, respectively) (P=.001 and P=.006, respectively) or with preclinical ILD (mean [SD], 11.4 [2.6] pg/mL and 3.63 [0.66] ng/mL, respectively) (P=.04 and P=.007, respectively). Compared with patients having stable RA-ILD, patients having progressive RA-ILD had statistically significantly higher frequencies of treatment using methotrexate and higher alveolar concentrations of interferon gamma and transforming growth factor beta(1) (P=.046, P=.04, and P=.04, respectively). CONCLUSIONS Asymptomatic preclinical ILD, which is detectable by HRCT, may be prevalent and progressive among patients having RA. Cigarette smoking seems to be associated with preclinical ILD in patients having RA, and treatment using methotrexate may be a risk factor for progression of preclinical ILD. Quantification of alveolar proteins indicates that potential pathogenic mechanisms seem to differ in patients having RA-ILD and symptomatic RAPF.

Acute Fibrinous and Organizing Pneumonia A Histologic Pattern of Lung Injury and Possible Variant of Diffuse Alveolar Damage

CONTEXT The histologic patterns of diffuse alveolar damage (DAD), bronchiolitis obliterans with organizing pneumonia (BOOP), and eosinophilic pneumonia (EP) are well-recognized histologic patterns of lung injury associated with an acute or subacute clinical presentation. We have recognized acute fibrinous and organizing pneumonia (AFOP) as a histologic pattern, which also occurs in this clinical setting but does not meet the classic histologic criteria for DAD, BOOP, or EP and may represent an underreported variant. OBJECTIVE To investigate the clinical significance of the AFOP histologic pattern and to explore its possible relationship to other disorders, including DAD and BOOP. DESIGN Open lung biopsy specimens and autopsy specimens were selected from the consultation files of the Armed Forces Institute of Pathology, which showed a dominant histologic pattern of intra-alveolar fibrin and organizing pneumonia. Varying amounts of organizing pneumonia, type 2 pneumocyte hyperplasia, edema, acute and chronic inflammation, and interstitial widening were seen. Cases with histologic patterns of classic DAD, BOOP, abscess formation, or eosinophilic pneumonia were excluded. To determine the clinical behavior of patients with this histologic finding, clinical and radiographic information and follow-up information were obtained. Statistical analysis was performed using Kaplan-Meier and chi(2) analysis. RESULTS Seventeen patients (10 men, 7 women) with a mean age of 62 years (range, 33-78 years) had acute-onset symptoms of dyspnea (11), fever (6), cough (3), and hemoptysis (2). Associations believed to be clinically related to the lung disease included definitive or probable collagen vascular disease (3), amiodarone (1), sputum culture positive for Haemophilus influenza (1), lung culture positive for Acinetobacter sp. (1), lymphoma (1), hairspray (1), construction work (1), coal mining (1), and zoological work (1). Six patients had no identifiable origin or association. Follow-up revealed 2 clinical patterns of disease progression: a fulminate illness with rapid progression to death (n = 9; mean survival, 0.1 year) and a more subacute illness, with recovery (n = 8). Histologic analysis and initial symptoms did not correlate with eventual outcome, but 5 of the 5 patients who required mechanical ventilation died (P =.007). CONCLUSIONS Acute fibrinous and organizing pneumonia is a histologic pattern associated with a clinical picture of acute lung injury that differs from the classic histologic patterns of DAD, BOOP, or EP. Similar to these patterns of acute lung injury, the AFOP pattern can occur in an idiopathic setting or with a spectrum of clinical associations. The overall mortality rate is similar to DAD and therefore may represent a histologic variant; however, AFOP appears to have 2 distinct patterns of disease progression and outcome. The need for mechanical ventilation was the only parameter that correlated with prognosis. None of the patients with a subacute clinical course required mechanical ventilation.

Effect of pirfenidone on the pulmonary fibrosis of Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) consists of oculocutaneous albinism, a platelet storage pool deficiency and, in patients with HPS1 gene mutations, a progressive, fatal pulmonary fibrosis. We investigated the safety and efficacy of an antifibrotic agent, pirfenidone (800 mg, t.i.d.), in treating 21 adult Puerto Rican HPS patients, including 20 homozygous for the same HPS1 mutation. Patients were examined every 4 months for up to 44 months in a randomized, placebo-controlled trial, with rate of change in pulmonary function values as outcome parameters. Using the complete data set of 130 patient admissions, a repeated measures model showed that 11 pirfenidone-treated patients lost FVC at a rate 5% of predicted ( approximately 400 mL) per year slower than 10 placebo-treated patients (p=0.001). A random coefficients model showed no significant difference. However, using data restricted to patients with an initial FVC >50% of predicted, both models showed the pirfenidone group losing FVC (p<0.022), FEV(1) (p<0.0007), TLC (p<0.001), and DL(CO) (p<0.122) at a rate approximately 8%/year slower than the placebo group. Clinical and laboratory side effects were similar in the two groups. Pirfenidone appears to slow the progression of pulmonary fibrosis in HPS patients who have significant residual lung function.

