PPG: Pathophysiology of Acute Lung Injury

Patients with acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) have impaired gas (oxygen and carbon dioxide) exchange due to altered alveolar epithelial function which results in accumulation of edema fluid leading to hypoxia. Alveolar hypoxia is common in patients with ALI and ARDS and contributes to alveolar epithelial dysfunction. Seasonal influenza infection affects a significant proportion of the population in the US and worldwide and while most patients infected with influenza A recover without sequelae, in some patients influenza virus may cause severe pneumonitis and ARDS. Alveolar epithelial cells are targets for influenza virus A, and thus play an important role in mounting the host immune response. Upon influenza A virus infection, alveolar epithelial cells release cytokines that contribute to the recruitment of monocytes and macrophages to the site of infection and also participate in virus clearance and thus limit the infection from spreading. However, the underlying mechanisms of these events are not completely understood. We hypothesize that, in addition of its barrier function, the alveolar epithelium plays an important effector role in protecting the lung from injury.

The first aim of this grant proposal seeks to elucidate the mechanisms that lead to the stabilization of Na,K-ATPase plasma membrane levels during hypoxia as an adaptation to severe stress thus promoting cell survival. We will study whether the degradation of PKCζ, which triggers the down-regulation of Na,K-ATPase, by the E3 ligase HOIL-1L decreases alveolar epithelial cell death and thus lung injury during chronic hypoxia. HOIL-1L is a member of the Linear Ubiquitination Assembly Complex (LUBAC); however, we propose that during hypoxia HOIL-1L acts independently of LUBAC. In studies proposed for the second specific aim we will assess whether LUBAC participates in the modulation of the inflammatory signaling intensity in the alveolar epithelium during influenza virus infection. We seek to understand the role of LUBAC in increasing the efficiency of NF-κB activation and interferon production. The third specific aim examines the mechanisms by which increased intracellular sodium concentration which occurs during inhibition of the Na,K-ATPase, prevents viral replication in alveolar epithelial cells and whether pharmacologic inhibition of the Na,K-ATPase by cardiotonic steroids such as ouabain and digoxin represents a protective mechanism by inhibiting viral replication. Understanding the mechanism(s) that lead to alveolar epithelial dysfunction caused by hypoxia and influenza virus infection will provide novel information which is of clinical relevance and has the potential for novel approaches in the treatment of patients with acute lung injury.

Faculty Associated With Project 1

Jacob I. Sznajder Project, Leader 
Aaron Ciechanover, Co-Investigator 
Laura Dada, Co-Investigator
Emilia Lecuona, Co-Investigator
Susanne Herold, Co-Investigator

Influenza A virus is a highly contagious virus that causes upper and lower respiratory tract infections resulting in 200,000 hospitalizations and 36,000 deaths in the United States annually, and new influenza strains generate recurring epidemics and pandemics with significant attributable morbidity and mortality. Infection with influenza A virus can result in the development of acute respiratory distress syndrome (ARDS). Acute lung injury (ALI) and ARDS often manifest as part of an inflammatory process resulting in the development of diffuse alveolar damage, capillary injury, and exudation of protein-rich fluid into the alveolar space. We will test the hypothesis that vimentin is required for the activation of the NLRP3 inflammasome, which leads to the development of lung viral pneumonia. We provide preliminary data that vimentin−/− mice are protected from lung viral pneumonia following infection with influenza A virus. Increasing evidence from the field of intermediate filament biology and our group suggests that these filamentous cytoskeleton structures play key roles in signal transduction pathways and provide a scaffold for the formation and activation of protein complexes in the cell, such as the NRLP3 inflammasome. We show that NLRP3 interacts with vimentin and that this protein-protein interaction is required for the processing and maturation of pro-IL-1β into biologically active IL-1β. Additionally, we provide preliminary data that vimentin is required for the interaction and translocation of NOD2 to the outer mitochondrial membrane, which results in the NOD2-mediated activation of IRF3 and interferon in IAV infected cells. We hypothesis that vimentin acts as scaffold for the assembly and activation of the NLRP3 inflammasome and that NOD2 protein interaction with vimentin is required for the activation of IRF3 signaling. We have formulated three interrelated specific aims to study the regulation of vimentin intermediate filaments in both in vivo and in vitro models of lung vial pneumonia: Specific Aim 1: To determine the mechanism by which vimentin, a type III intermediate filaments protein, contributes to activation of NLR proteins during influenza A virus-induced acute lung injury. Specific Aim 2: To define the protein domain(s) in vimentin required for interaction and activation of the NLRP3 inflammasome. Specific Aim 3: To determine whether the interaction between vimentin and NOD2 is required for the activation of IRF3 and interferon in IAV infected cells.

Faculty Associated With Project 2

Karen Ridge, Project Leader
Robert Goldman, Co-Investigator
Christian Stehlik, Co-Investigator

Approximately 200,000 people in the United States develop acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) annually. Despite research driven advances in therapy, 30%-40% of these patients will die.  ALI and ARDS often manifest as a part of an inflammatory process characterized by acute failure of the alveolar-capillary membrane, which allows an exudate of plasma to flood the alveolar space.  Influenza is the leading cause of death from an infectious cause in the United States and is an important cause of ARDS. Current influenza therapies inhibit viral proteins to prevent either replication or spreading of the virus while other strategies target host anti-viral responses. However, the high genetic variability of the influenza virus results in variants that escape strain-specific adaptive immune responses or that are resistant to antiviral agents. Thus, targeting host cell proteins rather than viral proteins might prove to be a more effective strategy.  Metabolism is an understudied area in acute lung injury and because viral metabolism requires highly conserved host proteins rather than viral proteins, targeting metabolic enzymes might provide a novel and effective strategy to prevent influenza-induced lung injury. We conducted a large scale metabolomics screen in primary human lung epithelial cells following infection with influenza A virus.  The goal of this grant is to mechanistically determine the changes in metabolic pathways in epithelial cells that are essential for influenza virus induced lung injury. 

