Publications by authors named "Jacob I Sznajder"

148 Publications

TRAF2 Is a Novel Ubiquitin E3 Ligase for the Na,K-ATPase β-Subunit That Drives Alveolar Epithelial Dysfunction in Hypercapnia.

Front Cell Dev Biol 2021 2;9:689983. Epub 2021 Jul 2.

Member of the German Center for Lung Research (DZL), Department of Internal Medicine, Justus Liebig University Giessen, Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany.

Several acute and chronic lung diseases are associated with alveolar hypoventilation leading to accumulation of CO (hypercapnia). The β-subunit of the Na,K-ATPase plays a pivotal role in maintaining epithelial integrity by functioning as a cell adhesion molecule and regulating cell surface stability of the catalytic α-subunit of the transporter, thereby, maintaining optimal alveolar fluid balance. Here, we identified the E3 ubiquitin ligase for the Na,K-ATPase β-subunit, which promoted polyubiquitination, subsequent endocytosis and proteasomal degradation of the protein upon exposure of alveolar epithelial cells to elevated CO levels, thus impairing alveolar integrity. Ubiquitination of the Na,K-ATPase β-subunit required lysine 5 and 7 and mutating these residues (but not other lysines) prevented trafficking of Na,K-ATPase from the plasma membrane and stabilized the protein upon hypercapnia. Furthermore, ubiquitination of the Na,K-ATPase β-subunit was dependent on prior phosphorylation at serine 11 by protein kinase C (PKC)-ζ. Using a protein microarray, we identified the tumor necrosis factor receptor-associated factor 2 (TRAF2) as the E3 ligase driving ubiquitination of the Na,K-ATPase β-subunit upon hypercapnia. Of note, prevention of Na,K-ATPase β-subunit ubiquitination was necessary and sufficient to restore the formation of cell-cell junctions under hypercapnic conditions. These results suggest that a hypercapnic environment in the lung may lead to persistent epithelial dysfunction in affected patients. As such, the identification of the E3 ligase for the Na,K-ATPase may provide a novel therapeutic target, to be employed in patients with acute or chronic hypercapnic respiratory failure, aiming to restore alveolar epithelial integrity.
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http://dx.doi.org/10.3389/fcell.2021.689983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8283768PMC
July 2021

Transcriptional Profiling of Monocytes Deficient in Nuclear Orphan Receptors and Reveals Distinct Signalling Roles Related to Antigen Presentation and Viral Response.

Front Immunol 2021 25;12:676644. Epub 2021 Jun 25.

School of Medicine, University College Dublin, Dublin, Ireland.

The nuclear receptor sub-family 4 group A (NR4A) family are early response genes that encode proteins that are activated in several tissues/cells in response to a variety of stressors. The NR4A family comprises , and of which and are under researched and less understood, particularly in the context of immune cells. NR4A expression is associated with multiple diseases e.g. arthritis and atherosclerosis and the development of NR4A-targetting molecules as therapeutics is a current focus in this research field. Here, we use a combination of RNA-sequencing coupled with strategic bioinformatic analysis to investigate the down-stream effects of and in monocytes and dissect their common and distinct signalling roles. Our data reveals that and depletion has a robust and broad-reaching effect on transcription in both the unstimulated state and in the presence of LPS. Interestingly, many of the genes affected were present in both the unstimulated and stimulated states revealing a previously unappreciated role for the NR4As in unstimulated cells. Strategic clustering and bioinformatic analysis identified both distinct and common transcriptional roles for and in monocytes. notably was linked by both bioinformatic clustering analysis and transcription factor interactome analysis to pathways associated with antigen presentation and regulation of MHC genes. in contrast was more closely linked to pathways associated with viral response. Functional studies further support our data analysis pointing towards preferential/selective roles for in the regulation of antigen processing with common roles for and evident with respect to cell migration. Taken together this study provides novel mechanistic insights into the role of the enigmatic nuclear receptors NR4A2 and NR4A3 in monocytes.
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http://dx.doi.org/10.3389/fimmu.2021.676644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8267906PMC
June 2021

Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease.

J Membr Biol 2021 Jun 10. Epub 2021 Jun 10.

Department of Internal Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Justus Liebig University, Klinikstrasse 33, 35392, Giessen, Germany.

The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na and K ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:β-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions.
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http://dx.doi.org/10.1007/s00232-021-00184-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192048PMC
June 2021

Targeting the Linear Ubiquitin Assembly Complex to Modulate the Host Response and Improve Influenza A Virus Induced Lung Injury.

Arch Bronconeumol (Engl Ed) 2020 09 14;56(9):586-591. Epub 2020 Sep 14.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Northwestern University, United States.

