Neutrophils are abundant immune cells that are recruited from the circulation in response to inflammation, infection, and injury. In the lung and airways, neutrophils serve as critical innate immune effectors of the host defense against pathogens yet also drive serious and important diseases with enormous public health relevance. Pneumonia, the most common cause of death due to infection worldwide, is characterized by a severe neutrophilic alveolitis. Lung transplantation, the only life-prolonging option for many chronic lung diseases, is limited by primary graft dysfunction (PGD)—a form of sterile neutrophilic alveolitis that occurs in the days following the procedure and is a major driver of poor outcomes in lung transplant recipients. Asthma is among the most common chronic diseases worldwide and carries an enormous public health burden concentrated in patients whose disease is driven by neutrophilic airways inflammation. During the pathogenesis and clinical course of these diseases, tissue and blood neutrophils respond to cellular and soluble signals from the bone marrow, circulation, and local lung microenvironments. A major challenge in ascertaining specific mechanisms that link these signals to the maturation, activation, migration, survival, and function of lung neutrophils has been the limited availability of patient lung samples combined with technical limitations to assessing their phenotype and function. Investigators in the proposed Neu-Lung Consortium have pioneered robust human lung sample collection schema and technological advances in single-cell profiling, ex vivo functional assessment, and computational immunology that now allow us to identify mechanisms underlying human lung neutrophil immunobiology during lung inflammation, injury and repair. The Neu-Lung investigators have generated substantial published and preliminary data supporting the unifying hypothesis that cellular and humoral signals originating from the circulation and from specific niches within the lung microenvironment drive heterogeneous neutrophil phenotypes and functions that determine the balance between inflammation, injury, and repair in patients with neutrophilic alveolar and airways diseases. This hypothesis will be tested with the support of Administrative and Scientific Cores in two Projects that will leverage ongoing, robust research programs in pneumonia, PGD, and asthma to examine mechanistic contributors to neutrophil pathobiology in humans:
Project 1. To determine whether CD11b/TLR/MYD88/NFκB signaling and systemic and tissue-derived signals (CSF3 and type I and II IFN) regulate neutrophil activation, migration, and function in patients with severe pneumonia and PGD.
Project 2. To determine whether airway recruitment and mucosal programming of neutrophils in severe asthma, occurs in a T2, T1, and T17 endotype-specific manner, and leads to neutrophil inflammatory, immunomodulatory, and proteolytic activities that drive airway epithelial remodeling.
Neutrophils are abundant yet poorly understood immune cells that drive lung diseases with enormous public health relevance. We hypothesize that cellular and humoral signals originating from the circulation and from specific niches within the injured lung microenvironment drive heterogeneous neutrophil phenotypes and functions that determine the balance between inflammation, injury, and repair in patients with neutrophilic lung and airways diseases. The Neu-Lung Consortium brings together experts in fundamental immunology, respiratory disease, clinical medicine, and computational biology who will synergize to test mechanistic hypotheses of neutrophilic disease pathogenesis using cutting-edge technologies applied to human lung, airway, and blood samples obtained from well-phenotyped patients with pneumonia, primary lung allograft dysfunction, and asthma.
Visit our website at https://www.feinberg.northwestern.edu/sites/immunobiology/index.html
Our U19 Center consists of four Cores and two Projects:
ADMINISTRATIVE CORE
EISENBARTH, STEPHANIE CAROLINE, Core Lead
The goals of the Administrative Core, located at the Northwestern University Feinberg School of Medicine, are to provide coordination and integration of all Projects and Cores across Northwestern, National Jewish and University of Calgary, as well as dissemination of resources, primary data, and scientific results with the other Cooperative Centers on Human Immunology and to the public. The Administrative Core will accomplish these objectives as outlined in four specific aims: 1) To support communication between Project Investigators, Core Leaders, other Cooperative Centers on Human Immunology Sites, and NIAID Program Staff. In addition, Core A will organize quarterly executive committee hybrid meetings, host an annual in-person scientific meeting of our multidisciplinary investigators, and will host an annual external advisory board review. 2) To provide a structure for the distribution of common resources, and regulatory and budgetary oversight for Project Investigators and Core Leaders. 3) To disseminate the discoveries made by Neu-Lung Consortium Investigators through support of publications and presentations, and to implement sharing of materials, protocols and raw data to other institutions and investigators, including NIAID-directed public databases/repositories. 4) To foster an environment of collaborative interdisciplinary research and mentoring of students, post-doctoral fellows, and investigators. Core A will be led by Dr. Stephanie Eisenbarth, an accomplished immunologist with expertise in both innate and adaptive immunity, and the inaugural director of the new Center for Human Immunobiology (CHI) at Northwestern. Core A will be supported by the expertise of Dr. G. R. Scott Budinger, Chief of Pulmonary and Critical Care Medicine at Northwestern and CHI advisory board member, and an institutionally supported CHI Research Program Coordinator. The primary mission of the CHI is to foster collaboration and provide resources for immunology-focused investigators at Northwestern. As part of that mission, monthly faculty working groups have been formed to identify scientific synergy amongst CHI labs. This proposal is a result of the “Innate Immunity in the Lung” working group, which brings together immunologists with expertise in neutrophil biology, lung pathology, and immune disease modeling. Core A will continue to foster the interactions of this group by hosting monthly hybrid meetings, an annual in-person scientific meeting and oversight of this U19 if awarded. The CHI at Northwestern will provide important resources for this proposal if funded as the breadth of human immunology grows on campus and new initiatives to support trainees focused on human immunology are implemented.
