Washington University

NIH Research Projects · FY 2026 · 2022-02

1 This is an application for a K01 award for Beth Prusaczyk, PhD, MSW, whose research focuses 2 on improving the health of vulnerable older adults, such as those with Alzheimer’s disease and 3 related dementias (ADRD) living in rural areas, by improving systems of care. The experiences 4 of those in rural areas with ADRD, including the older adults with ADRD, their caregivers, their 5 health and social service providers, and their community, are distinctly different from those in 6 urban areas. A lack of integration between services is a barrier to caregivers receiving information. 7 Providers desire more integrated services, such as having case managers at primary care offices 8 to help coordinate social services. Failure to improve the communication and care coordination 9 across rural ADRD networks as the aging population increases will compound these existing 10 disparities and result in poorer care for older adults with ADRD in rural settings. Health Information 11 Technology (HIT) tools, such as telehealth, clinical decision support, mobile applications, 12 electronic referrals or prescribing, and others, have been applied in rural areas to ADRD disease 13 risk, testing, and diagnosis, but have not yet been applied to communication and care 14 coordination. The purpose of this K01 is to build off preliminary data and use network analysis to 15 identifying communication and care coordination gaps in two rural ADRD care systems then, in 16 collaboration with local older adults with ADRD, their caregivers, their health and social service 17 providers, select and pilot test a HIT tool to fill those gaps. Specifically, this study will: 1) build 18 network models of communication and care coordination within two rural ADRD networks, and 19 identify factors related to communication and care coordination gaps; 2) assess the perceived 20 usability, feasibility, and acceptability of HIT tools designed to address communication and care 21 coordination in the networks and select a HIT tool to pilot test; and 3) pilot test the communication 22 and care coordination HIT tool in the network. Through this K01 award, Dr. Prusaczyk, who is a 23 health services researcher at Washington University School of Medicine in St. Louis (WUSM), will 24 improve upon and address gaps in her skill set through targeted training activities in: 1) advanced 25 network analysis; 2) HIT; and 3) rural ADRD research. These activities will be possible through 26 resources available at WUSM, including the Knight Alzheimer’s Disease Research Center and 27 the Institute for Informatics. Dr. Prusaczyk has the support of an outstanding mentorship team 28 with expertise in ADRD, network analysis, rural health services research, and HIT. This research 29 is significant because it will yield a comprehensive understanding of rural ADRD systems – a 30 critical context for which there is limited knowledge – and a possible solution for improvement.

NIH Research Projects · FY 2026 · 2022-02

ABSTRACT The development of tissue-specific autoimmunity is a chronic process in which finely programmed autoimmune responses progress and culminate in the target organ. Although the tissue environment is eventually dominated by the invasive responses, regulatory elements that function to oppose such pathogenic activities remain understudied. In type 1 diabetes (T1D), the insulin-producing β cells are targeted by self-reactive T cells infiltrating into the pancreatic islets. Less appreciated is that before the entry of the first T cells, the islet environment has established an intrinsic mechanism that restrains the onward autoimmune attack. Such regulation is mediated by a specific subset of the islet resident macrophages specialized in the clearance of apoptotic β cells, a process referred to as efferocytosis. The efferocytosis program induces anti-inflammatory responses and when enhanced, imposes strong immunoinhibitory functions, leading to profound protection from autoimmune diabetes. We hypothesize that the efferocytosis program may act as an original control mechanism installed in the normal islet environment regardless of autoimmune propensity. This innate mechanism is common in mice and humans and may considerably differ from other immunomodulatory elements (i.e., regulatory T cells (Tregs)), which are introduced along with the adaptive immune invasion. Therefore, examining efferocytosis in islets will provide conceptual advances to the biological and autoimmune events taking place in this important organ. Moreover, by analyzing the immunoinhibitory components associated with macrophage efferocytosis, this project will provide translational and therapeutical insights relevant to human T1D. In this proposal, we seek to thoroughly examine the efferocytic islet macrophages and define their role in regulating a complex autoimmune process.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY Triple-negative breast cancer (TNBC) has remained a considerable clinical challenge due to the lack of efficacious genetic targets. We need unique effective therapies and accurate biomarkers that can be used to predict patient responses in TNBC. We find that the ARF tumor suppressor is lost alongside p53 mutation in 60% of TNBC. Potentially stemming from the dual loss of ARF and p53, we have observed that type I IFN signaling is elevated in TNBC. We show that this IFN production is being kept in check by the ADAR1 enzyme. Notably, we discovered that ADAR1 is a novel binding partner for ARF. The central premise of this proposal is that the novel ARF-ADAR1 interaction provides key insights into how these two proteins function in the etiology of TNBC. The research application focuses on the role of this interaction in regulating the type I interferon response and sensitizing TNBC cells to cell death and immune recognition. The overarching hypothesis of the proposed research is that loss of ARF and p53 results in elevated type I IFN signaling and sensitizes cells to ADAR1 depletion. In Aim 1, we will define the functional interaction of ARF and ADAR1. In the absence of functional p53, ARF protein expression is induced. In this setting, we find that ARF can fully titrate all the cellular ADAR1 into ARF complexes. We will test the hypothesis that ARF mechanistically traps ADAR1 in the nucleolus to prevent ADAR1 from repressing the type I interferon pathway. In Aim 2, we present data that TNBC cells are sensitive to ADAR1 depletion. Importantly, this sensitivity is dependent on type I interferon signaling. We will test the hypothesis that activation of IFN production and signaling will combine with ADAR1 depletion to produce synthetic lethality in vitro and in vivo. We will utilize both agonists of the IFN pathway and synthetic ARF peptide mimics. In Aim 3, we will assess how the ARF-ADAR1 interaction influences the tumor microenvironment. While type I IFN release is a major component of the anti-viral response, chronic IFN- release by tumor cells can both alter the local immune environment and expression of PD-L1 on tumor cells. We will test the hypothesis that hyperactivation of the type I IFN pathway by ADAR depletion will result in gains in tumor infiltrating lymphocytes and elicit an anti-tumor immune response that will prevent metastasis. These studies are paramount to informing new approaches in treating TNBC through activation of IFN signaling in the tumor microenvironment.