Influenza virus infection is characterized by symptoms ranging from mild congestion and body aches to severe pulmonary edema and respiratory failure. While the majority of those exposed have minor symptoms and recover with little morbidity, an estimated 500,000 people succumb to IAV-related complications each year worldwide. In these severe cases, an exaggerated inflammatory response, known as "cytokine storm", occurs which results in damage to the respiratory epithelial barrier and development of acute respiratory distress syndrome (ARDS). Data from retrospective human studies as well as experimental animal models of influenza virus infection highlight the fine line between an excessive and an inadequate immune response, where the host response must balance viral clearance with exuberant inflammation. Current pharmacological modulators of inflammation, including corticosteroids and statins, have not been successful in improving outcomes during influenza virus infection. We have reported that the amplitude of the inflammatory response is regulated by Linear Ubiquitin Assembly Complex (LUBAC) activity and that dampening of LUBAC activity is protective during severe influenza virus infection. Therapeutic modulation of LUBAC activity may be crucial to improve outcomes during severe influenza virus infection, as it functions as a molecular rheostat of the host response. Here we review the evidence for modulating inflammation to ameliorate influenza virus infection-induced lung injury, data on current anti-inflammatory strategies, and potential new avenues to target viral inflammation and improve outcomes.
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http://dx.doi.org/10.1016/j.arbr.2020.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7489339PMC
September 2020

Resetting proteostasis with ISRIB promotes epithelial differentiation to attenuate pulmonary fibrosis.

Proc Natl Acad Sci U S A 2021 May;118(20)

Department of Medicine, Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611;

Pulmonary fibrosis is a relentlessly progressive and often fatal disease with a paucity of available therapies. Genetic evidence implicates disordered epithelial repair, which is normally achieved by the differentiation of small cuboidal alveolar type 2 (AT2) cells into large, flattened alveolar type 1 (AT1) cells as an initiating event in pulmonary fibrosis pathogenesis. Using models of pulmonary fibrosis in young adult and old mice and a model of adult alveologenesis after pneumonectomy, we show that administration of ISRIB, a small molecule that restores protein translation by EIF2B during activation of the integrated stress response (ISR), accelerated the differentiation of AT2 into AT1 cells. Accelerated epithelial repair reduced the recruitment of profibrotic monocyte-derived alveolar macrophages and ameliorated lung fibrosis. These findings suggest a dysfunctional role for the ISR in regeneration of the alveolar epithelium after injury with implications for therapy.
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http://dx.doi.org/10.1073/pnas.2101100118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8157939PMC
May 2021

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Am J Physiol Lung Cell Mol Physiol 2021 06 21;320(6):L1183-L1185. Epub 2021 Apr 21.

Northwestern University Feinberg School of Medicine, Chicago, Illinois.

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http://dx.doi.org/10.1152/ajplung.00170.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238444PMC
June 2021

Elevated CO modulates airway contractility.

Interface Focus 2021 Apr 12;11(2):20200021. Epub 2021 Feb 12.

Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL, USA.

Carbon dioxide (CO), a primary product of oxidative metabolism, can be sensed by eukaryotic cells eliciting unique responses via specific signalling pathways. Severe lung diseases such as chronic obstructive pulmonary disease are associated with hypoventilation that can lead to the elevation of CO levels in lung tissues and the bloodstream (hypercapnia). However, the pathophysiological effects of hypercapnia on the lungs and specific lung cells are incompletely understood. We have recently reported using combined unbiased molecular approaches with studies in mice and cell culture systems on the mechanisms by which hypercapnia alters airway smooth muscle contractility. In this review, we provide a pathophysiological and mechanistic perspective on the effects of hypercapnia on the lung airways and discuss the recent understanding of high CO modulation of the airway contractility.
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http://dx.doi.org/10.1098/rsfs.2020.0021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898145PMC
April 2021

The lung microenvironment shapes a dysfunctional response of alveolar macrophages in aging.

J Clin Invest 2021 Feb;131(4)

Department of Medicine, Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, Illinois, USA.

Alveolar macrophages orchestrate the response to viral infections. Age-related changes in these cells may underlie the differential severity of pneumonia in older patients. We performed an integrated analysis of single-cell RNA-Seq data that revealed homogenous age-related changes in the alveolar macrophage transcriptome in humans and mice. Using genetic lineage tracing with sequential injury, heterochronic adoptive transfer, and parabiosis, we found that the lung microenvironment drove an age-related resistance of alveolar macrophages to proliferation that persisted during influenza A viral infection. Ligand-receptor pair analysis localized these changes to the extracellular matrix, where hyaluronan was increased in aged animals and altered the proliferative response of bone marrow-derived macrophages to granulocyte macrophage colony-stimulating factor (GM-CSF). Our findings suggest that strategies targeting the aging lung microenvironment will be necessary to restore alveolar macrophage function in aging.
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http://dx.doi.org/10.1172/JCI140299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919859PMC
February 2021

Elevated CO Levels Delay Skeletal Muscle Repair by Increasing Fatty Acid Oxidation.

