Pulmonology
Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease. Recent findings have shown a marked metabolic reprogramming associated with changes in mitochondrial homeostasis and autophagy during pulmonary fibrosis. The microRNA-33 (miR-33) family of microRNAs (miRNAs) encoded within the introns of SREBP (sterol regulatory element binding protein) genes are master regulators of sterol and fatty acid (FA) metabolism. miR-33 controls macrophage immuno-metabolic response and enhances mitochondrial biogenesis, FA oxidation, and cholesterol efflux. Here, we show that miR-33 levels are increased in Broncho Alveolar Lavage (BAL) cells isolated from IPF patients compared to healthy controls. We demonstrate that specific genetic ablation of miR-33 in macrophages protects against bleomycin-induced pulmonary fibrosis. The absence of miR-33 in macrophages improves mitochondrial homeostasis and increases autophagy while decreasing inflammatory response after bleomycin injury. Notably, pharmacological inhibition of miR-33 in macrophages via administration of anti-miR-33 Peptide Nucleic Acids (PNA-33) attenuates fibrosis in different in vivo and ex vivo mice and human models of pulmonary fibrosis. Together, these studies elucidate a major role of miR-33 in macrophages in the regulation of pulmonary fibrosis and uncover a novel therapeutic approach to treat this disease.
Authors
Farida Ahangari, Nathan L. Price, Shipra Malik, Maurizio Chioccioli, Thomas Bärnthaler, Taylor S. Adams, Jooyoung Kim, Sai Pallavi Pradeep, Shuizi Ding, Carlos Cosme Jr, Kadi-Ann S. Rose, John E. McDonough, Nachelle R. Aurelien, Gabriel Ibarra, Norihito Omote, Jonas C. Schupp, Giuseppe DeIuliis, Julian A. Villalba Nunez, Lokesh Sharma, Changwan Ryu, Charles S. Dela Cruz, Xinran Liu, Antje Prasse, Ivan Rosas, Raman Bahal, Carlos Fernandez-Hernando, Naftali Kaminski
Abstract
In the progression phase of idiopathic pulmonary fibrosis (IPF) the normal alveolar structure of the lung is lost and replaced by remodeled fibrotic tissue and by bronchiolized cystic airspaces. Although these are characteristic features of IPF, knowledge of specific interactions between these pathological processes is limited. Here, the interaction of lung epithelial and lung mesenchymal cells was investigated in a co–culture model of human primary airway epithelial cells (EC) and lung fibroblasts (FB). Single–cell RNA sequencing (sc–RNA–seq) revealed that the starting EC population was heterogenous and enriched for cells with a basal cell signature. Furthermore, fractions of the initial EC and FB cell populations adopted distinct pro–fibrotic cell differentiation states upon co-cultivation, resembling specific cell populations that were previously identified in lungs of IPF patients. Transcriptomic analysis revealed active nuclear factor NF–kappa–B (NF–κB) signaling early in the co–cultured EC and FB cells and the identified NF–κB expression signatures were also found in “HAS1 High FB” and “PLIN2+ FB” populations from IPF patient lungs. Pharmacological blockade of NF–κB signaling attenuated specific phenotypic changes of EC and prevented FB–mediated interleukin–6 (IL6), interleukin–8 (IL–8) and C–X–C motif chemokine ligand 6 (CXCL6) cytokine secretion, as well as collagen alpha–1(I) chain (COL1A1) and alpha–smooth muscle actin (α–SMA) accumulation. Thus, we identified NF–κB as a potential mediator, linking epithelial pathobiology with fibrogenesis.