NIH Research Projects · FY 2026 · 2022-02

ABSTRACT Reactive lymphangiogenesis requires B cell-expressed lymphotoxin (LT)α. LTα signals through TNFR1 or TNFR2 when it is presented to cells as a LTα3 homotrimer, or it signals through LTβR when it is part of the LTa1b2 heterotrimeric complex. Lymphangitis has been described as a feature of Crohn's disease, but the nature and role of lymphangitis in the disease has not been elucidated. In resected tissue from Crohn's disease patients, outflow lymphatic trunks (called collecting lymphatic vessels) running from the ileum through the mesentery and on to draining lymph nodes appeared obstructed by tertiary lymphoid organs (TLOs) that formed around them. The TLOs were rich in B cells. In a mouse model of ileitis driven by a mutation in the mRNA locus for the cytokine TNF (TNF∆ARE mice) that increases TNF abundance when the gene is environmentally triggered, TLOs developed along mesenteric collecting lymphatics draining the ileum, like patients. The mice display marked blockade of lymph flow due to obstruction by the TLOs, which raised lymph pressure so that flow did not move forward but pushed backwards through lymphatic collaterals toward the gut. Backflow characterizes compromised valves. Indeed, TLOs mainly formed where lymphatic valves lost their identity and quiescence. Instead, these valve sites supported lymphangiogenesis that partially encapsulated expanding TLOs. Before lymphangiogenesis got underway, B cells accumulated around lymphatic valves. These B cells may be the source of cytokines that act on the lymphatic vessels. Indeed, data so far suggest that the action of TNF on lymphatic endothelium may be sufficient in vitro and in vivo to reprogram valve- associated genes critical to the maintenance of normal lymph flow. That is, early blockade with anti-TNF neutralizing mAb restores lymph flow and prevents progression of TLOs. At late stages in disease, anti-TNF therapy becomes less effective as the TLOs have greatly expanded and enlarged, but nonetheless anti-TNF still partially restores mesenteric lymph flow, suggesting that TNF remains a key, and possibly required, signal in disease progression. However, it remains unclear if the TLOs per se drive worsened ileitis. What would happen if the TLOs did not form but other inflammatory pathways were left intact? In preliminary data for this application, we have identified a means to prevent TLO formation: TLOs do not form in the absence of B cells. Furthermore, TNF and/or LTα expressed by B cells is required for the formation of TLOs in the ileitis-affected mesentery of TNFARE mice. These data cause us to hypothesize that TNF receptor ligands TNF and/or LTα derived from B cells act through TNF receptors on lymphatic endothelium to drive tertiary lymphoid organ (TLO) formation by skewing signals that normally maintain lymphatic valve integrity. We predict, in turn, that TLOs govern the pathophysiology of the disease itself by trapping inflammatory cells and mediators rather than allowing such mediators to disseminate and enhance systemic pathology. This model predicts a Catch-22 feature of TLOs: they protect against some forms of pathology but drive others.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY African American (AA) women are disproportionately affected by triple negative breast cancer (TNBC), a highly heterogeneous and aggressive subtype of breast cancer. We found that AA patients with TNBC have a higher risk of death from the disease than their European American (EA) counterparts, which is independent of their sociodemographic and clinical factors. Notably, this TNBC disparity is more obvious in patients receiving chemotherapy. However, the molecular mechanisms driving differential tumor response to chemotherapy and the consequent prognostic disparities in AA vs. EA TNBCs remains unknown. Most chemotherapy agents exert their cytotoxic effects through the induction of deadly DNA double-strand breaks (DSBs) that are repaired by two major mechanisms: error-free homologous recombination (HR) and error-prone non-homologous end- joining (NHEJ). Genomic stability and survival of tumor cells upon genotoxic treatments are known to depend on HR. Our pilot study showed that AA TNBCs tend to have higher expression of HR-related proteins and lower expression of NHEJ-related proteins compared with EA TNBCs. The AAA+ ATPase VCP has been suggested to influence the choice of DSB repair pathways in favor of HR as opposed to NHEJ. We recently reported that Ser784 phosphorylation of VCP is required for DNA damage repair and intra-tumor pSer784-VCP levels predict poor survival in TNBC patients, particularly those receiving chemotherapy. Our pilot data also showed that both total VCP protein and DNA damage-induced pSer784-VCP levels are higher in AA vs. EA TNBC samples. Based on these data, we hypothesize that AA TNBCs, relative to EA TNBCs, are intrinsically more capable of repairing chemotherapy-induced DSBs by HR as opposed to NHEJ due to differential expression levels and functionality of DSB repair factors including pSer784-VCP, which underlies their worse clinical outcomes. We propose three specific aims. First, we will confirm racial differences in protein expression of HR and NHEJ factors in tumor tissues obtained from two large cohorts of TNBC patients and examine their contribution to survival disparities. Second, we will experimentally compare HR and NHEJ efficiencies between AA and EA TNBC cell line and patient-derived mouse xenograft (PDX) models, and correlate the racial differences in DSB repair pathway choice and genotoxic chemotherapy effects. Third, we will examine the ability of pSer784-VCP to regulate DSB repair pathway choice and genotoxic chemotherapy effects in AA TNBC models. The proposed study will greatly improve our understanding of the molecular mechanisms underlying racial disparities in TNBC treatment response and outcomes. The results will also help create new disparity intervention strategies by establishing pSer784-VCP as a novel predictive biomarker and sensitizing target for genotoxic chemotherapy treatments in AA TNBCs. .