Front Physiol 2020 21;11:630910. Epub 2021 Jan 21.

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States.

Muscle dysfunction often occurs in patients with chronic obstructive pulmonary diseases (COPD) and affects ventilatory and non-ventilatory skeletal muscles. We have previously reported that hypercapnia (elevated CO levels) causes muscle atrophy through the activation of the AMPKα2-FoxO3a-MuRF1 pathway. In the present study, we investigated the effect of normoxic hypercapnia on skeletal muscle regeneration. We found that mouse C2C12 myoblasts exposed to elevated CO levels had decreased fusion index compared to myoblasts exposed to normal CO. Metabolic analyses of C2C12 myoblasts exposed to high CO showed increased oxidative phosphorylation due to increased fatty acid oxidation. We utilized the cardiotoxin-induced muscle injury model in mice exposed to normoxia and 10% CO for 21 days and observed that muscle regeneration was delayed. High CO-delayed differentiation in both mouse C2C12 myoblasts and skeletal muscle after injury and was restored to control levels when cells or mice were treated with a carnitine palmitoyltransfearse-1 (CPT1) inhibitor. Taken together, our data suggest that hypercapnia leads to changes in the metabolic activity of skeletal muscle cells, which results in impaired muscle regeneration and recovery after injury.
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http://dx.doi.org/10.3389/fphys.2020.630910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859333PMC
January 2021

Activation of p21 limits acute lung injury and induces early senescence after acid aspiration and mechanical ventilation.

Transl Res 2021 Jul 27;233:104-116. Epub 2021 Jan 27.

Instituto de Investigación Sanitaria del Principado de Asturias. Oviedo, Spain; Centro de Investigación Biomédica en Red (CIBER)-Enfermedades Respiratorias. Madrid, Spain; Instituto Universitario de Oncología del Principado de Asturias. Oviedo, Spain; Unidad de Cuidados Intensivos Cardiológicos. Hospital Universitario Central de Asturias. Oviedo, Spain; Departamento de Biología Funcional. Universidad de Oviedo. Oviedo, Spain. Electronic address:

The p53/p21 pathway is activated in response to cell stress. However, its role in acute lung injury has not been elucidated. Acute lung injury is associated with disruption of the alveolo-capillary barrier leading to acute respiratory distress syndrome (ARDS). Mechanical ventilation may be necessary to support gas exchange in patients with ARDS, however, high positive airway pressures can cause regional overdistension of alveolar units and aggravate lung injury. Here, we report that acute lung injury and alveolar overstretching activate the p53/p21 pathway to maintain homeostasis and avoid massive cell apoptosis. A systematic pooling of transcriptomic data from animal models of lung injury demonstrates the enrichment of specific p53- and p21-dependent gene signatures and a validated senescence profile. In a clinically relevant, murine model of acid aspiration and mechanical ventilation, we observed changes in the nuclear envelope and the underlying chromatin, DNA damage and activation of the Tp53/p21 pathway. Absence of Cdkn1a decreased the senescent response, but worsened lung injury due to increased cell apoptosis. Conversely, treatment with lopinavir and/or ritonavir led to Cdkn1a overexpression and ameliorated cell apoptosis and lung injury. The activation of these mechanisms was associated with early markers of senescence, including expression of senescence-related genes and increases in senescence-associated heterochromatin foci in alveolar cells. Autopsy samples from lungs of patients with ARDS revealed increased senescence-associated heterochromatin foci. Collectively, these results suggest that acute lung injury activates p53/p21 as an antiapoptotic mechanism to ameliorate damage, but with the side effect of induction of senescence.
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http://dx.doi.org/10.1016/j.trsl.2021.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7838583PMC
July 2021

Hypercapnia Regulates Gene Expression and Tissue Function.

Front Physiol 2020 20;11:598122. Epub 2020 Nov 20.

Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL, United States.

Carbon dioxide (CO) is produced in eukaryotic cells primarily during aerobic respiration, resulting in higher CO levels in mammalian tissues than those in the atmosphere. CO like other gaseous molecules such as oxygen and nitric oxide, is sensed by cells and contributes to cellular and organismal physiology. In humans, elevation of CO levels in tissues and the bloodstream (hypercapnia) occurs during impaired alveolar gas exchange in patients with severe acute and chronic lung diseases. Advances in understanding of the biology of high CO effects reveal that the changes in CO levels are sensed in cells resulting in specific tissue responses. There is accumulating evidence on the transcriptional response to elevated CO levels that alters gene expression and activates signaling pathways with consequences for cellular and tissue functions. The nature of hypercapnia-responsive transcriptional regulation is an emerging area of research, as the responses to hypercapnia in different cell types, tissues, and species are not fully understood. Here, we review the current understanding of hypercapnia effects on gene transcription and consequent cellular and tissue functions.
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http://dx.doi.org/10.3389/fphys.2020.598122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7715027PMC
November 2020

Hypercapnia: An Aggravating Factor in Asthma.

