BACKGROUND. Matrix metalloprotease 9 (MMP-9) is associated with inflammation and lung remodeling in chronic obstructive pulmonary disease (COPD). We hypothesized that elevated circulating MMP-9 represents a potentially novel biomarker that identifies a subset of individuals with COPD with an inflammatory phenotype who are at increased risk for acute exacerbation (AECOPD). METHODS. We analyzed Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) and Genetic Epidemiology of COPD (COPDGene) cohorts for which baseline and prospective data were available. Elevated MMP-9 was defined based on >95th percentile plasma values from control (non-COPD) sample in SPIROMICS. COPD subjects were classified as having elevated or nonelevated MMP-9. Logistic, Poisson, and Kaplan-Meier analyses were used to identify associations with prospective AECOPD in both cohorts. RESULTS. Elevated MMP-9 was present in 95/1,053 (9%) of SPIROMICS and 41/140 (29%) of COPDGene participants with COPD. COPD subjects with elevated MMP-9 had a 13%–16% increased absolute risk for AECOPD and a higher median (interquartile range; IQR) annual AECOPD rate (0.33 [0–0.74] versus 0 [0–0.80] events/year and 0.9 [0.5–2] versus 0.5 [0–1.4] events/year for SPIROMICS and COPDGene, respectively). In adjusted models within each cohort, elevated MMP-9 was associated with increased odds (odds ratio [OR], 1.71; 95%CI, 1.00–2.90; and OR, 3.03; 95%CI, 1.02–9.01), frequency (incidence rate ratio [IRR], 1.45; 95%CI, 1.23–1.7; and IRR, 1.24; 95%CI, 1.03–1.49), and shorter time-to-first AECOPD (21.7 versus 31.7 months and 14 versus 21 months) in SPIROMICS and COPDGene, respectively. CONCLUSIONS. Elevated MMP-9 was independently associated with AECOPD risk in 2 well-characterized COPD cohorts. These findings provide evidence for MMP-9 as a prognostic biomarker and potential therapeutic target in COPD. TRIAL REGISTRATION. ClinicalTrials.gov: NCT01969344 (SPIROMICS) and NCT00608764 (COPDGene). FUNDING. This work was funded by K08 HL123940 to JMW; R01HL124233 to PJC; Merit Review I01 CX000911 to JLC; R01 (R01HL102371, R01HL126596) and VA Merit (I01BX001756) to AG. SPIROMICS (Subpopulations and Intermediate Outcomes in COPD Study) is funded by contracts from the NHLBI (HHSN268200900013C, HHSN268200900014C,HHSN268200900015C HHSN268200900016C, HHSN268200900017C, HHSN268200900018C, HHSN268200900019C, and HHSN268200900020C) and a grant from the NIH/NHLBI (U01 HL137880), and supplemented by contributions made through the Foundation for the NIH and the COPD Foundation from AstraZeneca/MedImmune; Bayer; Bellerophon Therapeutics; Boehringer-Ingelheim Pharmaceuticals Inc.; Chiesi Farmaceutici; Forest Research Institute Inc.; GlaxoSmithKline; Grifols Therapeutics Inc.; Ikaria Inc.; Novartis Pharmaceuticals Corporation; Nycomed GmbH; ProterixBio; Regeneron Pharmaceuticals Inc.; Sanofi; Sunovion; Takeda Pharmaceutical Company; and Theravance Biopharma and Mylan. COPDGene is funded by the NHLBI (R01 HL089897 and R01 HL089856) and by the COPD Foundation through contributions made to an Industry Advisory Board composed of AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Novartis, Pfizer, Siemens, and Sunovion.
J. Michael Wells, Margaret M. Parker, Robert A. Oster, Russ P. Bowler, Mark T. Dransfield, Surya P. Bhatt, Michael H. Cho, Victor Kim, Jeffrey L. Curtis, Fernando J. Martinez, Robert Paine III, Wanda O’Neal, Wassim W. Labaki, Robert J. Kaner, Igor Barjaktarevic, MeiLan K. Han, Edwin K. Silverman, James D. Crapo, R. Graham Barr, Prescott Woodruff, Peter J. Castaldi, Amit Gaggar, the SPIROMICS and COPDGene Investigators
Patients with severe, treatment-refractory asthma are at risk for death from acute exacerbations. The cytokine IL17A has been associated with airway inflammation in severe asthma, and novel therapeutic targets within this pathway are urgently needed. We recently showed that IL17A increases airway contractility by activating the procontractile GTPase RhoA. Here, we explore the therapeutic potential of targeting the RhoA pathway activated by IL17A by inhibiting RhoA guanine nucleotide exchange factors (RhoGEFs), intracellular activators of RhoA. We first used a ribosomal pulldown approach to profile mouse airway smooth muscle by qPCR and identified Arhgef12 as highly expressed among a panel of RhoGEFs. ARHGEF12 was also the most highly expressed RhoGEF in patients with asthma, as found by RNA sequencing. Tracheal rings from Arhgef12-KO mice and WT rings treated with a RhoGEF inhibitor had evidence of decreased contractility and RhoA activation in response to IL17A treatment. In a house dust mite model of allergic sensitization, Arhgef12-KO mice had decreased airway hyperresponsiveness without effects on airway inflammation. Taken together, our results show that Arhgef12 is necessary for IL17A-induced airway contractility and identify a therapeutic target for severe asthma.