NIH Research Projects · FY 2026 · 2022-02

The long-term goal of this proposal is to define the contribution of altered epigenetic patterns and genome organization to the pathogenesis of acute myeloid leukemia. Acute myeloid leukemia (AML) is a devastating cancer that is initiated by somatic mutations in hematopoietic stem/progenitor cells. AML cells are also characterized by DNA methylation changes and altered gene expression patterns, but the relationships between AML mutations, DNA methylation, and transcriptional activity in AML are poorly understood. We have performed comprehensive epigenetic analysis to investigate the regulatory mechanisms that control expression of the HOX gene loci in AML cells, which encode transcription factors that maintain normal hematopoietic stem cell identity and promote self-renewal in AML. These studies have identified specific long-range three- dimensional (3D) genome interactions at the HOXA locus that are increased in AML vs. normal hematopoietic stem cells. Further analysis has showed that the loci involved in these interactions have AML-specific epigenetic changes suggesting they may be enhancers. We have extended these studies by performing a genome-wide analysis of DNA methylation and 3D genome architecture in primary AML samples. This demonstrated that AMLs with canonical mutations in either IDH1 or IDH2 have focal hypermethylation at enhancers that form direct interactions with genes relevant for AML pathogenesis, including MYC and ETV6. Based on these findings, we hypothesize that epigenetic changes at specific regulatory enhancers in AML cells can cause the dysregulation of genes that contribute to AML pathogenesis. Here we propose to test this hypothesis by performing detailed, mechanistic studies of enhancers and gene regulation in primary AML samples and AML cell line models. In Aim 1, we will use capture-HiC to perform in-depth studies of the HOXA locus in primary AML samples and AML cell lines that will define the relationships between AML mutations, enhancer interactions, and HOXA gene expression. We will then use massively parallel reporter assays, CRISPR/Cas9 mediated genome editing, and functional studies in vitro and in vivo to identify the specific enhancers and epigenetic pathways that regulate expression of HOXA genes. In Aim 2, we will use in situ HiC to define the 3D genome organization of primary AML samples with mutations in IDH1 and IDH2 that have focal DNA hypermethylation at enhancers. We will integrate these data with DNA methylation, chromatin profiling, and gene expression to determine how DNA methylation influences enhancer-promoter interactions and gene regulation in AML cells. Together, these studies will provide mechanistic insights into HOX gene regulation that may guide therapeutic approaches that target the HOX self-renewal pathway in AML cells, and determine the extent to which DNA methylation contributes to the leukemia phenotype by altering the function of regulatory enhancers.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY B cells have an important pathogenic role in neurological autoimmune disorders, such as multiple sclerosis (MS) and neuropsychiatric systemic lupus erythematosus (NP-SLE), one of the most disabling manifestations of SLE. The relevance of B cells in central nervous system (CNS) autoimmunity is underscored by the therapeutic efficacy of mAb-mediated B cell depletion in MS. How autoreactive B cells are generated and infiltrate the CNS remains enigmatical. The CNS is enclosed within three membranes: pia, arachnoid, and dura. Between the arachnoid and pia lies the subarachnoid space, which contains cerebrospinal fluid that harbors assorted immune cells, including B cells. During neuroinflammation, blood lymphocytes infiltrate the meninges to mount local humoral and/or cellular responses. Thus, meningeal B cells are thought to exclusively derive from the systemic circulation. However, we recently showed that in young adult mice meningeal B cells mainly derive from the bone marrow of cranial flat bones, known as calvaria, through special vascular channels. During aging, in contrast, age-associated-B cells (ABC) migrate from the periphery into the dura, where they may differentiate into Ig- secreting plasma cells. We hypothesize that meningeal B cells that derive from calvaria and differentiate locally are tolerant to CNS-Ag. By contrast, B cells that originate from the periphery and hence are not educated by the local antigenic milieu, may differentiate into autoreactive plasma cells upon CNS-Ag encounter. In Aim 1, we will investigate mechanisms of meningeal B cell tolerance to local antigens under steady-state. Preliminary data suggest that self-Ag experience during B cell development induces meningeal B cell depletion. Alongside, we will examine meningeal B cell activation upon foreign Ag encounter. Finally, we will investigate the impact of the microenvironment in dura B cell development, focusing on CXCL12 produced by dura fibroblasts. In Aim 2, we will investigate autoreactivity of meningeal B cells and plasma cells in the SWAP-70/DEF6 double knock-out (DKO) model of lupus. Preliminary data show accumulation of plasmablasts in the meninges of DKO mice. We will compare the transcriptional profiles and B cell receptor (BCR) repertoires of B cells and plasma cells from the dura and spleen to determine whether systemic B cells clones disseminate equally in lymphoid organs and meninges, or whether the CNS environment recruits specific clones that further differentiate into plasma cells. In parallel, DKO mice will be examined for behavioral alterations and CNS pathology. We will also identify the utmost expanded BCR clones in the dura of DKO mice and generate monoclonal antibodies to ascertain specificity for autoantigens. In Aim 3, we will obtain a single-cell transcriptomic profile of human dura immune cells isolated from autoptic specimens, filling a critical gap in our knowledge of human meninges. Overall, this proposal will advance our understanding of B cells in the CNS and mechanisms that promote neuroinflammation. To achieve this, we will leverage the complementary expertise of the Colonna lab, which studies neuroinflammation, and the Pernis lab, which studies autoimmunity in both humans and mouse models.

NIH Research Projects · FY 2025 · 2022-02

ABSTRACT Treatment Resistant Depression (TRD) in older adults is a leading cause of disability, excess mortality from suicide, and dementia. Cognitive problems and sleep disturbances are common, contributing to recurrence and poor long-term outcomes. Disrupted slow wave sleep is at the nexus of depression and cognitive dysfunction in older adults. Novel approaches to target this core pathophysiology are lacking. Our mechanistic project, Slow Wave Induction by Propofol to Eliminate Depression (SWIPED) Trial, is designed to elucidate the relationships between TRD, sleep disturbances, and cognitive impairments in older adults. Through personalized infusions targeting electroencephalographic (EEG) patterns, we aim for a systematic characterization of the relationships between the propofol-induced EEG slow waves, enhancement of slow wave sleep, and cognitive outcomes. Through the repurposing of propofol, this innovative proposal will establish whether EEG slow waves are a viable therapeutic target for novel antidepressant approaches. The project consists of two clinical trials (Phase 1 and Phase 2) in older adults with TRD. In Phase 1 we will enroll 15 individuals to receive a dose-finding propofol infusion, and then a second infusion at the dose determined to induce slow waves. This phase will establish that propofol both induces slow waves during the infusion and enhances slow wave sleep (SWS) on nights of sleep post-infusion. Then, in Phase 2 we will randomize 60 individuals to multiple infusions of propofol at either a dose that induces slow waves, or a subthreshold dose that does not induce slow waves, and we will examine short and long-term changes in post-infusion SWS as well as executive function, alertness, and depressive symptoms. This phase will test whether enhancement of SWS leads to improvements in cognitive function and mood. This work will enhance our understanding of core deficits contributing to poor mood and cognition in a population at risk for Alzheimer's disease and related dementias. With the rise in the aging population, we hope to provide translatable biomarkers and approaches for future precision medicine, with a long-term goal of improving public health and quality of life for those afflicted with TRD.

NIH Research Projects · FY 2025 · 2022-02

PROJECT SUMMARY The transcription factor Cone-Rod Homeobox (CRX ) is a master regulator of photoreceptor cell fate. Sequence variants in CRX can cause Retinitis Pigmentosa, Cone-Rod Dystrophy, and Leber Congenital Amaurosis, all inherited causes of vision loss and blindness. CRX is the only gene implicated in the pathogenesis of all three of these diseases, which present with both rod- and cone-centric phenotypes of varying age of onset and severity. Several CRX variants have been reported to cause severe dominant disease through antimorphic genetic interac- tions with wild-type CRX, and yet these mutations are adjacent to variants which are benign or only cause mild, recessive disease. Determining which mutations in CRX are pathogenic and quantifying their effect on functional activity is prerequisite to interpreting patient variation and predicting patient phenotypes. However, most variants in CRX are “Variants of Uncertain Significance” (VUS), meaning that insufficient clinical or functional evidence exists to determine their pathogenicity. Without a robust catalog of human genetic variation, advances in patient sequencing cannot be translated into clinical guidance or therapies for patients with uncharacterized variants. One potential solution to this challenge is Deep Mutational Scanning (DMS), which uses massive libraries of variant sequences in multiplexed assays to simultaneously measure the functional consequence of thousands of variants in a gene of interest in a single experiment. In DMS, each gene variant is assigned a quantitative functional pathogenicity score based on its activity in a molecular assay. This proposal will use DMS to simultaneously assay the transcriptional activity and abundance of all point substitutions, truncations, and frameshifts in CRX. The direct product of this work will be a “look-up table” listing the functional consequence of every CRX variant on protein activity and stability, which will be directly applicable to clinical variant classification and decision making. Given the retina's privileged location as a facile target for gene therapy, a catalog of human variation in CRX could inform the clinical management of patients afflicted with inherited retinopathies in the near term. Furthermore, this work will establish an extensible platform for additional DMS studies of other retinal transcription factors, broadly expanding our understanding of inherited retinal disease.

