Washington University

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY / ABSTRACT Acute kidney injury (AKI) is a common clinical disorder, with a total of more than 13 million people affected globally every year. It has a high associated morbidity and mortality, and there are currently no definitive treatments besides supportive care. One of the most common causes of AKI is ischemia reperfusion injury (IRI), characterized by acute tubular necrosis and intrarenal inflammation. Regulated cell death pathways have been increasingly implicated in the tubular injury and resulting inflammation of AKI, and necroptosis in particular plays a critical role during IRI. There are very few known host factors that regulate necroptosis. Preliminary data in this proposal has identified interferon-stimulated gene 15 (ISG15) as a novel regulator of necroptosis and inflammation. Mice lacking ISG15 display a complete susceptibility to AKI in an IRI model, which can be rescued by also eliminating the executioner of necroptosis, mixed-linage kinase domain like pseudokinase (MLKL). In addition, preliminary data suggest that ISG15 negatively regulates necroptosis in murine primary proximal tubule cells in vitro. The overall hypothesis is that ISG15 acts as a critical factor in the host response to renal IRI by regulating the magnitude and timing of necroptosis in proximal tubule cells and downstream inflammation in the kidney. In Aim 1, damage to the kidney epithelium and the magnitude and timing of necroptosis due to an 18-minute bilateral IRI in vivo will be determined, as well as the host inflammatory response in the kidney. In Aim 2, the cell type in which ISG15 is acting to limit injury will be identified, and the interaction of ISG15 with the key signaling complex of necroptosis will be determined. The overall objective of this proposal is to advance understanding of the role of cell death pathways during renal IRI and elucidate the novel role of ISG15 as a host protective factor regulating cell death. This information is critical to advancing our fundamental understanding of the pathophysiology of AKI and developing effective therapeutics. The proposed research and training plan will facilitate the applicant’s development of key knowledge and skills to become an independent physician-scientist. Dr. Deborah Lenschow, the sponsor of this work, has extensive experience studying disease pathogenesis and the innate immune response to tissue injury, and the co-sponsor Dr. Benjamin Humphreys has deep expertise in kidney biology, genomics, and injury. The institutional environment provides a rigorous and supportive intellectual atmosphere as well as collaborative experts in AKI and kidney injury pathogenesis. This fellowship will support the applicant in becoming an independent investigator and a practicing physician-scientist.

NIH Research Projects · FY 2025 · 2024-07

Abstract The overall goal of this research is to better understand how endogenous opioids control the analgesic properties of the central noradrenergic system. Endogenous opioid systems provide powerful inhibition of locus coeruleus noradrenergic neurons and acute activation of mu opioid receptors in the locus coeruleus is antinociceptive. Therefore, we hypothesized that this endogenous mu opioid receptor-mediated inhibition could be critical to how the locus coeruleus modulates pain. Using conditional knockout and rescue of locus coeruleus-mu opioid receptor signaling, we show that the presence of these receptors in locus coeruleus neurons following neuropathic injury can reverse the expression of mechanical allodynia and thermal hyperalgesia. However, it is it is unknown whether the locus coeruleus-mu opioid receptor system is differentially modulated in pain states. This research focuses on understanding the mechanisms by which endogenous opioids inhibit the locus coeruleus noradrenergic system to promote endogenous analgesia and how chronic neuropathic injury disrupts this system. The central hypothesis of this proposal is that loss of mu opioid receptor-mediated locus coeruleus inhibition following long-term neuropathic injury promotes and maintains chronic pain. The first aim of this proposal will use neurochemical and gene expression studies to determine how endogenous opioid ligand and receptor systems in the locus coeruleus evolve following long-term neuropathic injury. The second aim seeks to understand whether projections from the locus coeruleus to the medial prefrontal cortex are selectively disinhibited after injury. This aim seeks to determine whether opioid sensitivity in these neurons is decreased in chronic pain. To do so we will use a high-throughput calcium imaging assay and in vivo optogenetics test the function of locus coeruleus neurons that project to the medial prefrontal cortex. The final aim seeks to identify non-opioid strategies for inhibiting the locus coeruleus to discover new analgesic targets. These studies will define the role of locus coeruleus mu opioid receptors 1) in pain from neuropathic injury, 2) along projections to the medial prefrontal cortex, and 3) identify mechanisms to suppress pain-generating locus coeruleus activity. This information will be critical for translational research targeting the noradrenergic system in the treatment of pain and neuropsychiatric disorders.

NIH Research Projects · FY 2025 · 2024-07

Project Summary The 5-year survival rate for patients diagnosed with pancreatic ductal adenocarcinomas (PDACs) is 11%. This poor outcome is due to multiple factors, including rapid progression to metastatic disease, poor clinical responses to standard of care therapies, and no effective targeted or immunotherapeutic approaches1. The treatment refractory nature of PDAC is likely due in part to the profoundly fibrotic and immune suppressive tumor microenvironment (TME) that is a hallmark of this disease. Two major drivers of this TME include a dense fibrotic tumor stroma and a robust infiltration of tumor-supportive myeloid cells. PDAC contains phenotypically diverse cancer associated fibroblasts (CAFs) subsets. These subsets include myofibroblasts (myCAF), inflammatory fibroblasts and a small subset of antigen presenting CAFs. Recently, it has been proposed by some investigators that myCAFs may have tumor restraining properties, while other investigators have found myCAFs can promote tumor progression and treatment resistance. What is clear is that tumor restraining or tumor promoting features are likely phenotype and context dependent. We propose herein that cellular senescence may be distinguished by the tumor-promoting and restraining CAF subsets. Our overall hypothesis that: Stromal senescence plays a key role in driving tumor progression by altering tumor immune and ECM properties. To address this hypothesis, we will use state of the art biophysical and immunological techniques in human PDAC specimens, and state of the art genetically engineered mouse models for both PDAC and the study of senescent cells, to evaluate the following aims. Aim 1. Determine how biophysical properties of the extracellular matrix regulate the induction and function of senescent CAFs. Aim 2. Determine the impact of senescent CAFs on myeloid and dendritic cell driven immune surveillance. Aim 3. Determine the organ specific impact of senescent CAFs on metastatic progression. Significance: Understanding how the PDAC TME's regulate tumor immunity is critical to employing stromal modulatory therapy to enhance immunotherapeutics. This concept is central to these studies.