Valerie Fong, Austin Hsu, Esther Wu, Agnieszka P. Looney, Previn Ganesan, Xin Ren, Dean Sheppard, Sarah A. Wicher, Michael A. Thompson, Rodney D. Britt Jr., Y.S. Prakash, Mallar Bhattacharya
Pulmonary fibrosis and emphysema are irreversible chronic events after inhalation injury. However, the mechanism(s) involved in their development remain poorly understood. Higher levels of plasma and lung heme have been recorded in acute lung injury associated with several insults. Here, we provide the molecular basis for heme-induced chronic lung injury. We found elevated plasma heme in chronic obstructive pulmonary disease (COPD) (GOLD stage 4) patients and also in a ferret model of COPD secondary to chronic cigarette smoke inhalation. Next, we developed a rodent model of chronic lung injury, where we exposed C57BL/6 mice to the halogen gas, bromine (Br2) (400 ppm, 30 minutes), and returned them to room air resulting in combined airway fibrosis and emphysematous phenotype, as indicated by high collagen deposition in the peribronchial spaces, increased lung hydroxyproline concentrations, and alveolar septal damage. These mice also had elevated pulmonary endoplasmic reticulum (ER) stress as seen in COPD patients; the pharmacological or genetic diminution of ER stress in mice attenuated Br2-induced lung changes. Finally, treating mice with the heme-scavenging protein, hemopexin, reduced plasma heme, ER stress, airway fibrosis, and emphysema. This is the first study to our knowledge to report elevated heme in COPD patients and establishes heme scavenging as a potential therapy after inhalation injury.
Saurabh Aggarwal, Israr Ahmad, Adam Lam, Matthew A. Carlisle, Changzhao Li, J. Michael Wells, S. Vamsee Raju, Mohammad Athar, Steven M. Rowe, Mark T. Dransfield, Sadis Matalon
Proton secretion mediated by ATP12A protein on the surface of the airway epithelium may contribute to cystic fibrosis (CF) lung disease by favoring bacterial infection and airway obstruction. We studied ATP12A in fresh bronchial samples and in cultured epithelial cells. In vivo, ATP12A expression was found almost exclusively at the apical side of nonciliated cells of airway epithelium and in submucosal glands, with much higher expression in CF samples. This could be due to bacterial infection and inflammation, since treating cultured cells with bacterial supernatants or with IL-4 (a cytokine that induces goblet cell hyperplasia) increased the expression of ATP12A in nonciliated cells. This observation was associated with upregulation and translocation of ATP1B1 protein from the basal to apical epithelial side, where it colocalizes with ATP12A. ATP12A function was evaluated by measuring the pH of the apical fluid in cultured epithelia. Under resting conditions, CF epithelia showed more acidic values. This abnormality was minimized by inhibiting ATP12A with ouabain. Following treatment with IL-4, ATP12A function was markedly increased, as indicated by strong acidification occurring under bicarbonate-free conditions. Our study reveals potentially novel aspects of ATP12A and remarks its importance as a possible therapeutic target in CF and other respiratory diseases.
Paolo Scudieri, Ilaria Musante, Emanuela Caci, Arianna Venturini, Patrizia Morelli, Christine Walter, Davide Tosi, Alessandro Palleschi, Pablo Martin-Vasallo, Isabelle Sermet-Gaudelus, Gabrielle Planelles, Gilles Crambert, Luis J.V. Galietta
Fibrosis is characterized by persistent deposition of extracellular matrix (ECM) by fibroblasts. Fibroblast mechanosensing of a stiffened ECM is hypothesized to drive the fibrotic program; however, the spatial distribution of ECM mechanics and their derangements in progressive fibrosis are poorly characterized. Importantly, fibrosis presents with significant histopathological heterogeneity at the microscale. Here, we report that fibroblastic foci (FF), the regions of active fibrogenesis in idiopathic pulmonary fibrosis (IPF), are surprisingly of similar modulus as normal lung parenchyma and are nonlinearly elastic. In vitro, provisional ECMs with mechanical properties similar to those of FF activate both normal and IPF patient–derived fibroblasts, whereas type I collagen ECMs with similar mechanical properties do not. This is mediated, in part, by αvβ3 integrin engagement and is augmented by loss of expression of Thy-1, which regulates αvβ3 integrin avidity for ECM. Thy-1 loss potentiates cell contractility-driven strain stiffening of provisional ECM in vitro and causes elevated αvβ3 integrin activation, increased fibrosis, and greater mortality following fibrotic lung injury in vivo. These data suggest a central role for αvβ3 integrin and provisional ECM in overriding mechanical cues that normally impose quiescent phenotypes, driving progressive fibrosis through physical stiffening of the fibrotic niche.