J Clin Med 2020 Oct 5;9(10). Epub 2020 Oct 5.

Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL 60611, USA.

Asthma is a common chronic respiratory disorder with relatively good outcomes in the majority of patients with appropriate maintenance therapy. However, in a small minority, patients can experience severe asthma with respiratory failure and hypercapnia, necessitating intensive care unit admission. Hypercapnia occurs due to alveolar hypoventilation and insufficient removal of carbon dioxide (CO) from the blood. Although mild hypercapnia is generally well tolerated in patients with asthma, there is accumulating evidence that elevated levels of CO can act as a gaso-signaling molecule, triggering deleterious effects in various organs such as the lung, skeletal muscles and the innate immune system. Here, we review recent advances on pathophysiological response to hypercapnia and discuss potential detrimental effects of hypercapnia in patients with asthma.
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http://dx.doi.org/10.3390/jcm9103207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599850PMC
October 2020

Epithelial cell-specific loss of function of causes a spontaneous COPD-like phenotype and up-regulates expression in mice.

Sci Adv 2020 08 14;6(33):eabb7238. Epub 2020 Aug 14.

Department of Surgery, College of Medicine and University of Illinois Cancer Center, University of Illinois at Chicago, Chicago, IL 60612, USA.

Cigarette smoking, the leading cause of chronic obstructive pulmonary disease (COPD), has been implicated as a risk factor for severe disease in patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we show that mice with lung epithelial cell-specific loss of function of , which we identified as a negative regulator of nuclear factor κB (NF-κB) signaling, spontaneously develop progressive age-related changes resembling COPD. Furthermore, loss of Miz1 up-regulates the expression of , the receptor for SARS-CoV-2. Concomitant partial loss of κ prevented the development of COPD-like phenotype in -deficient mice. Miz1 protein levels are reduced in the lungs from patients with COPD, and in the lungs of mice exposed to chronic cigarette smoke. Our data suggest that Miz1 down-regulation-induced sustained activation of NF-κB-dependent inflammation in the lung epithelium is sufficient to induce progressive lung and airway destruction that recapitulates features of COPD, with implications for COVID-19.
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http://dx.doi.org/10.1126/sciadv.abb7238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7428331PMC
August 2020

Impaired phagocytic function in CX3CR1 tissue-resident skeletal muscle macrophages prevents muscle recovery after influenza A virus-induced pneumonia in old mice.

Aging Cell 2020 09 28;19(9):e13180. Epub 2020 Jul 28.

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.

Skeletal muscle dysfunction in survivors of pneumonia disproportionately affects older individuals in whom it causes substantial morbidity. We found that skeletal muscle recovery was impaired in old compared with young mice after influenza A virus-induced pneumonia. In young mice, recovery of muscle loss was associated with expansion of tissue-resident skeletal muscle macrophages and downregulation of MHC II expression, followed by a proliferation of muscle satellite cells. These findings were absent in old mice and in mice deficient in Cx3cr1. Transcriptomic profiling of tissue-resident skeletal muscle macrophages from old compared with young mice showed downregulation of pathways associated with phagocytosis and proteostasis, and persistent upregulation of inflammatory pathways. Consistently, skeletal muscle macrophages from old mice failed to downregulate MHCII expression during recovery from influenza A virus-induced pneumonia and showed impaired phagocytic function in vitro. Like old animals, mice deficient in the phagocytic receptor Mertk showed no macrophage expansion, MHCII downregulation, or satellite cell proliferation and failed to recover skeletal muscle function after influenza A pneumonia. Our data suggest that a loss of phagocytic function in a CX3CR1 tissue-resident skeletal muscle macrophage population in old mice precludes satellite cell proliferation and recovery of skeletal muscle function after influenza A pneumonia.
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http://dx.doi.org/10.1111/acel.13180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587460PMC
September 2020

Targeting the Linear Ubiquitin Assembly Complex to Modulate the Host Response and Improve Influenza A Virus Induced Lung Injury.

