Washington University

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT Head and neck cancer (HNSCC) afflicts mostly men. Female patients who develop HNSCC tend to be postmenopausal, with menopausal hormone therapy lowering their risk of developing the disease. We show that premenopausal female HNSCC patients have an improved response to therapy including immunotherapy (IO). Using clinical trial data, we show no difference in response to IO from testosterone. Similarly, our preclinical data show no IO response effect from gonadal castration. But 0.1 mg estradiol (E2) improves response in castrated male mice to that of female mice. Our overall objective is to understand how the premenopausal female hormone E2 contributes to progression and treatment resistance. Our data show that E2 had no effect on cancer cell viability, suggesting the tumor microenvironment (TME) as the site of action. E2 is known to play a major role in autoimmunity and inflammation, where it modulates dendritic cell (DC) and regulatory T cells (Treg) differentiation and function, and lymphatic vessel maturation. These immune cell populations regulate response to immunotherapy in HNSCC, but how they are affected by sex hormones remains largely uncharacterized. Our preclinical data also show that compared to male mice, females have increased DCs, less suppressive Tregs, and respond better to immunotherapeutic strategies with enhanced Teff function. Such response is removed with oophorectomy (OVX) but rescued with E2 of 0.1mg. OVX also changes the TME towards an immunosuppressive one with enhanced immunosuppressive Treg activity, similar to that of male mice. Finally, within the TME, OVX reduces the formation of high endothelial venules (HEVs) in the TME and draining lymph nodes (dLN), which enhance Teff cell priming and subsequent trafficking into the TME. We hypothesize that the sexually dimorphic response is driven by E2 acting via its receptors on Tregs and DCs to enhance antigen presentation and increase Teff activation, thus selectively determining response to immunotherapy. We also hypothesize that E2 will increase homing and trafficking of antigen specific T cells by enhancing HEV formation and maturation. In Aim1, we will interrogate the regulatory mechanisms of E2's effects on the DC biology by using genetically engineered mouse models (GEMMS), in vitro assays, and proteomic analysis to examine receptor subtypes and molecular mediators of DC differentiation, maturation, and antigen presentation. Aim 2 will dissect mechanisms of how E2's effect on Tregs can drive response and selection of sex-specific immunotherapies. We will test this with GEMMS, mouse chimeras, and pharmacological manipulation in in vivo and in vitro co-culture experiments, to identify signaling and downstream effectors. Focusing on T cell trafficking in Aim 3, we will examine E2's effect on HEV formation, and T cell egress from the dLN to the TME and systemic circulation using a combination of flow and immunofluorescence techniques in GEMMs and in vitro systems. These studies will collectively elucidate the basic mechanisms for advancing sex- directed novel biomarkers and therapeutics in HNSCC.