NIH Research Projects · FY 2025 · 2024-06

Project Summary/Abstract The human cerebral cortex supports extraordinary cognitive capacities, including unmatched social and technological complexity, elaborate cultural traditions, and language. It also plays a central role in diverse brain disorders that cause profound human suffering, including schizophrenia and Alzheimer's dementia, that may have only limited analogs in non-human species. One key adaptation is the dramatic expansion of the cortical sheet vs other primates, particularly in higher cognitive (association) areas. This expansion is not uniform, but it has proven challenging to accurately map the degree of expansion in different regions and to determine the relative contributions of expansion of evolutionarily conserved areas vs the emergence of new areas. Recent methodological advances make it feasible to generate substantially more accurate cortical expansion maps than heretofore possible. Evolutionary divergence in cortical organization presumably reflects changes in gene expression patterns responsible for the differentiation of cortical areas and the determination of areal size. Recent advances in spatially resolved single-cell transcriptomics have led to the discovery of hundreds of putative cell types whose diversity is a critical substrate of brain evolution. In this proposal both sets of advances will be leveraged to address both evolutionary expansion and transcriptomic cell type divergence in human vs macaque cortex. The proposed analyses will be empowered by active collaboration with multiple consortia, including the Non-human Primate Neuroimaging & Neuroanatomy Project (NHP-NNP), and the Human and Mammalian Brain Atlas (HMBA), a facet of the BRAIN Initiative Cell Atlas Network. State-of-the-art interspecies registration will be performed by integrating putative homologous regions, myelin maps, and resting state networks to derive a substantially more accurate map of the evolutionary expansion of the cortex in macaque vs human than has previously been reported. Comparing this map and other brain maps to patterns of transcription in humans and macaques will allow testing of whether (1) evolutionary expansion has a distinct transcriptional signature from other brain measures, (2) this signature is more evident in cell type distribution than in aggregate gene expression, and (3) human-enriched cell types (e.g., in layer 4) play a prominent role in cortical expansion and diversification. Accurately registered transcription and neuroimaging measures will provide new evidence for distinguishing evolutionally emergent areas from conserved areas that have undergone evolutionary expansion. Taken together, the proposed research will augment the translational potential of studies utilizing non-human primate models, enhance our understanding of the areal and cellular substrates of the complex behaviors, and provide new insights into possible mechanisms of primarily human mental and neurological disorders.

NSF Awards · FY 2024 · 2024-06

Recent advances in artificial intelligence (AI), perception, and decision-making theory have facilitated the development of autonomous agents (e.g., ground, and aerial robots) capable of collaborating on complex tasks such as delivery, search-and-rescue, transportation, and manufacturing, in unknown environments. To handle environmental uncertainty effectively, decision-making in multi-agent systems often relies on AI techniques, particularly reinforcement learning (RL). RL translates perceptual feedback and shared information into individual control decisions. However, RL-driven control strategies have been shown to be vulnerable to adversarial conditions, including small perceptual noise, compromised shared information, agent failures, and self-interested agents. Despite recent empirical efforts to evaluate and enhance the robustness of such strategies, obtaining theoretical guarantees remains an open problem and a crucial challenge for ensuring the safe deployment of AI-enabled cyber-physical systems (CPS). This project aims to tackle this challenge. Specifically, the project’s novelties are the design of certification, verification, and robust training algorithms for multi-agent RL (MARL) control policies. If successful, this project will establish the scientific underpinnings for certifiable robust AI-enabled CPS making them suitable for safety-critical applications in potentially adversarial environments. The broader impact encompasses societal applications, facilitating the deployment of CPS in disaster relief, manufacturing, and surveillance scenarios. Additionally, the project will contribute towards educational initiatives spanning K-12 through graduate-level. To achieve this research goal, three tightly coupled research directions are being pursued. The first objective focuses on training robust MARL control policies and theoretically certifying their robustness against adversarial high-dimensional sensory inputs. The second one focuses on assessing and enhancing the robustness of MARL control policies against adversarial communications and unexpected agent failures/removals. Extending these ideas to non-cooperative settings, the third research direction is explores the existence and computation of robust equilibria. Developing these fundamental analysis and synthesis capabilities for CPS necessitates designing new theoretical results drawing from formal methods, machine learning, and control theory. These research findings will be demonstrated on physical CPS testbeds and photorealistic simulators, using aerial and ground robot platforms, with particular emphasis on manufacturing and disaster relief applications. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-06

Project summary/Abstract Motile cilia are essential for lung defense, as evidenced by the genetic syndrome primary ciliary dyskinesia (PCD). PCD is characterized by impaired motile cilia resulting in respiratory distress at birth, followed by chronic sinopulmonary infection and bronchiectasis, which can lead to respiratory failure. There are no specific therapies for PCD, in part because key pathways for motile cilia biogenesis and pathogenesis are not defined. PCD has been linked to mutations in nearly 50 genes, which belong to three main groups: 1) Those encoding axonemal dynein proteins - the motors necessary for cilia beating, 2) axonemal structural proteins, and 3) cytoplasmic assembly and chaperon proteins. We have found that cells with variants in PCD genes have increased expression of genes related to cell stress, including cytokines and interleukins. To better define the expression profile of these cells, we used single cell RNA sequencing, and identified unique transcriptional changes in ciliated cells from PCD patients that differed from those from healthy individuals. We identified activation of the KEAP1-NRF2 pathway and differential gene expression of NRF2 target genes in PCD cells. The NRF2 pathway is a major regulator of stress in cells. We also identified GSTA2 as a novel cilia axonemal protein that was increased in PCD cells. GSTA2 is an enzyme that belongs to the glutathione pathway which is important for protection from toxins and oxidative stress. This finding indicate that cilia have a dedicated glutathione pathway. We hypothesize that GSTA2 plays a critical role in maintaining cilia function in health and disease, protecting the ciliated cells from endogenous and exogenous factors. The exact function and mechanism of GSTA2 and the NRF2 pathway in ciliated cells will be tested through the following Specific Aims: (1) Characterize and test the requirement for a glutathione system in normal and PCD cilia; (2) Determine the role of the KEAP1- NRF2 transcriptional program in the homeostasis of motile cilia and its activation in PCD. We will leverage primary culture nasal cells from patients with mutations in different classes of PCD genes seen at our PCD clinic. We will use advanced microscopy including expansion microscopy of native and tagged GSTA2 to determine the localization of GSTA2. To define the role of the NRF2 pathway in ciliated cells and PCD, we will determine the effects of pathway inhibition and activation in ciliated cells and measure changes in oxidative burden in cells. We will use transcriptomic and proteomic analysis to investigate these novel candidates and define responses to treatment. Finally, we will test these pathways using in vivo PCD models. Findings generated through the proposed studies will provide fundamental understanding of motile cilia function and provide novel therapeutic targets for PCD and motile cilia disease.

