The main objective of our U19 proposal is to define the contribution of airway epithelial cells to age-related dysfunction of the tissue-resident immune system, particularly in the context of lung responses to viral infections. Indeed, age is a major risk factor for increased susceptibility to infectious diseases, including severe seasonal influenza virus infection. Recently, this risk was brought into even greater focus by the COVID-19 pandemic, with poor outcomes disproportionately affecting individuals over 65 years of age. While it is well documented that aging impacts both innate and adaptive immunity, the mechanisms underlying these effects, especially in lung tissue, are not well understood. In addition, little is known about how the aged airway epithelium contributes to functional immune alterations during respiratory virus infections. Based on our preliminary studies, we hypothesize that age-related epithelial changes contribute to chronic inflammation and altered tissue- resident lung immunity. This hypothesis directly builds upon our current U19 lung immunity research program that is advancing the concept that the lung epithelium, as the first site of respiratory virus infection and replication, acts as a critical pathogen barrier both as a modulator and an effector of the immune system. The workhorses of our program are primary human ex vivo air-liquid-interface (ALI) cultures, derived from airway epithelial progenitors, which closely resemble in vivo airway epithelial cell composition and responses to viruses. ALI cultures allow for 1) modelling, in time and space, of interactions between airway epithelium, pathogenic viruses (influenza and SARS-CoV-2), and immune cells; 2) genetically altering ALI genomes to study specific genes/pathways; 3) measuring the magnitude and kinetics of transcriptional responses to inflammatory insults that are difficult to measure in vivo; and 4) studying epigenetic regulation, RNA splicing, and impact of the microbiome in immune responses. Our findings will be validated using precision tissue slice assays from uninvolved lung tissue. We will also continue increasing the complexity of our 3D lung models, by incorporating alveolar space, integrating immune cells, and leveraging 3D bioprinting. To achieve our objective, we structured our Center around: two integrated research Projects focused on epigenetic, transcriptional, and alternative splicing mechanisms that we propose lead to a skewed isoform repertoire in aging lung epithelial cells and dysfunctional tissue-resident immunity; a Technology Development Project that will create sophisticated cellular models and gene editing tools to support research Project objectives; a Sample Core for storage and distribution of human tissues; and a Data Science Core for integrative analysis and data dissemination. The Center brings together clinicians and experts in lung immunology, bioengineering, genomics, and computational biology to maximize our scientific impact. An Administrative Core will provide coordination, communication, and oversight for the program. The proposed studies will enable us to uncover the molecular mechanisms underlying immune dysfunction in the lung of older adults, potentially identifying new targets for preventive intervention.
Age is a major risk factor for increased susceptibility to infectious diseases, including respiratory virus infection such as influenza and SARS-CoV-2. This Center will define the contribution of airway epithelial cells to age- related dysfunction of the tissue-resident immune system, particularly in the context of lung responses to viral infections.
These discoveries could form the basis for next-generation therapeutics to treat lung diseases, an outcome of great public health importance given the range of infectious and other age-related immune diseases that originate in, or secondarily impact, the lung.