CLINICAL PHENOTYPING and MOLECULAR INTEGRATION CORE
WALUNAS, THERESA L., Core Lead
Our innovative application seeks to understand the how heterogeneity neutrophil populations in the lung impact pneumonia and asthma development and outcomes by intersecting large scale clinical data collected during the course of care for people with severe pneumonia with high-dimensional genomic, transcriptomic and functional data. The overall goal of the Clinical Phenotyping and Molecular Integration (CPMI) Core is to curate patient samples at participating sites and develop and implement strategies that support phenotypic and mechanistic characterization of patient participants by integrating information from electronic health record (EHR) data, multi- omics, spatial-transcriptomics and neutrophil functional studies. This core, which will sit at the heart of the Neu- Lung project, will have sites at both Northwestern University (NU) and National Jewish (NJH), which will be responsible for development of datasets for the pneumonia and asthma samples, respectively. While the NU core will focus on a set of previously collected samples and associated clinical data, the team at NJ will collect samples from asthma patients during the course of Neu-Lung. Both sites in the CPMI will then apply a similar analysis pipeline 1) clean and curate clinical samples and data, 2) integrate clinical and molecular profiling data for each patient and 3) apply effective machine learning strategies to develop models of neutrophil behavior in the context of their lung disease focus area. The design of this core is based on the premise that data captured during the course of clinical care that provides a thorough description of the patient and their environment are essential for understanding the results of functional and multi-omic analyses and identifying clinically and mechanistically relevant subpopulations of patients. Achieving the goals of this project will require tight integration of clinical and molecular profiling data and the development of novel machine learning strategies that can translate a complex set of multi-dimensional datasets into interpretable and actionable information to support clinical care and mechanistic studies. In particular, we will leverage tailor-made algorithms for an integrative analysis of multiple omics data sets with clinical that enable modeling of the underlying gene regulatory networks. Datasets and metadata will be archived in the appropriate NIAID and/or NCBI archives and computational tools will be made available for broader use through a GitHub repository. In addition, we will develop web-based tools that will allow Neu-Lung researchers to interact with the study data repository and both browse and analyze the collected data. Aim 1: Curate clinical samples and metadata from patients with severe pneumonia and lung transplant recipients. Aim 2: Collect and curate clinical samples from patients with asthma. Aim 3: Implement a data integration-analysis-modeling pipeline and web-based data analysis portal and disseminate data and code developed during the Neu-Lung program.
NEUTROPHIL PHYSIOLOGY CORE
MULLER, WILLIAM, Core Lead
The Neutrophil Physiology Core provides critical services to both Projects 1 and 2. The multi-omics studies provided by the other cores will provide valuable phenotyping information about our patients. The Neutrophil Physiology Core will perform functional assays and provide quantitative data to show how their neutrophils (PMN) are actually performing their roles in the innate immune response. Close communication with the PIs of the two Projects will ensure that the appropriate assays are carried out for each patient sample depending on the clinical context, especially in case of limited samples. PMN will be isolated from peripheral blood and bronchoalveolar lavage (BAL) samples collected simultaneously from the enrolled patients. Isolated PMN will be subjected to a number of assays to determine their ability to adhere to and move across physiologically relevant substrates in response to appropriate inflammatory responses (adhesion to endothelial cells, adhesion to respiratory epithelial cells, transendothelial migration, chemotaxis through extracellular matrix), engage in appropriate effector function responses (phagocytosis of opsonized particles, phagocytosis and killing of bacteria currently infecting the host, ROS generation, secretion of cytokines, release of granule content, generation of regulatory extracellular vesicles (EVs) and production of antibacterial NETs), and to respond to infection and tissue adaptation with relevant changes in metabolism (oxidative reactions, glycolysis, mitochondrial activation). The core will also co-culture patient PMN with patient T cells and endothelial/epithelial cells in physiologic assays to assess whether and how proper intercellular communication is impacted by disease. A parallel arm of the core located at our site at National Jewish Hospital in Denver will carry out similar studies comparing blood, (BAL), and unique sputum samples from their patients. To ensure rigor and reproducibility, endothelial cells are harvested regularly from human umbilical cords and never used beyond passage two. This assures that phenomena are not unique to any particular endothelial cell line. Endothelial cells are treated with the relevant cytokines and checked by flow cytometry and/or immunofluorescence to verify that they are responding appropriately to each stimulus. Similarly, respiratory epithelial cells are harvested from bronchial brushings and cultured in physiological air-liquid interface conditions in early passages. Differentiation will be confirmed by detection of ciliation, mucus production and junctional proteins using imaging approaches. The Core Leads and staff will meet weekly to discuss any issues that might arise regarding assay or reagent reproducibility or workflow. They will meet more frequently should any problems arise. The Core PIs will meet regularly with the PIs of the other cores and projects to review progress toward completion of the scientific Aims.