NSF Awards · FY 2024 · 2024-06

This doctoral dissertation award focuses on the "Goosefoot" plant, a crop which was domesticated at least 3,500 years ago, but is now extinct. This project analyzes ancient goosefoot seeds which were recovered from archaeological sites. The student will also study current day modern wild populations of the species. This research addresses not only locally specific questions about the original domestication process, but also serves as a case study for assessing how annual plants that exhibit plasticity (flexibility in growth and development) are domesticated. Recently, there have been discussions about the possibility of bringing goosefoot back as a crop or using it to genetically fortify quinoa, which could lead to a new variety of plants resilient to climate change challenges. At a theoretical level multi-disciplinary analysis will speak to broader disciplines concerned with plant domestication and evolutionary theory. New findings about the variability of seeds produced by modern, wild goosefoot populations complicates current theories about goosefoot domestication. Recent research establishes that variability in seed coat thickness of wild goosefoot is more common than previously assumed, and this is a key trait for understanding domestication of annual seed crops. This project is reframing the current narrative of goosefoot domestication by reanalyzing ancient goosefoot assemblages using the framework of adaptive transgenerational plasticity: the idea that parent plants can shape characteristics of their seeds in response to the environment, providing them with better odds of survival. The researchers are also studying the size and shape of ancient goosefoot seeds from several regions to understand if there were distinct local varieties of goosefoot in ancient times. They examine the size and shape of modern, wild goosefoot seeds for comparison, and collect seeds for future growth experiments. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-06

Opiates have been widely prescribed for pain management in the US, with a staggering 142 million prescriptions in 2020 alone. While these drugs effectively relieve pain by interacting with the mu opioid receptor (MOR), their complex pharmacology has led to concerning side effects such as abuse potential, respiratory depression, and addiction risks. In fact, opioid overdose has become a leading cause of death in the US, with more than 100,000 reported deaths in 2021. Consequently, there is a growing need to explore alternative treatments for pain and related conditions. One highly promising therapeutic target is the kappa opioid receptor (KOR), which not only addresses pain but also offers potential benefits in tackling affective disorders and addiction. This proposal aims to identify the mechanisms and regulators that control KOR signaling and potentially overcome the limitations and side effects associated with traditional opioids by targeting novel signal transducers. The KOR signals through seven Gαi subtypes (Gi1, Gi2, Gi3, GoA, GoB, Gz, and Ggustducin (Gg)) and two β-arrestins (β-arrestin1 and β-arrestin2). These G proteins consist of Gα, Gβ (Gβ1-5), and Gγ (Gγ1-13) subunits, forming heterotrimer complexes. Recently, we reported the structures of KOR in complex with four different G protein subtypes (Gi1, GoA, Gz, and Gg), which shed light on KOR receptor activation and signaling. Each G protein subtype has been found to play distinct roles in opioid-mediated responses. Functional characterization has also revealed significant differences among the four G protein subtypes. Moreover, our preliminary studies have identified that different combinations of Gβ and Gγ subunits can significantly influence the signaling profile of individual Gα proteins by affecting the stability of the Gα-Gβ-Gγ complex. Additionally, GPCR kinases (GRKs), such as GRK2, play a crucial role in terminating G protein signaling, promoting receptor internalization, and degradation. Interestingly, we have discovered that GRK2 not only directly interacts with KOR and phosphorylates it but also forms a stable complex with Gβ1Gγ2 and KOR, suggesting a previously unknown role of GRKs. Furthermore, using proximity labeling (APEX) combined with mass spectrometry (MS), we have identified ligand-specific signaling profiles engaged by specific G protein signaling. These findings indicate that individual signaling events mediated by different transducers may separately contribute to the therapeutic efficacy and side effects associated with KOR. Our central hypothesis is that the ligand-specific responses at KOR are determined by complex, non-traditional signaling networks at the cellular level. To test this hypothesis, we propose the following studies. Aim 1. Define the role of non-traditional regulators in KOR-G protein signaling. Aim 2. Identify the molecular determinants of GRK subtype selectivity in KOR signaling. Aim 3. Profile protein-protein interaction networks in ligand-dependent cellular signaling response of KOR. The goal of these studies will be a more detailed understanding of the molecular pharmacology of KOR, therefore providing the opportunity for gaining new insights into the chemical biology of KOR and access to new chemical modulators as opioid alternatives.

NIH Research Projects · FY 2025 · 2024-06

Project Summary Chiari malformation (CM) is a challenging disease that frequently presents during development in the pediatric age range. Despite its well-defined anatomic pathology, characterized by ectopic position of the cerebellar tonsils herniating through the foramen magnum, its clinical phenotype is variable. Here we propose an advanced neuroimaging study to seek novel explanations for CM symptomology through personalized connectomics using resting state functional MRI (rs-fMRI). In Aim 1, we will first capitalize on publicly shared data from the Adolescent Brain Cognitive Development (ABCD) Study for incidentally identified CM subjects and typically developing controls. In Aim 2, we will acquire identical ABCD protocol MRI data at presentation and longitudinal timepoints in both surgical and non-surgical subjects who are symptomatic and present for clinical care. We will replicate the imaging and cognitive testing from the ABCD Study in our clinical cohort for direct comparisons. We will also record the Chiari Severity Index (CSI) and Chiari Health Index in Pediatrics (CHIP) metrics, which have been developed and validated specific to CM, in our clinical cohort. Finally, in Aim 3, for a subset of participants we will obtain highly sampled longitudinal data at frequent timepoints after surgical intervention to better characterize the time course of functional connectivity (FC) changes, or potentially confirm if any lack of change could be dependent on timing of data collection. This final aim will be critical for future studies of similar design to optimize decisions on how long after surgery imaging should ideally be acquired to maximize effect measured and avoid missing a transient effect. The FC changes and correlations to natural history, symptoms, and response to treatment will be valuable as potential biomarkers of disease that could be used to guide treatment decisions for future clinical care. While this study is aimed at evaluating FC as a potential marker of cognitive dysfunction specific to CM, its methodology and design will be adapted to several other novel pathologies in the future as my research career develops. Furthermore, the results of this study will provide fundamental knowledge about the relationship between brain structure and function. Specifically, for CM these results relate mechanical compression from ectopic position of the cerebellar tonsils to FC in resting state networks throughout the brain and its related cognitive symptoms. In this manner, we are directly addressing the aims of the NINDS by assessing basic cognitive neuroscience fundamentals of disease in CM and its potential for translation to clinical care of neurologic disease. The data collection and analysis methods are part of a focused plan to advance my career development through training in acquisition of advanced fMRI data, surface-based analysis, and large data analysis from the ABCD Study dataset. In parallel, I will participate in course work including responsible conduct of research, scientific writing and communication, and cognitive neuroscience.