NIH Research Projects · FY 2026 · 2023-07

Project Summary Nonalcoholic steatohepatitis (NASH) is a progressive liver disease that is a leading cause of liver-related morbidity and mortality. Having NASH greatly increases the risk of developing end-stage liver disease such as cirrhosis and hepatocellular carcinoma. Despite the healthcare burden, there are no licensed drug therapies for NASH. NASH presents as hepatocyte damage, inflammation, and varying degrees of fibrosis, but the distinct mechanisms that drive this progression are unclear. However, evidence shows that infiltrating macrophages play a pivotal role in NASH progression. Macrophages are innate immune cells that can polarize into inflamma- tory phenotypes that exacerbate hepatic injury, or into resolving phenotypes that can mediate liver repair. Yet the mechanisms driving macrophage polarity in NASH progression are undefined. There is a critical need to understand the processes that dictate macrophage polarization in NASH progression in order to identify thera- peutic targets that decrease the burden of NASH. Our long-term goal is to understand the metabolic regulatory mechanisms dictating macrophage polariza- tion to develop treatments for NASH and its associated comorbidities. Importantly, macrophage polarization results in the metabolic rewiring of mitochondrial metabolism. This is not only a consequence of the polariza- tion process, but the accumulation of specific tricarboxylic acid (TCA) cycle-derived intermediates profoundly influences inflammatory and anti-inflammatory gene expression in macrophages. A central player in mitochon- drial metabolism is the mitochondrial pyruvate carrier (MPC), which transports pyruvate, a primary product of glycolysis, into the mitochondrial matrix where it can enter the TCA cycle. Previous evidence shows MPC inhi- bition alters intracellular TCA metabolites in metabolic cells like myocytes and hepatocytes, but the effect on macrophages is unknown. Furthermore, our lab has shown that novel MPC inhibitors currently used in clinical trials effectively reduce NASH in mice. Importantly, the effect of MPC inhibition in macrophages is undefined. Given the significance of intermediary metabolism in macrophage activation, and the significant role of the MPC in mitochondrial metabolism, the primary objective of this application is to [1] identify the mechanisms by which MPC contributes to macrophage activation and polarization, and [2] determine how specific deletion of MPC in macrophages influences NASH progression. Our central hypothesis is that MPC inhibition in mono- cyte-derived macrophages will reduce the inflammatory response and mitigate NASH development. Comple- tion of these studies will contribute to the understanding of mitochondrial metabolism in macrophage function and NASH progression, and highlight the clinical utility of macrophages in NASH.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Acute myeloid leukemia (AML) has thus far proven resistant to T cell redirecting therapies such as bispecific antibodies or chimeric antigen receptor (CAR) T cells, which is unexpected given the proven success of these in other hematologic malignancies. Preliminary studies show that the anti-tumor activity of CD19-targeting CAR T cells (CART19) is enhanced in the presence of myeloid cells, and that targeting these myeloid cells with CD33-targeting CART cells (CART33) leads to diminished long-term anti-tumor effect. Furthermore, removing CD33 from normal myeloid cells improves CART33 expansion and disease control. Based on these findings, the central hypothesis of this proposal is that bystander myeloid cells enhance CART cell activity, and directly targeting myeloid cells compromises this effect. The objective of this proposal is to delineate how myeloid cells influence CAR T cell behavior when they are targets of therapy, as compared to when they are simply bystanders, so that we can intervene within these interactions and improve CAR T cell therapy for AML. This will be achieved through the following specific aims: 1) Determine how bystander myeloid cells modulate CAR T cell function; 2) Determine how targeting myeloid cells changes CAR T cell profile. To this end, hematopoietic stem cells (HSCs) will be engineered through either viral gene transfer or CRISPR/Cas9 gene knockout to generate myeloid cells that are positive or negative for the antigen targeted by the CAR. The effects of wild-type or genetically modified myeloid cells on CAR T cells will be interrogated by in vitro culture and in vivo mouse models, using flow cytometry, single-cell RNA-seq, and functional studies. The innovations of this project are that it draws attention to host environmental factors that influence CAR T cell activity, and advances the concept that the nature of the cell being targeted can influence CAR T cell behavior. This research is significant because it will contribute to a better understanding of how CAR T cells work with the immune environment, and illuminate methods to intervene within these interactions to improve the outcomes of therapy. The long-term goal of this proposal is to establish the applicant Dr. Miriam Kim's career as an independent researcher focused on developing novel cell therapies for AML. The proposed research and career development plan will provide Dr. Kim with training in HSC/myeloid biology, immunology, and bioinformatics. Her primary mentor, Dr. John DiPersio, and co-mentors Drs. Robert Schreiber, Todd Fehniger and Carl DeSelm, offer complementary expertise in cell engineering and immunotherapy. Additionally, Dr. Kim's collaborators Dr. Li Ding and Dr. Feng Gao will contribute to developing her skills in bioinforrnatics and biostatistics. Furthermore, Washington University provides an ideal environment for Dr. Kim to successfully establish herself as an independent investigator.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Overall: Abstract Since its original description by Hans Chiari in the 1890s, Chiari type I malformation (CM) has been defined as caudal displacement (ectopia) of the cerebellar tonsils below the foramen magnum. This definition oversimplifies the complex range of craniovertebral junction pathology affecting more than 1% of the population and has little relevance to the neurological effects of CM. However, current clinical practice continues to rely heavily on this oversimplified and outdated definition of CM, with most management decisions based on a single, static 2D sagittal MRI image, failing to consider the dynamic pathophysiology at the interface of the cerebellum, brainstem, and spinal cord. Investigations from our group and others implicate a hyperdynamic pathophysiological state in CM, with turbulent CSF flow patterns, exaggerated pulsatile motion of the tonsils and brainstem, and dysfunction or injury to the cerebellum, brainstem, or spinal cord. The relationship of these changes at the CVJ to patient experience (pain and/or neurological deficits), cognitive or behavioral outcome, and CM-associated syringomyelia (SM, spinal cord cavitation), remains unclear and under-investigated. The Park-Reeves Chiari & Syringomyelia Center (PRCSC) at Washington University was established in 2011 and has become the international leader and vibrant focal point for research into CM. The Overarching Theme of this PRCSC Program Project Proposal is “Redefining Chiari Type I Malformation and its Impact on Neurological Outcome.” This proposal will leverage key strengths and scientific advances at Washington University to investigate the largest and most refined cohort of CM patients in the world. Four Cores [Administrative (AC), Clinical (CC), Genetic (GC), and Radiology (RC) Cores] will support 4 Projects: Genetic Underpinnings of CM and Effect on Brain Development [Haller, Project Lead (PL); Solnica-Krezel, Co-Lead (CL)]; Functional Connectivity, Brain Development, and Outcomes in CM [Dosenbach, PL; Roland and Marek, CL]; CSF-Mediated Pathophysiology and Microstructural Injury Determines Outcomes in CM [Strahle, PL; Song, CL]; and Redefining CM Using Genotype-Phenotype Relationships and Impact on Outcome [Limbrick, PL; Greenberg and Lu, CL]. Although these Cores and Projects each address unique Specific Aims, they will generate a wealth of multidimensional data from PRCSC participants to permit a cohesive, synergistic, and comprehensive examination of the overarching Aims of this application. Indeed, PRCSC as a whole is much greater than the sum of its Cores and Projects. Overall Specific Aim 1. Determine the genetic factors contributing to CM, brain development, and neurological function. Overall Specific Aim 2. Determine the pathophysiological basis for CM-related neurological effects. Overall Specific Aim 3: Create a novel CM framework to enhance treatment response and outcomes.