NIH Research Projects · FY 2025 · 2022-01

Project Summary The goal of this study is to test the efficacy of a theoretically informed, school-based intervention for sexual and gender minority (SGM, e.g., lesbian, gay, bisexual, transgender) adolescents, Proud & Empowered (P&E). Studies found that SGM students are 8 to 10 times more likely to experience victimization in schools than heterosexuals, with rates even higher among transgender youth. This bias-based victimization, part of which is commonly known as minority stress, has been cited as a participating factor in the substantial behavioral health disparities SGM face when compared to their heterosexual counterparts, such as depression, anxiety, self-harm, and suicidal ideation and attempt. These disparities are unique to SGM, as when compared to similarly victimized non-SGM peers, victimized SGM adolescents report significantly higher rates of suicide. When schools lack SGM bullying policies, SGM students are more likely to report suicidality than peers in schools with protective policies. Studies also indicate that SGM victimization is more common in schools that lack protective policies and resources such as gender and sexuality alliances (GSAs), SGM-specific antibullying guidelines, teacher and staff training, and openly supportive allies. Therefore, it is clear that any intervention for SGM youth must simultaneously (a) help SGM youth cope with the effects of minority stress and (b) work to reduce the likelihood of future victimization by addressing school-level factors. The P&E intervention seeks to address these outcomes through a novel multi-level school-based intervention. Supported by nine years of research including an NIH- supported feasibility study conducted at four schools (1R21MD013971), we will determine the interventions' efficacy by completing three specific aims: 1) Determine participant-level efficacy of the intervention in an RCT with 24 schools. 2) Determine the schoolwide intervention effects on (a) reporting of minority stress and behavioral health outcomes among all SGM students and (b) perceptions of school climate (i.e., norms, attitudes, beliefs, bullying behavior toward SGMA, policies) among all students. 3) Examine factors that may affect intervention success (e.g., fidelity of implementation, barriers or facilitators to implementation, school or student characteristics) to prepare the intervention for future dissemination. Following the completion of all ten P&E sessions, school-level factors will be addressed by student-led implementation of environmental change strategies at the school focused on key domains of school climate: safety, relationships, teaching and learning, and institutional environment. This innovative R01 application brings together a team of nationally recognized minority stress and prevention science experts and responds to a nationally established public health need for research from the National Academy of Medicine, the National Institutes of Health (NOT-MD-19-001), and the National Gay and Lesbian Task Force.

NIH Research Projects · FY 2026 · 2022-01

Project Summary/Abstract Actin assembly underlies and drives many biological phenomena. Barbed ends of actin filaments, the major sites of polymerization, are controlled by the heterodimeric actin capping protein (CP). CP is regulated by the direct binding of CPI-motif proteins and the protein V-1. These two classes of regulators, CPI-motif proteins and V-1, bind to opposite sides of CP, and they induce conformational changes in CP that allosterically antagonize the binding of the other class. We are studying the molecular biophysical mechanism of these allosteric regulators. We are also studying the physiological function of CP and its regulators, using biochemical reconstitution with purified components, along with molecular genetic perturbations of living cells. Our biochemical studies will test a novel hypothesis for how CP regulators function in cells. Cells contain stoichiometric amounts of V-1 in micromolar concentrations, sufficient to inhibit nearly all of the cellular CP. V-1 is highly diffusible, and V-1 sterically blocks the ability of CP to cap actin filaments. CPI-motif proteins are targeted to membranes, and their CPI motifs allosterically induce the dissociation of V- 1, thus activating CP locally at the membrane. We are testing this hypothesis by determining the molecular biophysical mechanism of the allostery, and by testing the functions of the CPI-motif proteins with respect to cell motility and migration. We have discovered key differences in the biochemical activities of different families of CPI-motif proteins, and we are now using that information to investigate the allosteric mechanism, by combining single-molecule FRET measurements with molecular dynamics simulations. In addition, we are using purified proteins and lipids in a biochemical reconstitution system that induces actin assembly at a surface, thereby mimicking actin polymerization at cellular membranes. We are also using our discoveries about biochemical activities of CPI motifs to test cellular functions of CPI-motif proteins, using chimeras constructed from different CPI- motif proteins with molecular genetic perturbations and real-time movies of the motility phenomena of living cells. Our cell motility assays employ a system of endothelial cell monolayers with transmigrating immune and cancer cells, mimicking the physiological process of transendothelial migration.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY/ABSTRACT Groundbreaking discoveries resulting in the “immuno-revolution” have established the importance of immunology across medical disciplines. Inflammation has long been considered as an important mechanism contributing to the progression of ischemic and nonischemic forms of heart failure. Countless studies have reported associations between numerous serum cytokines, adverse left ventricular remodeling, and patient outcomes in the settings of chronic heart failure and myocardial infarction. While these observations highlight the potential utility of suppressing inflammation in the heart, early clinical studies investigating anti- inflammatory therapies in patients who experienced a myocardial infarction (corticosteroids) or those with chronic heart failure (corticosteroids, TNF blockade) revealed disappointing results and dampened enthusiasm around further drug development. At that time, limited information existed regarding the precise immune cell types that promote disease and the signaling mechanisms that exert their effects. In recent years, a renewed interest in targeting the immune system in cardiovascular disease has emerged. This resurgence is powered by the discovery of the cellular mediators of inflammation in the heart and the identification of the mechanisms that orchestrate their activation and damaging effector functions. These findings exemplify the exciting, but unmet, opportunity to effectively target the immune system and improve outcomes for individuals with cardiac diseases. The proposed research program will built upon our laboratory’s prior accomplishments that have uncovered remarkable diversity amongst the composition and function of macrophages in the healthy, failing, and transplanted heart. We will integrate 3 active projects into a unified research program that aims to 1) dissect new biological mechanisms governing cardiac macrophage diversity and function, and 2) translate our findings into new diagnostic and therapeutic approaches to abrogate heart failure pathogenesis, potentiate functional recovery of the failing heart, and prevent heart transplant rejection. Key themes will include the roles of mechanosensing, inflammatory signaling, and monocyte fate specification in the pathogenesis of heart failure across disease etiologies, cardiac tissue repair and recovery, and heart transplant rejection.