TECHNOLOGY CORE
DULAI, PARAMBIR SINGH, Core Lead
An overarching goal of this proposal is to study neutrophil heterogeneity in the context of pulmonary infection and inflammation, and how neutrophil heterogeneity may influence injury and repair in both lung and airway diseases. To accomplish this complex matrix, we will need to provide ground truth assessments of neutrophil profiles to fully resolve their heterogeneity, alongside cohort level targeted assessments of neutrophil profiles linked back to patient phenotypes and disease outcomes. The Technology Core will support this by first defining compartmental (blood, airway, tissue) neutrophil heterogeneity through novel spatial biology approaches. Then we will apply a cascade approach to profiling by leveraging existing single-cell sequencing libraries with new spatial transcriptomics and proteomics datasets to guide targeted panel development and cohort level assessments of neutrophil heterogeneity. This will enable optimal and efficient integration of Projects 1 and 2 with Cores B and C to accomplish the overarching goal of this proposal – linking neutrophil heterogeneity to disease phenotypes and outcomes. The Technology Core will accomplish this through three specific aims: 1) Unbiased spatial transcriptomics and proteomics of lung and airway tissue will fully define tissue neutrophil heterogeneity in the context of tissue immune and epithelial heterogeneity across the spectrum of lung and airway disease. 2) Targeted compartmental immunophenotyping of neutrophils in the blood, airway, and pulmonary tissue at the cohort level will be accomplished through 10x Genomics Flex kit panels, multiparameter flow cytometry panels, and custom spatial plex panels, developed and guided by deep clinical and molecular phenotyping from Core B, physiology read outs from Core C, and unbiased spatial profiling. This will allow for deeper phenotyping and fully resolve associations between cohort level neutrophil heterogeneity and disease outcomes. 3) Spatial microbe and viral profiling, through custom probe technology, will be developed and applied to assess neutrophil-pathogen interactions and how this governs microenvironmental inflammation and/or fibrosis in Projects 1 and 2. Core D will be led by Parambir S. Dulai, Director of Precision Medicine and Spatial Biology in the Division of Gastroenterology and Hepatology at Northwestern University, who has successfully incorporated these approaches to study mucosal neutrophils and spatial immune-microbe interactions in colitis and applied this workflow for multi-center collaborative consortiums. Core D will be further supported by the expertise of Hiam Abdala-Valencia, Director of Integrative Genomics Metabolomics Core at Northwestern University, and Max Seibold, Director Computational Biology Program at University of Colorado.