Oxidative Stress Alters Syndecan-1 Distribution in Lungs with Pulmonary Fibrosis *□

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by severe, progressive fibrosis. Roles for inflammation and oxidative stress have recently been demonstrated, but despite advances in understanding the pathogenesis, there are still no effective therapies for IPF. This study investigates how extracellular superoxide dismutase (EC-SOD), a syndecan-binding antioxidant enzyme, inhibits inflammation and lung fibrosis. We hypothesize that EC-SOD protects the lung from oxidant damage by preventing syndecan fragmentation/shedding. Wild-type or EC-SOD-null mice were exposed to an intratracheal instillation of asbestos or bleomycin. Western blot was used to detect syndecans in the bronchoalveolar lavage fluid and lung. Human lung samples (normal and IPF) were also analyzed. Immunohistochemistry for syndecan-1 and EC-SOD was performed on human and mouse lungs. In vitro, alveolar epithelial cells were exposed to oxidative stress and EC-SOD. Cell supernatants were analyzed for shed syndecan-1 by Western blot. Syndecan-1 ectodomain was assessed in wound healing and neutrophil chemotaxis. Increases in human syndecan-1 are detected in lung homogenates and lavage fluid of IPF lungs. Syndecan-1 is also significantly elevated in the lavage fluid of EC-SOD-null mice after asbestos and bleomycin exposure. On IHC, syndecan-1 staining increases within fibrotic areas of human and mouse lungs. In vitro, EC-SOD inhibits oxidant-induced loss of syndecan-1 from A549 cells. Shed and exogenous syndecan-1 ectodomain induce neutrophil chemotaxis, inhibit alveolar epithelial wound healing, and promote fibrogenesis. Oxidative shedding of syndecan-1 is an underlying cause of neutrophil chemotaxis and aberrant wound healing that may contribute to pulmonary fibrosis.

Danazol Treatment for Telomere Diseases

BACKGROUND Genetic defects in telomere maintenance and repair cause bone marrow failure, liver cirrhosis, and pulmonary fibrosis, and they increase susceptibility to cancer. Historically, androgens have been useful as treatment for marrow failure syndromes. In tissue culture and animal models, sex hormones regulate expression of the telomerase gene. METHODS In a phase 1-2 prospective study involving patients with telomere diseases, we administered the synthetic sex hormone danazol orally at a dose of 800 mg per day for a total of 24 months. The goal of treatment was the attenuation of accelerated telomere attrition, and the primary efficacy end point was a 20% reduction in the annual rate of telomere attrition measured at 24 months. The occurrence of toxic effects of treatment was the primary safety end point. Hematologic response to treatment at various time points was the secondary efficacy end point. RESULTS After 27 patients were enrolled, the study was halted early, because telomere attrition was reduced in all 12 patients who could be evaluated for the primary end point; in the intention-to-treat analysis, 12 of 27 patients (44%; 95% confidence interval [CI], 26 to 64) met the primary efficacy end point. Unexpectedly, almost all the patients (11 of 12, 92%) had a gain in telomere length at 24 months as compared with baseline (mean increase, 386 bp [95% CI, 178 to 593]); in exploratory analyses, similar increases were observed at 6 months (16 of 21 patients; mean increase, 175 bp [95% CI, 79 to 271]) and 12 months (16 of 18 patients; mean increase, 360 bp [95% CI, 209 to 512]). Hematologic responses occurred in 19 of 24 patients (79%) who could be evaluated at 3 months and in 10 of 12 patients (83%) who could be evaluated at 24 months. Known adverse effects of danazol--elevated liver-enzyme levels and muscle cramps--of grade 2 or less occurred in 41% and 33% of the patients, respectively. CONCLUSIONS In our study, treatment with danazol led to telomere elongation in patients with telomere diseases. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT01441037.).

Abnormal lymphangiogenesis in idiopathic pulmonary fibrosis with insights into cellular and molecular mechanisms

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, debilitating respiratory disease whose pathogenesis is poorly understood. In IPF, the lung parenchyma undergoes extensive remodeling. We hypothesized that lymphangiogenesis is part of lung remodeling and sought to characterize pathways leading to lymphangiogenesis in IPF. We found that the diameter of lymphatic vessels in alveolar spaces in IPF lung tissue correlated with disease severity, suggesting that the alveolar microenvironment plays a role in the lymphangiogenic process. In bronchoalveolar lavage fluid (BALF) from subjects with IPF, we found short-fragment hyaluronic acid, which induced migration and proliferation of lymphatic endothelial cells (LECs), processes required for lymphatic vessel formation. To determine the origin of LECs in IPF, we isolated macrophages from the alveolar spaces; CD11b+ macrophages from subjects with IPF, but not those from healthy volunteers, formed lymphatic-like vessels in vitro. Our findings demonstrate that in the alveolar microenvironment of IPF, soluble factors such as short-fragment hyaluronic acid and cells such as CD11b+ macrophages contribute to lymphangiogenesis. These results improve our understanding of lymphangiogenesis and tissue remodeling in IPF and perhaps other fibrotic diseases as well.