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Our U19 Center consists of three Cores and three Projects:
ADMINISTRATIVE CORE
PALUCKA, ANNA KAROLINA, Core Lead
The goal of the Administrative (Admin) Core is to provide management, oversight and logistical support for the proposed Jackson Laboratory-Icahn School of Medicine at Mt. Sinai Collaborative Center on Human Immunology (JAX-ISMMS CCHI). The essential functions of the Admin Core are to: 1) facilitate communication and foster a strong collaborative environment among the Center Directors/Investigators; 2) establish the External Scientific Advisory Group (ESAG), coordinate annual ESAG meetings and implement their recommendations for improvement or advancement; 3) maintain alignment of the JAX-ISMMS CCHI with the goals and objectives of the broader CCHI program; 4) monitor the progress of the research and technology development Projects and Cores towards their specific research and resource development goals; 5) optimize resource allocation and fiscal management; 6) ensure swift resolution of any logistic, scientific and fiscal challenges; and 7) ensure efficient data sharing through ImmPort and policy compliance. JAX and ISMMS are home to several NIH-funded research centers and have a long history of successful contributions to NIH-funded consortium efforts. JAX and ISMMS also offer excellent capabilities, facilities and resources to support the Admin Core and the CCHI as a whole. The Admin Core will be led by Center Directors Drs. Karolina Palucka and Adolfo García-Sastre, both of whom have substantial experience in center leadership, and will be supported by an experienced and dedicated Program Manager. The Admin Core will promote the goals of the JAX-ISMMS CCHI and the broader program effort through the following Specific Aims: Aim 1. Optimize integration and function of JAX-ISMMS CCHI activities through strong administrative and scientific oversight. Aim 2. Enhance communication and collaboration among JAX-ISMMS CCHI investigators. Aim 3. Exercise oversight of data management and dissemination to ensure efficiency and policy compliance. Aim 4. Manage resource allocation and fiscal accountability.
DATA SCIENCE CORE
CHAUSSABEL, DAMIEN, Core Lead
The Jackson Laboratory-Icahn School of Medicine at Mt. Sinai Cooperative Center on Human Immunology (JAX- ISMMS CCHI) aims to study lung tissue-resident immunity and its decline in older individuals. To manage and analyze the large volumes of multimodal data generated across the different Projects and the Sample Core, a dedicated Data Science Core (DSC) will be established with three Specific Aims. First, we will capture, integrate, and disseminate JAX-ISMMS CCHI data (Aim 1). To this end, we will establish a central data repository following FAIR principles and coordinate with U19 investigators to ensure data interoperability and reusability. Data will be shared in compliance with NIH policies and our Data Management and Sharing Plan. Web applications will be developed for data access and visualization, and figure generation for presentations and publications. Critical for meeting the goal of the JAX-ISMMS through supporting hypothesis-driven Projects 1 and 2, we will identify multimodal molecular signatures and phenotypes associated with impaired airway epithelium response to viral infection as a function of age (Aim 2). We will develop and employ fixed module repertoires and perform integrative data analyses to identify age-related immune pathways in response to viral infection. Finally, we will collectively interpret signatures associated with age-related alteration in airway immune responsiveness and select candidates for downstream validation experiments (Aim 3). Workshops will be organized to facilitate collaborative interpretation of datasets generated by our U19 Center and to select candidates for targeted profiling and functional validation experiments. Dr. Damien Chaussabel, Principal Computational Scientist at JAX, will lead the DSC, supported by a team of Computational Scientists, a Bioinformatics Analyst, and a Data Wrangler. The DSC will work with the other Cores, research Projects, and the Technology Development Project to mine harmonized data across the eight JAX-ISMMS U19 geographical sites. In sum, the DSC’s activities are crucial for achieving the U19’s objective of identifying age-related epithelial cell pathways driving immune dysfunction in the lung.