Authors
Patrick Sieber, Anny Schäfer, Raphael Lieberherr, Silvia L Caimi, Urs Lüthi, Jesper Ryge, Jan H. Bergmann, Francois Le Goff, Manuel Stritt, Peter Blattmann, Bérengère Renault, Patrick Rammelt, Bruno Sempere, Diego Freti, Rolf Studer, Eric S. White, Magdalena Birker-Robaczewska, Maxime Boucher, Oliver Nayler
Abstract
Keratin expression dynamically changes in airway basal cells (BCs) following acute and chronic injury, yet the functional consequences of these changes on BC behavior remain unknown. In Bronchiolitis Obliterans (BO) following lung transplantation, BC clonogenicity declines which is associated with a switch from Keratin15 (Krt15) to Keratin14 (Krt14). We investigated the roles of these keratins using Crispr-KO in vitro and in vivo and found that Krt14-KO and Krt15-KO produce contrasting phenotypes in terms of differentiation and clonogenicity. Primary mouse Krt14-KO BCs failed to differentiate into club and ciliated cells, but had enhanced clonogenicity. By contrast, Krt15-KO did not alter BC differentiation, but impaired clonogenicity in vitro and reduced the number of label-retaining BCs in vivo following injury. Krt14, but not Krt15, bound the tumor suppressor Stratifin (Sfn). Disruption of Krt14, but not of Krt15, reduced Sfn protein abundance and increased expression of the oncogene dNp63a during BC differentiation, while dNp63a levels were reduced in Krt15-KO BCs. Overall, the phenotype of Krt15-KO BCs contrasts that of Krt14-KO and resembles the phenotype in BO with decreased clonogenicity, increased Krt14 and decreased dNp63a expression. This work demonstrates that Krt14 and Krt15 functionally regulate BC behavior which is relevant in chronic disease states like BO.
Authors
Vitaly Ievlev, Thomas J. Lynch, Kyle W. Freischlag, Caitlyn B. Gries, Anit Shah, Albert C. Pai, Bethany A. Ahlers, Soo Yeun Park, John F. Engelhardt, Kalpaj R. Parekh
Abstract
Dysfunction of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, is implicated in pulmonary disease pathogenesis, highlighting the importance of human in vitro models. However, AEC2-like cells in culture have yet to be directly compared to their in vivo counterparts at single cell resolution. Here, we perform head-to-head comparisons between the transcriptomes of fresh primary (1o) adult human AEC2s, their cultured progeny, and human induced pluripotent stem cell-derived AEC2s (iAEC2s). We find each population occupies a distinct transcriptomic space with cultured AEC2s (1o and iAEC2s) exhibiting similarities to and differences from freshly purified 1o cells. Across each cell type, we find an inverse relationship between proliferative and maturation states, with pre-culture 1o AEC2s being most quiescent/mature and iAEC2s being most proliferative/least mature. Cultures of either type of human AEC2 do not generate detectable alveolar type 1 cells in these defined conditions; however, a subset of iAEC2s co-cultured with fibroblasts acquires a “transitional cell state” described in mice and humans to arise during fibrosis or following injury. Hence, we provide direct comparisons of the transcriptomic programs of 1o and engineered AEC2s, two in vitro models that can be harnessed to study human lung health and disease.
Authors
Konstantinos-Dionysios Alysandratos, Carolina Garcia-de-Alba, Changfu Yao, Patrizia Pessina, Jessie Huang, Carlos Villacorta-Martin, Olivia T. Hix, Kasey Minakin, Claire L. Burgess, Pushpinder Bawa, Aditi Murthy, Bindu Konda, Michael F. Beers, Barry R. Stripp, Carla F. Kim, Darrell N. Kotton
Abstract
Mitochondria are dynamic organelles responsible for energy production and many processes central to cellular function. Alterations in mitochondrial function is associated with human fibrotic lung diseases, including idiopathic pulmonary fibrosis (IPF). Pulmonary fibrosis is characterized by stiffening of the extracellular matrix (ECM). Fibroblasts migrate in the direction of greater stiffness, a phenomenon termed durotaxis. The mechanically guided fibroblast migration could be a crucial step in the progression of lung fibrosis. In this study, we identified mitochondria as an important mechanotransducer at the intersection between extracellular mechanical signals and durotactic lung fibroblast migration. Primary human lung fibroblasts sense increasing matrix stiffness with a change of mitochondrial dynamics in favor of mitochondrial fission and increased production of ATP. Mitochondria polarize in the direction of a physiologically relevant stiffness gradient, with conspicuous localization to the leading edge, primarily lamellipodia and filopodia, of migrating lung fibroblasts. Matrix stiffness-regulated mitochondrial fission and durotactic lung fibroblast migration are mediated by a DRP1/MFF-dependent pathway. Importantly, we found that the DRP1/MFF pathway is activated in fibrotic lung myofibroblasts in both human IPF and bleomycin-induced mouse lung fibrosis. Our findings suggest that energy-producing mitochondria need to be sectioned via fission and repositioned in durotactic lung fibroblasts to meet the higher energy demand. This represents a new mechanism through which mitochondria may contribute to the progression of fibrotic lung diseases. Inhibition of durotactic migration of lung fibroblasts may play an important role in preventing the progression of IPF.