NSF Awards · FY 2024 · 2024-06

Non-technical Abstract: Polysaccharides are polymers of sugar molecules. Examples include cellulose found in cotton and wood, amylose in the starch found in bread and rice, and chitins found in the exoskeleton of insects. Today, polysaccharides are preferred renewable materials for many applications, such as packaging, healthcare, personal care, and agriculture. However, polysaccharides found in nature can vary greatly in structure and purity, making them difficult to study and use effectively. The structures and properties of synthetic polysaccharides could be precisely controlled, but existing methods for synthesizing polysaccharides are often impractical for large-scale production. The goal of this research project is to find a new way to design and make a variety of polysaccharides with precisely controlled structures from natural sugars. In addition, non-sugar units will be blended with sugar units in these polymers to match and even surpass the properties of natural polysaccharides. This project will also establish a meeting called Massachusetts-Missouri Macro-Materials Meeting (5M). This meeting will promote the communication and collaboration of polymer scientists in the states of Massachusetts and Missouri and the nearby regions, and encourage college students from diverse backgrounds to pursue careers in STEM. Technical Abstract: The overall goal of the proposed research is to develop novel polysaccharide-based soft materials. Toward this goal, novel approaches to precision polysaccharides through living cationic ring-opening polymerization (CROP) of anhydrosugars will be devised. Precision polysaccharides are synthetic polysaccharides consisting of native glycosidic linkage and well-controlled molecular weight, dispersity, side-chain groups, and chain-end groups. Living CROP of anhydrosugars will be used to generate precision polysaccharide homopolymers with various monosaccharide repeating units, side-chain groups, and molecular weights to establish a comprehensive structure-property understanding. Detailed reactivity studies in the copolymerization of the anhydrosugar monomers and non-carbohydrate monomers will lead to a novel class of statistical copolymers. The knowledge gained through these fundamental studies will enable the additive manufacturing of these materials, amplifying their molecular-scale structural features to the macroscopic level. The nano-to-macroscale understanding of the structure-property relationship will further advance the development of even more sophisticated block copolysaccharide elastomers and mixed graft copolymers with polysaccharide branches. As part of this project, a new Massachusetts-Missouri Macro-Materials Meeting (5M) will be developed to provide a platform to promote research, communication, and STEM education among researchers and college youths. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-06

Project summary Recent development in biomarker assays measuring amyloid beta and tau for Alzheimer’s disease (AD) and an accelerated approval of anti-amyloid antibody, Lecanemab, defined a new era of AD research. However, there are no specific fluid or imaging biomarkers for primary tauopathies including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and frontotemporal lobar degeneration (FTLD), making clinical trial designs challenging. We have recently identified specific forms of tau protein in cerebrospinal fluid (CSF) that reflect brain tau pathology and can identify subsets of primary tauopathies from healthy control groups and other tauopathies. In this study, we propose to define when and which tau proteoforms change during disease progression in familial and sporadic primary tauopathies. If successful, this study will facilitate accurate diagnosis of primary tauopathies, determining clinical trial eligibility, evaluating drug efficacies, and developing treatment based on underlying neuropathology in primary tauopathies. If successful, this study will start to realize personal medicine for primary tauopathies and bridge the knowledge gap between AD research.

NIH Research Projects · FY 2024 · 2024-06

ABSTRACT Tumor associated macrophages (TAMs) are key components of the tumor microenvironment and are associated with immunosuppression, poor prognosis and an inadequate response to immune checkpoint therapy (ICT) in different types of tumors. Reprogramming of TAMs towards a more phagocytic and “type 1” pro-inflammatory phenotype can improve tumor control. Thus, several molecules associated with the immunosuppressive phenotype in TAMs have been targeted in both tumor models and patients, alone or in combination with ICT. Recently, we showed that the human inhibitory receptor ILT3, also known as LILRB4, is an attractive target for TAM reprogramming in tumor therapy. We found that ILT3 is highly expressed in TAMs in various types of cancer. Moreover, we discovered that ILT3 recognizes fibronectin (Fn), which is ubiquitously expressed in the extracellular matrix and enriched in various primary tumors and metastatic sites. ILT3-Fn interaction polarizes macrophages and other myeloid cells, such as dendritic cells and monocytes, towards an immunosuppressive state, which debilitates their capacity to stimulate and activate T cells. Thus, the ILT3-Fn axis is a mechanism underlying stromal-driven inhibition of anti-tumor activity that represents a promising target to promote reprogramming of TAMs and myeloid cells in general in cancer. In this grant application we propose to determine the impact of ILT3 blockade on the control of primary and metastatic tumor models in vivo with a specific monoclonal antibody, mAb1, that blocks ILT3Fn interaction. Since ILT3 is a human receptor with a distantly related mouse paralogue, studying its function in vivo has been difficult. To date, the evidence of ILT3 immunosuppressive activity is based on expression data in human samples and in vitro assays, while the in vivo impact of ILT3 on myeloid cell functions and anti-tumor activity remains to be elucidated. To overcome this issue, we generated a transgenic mouse that carries a bacterial artificial chromosome encompassing a portion of human chromosome 19 that contains the ILT3 gene, together with its putative regulatory regions. Analysis of ILT3 expression in this mouse demonstrated a pattern consistent with that in humans among different tissues. In specific aim 1, we will test the impact of ILT3 blockade on tumor growth and anti-tumor immune responses in two models of sarcoma and colorectal carcinoma in vivo. We will further test the ability of ILT3 blockade to restore an effective response to ICT in a model of ICT resistance. In specific aim 2, we will test the impact of ILT3 blockade on the composition of pre-metastatic and metastatic niches in two models of lung metastases and, ultimately, on the formation of metastatic lesions. This proposal will advance our knowledge of the mechanisms regulating the immune landscape in primary and metastatic tumors and validate the anti-human ILT3 mAb as a therapeutic agent that can be brought to the clinic, either alone or in association with ICT.

NIH Research Projects · FY 2026 · 2024-06

ABSTRACT Type I interferons (IFNs) are important innate cytokines that influence local and systemic immune responses. They can play beneficial roles through their antiviral properties and ability to augment host immune responses; however, IFN responses can also have detrimental outcomes for the host. The factors that determine these differential outcomes are poorly understood. Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes severe, febrile arthritis and myositis. Type I IFNs are a key host response during alphavirus infection. Mice lacking the type I IFN receptor develop severe and rapidly fatal disease following infection with CHIKV or other alphaviruses. In studying this response, we have made several interesting observations. First, IFNs exert both beneficial and detrimental effects during CHIKV infection. Second, IFNs regulate the host response to CHIVK by signaling on non-hematopoietic cells, likely stromal cells and other structural cells such as fibroblasts and endothelial cells. The contribution of stromal cells to in vivo host immune responses remains poorly defined, even though these cell types are among the first cells to respond to vaccinations and vector-borne infectious diseases. Our proposed research will test the hypothesis that type I IFNs play distinct roles throughout the course of CHIKV infection due to the responses of distinct stromal cell populations. The studies proposed will utilize mouse genetics to conditionally delete IFN signaling from different stromal cells, temporal antibody blocking strategies, and RNA sequencing techniques to interrogate how IFNs impact these specific cells at different times during CHIKV infection. These findings have the potential to greatly expand our understanding of stromal cells IFN responses and how these cells contribute to shaping the tissue-specific immune response during infections and vaccinations.