NIH Research Projects · FY 2026 · 2022-01

Project Summary/Abstract Chemotherapy-induced peripheral neuropathy (CIPN) is a common, frequently dose-limiting side-effect of chemotherapeutic drugs. CIPN can be excruciatingly painful, profoundly debilitating, cause permanent disability, and lead some patients to elect to end life-saving treatment. In contrast to other side effects, CIPN frequently lasts well beyond the duration of treatment and can cause permanent disability. Consequently, therapies are urgently needed as they would not only enhance the quality of life of cancer patients both during and after treatment, but also improve cancer therapy by permitting effective chemotherapeutic dosing. To address this need we have developed mechanism-based interventional strategies for CIPN. Chemotherapy-induced neuropathies are characterized by axonal degeneration, which leads to the unpleasant symptoms of neuropathies. We have shown that vincristine and bortezomib, two widely used chemotherapeutic agents with different mechanisms of action act via the neuronal protein SARM1, the central executioner of a genetically encoded axon degeneration program. Activated SARM1 cleaves the metabolic cofactor NAD+, leading to local NAD+ depletion, followed by metabolic collapse and axon fragmentation. We here present several new strategies to block this final common pathway to axon degeneration. We generated a SARM1 dominant/negative that potently inhibits SARM1 function and axon degeneration. We will utilize adeno- associated virus (AAV) -mediated expression of a SARM1 dominant-negative to block SARM1 activity and will assess the effect of SARM1-dominant/negative on axon degeneration, neuroinflammation and functional outcomes. We have shown in vitro that boosting the synthesis of NAD+ strongly protects against vincristine and bortezomib-induced axon degeneration. We will use virus-mediated expression of enzymes of the NAD+ salvage pathway to boost NAD+ synthesis, which counters the axon destructive effects of SARM1. As a further step to translation to the clinic, we will evaluate in mouse models of cancer whether our therapeutic strategies interact with the cancer or chemotherapy and are effective in cancer-bearing mice. Success of our experiments will lead directly to clinically viable means to prevent and treat CIPN.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY There are clear limitations to the current approach to prostate cancer (PCa) diagnosis. Approximately half of the men who undergo a transrectal prostate biopsy—an extremely uncomfortable, invasive procedure with significant risk including sepsis—are not found to have PCa. For those who have PCa, many have indolent cancers that are best managed with active surveillance (AS), which requires annual repeat biopsies due to a lack of accurate noninvasive tools. Biomarkers and prostate magnetic resonance imaging (MRI) have been increasingly used to attempt to address this problem. However, the currently available tools are not accurate enough alone or in combination to forgo biopsy. We have developed a new MRI sequence (diffusion basis spectrum imaging) and a method of analyzing these imaging metrics—diffusion histology imaging (DHI)—that may overcome the limitations of conventional MRI interpretation. Preliminary data demonstrates high accuracy of DHI to predict prostate biopsy results (presence of cancer and grade of cancer when present). We aim to apply DHI to patients in two distinct clinical settings: Aim 1, initial biopsy for PSA screening, and Aim 2, repeat biopsy for known indolent PCa managed with AS. We also plan for Aim 3 to update our DHI model based on the data obtained in these aims, then recruit and test the updated DHI model in an independent group of patients undergoing PSA screening. We hypothesize that DHI will allow for accurate and non-invasive diagnosis of PCa, and thus reduce unnecessary biopsies. In our proposed studies, the men will have had biomarker testing, then receive a clinical prostate MRI (conventional sequences) with the DBSI imaging protocol added onto it prior to biopsy. The DBSI imaging will be analyzed post-acquisition by our DHI model. Note that the DBSI protocol will add just a few minutes to the total duration of the clinical MRI and will not significantly impact the patient or the clinical imaging workflow. In parallel to conventional MRI interpretation and biopsy per clinical care, our team will perform DHI analysis on the MRI images. By comparing DHI to biomarkers and conventional MRI against the histopathologic gold standard (biopsy) in a prospective manner, we will determine if DHI can be used to noninvasively diagnose and monitor PCa; therefore, supporting the clinical translation of DHI to be used as an alternative to invasive biopsies.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY There is a fundamental gap in understanding the pathophysiology of isolated adult-onset dystonia due to genotypic and phenotypic heterogeneity. Determining common pathophysiologic mechanisms across dystonia subtypes is a critical step toward developing more generalizable and effective therapies. Emerging evidence points to striatal cholinergic interneurons (ChIs) playing a key role in the pathophysiology of dystonia, including biochemical and network-level dysfunction. In addition to offering a common therapeutic target, our findings are likely to benefit gene discovery and research on other disabling, less common forms of dystonia. Over the long term, results could guide researchers to perform greater in depth histopathological and biochemical studies in the brain that can lead to identification of new targets for therapeutic intervention. Our goal is to apply a recently developed PET radioligand, [18F]VAT, which possesses high selectivity for vesicular acetylcholine transporters, to investigate striatal ChIs. Structural and resting state functional MRI will examine the relationship of striatal ChIs to related brain networks across different focal dystonia subtypes. Identification of network-level changes across dystonia subtypes will provide better understanding of common pathophysiology and could potentially provide means to assess target engagement for future therapeutic interventions. The central hypothesis is that striatal ChIs contribute to a common pathophysiological mechanism in humans with isolated adult-onset focal dystonia, and that cholinergic integrity relates to striatal functional connectivity and clinical features of dystonia. The rationale for the proposed research is the likely involvement of striatal ChIs in a) normal motor control and b) animal models of dystonia. ChIs are autonomously active and mediate a baseline presynaptic inhibitory tone on striatal medium spiny neurons against the excitatory cortical drive, likely via modulation of dopamine release via presynaptic cholinergic receptors on nigrostriatal dopaminergic terminals. They also receive input from intralaminar thalamic neurons innervated by cerebellar afferents, that when dysfunctional may contribute to dystonic phenotypes. Thus, ChIs may contribute to a common pathogenic mechanism involving cortico-striata-thalamic or cerebello-thalamo-striatal networks. This hypothesis will be tested by pursuing three specific aims: Determine if 1) dysfunction of striatal ChIs is a common mechanism across isolated dystonia subtypes; 2) common aberrations in functional striatal brain networks underlie the isolated dystonia subtypes; 3) clinical characteristics relate to markers of ChIs and brain network dysfunction across different isolated adult-onset focal dystonias. This approach is innovative as it uses comprehensive multimodal imaging to investigate novel PET- measured cholinergic integrity and related functional networks. The proposed research is significant because it is expected to critically advance the understanding of dystonia. Ultimately, such knowledge may provide critical rationale to identify and test new therapeutic targets and better understand other disabling forms of dystonia.