Vincent F. Fiore, Simon S. Wong, Coleen Tran, Chunting Tan, Wenwei Xu, Todd Sulchek, Eric S. White, James S. Hagood, Thomas H. Barker
Severe influenza (IAV) infection can develop into bronchopneumonia and edema, leading to acquired respiratory distress syndrome (ARDS) and pathophysiology. Underlying causes for pulmonary edema and aberrant fluid regulation largely remain unknown, particularly regarding the role of viral-mediated mechanisms. Herein, we show that distinct IAV strains reduced the functions of the epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane regulator (CFTR) in murine respiratory and alveolar epithelia in vivo, as assessed by measurements of nasal potential differences and single-cell electrophysiology. Reduced ion channel activity was distinctly limited to virally infected cells in vivo and not bystander uninfected lung epithelium. Multiple lines of evidence indicated ENaC and CFTR dysfunction during the acute infection period; however, only CFTR dysfunction persisted beyond the infection period. ENaC, CFTR, and Na,K-ATPase activities and protein levels were also reduced in virally infected human airway epithelial cells. Reduced ENaC and CFTR led to changes in airway surface liquid morphology of human tracheobronchial cultures and airways of IAV-infected mice. Pharmacologic correction of CFTR function ameliorated IAV-induced physiologic changes. These changes are consistent with mucous stasis and pulmonary edema; furthermore, they indicate that repurposing therapeutic interventions correcting CFTR dysfunction may be efficacious for treatment of IAV lung pathophysiology.
Jeffrey D. Brand, Ahmed Lazrak, John E. Trombley, Ren-Jay Shei, A. Timothy Adewale, Jennifer L. Tipper, Zhihong Yu, Amit R. Ashtekar, Steven M. Rowe, Sadis Matalon, Kevin S. Harrod
Sensitization to Aspergillus species is associated with allergic respiratory diseases. Allergen immunotherapy with nonstandardized Aspergillus extracts is commonly used as therapy in these patients. Unfortunately, no method exists to measure the relevant allergen protein content in diagnostic and therapeutic extracts. Thus, there is a critical need for Aspergillus extract standardization. We hypothesized that development of Aspergillus-specific human IgE mAbs would allow for the characterization of the relevant human allergenic epitopes among currently available commercial Aspergillus fumigatus extracts. Patients with allergic bronchopulmonary mycosis were recruited from Vanderbilt University Medical Center. IgE antibody–secreting B cells were grown and immortalized using human hybridoma techniques first described here. Twenty-six human Aspergillus-reactive IgE mAbs were used as capture and detection reagents to characterize the Aspergillus allergen content of commercial extracts. We found extreme variability in the specificity and quantity of their protein targets. Just 4 mAbs reacted with all available extracts, and only 1 of 4 extracts contained the major allergen Asp f 1. This degree of variability will almost certainly affect the efficacy of these reagents when used in diagnosis and treatment. Human IgE mAbs represent an innovative tool for the evaluation of relevant human allergenic epitopes, which may assist in future development and long-term standardization of mold extracts.
Mark A. Wurth, Azadeh Hadadianpour, Dennis J. Horvath, Jacob Daniel, Olivia Bogdan, Kasia Goleniewska, Anna Pomés, Robert G. Hamilton, R. Stokes Peebles Jr., Scott A. Smith
The acute respiratory distress syndrome (ARDS) causes an estimated 70,000 US deaths annually. Multiple pharmacologic interventions for ARDS have been tested and failed. An unmet need is a suitable laboratory human model to predictively assess emerging therapeutics on organ function in ARDS. We previously demonstrated that the small molecule BC1215 blocks actions of a proinflammatory E3 ligase–associated protein, FBXO3, to suppress NF-κB signaling in animal models of lung injury. Ex vivo lung perfusion (EVLP) is a clinical technique that maintains lung function for possible transplant after organ donation. We used human lungs unacceptable for transplant to model endotoxemic injury with EVLP for 6 hours. LPS infusion induced inflammatory injury with impaired oxygenation of pulmonary venous circulation. BC1215 treatment after LPS rescued oxygenation and decreased inflammatory cytokines in bronchoalveolar lavage. RNA sequencing transcriptomics from biopsies taken during EVLP revealed robust inflammatory gene induction by LPS with a strong signal for NF-κB–associated transcripts. BC1215 treatment reduced the LPS induction of genes associated with inflammatory and host defense gene responses by Gene Ontology (GOterm) and pathways analysis. BC1215 also significantly antagonized LPS-mediated NF-κB activity. EVLP may provide a unique human platform for preclinical study of chemical entities such as FBXO3 inhibitors on tissue physiology.