NIH Research Projects · FY 2025 · 2024-06

Abstract Human immunodeficiency virus (HIV) is a major global health challenge. According to UNAIDS data 2020, approximately 38 million people across the world are living with HIV. Antiretroviral therapy (ART) is not able to clear the viral reservoir, leaving HIV-1 as an uncurable chronic disease. Despite viral suppression by ART, people living with HIV (PLWH) have increased risks for developing chronic lung diseases, such as chronic obstructive pulmonary disease, emphysema, asthma, primary lung cancer, and pulmonary arterial hypertension. Previous studies have demonstrated that chronic lung inflammation predisposes PLWH to pulmonary complications. Efforts to better understand the mechanisms underlying HIV-related lung diseases are needed. The lung microbiome (i.e., a collection of all bacteria residing in the airway) plays a significant role in modulating lung immunity and disease pathogenesis. Accumulating evidence has supported that PLWH on suppressive ART harbor an altered lung microbiome compared to people without HIV infection. The enrichment of bacteria in the genera of Prevotella spp., Veillonella spp., and Streptococcus spp. in the PLWH may contribute to chronic lung inflammation. Human population-based lung microbiome studies have been mostly cross-sectional. They are confounded by genetic and environmental factors and lack mechanistic insights. Longitudinal studies monitoring the dynamics of lung inflammation and microbial alterations are needed to understand the crosstalk between dysregulated immune pathways and lung microbial dysbiosis during HIV infection. Here we propose to use an advanced humanized mouse model to better understand lung inflammation and microbial dysbiosis driven by HIV infection. We have two specific aims to perform. Specific Aim 1 is to characterize the dynamics of lung microbial alterations and immune perturbations at cellular and transcriptional levels in humanized mice from the acute to chronic infection stages. We will also investigate how ART influence lung immunity and lung microbiome. Specific Aim 2 is to examine the role of lung bacterial dysbiosis on the lung immune system and develop novel approaches to reduce or resolve lung inflammation. We expect that our results will provide novel insights into the crosstalk between pulmonary immune tone and the microbiome in the context of HIV infection and pave the way for further mechanistic studies to better understand HIV-associated lung complications.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY/ABSTRACT The role that chronic viral infection plays in shaping the immune system and responses to subsequent infections is critically important but understudied. It is estimated that >80% of adults harbor at least 5 latent herpesviruses. Gammaherpesviruses establish latency in immune cells, further highlighting the importance of understanding how latency impacts host immune responses to subsequent infections. Currently there is a substantial knowledge gap in the role played by latency and/or mechanisms by which prior gammaherpesvirus infection contributes to subsequent immune responses, particularly in the lung. Despite the known deleterious effects of herpesvirus infection on human health, emerging evidence suggests the possibility the latent herpesvirus infection may actually protect from secondary infections. Therefore, to uncover key mechanisms by which latency impacts subsequent immune responses, I propose the following: I will test the premise that gammaherpesvirus latency impacts host protection during subsequent pulmonary infections that are prevalent across Kingdoms and dissect how latently infected immune cells intrinsically respond to heterologous pathogens. I will also delineate the impact of the microbiota on establishment and maintenance of herpesvirus latency given the critical impact of the microbiota on immune responses and the broad use of potent antibiotics in medical practice. These distinct but equally important directives will fill key knowledge gaps. At the completion, I expect to define the role of latency and reactivation on human immune responsiveness and develop a framework to define how the balance between microbiota and secondary infections are informed by latency. Moreover, this study is clinically relevant and fundamentally significant as these findings have the potential to define novel mechanisms and pathways important for pathogen control and tissue repair. In alignment with the Stephen I. Katz funding mechanism, this proposal represents a new direction for my research program. While I have previously studied cytokine regulation of herpesvirus latency during my doctoral thesis and identified protective microbial metabolites in pulmonary infection as a postdoctoral fellow, I have not investigated how viral latency intrinsically impacts immune cell function during secondary pulmonary infections or how the microbiota influences the establishment and maintenance of latency. This new investigation will require acquisition of novel tools and technologies that will reshape my research program and enhance its scope and impact. I am perfectly positioned to direct and execute this unique line of investigation as I have expertise across multiple relevant domains including virology, microbiology, immunology, and critical care medicine. Additionally as an Early Stage Investigator, I have established collaborations with experts across diverse domains of microbial pathogenesis.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY Celiac disease (CeD) is an immune-mediated enteropathy that occurs when genetically susceptible individuals consume gluten. CeD affects >1% of the US population, and its prevalence is rising. HLA-DQ2 and/or DQ8 haplotypes are necessary for the development of CeD. However, only ~3% of individuals with these risk alleles develop CeD, suggesting a role for environmental factors and gene-by-environment interactions in the initiation and pathogenesis of this disease. Diagnosis of this lifelong disease obligates strict adherence to a restrictive diet, which markedly reduces quality of life. Potential celiac disease (PCeD) is used to describe individuals with HLA-DQ2/HLA-DQ8 risk alleles and circulating antibodies to tissue transglutaminase and endomysium, and yet normal duodenal villous architecture. PCeD can progress to CeD (PCeD-P), revert to seronegativity, or remain persistently seropositive without abnormal small bowel histology (PCeD that is static (PCeD-S)). The PCeD population provides a unique ability to define the cascade of microbial and host factors culminating in CeD. Insight into the mechanisms that lead to the loss of gluten tolerance and resulting tissue injury will provide reliable markers of disease progression, aid in case management, and offer an opportunity to prevent the progression to mucosal injury. To do this, we will recruit children with suspected CeD based on positive serology and children with negative celiac serology undergoing upper endoscopy at Washington University and University of Alabama. This will allow us to compare paired plasma, stool, duodenal biopsies, duodenal fluid aspirates, and clinical metadata from four gluten consuming groups (newly diagnosed treatment naïve CeD, PCeD-P, PCeD-S, and non-CeD controls). To determine if there are transcriptomic signatures in children with PCeD who subsequently develop histologically proven CeD (PCeD-P), we will perform spatial transcriptomics on biopsies from PCeD participants and sex, age, and race matched CeD and non-CeD controls. Previously, we demonstrated that a subset of CD8+ T cells expressing killer cell immunoglobulin-like receptors (KIR) are increased in frequency in the blood and duodenum of adults with CeD and suppress pathogenic gliadin specific CD4+ T cells. We will determine the frequency of KIR+CD8+ T cells, gliadin specific CD4+ T cells, and the ratio of these cells among CeD, PCeD, and non-CeD controls. Additionally, we will determine the transcriptomic and TCR clonality shifts within KIR+CD8+ T cells and associate these to CeD progression. Understanding the interplay between CD4+ T cells and CD8+ T cells will help in assessing the contribution of adaptive immune system in CeD progression in children. We do not know if microbial differences drive small bowel CeD inflammation, or vice-versa. By systematically comparing the gut (small bowel and stool) bacterial microbiome and virome of children with biopsy confirmed CeD, PCeD-P, PCeD-S, and non-CeD controls we will define the microbial cascade of events leading to progression in susceptible individuals. This prospective interrogation of CeD at a uniquely instructive point in the disease process will provide novel and informative insight into the evolution of tissue injury in CeD.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY/ABSTRACT The long-term goal of this proposal is to train the applicant to become an independent, academic physician- scientist studying immune responses against lung cancer. The principal investigator (PI) has previously obtained Ph.D. training in biochemistry and molecular biology, as well as clinical training in internal medicine and hematology/oncology. He is ABIM board-certified in medical oncology. This application describes a 5- year career development program that will provide the PI a mentored educational experience with the aim of developing new scientific expertise in tumor immunology, molecular profiling, high-dimensional analysis, computational biology, and mouse models of human lung cancer. At the conclusion of the award period, the PI will have acquired the skills necessary to achieve his goal of becoming an independent investigator in an academic medical center studying lung cancer immunobiology and immunotherapy and caring for patients with thoracic malignancies. This research project will capitalize on the expertise and environment of Washington University in St. Louis, which has a long-track record of developing and supporting physician-scientists. Dr. David DeNardo will mentor the PI’s scientific and career development. Dr. DeNardo's work is responsible our critical understanding of how the tumor microenvironment of pancreatic cancer leads to impaired immune surveillance and his work has led to several clinical trials on improving immune-based therapies in pancreatic cancer. An advisory committee of scientists will provide additional scientific and career guidance. Lung cancer is the most common cause of cancer-related mortality worldwide. Immune checkpoint inhibition, though revolutionary, is beneficial for a subset of patients with metastatic non-small cell lung cancer (NSCLC). Unfortunately, the responses are often not durable with nearly 80% of those diagnosed with advanced stage NSCLC succumbing to their disease within 5 years of diagnosis. The tissue microenvironment of human NSCLC is characterized by a dense stromal network. Our laboratory has demonstrated that CAFs can impair response to anti-tumor immunity in pancreatic cancer as well as in models of NSCLC. We have found that targeting fibrosis via TGFbR inhibition can improve responses to immune checkpoint inhibition but only in the setting of chemotherapy. In this proposal, we will test the hypothesis that fibrosis impairs both T-cell priming and antigen trafficking/presentation and these are restored when TGFbR inhibition is combined with chemotherapy and immunotherapy. For these studies, we will utilize a genetically engineered mouse model of human lung adenocarcinoma, the most common type of NSCLC, which expresses ovalbumin, which will enable us to assess immune responses. The identification of the cellular and molecular mechanisms by which TGFbR inhibition restores anti-tumor immunity when combined with chemotherapy and immunotherapy has clinically relevant implications with respect to the development of novel immunotherapy strategies for patients with NSCLC.