NEUTROPHIL RESPONSES DRIVE DISEASES of the ALVEOLAR SPACE
MISHARIN, ALEXANDER, Project Lead
Lower respiratory tract infection (pneumonia) causes almost 80% of deaths due to infection. For many otherwise fatal lung diseases, lung transplantation is the only curative option yet carries a dismal 5-year survival of 50%. Primary graft dysfunction following lung transplantation is responsible for the bulk of the early mortality following lung transplantation and is the dominant risk factor for chronic lung allograft dysfunction, the major barrier to long term survival in lung transplant recipients. We and others determined that a profound neutrophilic alveolitis is central to both pneumonia and PGD, yet mechanisms underlying the pathobiology of neutrophilic lung injury in these important diseases remain unclear. At Northwestern, we have created robust research programs engaged in high-volume, serial bronchoalveolar lavage fluid, lung tissue, and blood sample collection from patients with pneumonia as part of routine clinical care as well as from donor lungs throughout the course of lung transplantation. Hence, we are well-positioned to test mechanistic questions about the activation, migration, and function of lung neutrophils in well-phenotyped patients with neutrophilic alveolitis. Historically, neutrophils have been viewed as a homogenous, single-function population; however, our preliminary data point to substantial heterogeneity in lung and blood neutrophils. For example, we identified heterogeneity in expression of the integrin CD11b, which we found serves as a negative regulator of inflammatory signaling. We also observed in preliminary data that type I and II interferons (IFNs) have different expression levels in the circulation versus in the alveolar space and that lung epithelial and endothelial cells are sources of the key neutrophil cytokine CSF3. Hence, we hypothesize that CD11b/TLR/MYD88/NFκB signaling and systemic and tissue-derived signals (CSF3 and type I and II IFN) regulate neutrophil activation, migration, and function in patients with severe pneumonia and PGD. To test our hypothesis in humans, we will perform systematic sampling and multidimensional assessment using single-cell sequencing and spatial profiling of neutrophils in the circulation and alveolar compartments of hundreds of patients with severe pneumonia and PGD. We will integrate these molecular datasets with ex vivo functional assays and deep clinical phenotypes. In Aim 1, we will determine whether CD11b/TLR/MYD88/NFκB signaling and type I and II IFN control neutrophil activation, migration, and function in patients with severe pneumonia and PGD. In Aim 2, we will determine whether CSF3 generated within the alveolus determines neutrophil maturation, activation, migration, and function and whether arginase- 1 and BAFF link persistent neutrophilia to adaptive immunity in patients with pneumonia and PGD. In both Aims, we will use cutting-edge computational strategies to test whether these pathways are associated with disease etiology, steroid responsiveness, age, and mortality in patients with severe pneumonia and PGD. We will address critical knowledge gaps by investigating neutrophil activation, migration, and function in patients with severe pneumonia and PGD with the goal of therapeutic targets for these devastating diseases.
NEUTROPHIL RESPONSES DRIVE SEVERE ASTHMA
SEIBOLD, MAX A, Project Lead
Asthma is the most common chronic lung disease worldwide. Importantly, over half of the $3.1B spent in the U.S. each year on asthma health care costs derive from less than 10% of patients, those who experience severe disease. Roughly, half of patients with severe asthma exhibit eosinophilic airway inflammation driven by type 2 (T2) cytokines (IL-4, IL-5, and IL-13). T2 inhibitor drugs are highly effective in blocking exacerbations among T2 patients with eosinophil-predominant disease. However, the remaining 50% with non-T2 disease are without treatment options and have poorly understood pathobiology. This represents the greatest unmet need in asthma. We have found non-T2 asthma includes those with interferon-γ-driven T1 and IL-17-driven T17 inflammation. A central feature of these T1 and T17 endotypes is persistent airway neutrophilia, accompanied by airway obstruction and remodeling. Moreover, we find that a subgroup of T2 patients also exhibit airway neutrophilia. At National Jewish, we have integrated translational research infrastructure into our high-volume clinical asthma program, allowing for the collection of airway epithelium and biopsies, bronchoalveolar lavage, sputum cells, and blood from patients with severe asthma. These biosamples collected from well-phenotyped patients provide the opportunity to evaluate mechanisms controlling the infiltration, activation, and function of airway neutrophils in severe asthma. Here, we challenge the notion that neutrophils are a static, homogeneous population, playing a simple pathogen response role in the airway. Rather our data suggest that neutrophil subsets could be differentially recruited to, and programmed by, the airways of T2, T1, and T17 asthmatics, through their expression of different combinations of neutrophil chemoattractant genes (CXCL1/3/8, C3), survival/maturation genes (CSF3), and activation genes (IFNG, IL17). At the same time, we find that neutrophils are potent producers of cytokines (e.g. IL-1α/β, IL-6) that induce airway remodeling and epithelial dysfunction. Therefore, we hypothesize that airway recruitment and mucosal programming of neutrophils in severe asthma occur in a T2, T1, and T17 endotype-specific manner, resulting in unique neutrophil inflammatory, immunomodulatory, and proteolytic activities that drive airway epithelial remodeling. To test this hypothesis, we will perform single-cell and spatial transcriptomic sequencing of circulating and airway neutrophils from well-phenotyped severe asthma patients. These in vivo molecular data will be paired with data from ex vivo functional assays and from the modeling of neutrophil-epithelial circuits using airway organoid cultures. In Aim 1 we will determine whether T2, T1, and T17 airway mucosal niches direct differential recruitment of circulating neutrophil heterogeneity through CXCL1/3/5/8, C5, and LTB4. In Aim 2 we will determine the role of T2, T1, and T17 airway mucosal programming factors (CSF3, IFN-γ, and IL-17) in the modulation of neutrophil activation and function. In Aim 3 we will determine neutrophil inflammatory, immunomodulatory, and protease activities, which lead to epithelial remodeling and dysfunction in T2, T1, and T17 airway mucosal niches.