NIH Research Projects · FY 2025 · 2023-07

Project Abstract Prostate cancer (PC) patient although initially respond to androgen deprivation therapy, most patients develop the resistance developing a stage referred to as the Castration Resistant Prostate Cancer (CRPC). Prostate cancer is a non-inflamed or “Immune desert” tumor where no immune infiltrate is observed, suggesting that failure has occurred somewhere in the process of T-cell priming, or T-cell trafficking back to the tumor. Not surprisingly, prostate cancer is highly refractory to immune checkpoint blockade (ICB) therapies exhibiting marginal efficacy in clinical trials, both as a single agent or in combination with other agents. Precisely how prostate cancer enforce evasion of anti-tumor immune response is not fully understood. Previously, we uncovered that a non-receptor tyrosine kinase, ACK1 deposited novel pY88-H4 epigenetic marks in AR gene enhancer, regulating AR/AR-V7 expression. Building on this discovery, we developed a new ACK1 small molecule inhibitor, (R)-9b, which suppressed enzalutamide-resistant tumor growth. To examine ACk1 signaling further, we generated a viable conditional ACK1 knockout (KO) mice, and noticed a significant increase in activated CD4+ and CD8+ T cells in KO mice, causing loss of syngeneic prostate tumor growth. The subsequent studies have revealed a crucial role for ACK1 kinase in the initiation of T cell antigen receptor (TCR) signaling by phosphorylation of CSK at a previously unknown site, Tyr18. CSK phosphorylated LCK at Tyr505 promoting auto-inhibition, inhibiting an adaptive immune response. Thus, ACK1 KO mice, or the (R)-9b injected mice exhibited increased CD4+ and CD8+ T cells activation and inhibition of tumor growth. In addition, (R)-9b functionally reinvigorated peripheral blood mononuclear cells (PBMCs) of the CRPC patients to mount robust immune response against CRPC organoids. Together, these data indicate that (R)-9b fulfills a unique niche, wherein it not only suppresses AR/AR-V7 within the tumor cells, but also activates host immune system to mount a robust `dual’ anti-tumor response. The specific aims of this project are: Aim 1: Examine roles of ACK1-mediated CSK Tyr18-phosphorylation in T cell quiescence Aim 2: Interrogate role of renewed pACK1/pY18-CSK/pY505-LCK signaling in silencing of anti-tumor immune response Aim 3: Evaluate therapeutic efficacy of (R)-9b in models of prostate cancer