SAMPLE CORE
PALUCKA, ANNA KAROLINA, Core Lead
The main function of the Sample Core is to provide Research Projects 1 and 2, and the Technology Development Project with human specimens to support the proposed research of this CCHI. The Core will manage the collection of coded, appropriately consented tissue samples and their distribution. The Core will critically intersect all Projects and Cores of the JAX-ISMMS CCHI program; as such, it will be an integral part of the program in the execution of the proposed research, as well as in the provision of ongoing sample and data management throughout the life of each Project. The Core will: 1) serve as a central resource for CCHI investigators to obtain appropriate lung, blood, bone marrow, microbiome (oral and nasopharyngeal samples for future follow-up studies) and associated demographic data for research purposes; 2) ensure that samples were acquired consistent with all applicable regulations for the use of human tissues in research, including human subjects consent; and 3) ensure that all specimens and associated information generated by each Project will be systematically cataloged, tracked, and stored. The Core will also generate and distribute air-liquid interface (ALI) cultures for experiments. The Core will draw on the extensive expertise of the Core Leader, Dr. Karolina Palucka, and her team in sample procurement and management; on the sample storage and data management infrastructure at The Jackson Laboratory for Genomic Medicine; and on the robust collaborations established with the CCHI’s clinical partners at the Hartford HealthCare Cancer Institute at Hartford Hospital (Andrew Salner, M.D., and Peter Yu, M.D.), Nationwide Children’s Hospital (Mark Peeples, Ph.D.), and Benaroya Research Institute (Carmen Mikacenic, M.D.). The Specific Aims of the Sample Core are: Aim 1: Oversee sample collection, acquisition, and processing; Aim 2: Manage sample storage, tracking, and distribution; Aim 3: Generate and distribute ALI cultures; and Aim 4: Oversee personnel training and compliance.
MODULATION OF LUNG IMMUNITY BY EPITHELIAL CELLS FROM OLDER INDIVIDUALS
PALUCKA, ANNA KAROLINA, Project Lead
The main objective of our U19 proposal is to define the contribution of airway epithelial cells to age-related dysfunction of the tissue-resident immune system, particularly in the context of lung responses to viral infections. Indeed, age is a risk factor for increased susceptibility to infectious diseases, and was most recently brought to focus by the COVID-19 pandemic, with poor outcomes disproportionately affecting individuals 65 years and older. Recently, we found that differentiated airway epithelial cells from older individuals have a baseline transcriptional program that is characterized by enhanced inflammation and is very distinct from young adult airway epithelium. Moreover, in response to influenza virus infection, young tissues induced a more potent interferon antiviral signature compared to old epithelium. Thus, we hypothesize that age-related epithelial changes contribute to chronic inflammation and altered tissue-resident lung immunity. We will leverage ex vivo air-liquid-interface (ALI) cultures, derived from airway epithelial progenitors, to study the effects of aging on airway epithelial viral responses and ensuing anti-viral immunity. We will determine how age impacts the response of broncho- epithelial cells (BECs) to virus, the fate of viral antigen, and how CD8+ T-cell immunity is modulated in aging tissues (Aim 1). Through mechanistic studies of antigen presentation and immune responses, coupled with deep analysis of epithelial-cell intrinsic mRNA isoform repertoire (Aim 2), our goal is to understand the molecular mechanisms that shape immunological status and early response to virus in epithelial cells and how their alterations in older individuals predispose to excessive inflammation and disease during viral infection. We will apply innovative technologies including 3D human lung tissues, functional studies based on precision-cut lung slices, spatial transcriptomics, as well as long-read RNA-sequencing (LR-seq) to address our hypothesis. Isoforms and functional endpoints will be analyzed in the context of the epigenetic status of ALI cultures influenced by the microbiome (Project 2) to identify genes and pathways dysregulated with aging. Integrative, multimodal data analyses will be carried out by the Data Science Core. Genes and isoforms selected during studies herein will be edited in Tech Dev Aim 3 in progenitor cells or induced pluripotent stem cells (iPSCs), thereby enabling functional validation in subsequent ALI cultures. Ultimately, our results will guide functional validation of a fully developed breathing lung model in Tech Dev Aim 1 for future studies of human lung immunology. The proposed studies will enable us to uncover the molecular mechanisms underlying immune dysfunction in the lung of older adults, potentially identifying new targets for preventive intervention.