Arch Bronconeumol (Engl Ed) 2020 Sep 13;56(9):586-591. Epub 2020 May 13.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Northwestern University, United States. Electronic address:

Influenza virus infection is characterized by symptoms ranging from mild congestion and body aches to severe pulmonary edema and respiratory failure. While the majority of those exposed have minor symptoms and recover with little morbidity, an estimated 500,000 people succumb to IAV-related complications each year worldwide. In these severe cases, an exaggerated inflammatory response, known as "cytokine storm", occurs which results in damage to the respiratory epithelial barrier and development of acute respiratory distress syndrome (ARDS). Data from retrospective human studies as well as experimental animal models of influenza virus infection highlight the fine line between an excessive and an inadequate immune response, where the host response must balance viral clearance with exuberant inflammation. Current pharmacological modulators of inflammation, including corticosteroids and statins, have not been successful in improving outcomes during influenza virus infection. We have reported that the amplitude of the inflammatory response is regulated by Linear Ubiquitin Assembly Complex (LUBAC) activity and that dampening of LUBAC activity is protective during severe influenza virus infection. Therapeutic modulation of LUBAC activity may be crucial to improve outcomes during severe influenza virus infection, as it functions as a molecular rheostat of the host response. Here we review the evidence for modulating inflammation to ameliorate influenza virus infection-induced lung injury, data on current anti-inflammatory strategies, and potential new avenues to target viral inflammation and improve outcomes.
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http://dx.doi.org/10.1016/j.arbres.2020.04.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218391PMC
September 2020

High CO Levels Impair Lung Wound Healing.

Am J Respir Cell Mol Biol 2020 08;63(2):244-254

Division of Pulmonary and Critical Care Medicine, and.

Delayed lung repair leads to alveolopleural fistulae, which are a major cause of morbidity after lung resections. We have reported that intrapleural hypercapnia is associated with delayed lung repair after lung resection. Here, we provide new evidence that hypercapnia delays wound closure of both large airway and alveolar epithelial cell monolayers because of inhibition of epithelial cell migration. Cell migration and airway epithelial wound closure were dependent on Rac1-GTPase activation, which was suppressed by hypercapnia directly through the upregulation of AMP kinase and indirectly through inhibition of injury-induced NF-κB-mediated CXCL12 (pleural CXC motif chemokine 12) release, respectively. Both these pathways were independently suppressed, because dominant negative AMP kinase rescued the effects of hypercapnia on Rac1-GTPase in uninjured resting cells, whereas proteasomal inhibition reversed the NF-κB-mediated CXCL12 release during injury. Constitutive overexpression of Rac1-GTPase rescued the effects of hypercapnia on both pathways as well as on wound healing. Similarly, exogenous recombinant CXCL12 reversed the effects of hypercapnia through Rac1-GTPase activation by its receptor, CXCR4. Moreover, transgenic murine recipients of orthotopic tracheal transplantation were protected from hypercapnia-induced inhibition of tracheal epithelial cell migration and wound repair. In patients undergoing lobectomy, we found inverse correlation between intrapleural carbon dioxide and pleural CXCL12 levels as well as between CXCL12 levels and alveolopleural leak. Accordingly, we provide first evidence that high carbon dioxide levels impair lung repair by inhibiting epithelial cell migration through two distinct pathways, which can be restored by recombinant CXCL12.
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http://dx.doi.org/10.1165/rcmb.2019-0354OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397765PMC
August 2020

Hypercapnia Impairs Na,K-ATPase Function by Inducing Endoplasmic Reticulum Retention of the β-Subunit of the Enzyme in Alveolar Epithelial Cells.

Int J Mol Sci 2020 Feb 21;21(4). Epub 2020 Feb 21.

Department of Internal Medicine, Justus Liebig University, Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), 35392 Giessen, Germany.

Alveolar edema, impaired alveolar fluid clearance, and elevated CO levels (hypercapnia) are hallmarks of the acute respiratory distress syndrome (ARDS). This study investigated how hypercapnia affects maturation of the Na,K-ATPase (NKA), a key membrane transporter, and a cell adhesion molecule involved in the resolution of alveolar edema in the endoplasmic reticulum (ER). Exposure of human alveolar epithelial cells to elevated CO concentrations caused a significant retention of NKA-β in the ER and, thus, decreased levels of the transporter in the Golgi apparatus. These effects were associated with a marked reduction of the plasma membrane (PM) abundance of the NKA-α/β complex as well as a decreased total and ouabain-sensitive ATPase activity. Furthermore, our study revealed that the ER-retained NKA-β subunits were only partially assembled with NKA α-subunits, which suggests that hypercapnia modifies the ER folding environment. Moreover, we observed that elevated CO levels decreased intracellular ATP production and increased ER protein and, particularly, NKA-β oxidation. Treatment with α-ketoglutaric acid (α-KG), which is a metabolite that has been shown to increase ATP levels and rescue mitochondrial function in hypercapnia-exposed cells, attenuated the deleterious effects of elevated CO concentrations and restored NKA PM abundance and function. Taken together, our findings provide new insights into the regulation of NKA in alveolar epithelial cells by elevated CO levels, which may lead to the development of new therapeutic approaches for patients with ARDS and hypercapnia.
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http://dx.doi.org/10.3390/ijms21041467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073107PMC
February 2020

Elevated CO regulates the Wnt signaling pathway in mammals, Drosophila melanogaster and Caenorhabditis elegans.