Characterization and peripheral blood biomarker assessment of anti–Jo‐1 antibody–positive interstitial lung disease

OBJECTIVE Using a combination of clinical, radiographic, functional, and serum protein biomarker assessments, this study was aimed at defining the prevalence and clinical characteristics of interstitial lung disease (ILD) in a large cohort of patients with anti-Jo-1 antibodies. METHODS A review of clinical records, pulmonary function test results, and findings on imaging studies determined the existence of ILD in anti-Jo-1 antibody-positive individuals whose data were accumulated in the University of Pittsburgh Myositis Database from 1982 to 2007. Multiplex enzyme-linked immunosorbent assays (ELISAs) for serum inflammation markers, cytokines, chemokines, and matrix metalloproteinases in different patient subgroups were performed to assess the serum proteins associated with anti-Jo-1 antibody-positive ILD. RESULTS Among the 90 anti-Jo-1 antibody-positive individuals with sufficient clinical, radiographic, and/or pulmonary function data, 77 (86%) met the criteria for ILD. While computed tomography scans revealed a variety of patterns suggestive of underlying usual interstitial pneumonia (UIP) or nonspecific interstitial pneumonia, a review of the histopathologic abnormalities in a subset of patients undergoing open lung biopsy or transplantation or whose lung tissue was obtained at autopsy (n = 22) demonstrated a preponderance of UIP and diffuse alveolar damage. Analysis by multiplex ELISA yielded statistically significant associations between anti-Jo-1 antibody-positive ILD and elevated serum levels of C-reactive protein (CRP), CXCL9, and CXCL10, which distinguished this disease entity from idiopathic pulmonary fibrosis and anti-signal recognition particle antibody-positive myositis. Recursive partitioning further demonstrated that combinations of these and other serum protein biomarkers can distinguish these disease subgroups at high levels of sensitivity and specificity. CONCLUSION In this large cohort of anti-Jo-1 antibody-positive individuals, the incidence of ILD approached 90%. Multiplex ELISA demonstrated disease-specific associations between anti-Jo-1 antibody-positive ILD and serum levels of CRP as well as the interferon-gamma-inducible chemokines CXCL9 and CXCL10, highlighting the potential of this approach to define biologically active molecules contributing to the pathogenesis of myositis-associated ILD.

Peribronchiolar metaplasia: A common histologic lesion in diffuse lung disease and a rare cause of interstitial lung disease: Clinicopathologic features of 15 cases

Peribronchiolar metaplasia (PBM) is a histologic lesion consisting of peribronchiolar metaplasia (PBM) of bronchiolar-type epithelium. Although widely recognized, PBM has received little attention in the pathologic literature and is not known to have clinical significance. We identified 15 cases in which PBM was the only major histologic finding in surgical lung biopsies from patients with interstitial lung disease (PBM-ILD), and we reviewed the clinical, imaging, and pathologic findings. The mean age was 57 years (range, 44-74 years) with 13 females and 2 males. One patient had been a welder with fume and asbestos exposure; another had pigeon exposure. Smoking history was available for 13 patients: three current smokers, one cocaine user, two former smokers, and seven never smokers. Three patients had collagen vascular disease. One had elevated serum antinuclear antibody titers. Pulmonary function data were available for 10 patients: one obstructive, five restrictive, two mixed obstructive and restrictive, and two normal. Computerized tomography in 7 patients showed mosaic attenuation in 3 patients and air trapping in 1 patient; no bronchiectasis, septal lines, or honeycombing were seen in any cases. All 11 patients with available follow-up are alive; 4 of them have experienced symptomatic improvement (follow-up, 0.6-6.9 years; mean, 2.4 years). PBM was found focally in other interstitial lung diseases, which were assessed for this lesion: 59% of usual interstitial pneumonia (17 of 29), 50% of nonspecific interstitial pneumonia (10 of 20), desquamative interstitial pneumonia (3 of 6), hypersensitivity pneumonitis (9 of 18), and 11% of respiratory bronchiolitis (2 of 18). In summary, PBM is a common histologic finding in various interstitial lung disorders. It is rarely the sole major lung biopsy finding in patients presenting with interstitial lung disease (PBM-ILD). Patients are mostly older women, with mild symptoms and CT findings. Survival appears to be favorable.