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
Chronic obstructive pulmonary disease (COPD), associated with cigarette smoke–induced (CS-induced) emphysema, contributes significantly to the global health care burden of disease. Although chronic kidney disease (CKD) may occur in patients with COPD, the relationship between COPD and CKD remains unclear. Using a murine model of experimental COPD, we show that chronic CS exposure resulted in marked kidney injury and fibrosis, as evidenced by histological and ultrastructural changes, altered macrophage subpopulations, and expression of tissue injury, fibrosis, and oxidative stress markers. CS induced mitochondrial dysfunction, and increased autophagic flux in kidney tissues and in kidney tubular epithelial (HK-2) cells, as determined by LC3B turnover assays. Mice heterozygous for Beclin-1 (Becn1+/–) were protected from the development of kidney tissue injury and renal fibrosis in response to CS exposure, and displayed impaired basal and inducible mitochondrial turnover by mitophagy. Interestingly, CS caused a reduction of Beclin-1 expression in mouse kidneys and kidney tubular epithelial cells, attributed to increased autophagy-dependent turnover of Beclin-1. These results suggest that Beclin-1 is required for CS-induced kidney injury and that reduced levels of Beclin-1 may confer renoprotection. These results identify the kidney as a target for CS-induced injury in COPD and the Beclin-1–dependent autophagy pathway as a potential therapeutic target in CKD.
Maria A. Pabón, Edwin Patino, Divya Bhatia, Jocelyn Quintero, Kevin C. Ma, Eli J. Finkelsztein, Juan C. Osorio, Faryal Malick, Francesca Polverino, Caroline A. Owen, Stefan W. Ryter, Augustine M.K. Choi, Suzanne M. Cloonan, Mary E. Choi
Pneumonia represents the leading infectious cause of death in the United States. Foxp3+ regulatory T cells promote recovery from severe pneumonia in mice, but T cell responses in patients with pneumonia remain incompletely characterized because of the limited ability to serially sample the distal airspaces and perform multidimensional molecular assessments on the small numbers of recovered cells. As T cell function is governed by their transcriptional and epigenetic landscape, we developed a method to safely perform high-resolution transcriptional and DNA methylation profiling of T cell subsets from the alveoli of critically ill patients. Our method involves nonbronchoscopic bronchoalveolar lavage combined with multiparameter fluorescence-activated cell sorting, unsupervised low-input RNA-sequencing, and a modified reduced-representation bisulfite sequencing protocol. Here, we demonstrate the safety and feasibility of our method and use it to validate functional genomic elements that were predicted by mouse models. Because of its potential for widespread application, our techniques allow unprecedented insights into the biology of human pneumonia.
James M. Walter, Kathryn A. Helmin, Hiam Abdala-Valencia, Richard G. Wunderink, Benjamin D. Singer
Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease causing fibrotic remodeling of the peripheral lung, leading to respiratory failure. Peripheral pulmonary epithelial cells lose normal alveolar epithelial gene expression patterns and variably express genes associated with diverse conducting airway epithelial cells, including basal cells. Single-cell RNA sequencing of pulmonary epithelial cells isolated from IPF lung tissue demonstrated altered expression of LncRNAs, including increased MEG3. MEG3 RNA was highly expressed in subsets of the atypical IPF epithelial cells and correlated with conducting airway epithelial gene expression patterns. Expression of MEG3 in human pulmonary epithelial cell lines increased basal cell–associated RNAs, including TP63, KRT14, STAT3, and YAP1, and enhanced cell migration, consistent with a role for MEG3 in regulating basal cell identity. MEG3 reduced expression of TP73, SOX2, and Notch-associated RNAs HES1 and HEY1, in primary human bronchial epithelial cells, demonstrating a role for MEG3 in the inhibition of genes influencing basal cell differentiation into club, ciliated, or goblet cells. MEG3 induced basal cell genes and suppressed genes associated with terminal differentiation of airway cells, supporting a role for MEG3 in regulation of basal progenitor cell functions, which may contribute to tissue remodeling in IPF.
Jason J. Gokey, John Snowball, Anusha Sridharan, Joseph P. Speth, Katharine E. Black, Lida P. Hariri, Anne-Karina T. Perl, Yan Xu, Jeffrey A. Whitsett
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