NIH Research Projects · FY 2025 · 2024-06

ABSTRACT The TGFβ pathway plays a critical role in maintaining the homeostasis and overall health of articular cartilage. However, the diminished expression of the TGFβ receptor (Tgfbr2) in osteoarthritis (OA) cartilage hampers the utilization of TGF-β1 as a therapeutic treatment for OA. Therefore, the goal of this proposal is to identify the key downstream mediators as well as potential therapeutic targets that are regulated by TGFβ1. We performed an unbiased multi-omics screening combining RNA-seq, ATAC-seq as well as metabolomics, and establish that TGFβ1 maintains articular chondrocyte homeostasis at least partially through suppression of lipid metabolism. We have shown that Nuclear Factor I A (NFIA) is a crucial transcription factor in the regulation of lipid metabolism in articular chondrocytes. Specifically, TGFβ1 reduces Nfia expression in murine articular chondrocyte, while increased Nfia expression is observed in Tgfbr2 loss-of-function (LOF) chondrocytes and human OA articular chondrocytes. Mechanistically, Nfia physically binds to the promoter regions of Acetyl-CoA carboxylase a (Acaca) and Carnitine palmitoyltransferase 2 (Cpt2) genes, which are rate-limiting enzymes for fatty acid synthesis and oxidation, respectively. Consequently, Nfia overexpression in articular chondrocytes induces the expression of both Acaca and Cpt2. Conversely, Nfia inhibition results in the suppression of the elevated expression of Acaca and Cpt2 in Tgfbr2 LOF chondrocytes. More importantly, NFIA LOF restores lipid metabolism and cellular homeostasis in human OA articular chondrocytes, and Nfia gene ablation in articular cartilage attenuates injury-induced OA progression in mice. Thus, Nfia represents a potential therapeutic target for OA. Based on these novel findings, our central hypothesis proposes that TGFβ1 maintains articular cartilage homeostasis by suppressing lipid metabolism through Nfia inhibition, and lipid metabolism is a viable and highly innovative target for OA treatment. Two Specific Aims are proposed. Specific Aim 1 will establish Nfia as a downstream mediator of the TGFβ pathway in regulating articular chondrocyte lipid metabolism and homeostasis in mice. Complementary in vitro and in vivo genetic approaches along with metabolomics analyses will be employed to establish TGFβ/Nfia-regulated lipid metabolism as a critical pathway necessary for articular chondrocyte homeostasis. Specific Aim 2 will define Nfia-mediated lipid metabolism as a potential therapeutic target for OA treatment. We will investigate if cartilage specific Nfia LOF abolishes the increase in lipid metabolism and attenuates OA progression associated with obesity and aging in mice. Single cell RNA-seq and metabolomics will be used to establish the mechanism behind the whole joint protection. In summary, this proposal will advance our understanding of mechanisms regulating lipid metabolism in OA disease and define Nfia mediated lipid metabolism as a novel target to treat OA.

NIH Research Projects · FY 2026 · 2024-06

SUMMARY. Chikungunya virus (CHIKV) is an emerging mosquito-transmitted alphavirus that causes an abrupt onset of fever with severe joint and muscle pain. In a significant fraction of patients, chronic and debilitating arthritis can develop and persist for months to years, with recent epidemiological projections suggesting there are more than 400,000 patients in the Western Hemisphere alone with chronic CHIKV musculoskeletal disease. Patients affected by chronic CHIKV disease show elevated synovial proinflammatory cytokines and infiltrating cells including monocytes and CD4+ T cells. In addition, persistent CHIKV RNA is detected in human patients, non-human primates, and mice for months to years after infection. To identify the cells that harbor this RNA during the chronic phase of disease, we recently engineered a recombinant CHIKV to express Cre recombinase and demonstrated that the infection of tdTomato reporter mice resulted in persistent tdTomato+ cells. To date, our analysis has revealed that fibroblasts and macrophages are key cell types that harbor viral RNA in the chronic phase. Therefore, this system allows us to test the hypothesis that during chronic disease, a subset of CHIKV infected cells survive infection and harbor persistent, non-productively replicating viral RNA that functions as a pathogen associated molecular pattern (PAMP) to drive persistent inflammation. The premise of this proposal is to utilize this CHIKV lineage tracing system in a murine model of CHIKV arthritis to identify, isolate, and characterize the cells that are infected, survive CHIKV infection, and harbor viral RNA. We will test the hypothesis that this persistent RNA activates pattern recognition receptors during acute and chronic disease driving inflammatory phenotypes in both macrophages and fibroblasts. These studies will provide important new insight into the pathogenesis of chronic CHIKV arthritis and possibly create new avenues for therapeutic interventions.