MODULATION OF EPITHELIAL MEMORY BY THE MICROBIOME
Oh, JULIA S, Project Lead
The goal of Project 2 is to understand how age-related declines in lung immunity and antiviral response relate to corresponding changes in the epigenome and respiratory microbiome. Older adults have increased susceptibility to viral infection and adverse outcomes. Multiple factors contribute to this increased susceptibility, including changes in innate immune memory and consequent changes in interactions with commensal microbiota, which play an important role in the morphogenesis and maintenance of respiratory immunity. Numerous epigenetic changes have been associated with aging-related disease. Our overarching hypothesis is that age-related epigenetic changes in the lung epithelium are linked with an altered response to the commensal microbiome, leading to chronic inflammation and altered tissue immunity to respond to viral infection. In Project 1, we observed significant transcriptional differences in lung progenitors from older vs. younger individuals. We seek here to determine, using bulk and single-cell genome-wide chromatin accessibility assays of air-liquid interface (ALI) cultures developed from these older vs. younger lung progenitors, to what degree epigenetic modifications drive these transcriptional changes and which are associated with altered viral response (Aim 1). Then, we will investigate to what degree these aging-associated modifications affect interactions with microbial stimuli (Aim 2), stimulating old/young ALI cultures with genetically diverse microbes, examining phenotype and transcriptional response following colonization. Finally, we will investigate how the collective changes in epithelial immunity resulting from age and differential microbial response affect interactions with tissue-resident immune cells and collective influenza response (Aim 3), leveraging the Technology Development Project’s models integrating alveolar macrophages into ALI cultures. We anticipate that these data will pinpoint microbial and epigenetic mechanisms contributing to lung immune aging, and how in turn these changes affect viral response.
TECHNOLOGY DEVELOPMENT PROJECT – INCREASING THE COMPLEXITY OF EX VIVO HUMAN AIRWAY MODELS FOR STUDYING IMMUNE RESPONSE TO VIRAL INFRECTION
WILLIAMS, ADAM, Project Lead
The goal of the Technology Development (Tech Dev) Project is to develop approaches and tools that address the needs of The Jackson Laboratory-Icahn School of Medicine at Mt. Sinai Cooperative Center on Human Immunology (JAX-ISMMS CCHI) and that also advance the capabilities of the broader scientific community to tackle fundamental mechanistic questions regarding human lung immunity and human immune-epithelial cell interactions. Major questions related to lung immune function remain unanswered—such as the cell-to-cell interactions between immune and lung epithelial cells that shape responses to foreign agents, and how age- dependent changes in splicing and epigenetic modifications impact tissue immunity and inflammation. A significant technical barrier to studying human immune-lung dynamics is the sheer complexity of the human lung and the dynamic interactions between the lung epithelium and resident immune cells such as macrophages, dendritic cells, and T cells. This complexity cannot be easily modeled in animal systems or using deceased human lung tissue. To surmount these challenges, the Tech Dev Project will focus on three aspects to further increase complexity and utility of an innovative human tissue platform which we have pioneered in our current Center: establish three-dimensional (3D) bioprinted ventilated and vascularized models of the lung and upper respiratory environment for investigating the functional lung-immune interactome during exposure to viral agents (Aim 1); develop improved methods for viral infection and co-culture of immune cells in lung models (Aim 2); and develop methods for, and perform, editing of airway epithelial cells, virus, and commensal bacteria for inclusion in lung models (Aim 3). Epithelial editing will entail development of tools to alter isoform usage (for Project 1), epigenetic modifications (for Project 2), and generating conditional alleles in primary human airway epithelial cells and induced-pluripotent stem cells (iPSCs). For viral editing, we will engineer various reporter influenza viruses to enable tracking of active versus history of infection. For bacterial editing, we will alter production of candidate immune modulating metabolites from commensal bacteria (for Project 2) by perturbing their associated metabolic pathways either by using our CRISPRi toolkit or via overexpression. Each of these Aims addresses a specific unmet need for the Center and will enable us to study human lung immunity within a dynamic and physiologically relevant microenvironment and to interrogate specific cell types and molecular pathways predicted to respond to viral infections. Through these efforts, the JAX-ISMMS CCHI will be equipped to address previously inaccessible questions related to lung-immune dynamics, towards a more mechanistic understanding of lung immune function.