Faculty Associated With Project 3

Navdeep S Chandel, Project Leader 
Scott G. R. Budinger, Co-Investigator
Curt Horvath, Co-Investigator

The Administrative Core, designated as Core A, is housed in the McGaw building Mezzanine level, where the offices of the Pulmonary and Critical Care Medicine Division at Northwestern University, Feinberg School of Medicine are located. The Core organizes and supervises all the administrative aspects of the Program Project and provides leadership, coordination, and the necessary infrastructure to foster interactions between investigators.

Coordinated administrative services by Core A facilitate scientific interactions among the investigators involved in each project and core. The core organizes bi-weekly research seminars on Mondays, which will allow Program Project Grant investigators to present their "work in progress." This improves the overall effectiveness of this Program Project Grant and, importantly, ensures synergistic interactions among the researchers from diverse disciplines involved in this PPG. In addition to fostering frequent interaction, Core A provides the following resources to the investigators:

• Provide statistical and data processing support.
• Administer the use of histology and pathology cores.
• Ensure the PPG investigators meet the highest academic standards and its stated aims though periodic review by the internal and external review committees.
• Optimize utilization of shared supplies and services while maintaining quality and economies of scale.
• Develop an electronic file of reports, website, publications, and presentations of the PPG.
• Facilitate the use of common and core resources by all projects and cores.
• Centralize all administrative actions and financial reports.
• Prepare the scientific and financial reports required by Northwestern University and NHLBI.

Faculty

Jacob I. Sznajder, Core Leader
Anjana Yeldandi, Co-Investigator
Mary Kwasny, Co-Investigator

The Cell Culture and Translational Core, designated as Core B, is a centralized facility that provides primary alveolar epithelial cells and secondary alveolar epithelial cell lines to each of the projects within the program project. Centralization of the cell culture facilities ensures that a continuous supply of high quality and purity alveolar epithelial cells is available to each of the projects.

The Cell Culture Core B is responsible for the isolation and maintenance of primary cultures of human, rat, and mouse alveolar epithelial type 2 cells, rat alveolar epithelial type I cells, rat and mouse lung fibroblasts, rat and mouse alveolar and peritoneal macrophages, and rat lipofibroblasts. The Core uses standard techniques previously described by our group. The core purchase and propagate human, rat, and mouse alveolar epithelial cell lines. The purity of the cell cultures is verified using a variety of immunological and functional assays.

The Core B also provides rat and mouse precision-cut lung slices. Lung slices are provided using a Leica VT1000 S vibrating blade microtome vibratome and are viable up to 48 hours in culture.

The cell culture facility personnel have extensive experience in the isolation of all the different cell types as well as general cell culture techniques. The consolidation of the cell culture facilities provides an economical means of isolating and culturing lung cells.

Faculty Associated With Core B

Karen M Ridge , Core Leader
Emilia Lecuona, Co-Leader
Richard Wunderink, Co-Investigator

The purpose of this PPG is to determine how the alveolar epithelium alone and in coordination with inflammatory cells in the lung contributes to the development of acute lung injury. In the first cycles of this PPG, the project investigators made important discoveries, which have enhanced our understanding of the pathophysiology of acute lung injury. All of these discoveries were supported by murine models provided by this Core. Dr. Sznajder (Project 1) used mice genetically deficient in HOIL specifically in the alveolar epithelium to identify a critical role for LUBAC in the development of lung injury. Dr. Ridge (Project 2) used mice globally deficient in vimentin to show that this intermediate filament protein serves as a scaffold required for activation of the inflammasome. Dr. Chandel (Project 3) has used mice harboring cell specific knockouts of key metabolic proteins to demonstrate that alterations in metabolism play a causal role in the pathogenesis of ALI. In this renewal application, all of the project investigators have designed careful gain and loss-of-function experiments that use murine genetics to target putative genes/proteins/pathways involved in the development of acute lung injury in specific cells and tissues. To examine the effects of these interventions, the project investigators have identified a need for accurate breeding and genotyping of these murine strains and the performance of reliable, reproducible and complementary measurements to determine the severity of the resulting lung injury. To accomplish these goals, the murine phenotyping core will pursue three specific aims. 

• Aim 1. To perform breeding and genotyping of all of the murine strains, including the generation of tissue and cell type-specific knockout animals required by the project investigators.
• Aim 2. To provide investigators with several well-characterized models of acute lung injury.
• Aim 3. To provide quantitative, complementary measurements of lung inflammation, lung injury and lung regeneration following the induction of lung injury.

Support from the experienced personnel in the Core will provide resources that are unlikely to be achieved without support from the PPG mechanism. Careful attention to murine breeding techniques and rapid genotyping will accelerate the generation of tissue specific strains and reduce breeding costs. The highly reproducible murine model of influenza A pneumonia will provide a platform for the study of acute lung injury in each of the three projects. Findings in this model can be further examined in the other models of lung injury and fibrosis provided by the Core. Standardized, accurate, controlled and blinded measures of outcomes in the influenza A pneumonia and other models will allow investigators to compare the effects of their respective genes/pathways of interest in the same animal model opening new avenues for synergy.  

1. Misharin AV, Morales-Nebreda L, Mutlu GM, Budinger GRS, Perlman H. Flow Cytometric Analysis of the Macrophages and Dendritic Cell Subsets in the Mouse Lung. American Journal of Respiratory Cell and Molecular Biology 2013.

Faculty Associated With Core C

GR Scott Budinger, Core Leader
Gökhan Mutlu, Co-Leader