NIH Research Projects · FY 2026 · 2024-06

Abstract Virtually every step of HIV-1 replication as well as numerous cellular antiviral defense mechanisms are regulated by viral and cellular RNA-binding proteins (RBPs) that recognize distinct sequence or structural features on viral RNAs. One such interaction takes place between the HIV-1 major structural protein, Gag, and the viral genomic RNA (gRNA). Through its nucleocapsid (NC) domain, Gag selects two copies of an unspliced positive strand gRNA from a pool of cellular and spliced viral mRNAs in excess and orchestrates key steps of virion assembly. How gRNAs are selected for packaging, why only a dimer is packaged in a single virus particle and whether NC-gRNA interactions guide other RBPs that target the same gRNA remain poorly understood. Previous CLIP studies demonstrated that Gag predominantly binds to guanosine-rich sequences in the cytosol, but its binding preference shifts towards sequences with adenosine-rich nucleotide composition (including binding sites on cellular mRNAs) at the plasma membrane where virions assemble. Here we propose to formally test whether adenosine-richness of the HIV-1 gRNA facilitates its selective packaging through complementary genetic and biochemical approaches (Aim 1). According to a widely accepted model, recruitment of a dimeric gRNA to the plasma membrane nucleates virion assembly, resulting in progressive recruitment of Gag molecules from the cytosol. As the late arriving pool of Gag molecules at the nucleation site needs to be devoid of RNAs, we hypothesize that affinity of Gag towards cognate RNAs is a key parameter that underpins dimeric gRNA packaging. In Aim 2, we propose to disrupt this balance by generating Gag chimeras with altered affinity/avidity towards target RNAs and determine the impact on dimeric gRNA trafficking to the PM, gRNA packaging and virus particle assembly. In Aim 3, using the Gag chimeric viruses as tools, we propose to target key NC functions and determine the NC- and gRNA-dependence of other viral and host RBPs that are critical regulators of HIV-1 replication and host defenses. Specifically, we propose to examine whether members of the antiviral APOBEC3 protein family infiltrate into virions through binding to the viral gRNA or other virion- incorporated host RNAs, such as 7SL. As part of this aim, Gag chimeras that can package gRNA independent of NC will be used to test the dependence of integrase-gRNA binding, an event critical for proper virion maturation, on NC and NC-gRNA interactions. Understanding the interplay between multiple RBPs that target the same gRNA is highly relevant to our fundamental understanding of HIV-1 replication and virus-host interactions. Currently, no antiretrovirals target gRNA packaging and assembly despite these steps presenting a clear vulnerability for HIV-1. Our preliminary studies demonstrate that Gag-gRNA interactions are finely tuned and can be targeted. Proposed work is thus significant as it may guide the development of NC- or gRNA-targeted drugs that abrogate gRNA packaging, virion assembly and infectivity.

NIH Research Projects · FY 2026 · 2024-06

TITLE: Combined Imaging and RNA Analyses to Develop Cervical Cancer Biomarkers ABSTRACT Despite significant advances in disease prevention and screening, cervical cancer continues to be an important worldwide public health problem. Treating cervical cancer patients with personalized strategies can potentially improve the chance of survival. Predicting early in treatment whether a tumor is likely to be responsive is one of the most challenging yet important tasks for stratifying cervical cancer patients and supporting personalized treatment strategies to improve cancer patient care. Various unimodal data, including ribonucleic acids (RNAs), radiologic and histologic imaging, and clinicopathologic data, have been employed for predicting cervical cancer treatment response and patient outcome. Each type of unimodal data analyzes tumor phenotypes from a different point of view and provides valuable while limited prognostic information. We and others have shown that RNAs are promising biomarkers and play critical regulatory roles in cervical cancer. Radiologic imaging biomarkers have shown promise in stratifying patients with favorable and unfavorable prognosis for multiple tumor sites. Their non-invasive characteristics also allow for convenient and longitudinal monitoring of tumor progression and heterogeneous response during the treatment course. Moreover, histologic images provide key information about microscopic structure of cells and tissues of organisms. Recent reports and our preliminary studies have shown that histologic imaging biomarkers, can aid in clinical decision-making by identifying metastases, subtyping and grading tumors, and predicting clinical outcomes. Clinicopathologic biomarkers show prognostic value through retrospective studies. Still, many cervical cancer patients have tumor recurrence despite favorable prognosis by these biomarkers individually. The major goal of this study is to develop a comprehensive and robust computational model for prediction of cervical cancer treatment response and outcomes. We will integrate our recently developed advanced learning- based techniques to build prognostic models using about 600 cervical patient cases collected from two institutions. The prognostic model will form a solid basis for individualized care of cervical cancer patients. Moreover, our work is expected to discover the correlations among multimodal data, leading to dynamic patient stratification to support adaptive treatment strategies in the future.

NIH Research Projects · FY 2026 · 2024-06

Abstract Interstitial cystitis/bladder pain syndromes (IC/BPS) are a debilitating condition with unknown etiology. The spinal cord has long been identified as a critical site in integrating both non-noxious (bladder function) and noxious (intense bladder pressure or discomfort) sensory information from the bladder. These spinal circuits are known to exert a tighter control over bladder voiding and storage under naïve state. While the role of lumbosacral spinal cord neural circuitry in micturition and bladder sensation has long been studied, the precise cell types in the spinal cord that contribute to processing micturition and bladder nociception remains poorly understood. In preliminary studies, we have discovered two unique cell populations in the spinal cord that play a role in micturition and bladder nociception. Here we propose to build on this preliminary work to methodically dissect the roles of these spinal neurons in the voiding regulation and bladder nociception in pathological cystitis state. We will determine if cystitis leads to maladaptive changes in these spinal cord neurons. Could manipulating activity of these spinal cord neurons potentially attenuate bladder symptoms associated with pathological cystitis state? Completing these proposed studies, will advances our understanding of spinal circuits in cystitis and will provide a possible entry point into the spinal circuits for development of possible therapeutic drugs in treatment of the IC/BPS.