Authors
Ting Guo, Chun-sun Jiang, Shan-Zhong Yang, Yi Zhu, Chao He, A. Brent Carter, Veena B. Antony, Hong Peng, Yong Zhou
Abstract
Clinical outcomes after lung transplantation, a life-saving therapy for patients with end-stage lung diseases, are limited by primary graft dysfunction (PGD). PGD is an early form of acute lung injury with no specific pharmacologic therapies. Here, we present a large multicenter study of plasma and bronchoalveolar lavage (BAL) samples collected on the first post-transplant day, a critical time for investigations of immune pathways related to PGD. We demonstrated that ligands for NKG2D receptors were increased in the BAL from participants who developed severe PGD and were associated with increased time to extubation, prolonged intensive care unit length of stay, and poor peak lung function. Neutrophil extracellular traps (NETs) were increased in PGD and correlated with BAL TNF-α and IFN-γ cytokines. Mechanistically, we found that airway epithelial cell NKG2D ligands were increased following hypoxic challenge. Natural killer (NK) cell killing of hypoxic airway epithelial cells was abrogated with NKG2D receptor blockade, and TNF-α and IFN-γ provoked neutrophils to release NETs in culture. Together, these data support an aberrant NK cell-neutrophil axis in human PGD pathogenesis. Early measurement of stress ligands and blockade of the NKG2D receptor hold promise for risk stratification and management of PGD.
Authors
Daniel R. Calabrese, Tasha Tsao, Mélia Magnen, Colin Valet, Ying Gao, Beñat Mallavia, Jennifer J. Tian, Emily A. Aminian, Kristin M. Wang, Avishai Shemesh, Elman B. Punzalan, Aartik Sarma, Carolyn S. Calfee, Stephanie A. Christenson, Charles R. Langelier, Steven R. Hays, Jeff A. Golden, Lorriana E. Leard, Mary E. Kleinhenz, Nicholas A. Kolaitis, Rupal J. Shah, Aida Venado, Lewis L. Lanier, John R. Greenland, David M. Sayah, Abbas Ardehali, Jasleen Kukreja, S. Sam Weigt, John A. Belperio, Jonathan P Singer, Mark R. Looney
Abstract
Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled alveolar inflammation. We hypothesize that specific enzymes of the glycolytic pathway could function as key regulators of alveolar inflammation. Therefore, we screened isolated alveolar epithelia from mice exposed to ALI induced by injurious ventilation to assess their metabolic responses. These studies pointed us towards a selective role for isoform 3 of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Pharmacologic inhibition or genetic deletion of Pfkfb3 in alveolar epithelia (Pfkfb3loxp/loxp SPC-ER-Cre+ mice) was associated with profound increases in ALI during injurious mechanical ventilation or acid installation. Studies in genetic models linked Pfkfb3 expression and function to hypoxia-inducible factor Hif1a. Intra-tracheal pyruvate instillation not only reconstituted Pfkfb3loxp/loxp or Hif1aloxp/loxp SPC ER Cre+ mice, but pyruvate was also effective in ALI treatment of wild-type mice. Finally, proof-of-principle studies in human lung biopsies confirmed increased PFKFB3 staining in injured lungs and co-localized PFKFB3 to alveolar epithelia. These studies reveal a specific role for PFKFB3 in counter-balancing alveolar inflammation and lay the groundwork for novel metabolic therapeutic approaches during ALI.