NIH Research Projects · FY 2026 · 2022-01

Of the 33,330 men who died from prostate cancer in United States last year, over 80% of these patients presented with localized disease. Thus, a majority of PCa patients are diagnosed at a potentially curable stage and are often treated with radical prostatectomy (RP). Following RP, patients with aggressive disease face the risk of prostate cancer recurrence, which manifests as persistently elevated or increasing serum PSA. While salvage radiation therapy (RT) represents a standard treatment option for post-surgical recurrences, it results in long-term disease control in only 30-40% of patients. Thus, the post-surgical recurrence state presents multiple opportunities to improve patient care by addressing three critical challenges: (1) determining which patients will benefit from the addition of androgen deprivation therapy (ADT), (2) identifying which patients treated with RT and ADT will require further treatment intensification, and (3) identifying the appropriate treatment intensification strategy for RT and ADT treated patients. Since androgen-directed therapies represent the backbone of treatment for PCa (that have progressed through local therapies) the RTOG 96-01 and RTOG 05-34 phase III clinical trials represent a unique resource of banked prostatectomy cohorts from patients that were randomized to the presence or absence of treatment with ADT. For patients whose clinical and pathologic features place them at highest risk of dying from PCa, these landmark trials have defined the standard of care by showing that the addition of ADT to RT resulted in significant improvements to patient survival compared to RT alone. This presents an unparalleled opportunity to develop and validate predictive and prognostic biomarkers addressing multiple unmet clinical needs throughout patient care following surgery. Aim 1 will focus on using high-throughput DNA and RNA sequencing to develop a predictive classifier for identifying which patients would benefit from ADT using patients from RTOG 96-01 and 05-34. In Aim 2 we will develop and validate a prognostic classifier that integrates genomic and clinicopathologic data for PCa patients treated with RT+ADT that may benefit from treatment intensification. Aim 3 will focus on identifying RT+ADT patients requiring treatment intensification that could benefit from receiving pelvic lymph node radiation therapy using patients from RTOG 05-34. The proposed study would have significant impact by developing and optimizing prognostic and predictive biomarkers that could have enormous potential for rapid clinical translation to personalize therapy and transform the management of PCa patients following surgery.

NIH Research Projects · FY 2025 · 2022-01

Proposal Summary/Abstract While there have been significant advances in the detection and treatment of breast cancer, mortality due to metastatic disease recurrence remains unacceptably high and a leading cause of death. Furthermore, chemotherapies with intolerable side effects remain the standard of care for metastatic breast cancer patients, which is highly detrimental to quality of life. Advances in molecular targeting cancer imaging offer a window into underlying tumor biology and support the use of individualized targeted therapies that can spare patients over- treatment with harsh chemotherapies. However, most molecular imaging probes typically target either cancer cells or, more recently, a single cell type in the tumor microenvironment. These approaches are inherently limited in the information they can provide. Optimal treatment planning relies on a complete picture of disease state, including microscopic regions of residual disease and understanding the potential for recurrence based on a lesion’s specific molecular traits. As such, the goal of this proposal is to provide the foundation for a clinically useful means to image and understand disease status before, during, and after treatment of primary breast cancer and metastatic disease. We postulate that molecular imaging of both the microenvironment and cancer cells simultaneously will provide more complete detection of diseased regions and an understanding of dynamic changes in tumor microenvironment that are needed to support personalized therapy. We propose: (Aim1) A bispecific strategy targeting both breast cancer cells and cancer-associated fibroblasts in the tumor microenvironment to enhance tumor detection. (Aim 2) Differentially targeting cancer cells and cancer-associated fibroblasts with near-infrared molecular probes with similar spectral properties but different fluorescent lifetime values to extract tumor-stroma ratio noninvasively, which is a prognostic indicator in breast cancer. This non-invasive method of determining the relative abundance of malignant cells and cancer- associated fibroblasts will be compared with the traditional histologic determination of tumor-stroma ratio. At the completion of this study, we expect to have established a signal amplification method to detect small cancerous lesions and a non-invasive approach to map cancer-stromal ratio to understand how the dynamic changes in the tumor microenvironment affect cancer biology.

NIH Research Projects · FY 2026 · 2022-01

Our previous study of more than 10 thousand tumors across 33 cancer types identified over 800 germline predisposition variants in both tumor suppressors and oncogenes. Although the impact of some of these variants on cancer onset, incidence rate, and clonality have been documented, the effects of these variants (especially when compared to somatic mutations) on molecular characteristics of cancer cells, contributions of non-cancer cells having germline variants, and treatment responses, are far less studied. Our recent single cell(sc) /single nucleus(sn) RNA-seq analyses of cancer samples provided comprehensive expression profiles at a single cell resolution and revealed the expression of key cancer predisposition genes, such as BRCA 112, VHL, BAP1, and c-MET in many non-cancer stromal and immune cells in the tumor microenvironment (TME). Our pilot analyses also revealed significant differential gene expression in cancer and non-cancer cells based on the nature of the driver event being germline or somatic. We hypothesize that tumors with certain germline predisposition variants in tumor suppressors and oncogenes may show differences in tumor progression and treatment responses from those tumors with somatic mutations in the same genes due to differential influence on mutational, transcriptomic, and proteomic profiles of the cancer cells and potentially distinctive contributions from non-cancer cells having those germline changes. To take advantage of the considerable progress over the last few years, namely newly accumulated cancer sequencing data, advances in single cell omics, patient-derived xenografts (PDX), and tumor genetic models, we propose to test these hypotheses by performing the following: compare germline predisposition variants and somatic mutations to dissect their differential biological impacts and interactions using computational analysis (Aim 1); perform single nucleus RNA-seq/ATAC-seq, spatial transcriptomics, and multiplex imaging analysis of human cancer samples to reveal the differential roles of germline predisposition variants and somatic mutations in tumor cells and the TME (Aim 2); use PDX and genetic cancer models to investigate potential functional differences between germline predisposition variants and somatic mutations in tumor cells, TME, and treatment responses (Aim 3). Results from this study will advance our understanding of the unique contributions of germline variants to cancer cells and TME alike, improve genetic counseling and prognosis, and provide guidance for differential treatment of tumors carrying germline variants vs somatic mutations in key cancer driver genes.

NIH Research Projects · FY 2026 · 2022-01

Project Summary/Abstract Treatment of early stage breast cancer has substantially improved but preventing metastasis remains a more elusive target. Obesity, which is endemic in our society, is associated with an increased risk of breast cancer and accelerated metastasis. The means by which obesity compromises survival of breast cancer patients is, however, poorly understood. Liver is among the most common sites of breast cancer metastasis and its occurrence is generally associated with poor prognosis. Liver health is closely related to weight as obesity is the major cause of fatty liver disease, estimated to be present in 1/4 to 1/3 of Americans. We discovered that fatty liver disease markedly increases liver metastasis, in mice, by providing fuel to tumor cells thereby accelerating their growth. Examination of human liver biopsies and MRI analysis suggests the same is true in women with metastatic breast cancer. Thus, our first goal is to determine if treating fatty liver disease reduces breast cancer liver metastasis and improves its response to chemotherapy. To identify potential new treatments for liver metastasis we will explore the mechanisms by which fatty liver disease stimulates tumor growth. Finally, we will ask if human breast cancers exhibit the same liver metastatic properties as those arising in mice. If our conclusion proves true, treatment of breast cancer will universally require prevention and treatment of fatty liver disease and therefore impact all affected women. Prevention and treatment of fatty liver disease in breast cancer patients, however, may likely reduce liver metastasis and therefore prolong survival. Because fatty liver disease is most often the product of obesity, educating patients about calorie intake, which can be instituted immediately, may have significant effects on breast cancer prognosis.