Nathaniel M. Weathington, Diana Álvarez, John Sembrat, Josiah Radder, Nayra Cárdenes, Kentaro Noda, Qiaoke Gong, Hesper Wong, Jay Kolls, Jonathan D’Cunha, Rama K. Mallampalli, Bill B. Chen, Mauricio Rojas
BACKGROUND. The PD-1–blocking antibody nivolumab persists in patients several weeks after the last infusion. However, no study has systematically evaluated the maximum duration that the antibody persists on T cells or the association between this duration and residual therapeutic efficacy or potential adverse events. METHODS. To define the duration of binding and residual efficacy of nivolumab after discontinuation, we developed a simplified strategy for T cell monitoring and used it to analyze T cells from peripheral blood from 11 non–small cell lung cancer patients previously treated with nivolumab. To determine the suitability of our method for other applications, we compared transcriptome profiles between nivolumab-bound and nivolumab-unbound CD8 T cells. We also applied T cell monitoring in 2 nivolumab-treated patients who developed progressive lung tumors during long-term follow-up. RESULTS. Prolonged nivolumab binding was detected more than 20 weeks after the last infusion, regardless of the total number of nivolumab infusions (2–15 doses) or type of subsequent treatment, in 9 of the 11 cases in which long-term monitoring was possible. Ki-67 positivity, a proliferation marker, in T cells decreased in patients with progressive disease. Transcriptome profiling identified the signals regulating activation of nivolumab-bound T cells, which may contribute to nivolumab resistance. In 2 patients who restarted nivolumab, T cell proliferation markers exhibited the opposite trend and correlated with clinical response. CONCLUSIONS. Although only a few samples were analyzed, our strategy of monitoring both nivolumab binding and Ki-67 in T cells might help determine residual efficacy under various types of concurrent or subsequent treatment. TRIAL REGISTRATION. University Hospital Medical Information Network Clinical Trials Registry, UMIN000024623. FUNDING. This work was supported by Japan Society for the Promotion of Science KAKENHI (JP17K16045, JP18H05282, and JP15K09220), Japan Agency for Medical Research and Development (JP17cm0106310, JP18cm0106335 and JP18cm059042), and Core Research for Evolutional Science and Technology (JPMJCR16G2).
Akio Osa, Takeshi Uenami, Shohei Koyama, Kosuke Fujimoto, Daisuke Okuzaki, Takayuki Takimoto, Haruhiko Hirata, Yukihiro Yano, Soichiro Yokota, Yuhei Kinehara, Yujiro Naito, Tomoyuki Otsuka, Masaki Kanazu, Muneyoshi Kuroyama, Masanari Hamaguchi, Taro Koba, Yu Futami, Mikako Ishijima, Yasuhiko Suga, Yuki Akazawa, Hirotomo Machiyama, Kota Iwahori, Hyota Takamatsu, Izumi Nagatomo, Yoshito Takeda, Hiroshi Kida, Esra A. Akbay, Peter S. Hammerman, Kwok-kin Wong, Glenn Dranoff, Masahide Mori, Takashi Kijima, Atsushi Kumanogoh
The pathogenetic mechanisms underlying the pathologic fibrosis in diseases such as idiopathic pulmonary fibrosis (IPF) are poorly understood. To identify genetic factors affecting susceptibility to IPF, we analyzed a murine genetic model of IPF in which a profibrotic cytokine (TGF-β1) was expressed in the lungs of 10 different inbred mouse strains. Surprisingly, the extent of TGF-β1–induced lung fibrosis was highly strain dependent. Haplotype-based computational genetic analysis and gene expression profiling of lung tissue obtained from fibrosis-susceptible and -resistant strains identified laminin α1 (Lama1) as a genetic modifier for susceptibility to IPF. Subsequent studies demonstrated that Lama1 plays an important role in multiple processes that affect the pulmonary response to lung injury and susceptibility to fibrosis, which include: macrophage activation, fibroblast proliferation, myofibroblast transformation, and the production of extracellular matrix. Also, Lama1 mRNA expression was significantly increased in lung tissue obtained from IPF patients. These studies identify Lama1 as the genetic modifier of TGF-β1 effector responses that significantly affects the development of pulmonary fibrosis.
Chang-Min Lee, Soo Jung Cho, Won-Kyung Cho, Jin Wook Park, Jae-Hyun Lee, Augustine M. Choi, Ivan O. Rosas, Ming Zheng, Gary Peltz, Chun Geun Lee, Jack A. Elias
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