Sci Rep 2019 12 3;9(1):18251. Epub 2019 Dec 3.

Division of Pulmonary and Critical Care, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States of America.

Carbon dioxide (CO) is sensed by cells and can trigger signals to modify gene expression in different tissues leading to changes in organismal functions. Despite accumulating evidence that several pathways in various organisms are responsive to CO elevation (hypercapnia), it has yet to be elucidated how hypercapnia activates genes and signaling pathways, or whether they interact, are integrated, or are conserved across species. Here, we performed a large-scale transcriptomic study to explore the interaction/integration/conservation of hypercapnia-induced genomic responses in mammals (mice and humans) as well as invertebrates (Caenorhabditis elegans and Drosophila melanogaster). We found that hypercapnia activated genes that regulate Wnt signaling in mouse lungs and skeletal muscles in vivo and in several cell lines of different tissue origin. Hypercapnia-responsive Wnt pathway homologues were similarly observed in secondary analysis of available transcriptomic datasets of hypercapnia in a human bronchial cell line, flies and nematodes. Our data suggest the evolutionarily conserved role of high CO in regulating Wnt pathway genes.
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http://dx.doi.org/10.1038/s41598-019-54683-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890671PMC
December 2019

Linear ubiquitin assembly complex regulates lung epithelial-driven responses during influenza infection.

J Clin Invest 2020 03;130(3):1301-1314

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Northwestern University, Chicago, Illinois, USA.

Influenza A virus (IAV) is among the most common causes of pneumonia-related death worldwide. Pulmonary epithelial cells are the primary target for viral infection and replication and respond by releasing inflammatory mediators that recruit immune cells to mount the host response. Severe lung injury and death during IAV infection result from an exuberant host inflammatory response. The linear ubiquitin assembly complex (LUBAC), composed of SHARPIN, HOIL-1L, and HOIP, is a critical regulator of NF-κB-dependent inflammation. Using mice with lung epithelial-specific deletions of HOIL-1L or HOIP in a model of IAV infection, we provided evidence that, while a reduction in the inflammatory response was beneficial, ablation of the LUBAC-dependent lung epithelial-driven response worsened lung injury and increased mortality. Moreover, we described a mechanism for the upregulation of HOIL-1L in infected and noninfected cells triggered by the activation of type I IFN receptor and mediated by IRF1, which was maladaptive and contributed to hyperinflammation. Thus, we propose that lung epithelial LUBAC acts as a molecular rheostat that could be selectively targeted to modulate the immune response in patients with severe IAV-induced pneumonia.
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http://dx.doi.org/10.1172/JCI128368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269564PMC
March 2020

Cardiac glycosides decrease influenza virus replication by inhibiting cell protein translational machinery.

Am J Physiol Lung Cell Mol Physiol 2019 06 20;316(6):L1094-L1106. Epub 2019 Mar 20.

Division of Pulmonary and Critical Care, Feinberg School of Medicine, Northwestern University , Chicago, Illinois.

Cardiac glycosides (CGs) are used primarily for cardiac failure and have been reported to have other effects, including inhibition of viral replication. Here we set out to study mechanisms by which CGs as inhibitors of the Na-K-ATPase decrease influenza A virus (IAV) replication in the lungs. We found that CGs inhibit influenza virus replication in alveolar epithelial cells by decreasing intracellular potassium, which in turn inhibits protein translation, independently of viral entry, mRNA transcription, and protein degradation. These effects were independent of the Src signaling pathway and intracellular calcium concentration changes. We found that short-term treatment with ouabain prevented IAV replication without cytotoxicity. Rodents express a Na-K-ATPase-α1 resistant to CGs. Thus we utilized Na-K-ATPase-α-sensitive mice, infected them with high doses of influenza virus, and observed a modest survival benefit when treated with ouabain. In summary, we provide evidence that the inhibition of the Na-K-ATPase by CGs decreases influenza A viral replication by modulating the cell protein translational machinery and results in a modest survival benefit in mice.
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http://dx.doi.org/10.1152/ajplung.00173.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620673PMC
June 2019

Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis.

Am J Respir Crit Care Med 2019 06;199(12):1517-1536

4 Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois; and.