NIH Research Projects · FY 2025 · 2021-12

PROJECT SUMMARY: α1-antitrypsin deficiency (ATD) liver disease is one of the most common genetic causes of liver disease in children and adults. The only currently available treatment is liver transplantation. The pathobiology of the liver disease begins with a point mutation in α1-antitrypsin (AT), one of the most abundant secretory glycoproteins of the liver. The variant, α1-antitrypsin Z (ATZ), is prone to misfolding and that leads to its accumulation within the early part of the secretory pathway of liver cells. Most of the ATZ accumulates in the endoplasmic reticulum (ER) as polymers and aggregates, and we now know that it is this accumulation of polymerogenic, aggregation-prone ATZ that initiates the process of liver damage by a gain-of-toxic function mechanism. Very little is known about the pathobiological steps after accumulation of ATZ that result in liver damage but it is assumed that liver cell function becomes impaired with stereotypical fibrogenic consequences. Marked alterations of mitochondria have been observed in liver cells of human ATD patients and the PiZ mouse model of ATD, leading to speculation that mitochondrial dysfunction is at least part of the final steps in the demise of liver cell function that characterizes severe ATD liver disease. Over the years we have learned that only a sub-group of homozygotes for ATZ develop progressive liver disease and the majority completely escape clinical effects. This observation has led to the recognition that genetic and environmental modifiers play an important role in the pathobiological effects of ATZ. Work led by the Perlmutter laboratory has shown that the intracellular degradation pathway known as autophagy is a key determinant of ATZ accumulation in liver cells and that drugs which enhance the autophagic degradation of ATZ decrease hepatic fibrosis in animal models, including the ATZ nematode and PiZ mouse models. Other recent studies have shown specificity for the molecular pathways involved in autophagy of specific organelles, and the term `ER-phagy' has recently been recognized as at least part of the process by which ATZ is specifically degraded. Furthermore, a very important new study has shown that at least one ER-phagy pathway is regulated by oxidative phosphorylation genes and mitochondrial function. Based on these considerations and new preliminary data described in the proposal, we now believe that mitochondrial impairment is a key part of the pathobiology of ATD liver disease in two ways, impaired liver cell energy metabolism and reduced autophagic response, and, therein, that mitochondrial function is a very appealing target for potential therapeutic interventions. In this grant we propose to investigate the effects of ATZ accumulation on mitochondrial function to better understand the mechanism by which liver is damaged and to investigate whether mitochondrial function can be targeted for therapy. Our overarching goal with these studies is to provide a basis for clinical trials of human ATD liver disease that target mitochondrial dysfunction.

NIH Research Projects · FY 2026 · 2021-12

PROJECT SUMMARY/ABSTRACT Mutations in telomerase and telomere attrition are major risk factors for liver fibrosis and its progression to hepatocellular carcinoma (HCC). However, due to a lack of adequate models and intrinsic difficulties in studying human telomerase in physiologically relevant cells, the molecular mechanisms responsible for liver fibrosis and cancer in settings of DNA damage arising from short telomeres remain elusive. While telomerase knockout mice corroborate the importance of telomere maintenance and DNA repair for liver function, the molecular mechanisms that govern liver abnormalities in patients with damaged telomeres are still unknown. Likewise, the specific signaling pathways that trigger failure of hepatic cells following telomere shortening and accumulation of DNA damage remain to be determined. In addition, mutations in the promoter region of the telomerase reverse transcriptase component (TERT) have been described as the initial and most prevalent mutation in HCC. While these mutations have been shown to reactivate telomerase, the functional relevance of this process during failure and transformation of hepatic cells has yet to be interrogated. The focus of this proposal is to use human pluripotent stem cells as a novel platform to understand the detrimental effects of mutant telomerase, telomere shortening and accumulation of DNA damage in different hepatic cell lineages. We have previously generated isogenic hPSC lines harboring several disease-specific mutations in telomerase and have successfully derived telomerase-mutant human hepatocytes and hepatic stellate cells in vitro, following established protocols that recapitulate the in vivo development of these lineages. Here, two specific aims are proposed that utilize this platform to understand the molecular consequences of telomere erosion, DNA damage, and telomerase impairment for the function of hepatic cells, and to determine their role during early stages of transformation. In Aim 1 we will determine the role of telomere shortening and DNA damage accumulation during fibrotic failure of different hepatic cell lineages with impaired telomerase. We will determine the extent to which mitigation of DNA damage, reactivation of HNF4α, and modulation p53 prevent fibrotic triggering in telomerase-mutant hepatocytes with variable telomere lengths. As liver fibrosis and its progression to HCC are multicellular responses we will determine the role of progressive telomere shortening during the direct and the paracrine fibrotic activation of hepatic stellate cells. In Aim 2, we will investigate the molecular consequences of mutations in the TERT promoter region during progression of HCC, in settings of exacerbated DNA damage due to eroded telomeres. Specifically, we will analyze the biochemical and functional consequences of mutations in the TERT promoter region for hepatocyte function and immortalization. These studies will determine the molecular mechanisms of liver fibrosis and its progression to HCC in settings of mutant telomerase and DNA damage. Our unique tools, combined with our expertise in telomerase, DNA repair, and stem cell biology puts us in an ideal position to make a significant impact in this field.

NIH Research Projects · FY 2025 · 2021-12

Project Summary Pediatric patients are more vulnerable to radiation exposure when compared to adults. Each year, 2.2 million pediatric head computed tomography (CT) scans utilizing ionizing radiation are performed in the United States. Head trauma and craniosynostosis are two of the most common pediatric conditions requiring head CT scans. Multiple CT scans are often performed during clinical follow-up, exacerbating the cumulative risk of radiation exposure. Head trauma is common in children, frequently resulting in a skull fracture. Craniosynostosis is a congenital disability defined by a prematurely fused cranial suture. Standard clinical care for pediatric patients with head trauma or craniosynostosis employs 3D high-resolution cranial CT images to identify cranial fractures or cranial suture patency. The National Cancer Institute reported that radiation exposure from multiple head CT scans in children has the potential to triple the risk of leukemia and brain cancer due to radiosensitivity of their bone marrow and brain tissue. Magnetic resonance imaging (MRI) is a safe alternative without ionizing radiation. Existing “black bone” MRI methods rely on a diminished bone signal in a standard gradient echo scan to image the skull. Though these methods have shown encouraging results, they have not translated into clinical practice due to several challenges: motion artifacts, long acquisition time, and subjective manual image processing. Since pediatric patient movement is very common, sedation has been routinely used to minimize motion artifacts in an MR scan. Unfortunately, sedation is associated with risks including developmental delay and cardiopulmonary complications. It takes several minutes to acquire high-resolution MR images, which can be challenging for pediatric subject compliance and limits clinical adoption. Due to poor signal contrast between bone and its surrounding tissues in MR images, existing manual signal intensity-based approaches are challenging and not suitable for clinical translation. Our primary goal is to develop novel MR techniques to provide CT-equivalent 3D high-resolution cranial bone imaging. Four specific aims are proposed: 1) develop motion correction to address head motion in unsedated pediatric patients; 2) develop an MR image reconstruction method regularized by a deep-learning prior to reduce MR acquisition time to 1 minute or below; 3) develop a 3D Bayesian neural network to estimate pseudo-CT (pCT) and uncertainty maps from MRI for robust and automated image post-processing; and 4) determine the clinical utility of pCT in identifying cranial fractures and cranial suture patency. This study will have a profound impact on pediatric health by removing the risks associated with radiation and sedation.