NIH Research Projects · FY 2024 · 2024-06

PROJECT SUMMARY Clostridioides difficile infection (CDI) is a major cause of healthcare-related mortality and a significant public health burden in the US. C. difficile (Cd) remains a persistent cause of morbidity and mortality in healthcare settings, in part because many patients are asymptomatic for CDI yet colonized with Cd. These patients outnumber CDI patients, can transmit Cd and progress to CDI (especially in the context of antibiotic exposure). Importantly, the clinical outcomes in Cd-colonized patients exists on a spectrum influenced both by the microbial community and the virulence of the Cd strain. Critically, conventional animal models of CDI do not recapitulate microbiome and pathogen variation seen in asymptomatically-colonized patients, thus requiring the development of novel animal models to study this patient population. The long-term goal of this research is to identify opportunities for novel therapeutic intervention or pathogen surveillance by better understanding and predicting Cd-associated clinical outcomes. The objective of this proposal is to use clinically-relevant animal models to 1) investigate the extent to which commensal microbiota protect the host from diverse Cd strains, 2) predict microbiome vulnerabilities to antibiotic-induced CDI, and 3) identify microbiome features that synergize with prebiotic administration. The central hypothesis of this work is that the composition of the commensal microbiota plays a central role in determining host disease severity. The specific aims of this proposal are to: 1) investigate the in vivo role of commensal Eubacteriaceae in microbiome-based protection against Cd infection and 2) identify microbiome correlates of antibiotic-induced CDI and prebiotic synergy. The proposal will use microbiome-humanized models of Cd colonization/infection that integrate both clinically-represented Cd strains and patient-derived bacterial communities to understand the microbiota's impact on host inflammation, community metabolism, and Cd proliferation. This research will spur the development of innovative treatment and diagnostic approaches to mitigating CDI. Dr. Dantas will oversee the project, provide direct mentoring on statistical modeling of multi-omics data, and help Dr. Fishbein's transition to independence through support of networking strategies. Dr. Fishbein has prepared a Scholarly Advisory Committee along with other Significant Contributors with expertise in host-pathogen interactions, gut microbiome-pathogen dynamics, and intestinal inflammation. The training plan combines primary mentorship, committee interactions, formal coursework (at the University and externally), and seminar/conference presentations to expand Dr. Fishbein's technical and conceptual foundations in the microbiome field. This award will facilitate Dr. Fishbein's acquisition of independent funding, enabling her transition to an independent research program at a research-intensive academic institution.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Pathogenic bacteria engage in competition with their hosts for resources. Hosts actively deny resources — especially transition metals — to invading bacteria to suppress their growth. This is a strategy called nutritional immunity. Understanding how bacterial populations use metals and how their metal acquisition relates to their central metabolism is critical to understanding this host-microbe competition and developing new therapeutic strategies. We currently have a poor understanding both of bacterial metal requirements, and of how bacterial metal concentrations relate to their central metabolic strategies. The long-term aim of this project is to better understand the fundamental nature of the relationship between the metal content of individual bacterial cells within populations of genetically homogenous individuals, and how this relates to variations in their expression of central metabolism. The first aim of this project is to test the hypothesis that the metal concentrations of individual bacterial cells correlate with their central metabolic strategy. This is likely to be true, since enzymes involved in central metabolism require metal cofactors, but it has never been demonstrated for individual cells. To approach this question, we will develop a secondary-ion mass spectrometry (SIMS) imaging method capable of simultaneously tracking metabolism and metal content in hundreds of individual bacterial cells. This is possible due to recent developments in SIMS that have improved sensitivity and throughput to levels capable of investigating bacterial populations. This technique is capable of measuring single-cell bacterial metallomes, while simultaneously measuring abundances of isotope labels that were experimentally provided to trace central metabolism (stable isotope probing). The second aim of this project is to test the hypothesis that the phenotypic traits examined in Aim 1 are correlated with patterns of gene expression. Currently available methods are capable of revealing patterns of gene expression in single bacterial cells using amplified hybridization techniques coupled to fluorescent reporters. We will modify this technique to use a halogen reporter that is capable of being visualized by SIMS. The abundance of the halogen tag in single cells can be measured, correlating targeted patterns of gene expression with single-cell bacterial metallomes and bacterial metabolism. This is significant because it will help us understand fundamental relationships between resource abundance and central metabolism, and population strategies for allocating resources and metabolism among individual bacterial cells.

NIH Research Projects · FY 2026 · 2024-06

ABSTRACT Recent studies in model organisms have demonstrated that the intertissue communications play a critical role in the regulation of aging and longevity. In mammals, we have demonstrated that the intertissue communication between the hypothalamus and adipose tissue, particularly mediated by extracellular vesicles-contained extracellular nicotinamide phosphoribosyltrasferase (eNAMPT), the rate-limiting NAD+ biosynthetic enzyme in mammals, functions to counteract age-associated physiological decline and promote longevity in mice. Most recently, we have demonstrated that Ppp1r17-positive neurons in the dorsomedial hypothalamus (DMHPpp1r17 neurons) regulate white adipose tissue (WAT) function, including lipolysis and eNAMPT secretion, through the sympathetic nervous system (SNS), and the feedback loop between DMHPpp1r17 neurons and WAT plays a critical role in the regulation of aging and longevity in mice. Our preliminary results suggest that this critical feedback loop between DMHPpp1r17 neurons and WAT wanes over age, which is one of the key triggers for aging. Indeed, chemogenetic stimulation of DMHPpp1r17 neurons in aged mice significantly ameliorates multiple aging phenotypes, decreases age-associated mortality rate, and extends longevity. However, why and how this critical feedback loop wanes over age remains unknown. In this research proposal, we hypothesize that adipose tissue starts decreasing adipose-resident immune cells, particularly type 2 innate lymphoid cells (ILC2s), and increases cellular senescence, causing WAT dysfunction and decreasing the content and the secretion of adipose EVs. Such WAT dysfunction then affects the regulation of Ppp1r17 function in DMHPpp1r17 neurons, affecting their function and accelerating WAT dysfunction through decreased SNS function. Maintaining this hypothalamus- WAT feedback loop is critical to counteract age-associated physiological decline and promote lifespan in mammals. To address this hypothesis, we propose the following three SPECIFIC AIMs: SPECIFIC AIM (1) will examine the effect of miR-20a, a microRNA species in adipose EVs, on the expression of Prkg1 in DMHPpp1r17 neurons during aging. SPECIFIC AIM (2) will elucidate how adipose immune cells are dysregulated during aging. We will particularly focus on ILC2s, which are dramatically reduced in adipose tissue during aging. SPECIFIC AIM (3) will address whether restoring ILC2 function by transplanting young ILC2s could delay aging and extend lifespan in mice. The anticipated outcome of the proposed research will advance our understanding of the importance of intertissue communications in mammalian aging and longevity control and open a new opportunity to develop an effective anti-aging intervention based on the intertissue communication between the hypothalamus and WAT.