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY SARS-CoV-2 has led to unprecedented disruptions to society, killing more than 6 million people worldwide. It is a respiratory virus whose main mode of transmission is via respiratory droplets and aerosols. Social distancing and vaccines have greatly decreased the rate of infection and transmission. Despite these efforts, SARS-CoV- 2 transmission has continued. Moreover, different variants of concern, harboring signature mutations in the virus attachment Spike protein, have emerged. How these changes affect transmission of SARS-CoV-2 in naïve, infected and immunized individuals is not known. The transmission bottleneck is defined as the number of unique virus particles that establish an infection in the recipient host. This number is important as it determines the rate of evolution of the virus and the immune threshold required for protection from infection. This application will use barcoded or tagged SARS-CoV-2 to quantify how many virus particles establish an infection in the recipient host. We will use the Syrian hamster SARS-CoV-2 airborne transmission model to define how the innate and adaptive immune response in the donor and recipient host effect the number of unique transmission events. Using genetically modified hamsters that are deficient in their type I or III interferon response will be used to measure the role of innate immunity on SARS-CoV-2 transmission to the upper respiratory tract (URT) and subsequent dissemination of the virus to lower respiratory tract (LRT). Transmission and dissemination of SARS-CoV-2 will also be quantified in recipient animals that were previously infected with SARS-CoV-2, immunized with mucosal and systemic COVID-19 vaccines, or received neutralizing IgG and IgA antibodies. Finally, this application will measure the impact of immune escape on the transmission bottleneck in immune recipients. Fundamental insights into respiratory virus transmission and dissemination, transmission bottleneck and defining correlates of protection in the URT and LRT will inform future vaccine efforts against respiratory viruses.

NIH Research Projects · FY 2026 · 2023-07

When individuals choose between multiple options, decisions are based on subjective preferences. This behavior is specifically disrupted in a variety of mental and neurological disorders including frontotemporal dementia, major depression and drug addiction. Thus to better understand the origins of these disorders and to pave the way for treatments it is critical to understand the neural underpinnings of this behavior. Preference-based decisions are observed in many species, including non-human primates and rodents. Evidence from neurophysiology in non-human primates, functional imaging in humans and lesions in multiple species establishes a link between decision making and the orbitofrontal cortex (OFC). In particular, previous research in the PI’s lab examined the activity of single neurons in the OFC of monkeys choosing between different liquid rewards. This work identified three groups of cells encoding the identity and values of offered and chosen goods. The variables encoded in OFC capture both the input and the output of the choice process, suggesting that the cell groups identified in this area constitute the building blocks of a circuit in which decisions are formed. A series of theoretical and experimental results support this hypothesis, but many aspects of this circuit are poorly understood. For example, we don’t know whether different cell groups identified in OFC correspond to different anatomical cell types, whether they populate different cortical layers, and how they are connected with each other and with other brain regions. To address these and related questions, we brought our investigation to a genetically tractable species – the mouse. In a first study, we developed a mouse model of preference-based decisions and we demonstrated that optogenetic inactivation of the lateral orbital area (LO) disrupts this behavior. Next, we developed a protocol for two-photon (2P) calcium (Ca2+) imaging of area LO in mice performing choices. We thus identified three groups of neurons analogous to those previously found in primates. The overarching goal of this proposal is to link the functional cell groups identified in LO to anatomically defined cell types. To do so, we will combine 2P Ca2+ imaging with genetic markers and viral tracing. In a series of experiments, we will assess (Aim 1) whether different cell groups defined in relation to choice behavior reside preferentially in different layers, (Aim 2) whether different cell groups are differentially connected with sensory and/or motor regions, and (Aim 3) the relation between functional cell groups and inhibitory interneurons. In addition, (Aim 4) we will use statistical modeling to assess the effective connectivity between different cell groups. This is a collaboration between Dr. Padoa-Schioppa (PI), an expert in neural mechanisms underlying decision making, and Dr. Holy (co-I), an expert in mouse olfaction and optical imaging. The experiments proposed here will break new ground and extend previous work in a new and promising direction. If successful, our research will shed light on a fundamental brain function. It will also lay the ground to pursue numerous other questions. Preliminary results obtained for each specific aim demonstrate high likelihood of success.