Authors
Christine U. Vohwinkel, Nana Burns, Ethan Coit, Xiaoyi Yuan, Eszter K. Vladar, Christina Sul, Eric P. Schmidt, Peter Carmeliet, Kurt Stenmark, Eva S. Nozik, Rubin M. Tuder, Holger K. Eltzschig
Abstract
The fluid covering the surface of airway epithelia represents a first barrier against pathogens. The chemical and physical properties of the airway surface fluid are controlled by the activity of ion channels and transporters. In cystic fibrosis (CF), loss of CFTR chloride channel function causes airway surface dehydration, bacterial infection, and inflammation. We investigated the effects of IL-17A plus TNF-α, two cytokines with a relevant role in CF and other chronic lung diseases. Transcriptome analysis revealed a profound change with upregulation of several genes involved in ion transport, anti-bacterial defense, and neutrophil recruitment. At the functional level, bronchial epithelia treated in vitro with the cytokine combination showed upregulation of ENaC sodium channel, ATP12A proton pump, ADRB2 beta-adrenergic receptor, and SLC26A4 anion exchanger. The overall result of IL-17A/TNF-α treatment was hyperviscosity of the airway surface as demonstrated by fluorescence recovery after photobleaching (FRAP) experiments. Importantly, stimulation with a beta-adrenergic agonist switched airway surface to a low viscosity state in non-CF but not in CF epithelia. Our study suggests that CF lung disease is sustained by a vicious cycle in which epithelia cannot exit from the hyperviscous state thus perpetuating the proinflammatory airway surface condition.
Authors
Daniela Guidone, Martina Buccirossi, Paolo Scudieri, Michele Genovese, Sergio Sarnataro, Rossella De Cegli, Federico Cresta, Vito Terlizzi, Gabrielle Planelles, Gilles Crambert, Isabelle Sermet-Gaudelus, Luis J.V. Galietta
Abstract
Intravenous administration of a high affinity carbon monoxide (CO)-binding molecule, recombinant neuroglobin, can improve survival in CO poisoning mouse models. The current study aims to understand how biochemical variables of the scavenger determine the CO removal from the RBCs by evaluating three readily available hemoproteins, 2,3-diphosphoglycerate stripped human hemoglobin (StHb), N-ethylmaleimide modified hemoglobin (NEMHb), and equine myoglobin (Mb). These molecules efficiently sequester CO from hemoglobin in erythrocytes in vitro. A kinetic model was developed to predict the CO binding efficacy for hemoproteins, based on their measured in vitro oxygen and CO binding affinities, suggesting that the therapeutic efficacy of hemoproteins for CO poisoning relates to a high M value, which is the binding affinity for CO relative to oxygen (KA,CO/KA,O2). In a lethal CO poisoning mouse model, StHb, NEMHb, and Mb improved survival by 100%, 100%, and 60%, respectively, compared with saline controls, and were well tolerated in 48-hour toxicology assessments. In conclusion, both StHb and NEMHb have high CO binding affinities and M values and scavenge CO efficiently in vitro and in vivo, highlighting their therapeutic potential for point-of-care antidotal therapy of CO poisoning.
Authors
Qinzi Xu, Jason J. Rose, Xiukai Chen, Ling Wang, Anthony W. DeMartino, Matthew R. Dent, Sagarika Tiwari, Kaitlin Bocian, Xueyin N. Huang, Qin Tong, Charles F. McTiernan, Lanping Guo, Elmira Alipour, Trevor C. Jones, Kamil Burak Ucer, Daniel B. Kim-Shapiro, Jesus Tejero, Mark T. Gladwin
Abstract
Pulmonary fibrosis is a chronic and progressive interstitial lung disease associated with the decay of pulmonary function leading to a fatal outcome. As an essential epigenetic regulator of DNA methylation, the involvement of Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) in fibroblast activation remains largely undefined in pulmonary fibrosis. In the present study, we found that growth factor–β1(TGF-β1)-mediated upregulation of UHRF1 repressed Beclin1 via its promoter methylation induction which finally results in fibroblast activation and lung fibrosis both in vitro and in vivo. Moreover, knockdown of UHRF1 significantly arrested fibroblast proliferation and reactivated Beclin 1 in lung fibroblasts. Henceforth, intravenous administration of UHRF1 siRNA-loaded liposomes significantly protected mice against experimental pulmonary fibrosis. Accordingly, our data suggested that UHRF1 might be a novel potential therapeutic target in the pathogenesis of pulmonary fibrosis.
Authors
Demin Cheng, Yue Wang, Ziwei Li, Haojie Xiong, Wenqing Sun, Sichuan Xi, Siyun Zhou, Yi Liu, Chunhui Ni
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