The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.
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http://dx.doi.org/10.1164/rccm.201712-2410OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580683PMC
June 2019

Influenza A Virus Infection Induces Muscle Wasting via IL-6 Regulation of the E3 Ubiquitin Ligase Atrogin-1.

J Immunol 2019 01 7;202(2):484-493. Epub 2018 Dec 7.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611;

Muscle dysfunction is common in patients with adult respiratory distress syndrome and is associated with morbidity that can persist for years after discharge. In a mouse model of severe influenza A pneumonia, we found the proinflammatory cytokine IL-6 was necessary for the development of muscle dysfunction. Treatment with a Food and Drug Administration-approved Ab antagonist to the IL-6R (tocilizumab) attenuated the severity of influenza A-induced muscle dysfunction. In cultured myotubes, IL-6 promoted muscle degradation via JAK/STAT, FOXO3a, and atrogin-1 upregulation. Consistent with these findings, and mice had attenuated muscle dysfunction following influenza infection. Our data suggest that inflammatory endocrine signals originating from the injured lung activate signaling pathways in the muscle that induce dysfunction. Inhibiting these pathways may limit morbidity in patients with influenza A pneumonia and adult respiratory distress syndrome.
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http://dx.doi.org/10.4049/jimmunol.1701433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324970PMC
January 2019

Inflammatory pathways are upregulated in the nasal epithelium in patients with idiopathic pulmonary fibrosis.

Respir Res 2018 Nov 26;19(1):233. Epub 2018 Nov 26.

Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Tlalpan 4502, CP 14080, Mexico City, Mexico.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive scarring of the lung parenchyma, leading to respiratory failure and death. High resolution computed tomography of the chest is often diagnostic for IPF, but its cost and the risk of radiation exposure limit its use as a screening tool even in patients at high risk for the disease. In patients with lung cancer, investigators have detected transcriptional signatures of disease in airway and nasal epithelial cells distal to the site of disease that are clinically useful as screening tools. Here we assessed the feasibility of distinguishing patients with IPF from age-matched controls through transcriptomic profiling of nasal epithelial curettage samples, which can be safely and repeatedly sampled over the course of a patient's illness. We recruited 10 patients with IPF and 23 age-matched healthy control subjects. Using 3' messenger RNA sequencing (mRNA-seq), we identified 224 differentially expressed genes, most of which were upregulated in patients with IPF compared with controls. Pathway enrichment analysis revealed upregulation of pathways related to immune response and inflammatory signaling in IPF patients compared with controls. These findings support the concept that fibrosis is associated with upregulation of inflammatory pathways across the respiratory epithelium with possible implications for disease detection and pathobiology.
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http://dx.doi.org/10.1186/s12931-018-0932-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6257973PMC
November 2018

Metformin Targets Mitochondrial Electron Transport to Reduce Air-Pollution-Induced Thrombosis.

Cell Metab 2019 02 11;29(2):335-347.e5. Epub 2018 Oct 11.

Department of Medicine and Pulmonary and Critical Care Medicine, Northwestern University, 240 E Huron Street, M300, Chicago, IL 60611, USA.

Urban particulate matter air pollution induces the release of pro-inflammatory cytokines including interleukin-6 (IL-6) from alveolar macrophages, resulting in an increase in thrombosis. Here, we report that metformin provides protection in this murine model. Treatment of mice with metformin or exposure of murine or human alveolar macrophages to metformin prevented the particulate matter-induced generation of complex III mitochondrial reactive oxygen species, which were necessary for the opening of calcium release-activated channels (CRAC) and release of IL-6. Targeted genetic deletion of electron transport or CRAC channels in alveolar macrophages in mice prevented particulate matter-induced acceleration of arterial thrombosis. These findings suggest metformin as a potential therapy to prevent some of the premature deaths attributable to air pollution exposure worldwide.
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http://dx.doi.org/10.1016/j.cmet.2018.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365216PMC
February 2019

Hypercapnia Alters Expression of Immune Response, Nucleosome Assembly and Lipid Metabolism Genes in Differentiated Human Bronchial Epithelial Cells.

Sci Rep 2018 09 10;8(1):13508. Epub 2018 Sep 10.

Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America.