NIH Research Projects · FY 2026 · 2021-12

Project Summary Little is known about the mechanisms that determine daily rhythms in rest-activity. We recently found that cells within the motor cortex can be synchronized to daily cycles of glucocorticoids. We hypothesize that daily suprachiasmatic nucleus activity and corticosterone secretion entrain circadian rhythms in cortical neurons and astrocytes to regulate daily patterns of behavior. We will take advantage of new high-throughput, machine learning and the ability to record and manipulate gene expression and calcium levels in specific cell types in mice. Combining these methods with two-color, real-time imaging of gene expression simultaneously in neurons and astrocytes, we will determine the roles of cortical neurons and glial in distinct daily activities.

NIH Research Projects · FY 2026 · 2021-11

PROJECT SUMMARY Neutrophils are essential for host defense against bacteria, however toxic neutrophil mediators such as reactive oxygen radicals, granule enzymes and neutrophil extracellular traps contribute the pathogenesis of acute and chronic inflammatory diseases. While the ability of neutrophils to phagocytose and kill pathogens has been known for over 100 years, our knowledge of the molecular mechanisms guiding neutrophil activation lags significantly behind that of other immune cells, precluding the development of strategic therapeutic interventions targeting neutrophils. Our long-term goal is to define the mechanisms by which ion channels and associated signaling pathways regulate neutrophil activation, and to leverage this knowledge to modify disease. Calcium signals initiated via store-operated calcium entry (SOCE) are required for neutrophil activation. The cell membrane potential directly influences influx of positively charged calcium ions. While the functional role of the cell membrane potential has been extensively studied in excitable cells, little is known about how the membrane potential modifies cellular processes in neutrophils in the context of development and inflammation. This proposal is based on four fundamental observations from our laboratory: 1) ORAI1 and ORAI2 calcium channels are critical for neutrophil SOCE and host defense during infection with S. aureus. 2) Calcium responses in mouse neutrophils are heterogenous, with the magnitude of the calcium response modulated in part by differential regulation of the cell membrane potential. 3) Expression of the calcium- activated potassium channel KCa3.1 (Kcnn4) drives cell hyperpolarization and enhanced SOCE in a subset of neutrophils. 4) The membrane potential-SOCE relationship is modulated during neutrophil development and emergency granulopoiesis. Moreover, we have observed that a S. aureus pore-forming toxin manipulates the neutrophil membrane potential and SOCE. Together these observations illustrate that the membrane potential is a key modifier of calcium-dependent neutrophil function, and suggest that this pathway is a strategic target of human pathogenic bacteria that secrete pore-forming cytotoxins to overcome host neutrophil defenses. The objective of this proposal is to characterize the mechanisms by which membrane potential regulates SOCE during neutrophil development and inflammation. We will test the central hypothesis that the membrane potential is a critical modifier of neutrophil calcium signaling in mature neutrophils and homeostatic and emergency granulopoiesis. In Aims 1 and 2 we will investigate the role of KCa3.1 in neutrophil SOCE and calcium-dependent activation in mature and developing neutrophils. In Aim 3 we will expand these studies to investigate how exogenous manipulation of the cell membrane potential by bacterial pore-forming toxins disrupts calcium-dependent neutrophil function. The insight derived from these studies is anticipated to engender new clinical opportunities for modulation of neutrophil-dependent infectious and inflammatory disease, and potentially inform a broader understanding of membrane potential in immune cell function.

NIH Research Projects · FY 2026 · 2021-11

SUMMARY Urinary tract infections (UTIs) affect 15 million women in the United States every year and treatment for UTIs is becoming more difficult due to high rates of antibiotic resistance. Further, UTIs are highly recurrent. Between 20 and 40% of UTI episodes are followed by recurrent UTIs (rUTIs), with some women suffering as many as 6 or more recurrences per year. Uropathogenic Escherichia coli (UPEC) is the major causative agent of UTIs. Antibiotic resistance within UPEC isolates is rising, and the emergence of extended-spectrum beta-lactamase producing and fluoroquinolone resistant strains is a serious public health concern. Type 1 pili, tipped with the mannose binding FimH adhesin have been shown to be essential for bladder colonization and UTI pathogenesis in multiple mouse models. FimH mediates binding to mannosylated uroplakins lining the bladder surface to facilitate colonization and invasion into bladder cells where they rapidly replicate into intracellular bacterial communities that protect UPEC from immune cells and antibiotics. In addition, FimH facilitates the ability of UPEC to establish a reservoir in the GIT, from where they can seed UTIs by ascending from the periurethral area into the bladder. While UPEC are genetically variable, FimH is part of the core E. coli genome, although rare strains have been found with mutations in the type 1 operon. Immunization against FimH protects against UPEC UTI in murine and monkey cystitis models and a FimH-based vaccine has been allowed by the FDA for patients suffering from multi-drug resistant UPEC. In animal models, protection is antibody-mediated, as FimH- specific IgG antibodies are found in the urine from protected animals and can protect from UTI through passive transfer. Intriguingly, UPEC abundance in the gut is increased at the time of symptomatic UTI, suggesting that gut colonization is a key step in the rUTI cycle. Additionally, studies in this proposal show that eliciting a mucosal antibody response against FimH can reduce UPEC colonization of the gut. In light of these findings, this proposal addresses the hypothesis that anti-FimH induced antibodies can combat rUTI by two distinct mechanisms: i) prevention of UPEC binding to the uroepithelium; and ii) interference with UPEC colonization of the GIT, thereby lowering the likelihood of UPEC introduction into the urinary tract. The aims of this proposal are to: i) determine how mucosal vaccination against FimCH reduces UPEC gut colonization (Aim 1); ii) exploit vaccine induced B cell responses to isolate monoclonal antibodies (mAbs) to FimH and determine their epitope specificity (Aim 2); and iii) use these mAbs in mouse models of GIT colonization and cystitis in order to elucidate mechanisms of protection (Aim 3). The research plan will unravel the mechanisms by which anti-FimH antibodies may function to prevent UTIs by directly blocking bladder binding and indirectly by interfering with UPEC GIT colonization. These results will inform the rational targeting of the uropathogens that affect millions of people with rUTIs. In addition, our approach enables the rapid generation of anti-UPEC human mAbs that can be used for therapeutics and diagnostics.