Hypercapnia, the elevation of CO in blood and tissues, commonly occurs in severe acute and chronic respiratory diseases, and is associated with increased risk of mortality. Recent studies have shown that hypercapnia adversely affects innate immunity, host defense, lung edema clearance and cell proliferation. Airway epithelial dysfunction is a feature of advanced lung disease, but the effect of hypercapnia on airway epithelium is unknown. Thus, in the current study we examined the effect of normoxic hypercapnia (20% CO for 24 h) vs normocapnia (5% CO), on global gene expression in differentiated normal human airway epithelial cells. Gene expression was assessed on Affymetrix microarrays, and subjected to gene ontology analysis for biological process and cluster-network representation. We found that hypercapnia downregulated the expression of 183 genes and upregulated 126. Among these, major gene clusters linked to immune responses and nucleosome assembly were largely downregulated, while lipid metabolism genes were largely upregulated. The overwhelming majority of these genes were not previously known to be regulated by CO. These changes in gene expression indicate the potential for hypercapnia to impact bronchial epithelial cell function in ways that may contribute to poor clinical outcomes in patients with severe acute or advanced chronic lung diseases.
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http://dx.doi.org/10.1038/s41598-018-32008-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131151PMC
September 2018

Hypercapnia increases airway smooth muscle contractility via caspase-7-mediated miR-133a-RhoA signaling.

Sci Transl Med 2018 09;10(457)

Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL 60611, USA.

The elevation of carbon dioxide (CO) in tissues and the bloodstream (hypercapnia) occurs in patients with severe lung diseases, including chronic obstructive pulmonary disease (COPD). Whereas hypercapnia has been recognized as a marker of COPD severity, a role for hypercapnia in disease pathogenesis remains unclear. We provide evidence that CO acts as a signaling molecule in mouse and human airway smooth muscle cells. High CO activated calcium-calpain signaling and consequent smooth muscle cell contraction in mouse airway smooth muscle cells. The signaling was mediated by caspase-7-induced down-regulation of the microRNA-133a (miR-133a) and consequent up-regulation of Ras homolog family member A and myosin light-chain phosphorylation. Exposure of wild-type, but not caspase-7-null, mice to hypercapnia increased airway contraction and resistance. Deletion of the gene prevented hypercapnia-induced airway contractility, which was restored by lentiviral transfection of a miR-133a antagonist. In a cohort of patients with severe COPD, hypercapnic patients had higher airway resistance, which improved after correction of hypercapnia. Our data suggest a specific molecular mechanism by which the development of hypercapnia may drive COPD pathogenesis and progression.
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http://dx.doi.org/10.1126/scitranslmed.aat1662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889079PMC
September 2018

Ubiquitin-proteasome signaling in lung injury.

Transl Res 2018 08 23;198:29-39. Epub 2018 Apr 23.

Pulmonary and Critical Care Division, Northwestern Feinberg School of Medicine, Chicago, Illinois. Electronic address:

Cell homeostasis requires precise coordination of cellular proteins function. Ubiquitination is a post-translational modification that modulates protein half-life and function and is tightly regulated by ubiquitin E3 ligases and deubiquitinating enzymes. Lung injury can progress to acute respiratory distress syndrome that is characterized by an inflammatory response and disruption of the alveolocapillary barrier resulting in alveolar edema accumulation and hypoxemia. Ubiquitination plays an important role in the pathobiology of acute lung injury as it regulates the proteins modulating the alveolocapillary barrier and the inflammatory response. Better understanding of the signaling pathways regulated by ubiquitination may lead to novel therapeutic approaches by targeting specific elements of the ubiquitination pathways.
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http://dx.doi.org/10.1016/j.trsl.2018.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986356PMC
August 2018

Future Research Directions in Pneumonia. NHLBI Working Group Report.

Am J Respir Crit Care Med 2018 07;198(2):256-263

26 Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts.

Pneumonia is a complex pulmonary disease in need of new clinical approaches. Although triggered by a pathogen, pneumonia often results from dysregulations of host defense that likely precede infection. The coordinated activities of immune resistance and tissue resilience then dictate whether and how pneumonia progresses or resolves. Inadequate or inappropriate host responses lead to more severe outcomes such as acute respiratory distress syndrome and to organ dysfunction beyond the lungs and over extended time frames after pathogen clearance, some of which increase the risk for subsequent pneumonia. Improved understanding of such host responses will guide the development of novel approaches for preventing and curing pneumonia and for mitigating the subsequent pulmonary and extrapulmonary complications of pneumonia. The NHLBI assembled a working group of extramural investigators to prioritize avenues of host-directed pneumonia research that should yield novel approaches for interrupting the cycle of unhealthy decline caused by pneumonia. This report summarizes the working group's specific recommendations in the areas of pneumonia susceptibility, host response, and consequences. Overarching goals include the development of more host-focused clinical approaches for preventing and treating pneumonia, the generation of predictive tools (for pneumonia occurrence, severity, and outcome), and the elucidation of mechanisms mediating immune resistance and tissue resilience in the lung. Specific areas of research are highlighted as especially promising for making advances against pneumonia.
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http://dx.doi.org/10.1164/rccm.201801-0139WSDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058989PMC
July 2018