NIH Research Projects · FY 2024 · 2023-07

Project Summary/Abstract. Between 3-5% of adults harbor an intracranial aneurysm1, and subarachnoid hemorrhages (SAH) resulting from ruptured aneurysms are associated with a mortality rate of 10-25% with an additional 30% of patients suffering permanent disability2. Inflammation plays a central role, driving the morbidity associated with SAH. Despite understanding the important role of inflammation in morbidity following SAH, the current barrier is that there is no effective methodology to modulate the deleterious inflammatory response in patients following SAH. Due to this gap, there is a critical need for a novel approach to immunomodulation that can be safely, rapidly, and effectively deployed in SAH patients. Vagus nerve stimulation (VNS) provides a novel, non-pharmacologic approach to immunomodulation. Studies have demonstrated VNS to reduce systemic inflammatory markers3 and VNS has had early success treating inflammatory conditions such as arthritis4, sepsis5, and inflammatory bowel diseases6,7. Our long-term goal is to translate the use of non-invasive transcutaneous auricular VNS (taVNS) to reduce morbidity and improve outcomes in patients following spontaneous SAH. The overall objectives for this application in pursuit of achieving this goal are to (i) demonstrate the impact taVNS has on a key inflammatory marker in the blood and CSF in patients following SAH, and (ii) determine if taVNS reduces the incidence of inflammation-mediated sequelae of SAH by performing a prospective, randomized controlled trial. Our central hypothesis is that implementing taVNS in the acute period following spontaneous SAH will attenuate the expected inflammatory response to hemorrhage and will curtail morbidity associated with inflammatory-mediated clinical endpoints (i.e., vasospasm, hydrocephalus). Our hypothesis has been formulated based on preliminary findings where our team applied taVNS in a cohort of SAH patients leading to reductions in white blood cell count, TNF- α, and IL-6 in the CSF, as well as early evidence of decreased vasospasm and chronic hydrocephalus. Guided by this preliminary data, we will test our central hypothesis with the following specific aims: 1) Define the impact that transcutaneous auricular vagus nerve stimulation has on a key subarachnoid hemorrhage-induced inflammatory marker, TNF-α, in the serum and cerebrospinal fluid (CSF), 2) Determine that taVNS following SAH reduces angiographic vasospasm, and 3) Determine that taVNS following SAH reduces chronic hydrocephalus. We will perform a randomized controlled study on aneurysmal SAH patients where we will test whether twice daily treatment with taVNS compared to sham will alter these physiologic and clinical endpoints. This project is innovative because it diverges from the pharmacologic status quo by harnessing a novel non-invasive neuromodulatory approach and its known anti- inflammatory effects to alter the pathophysiology of SAH. The development of a new and effective intervention in SAH will be significant because this work will create the foundation of knowledge for advancing non-invasive taVNS to reduce the societal and personal burden of post-SAH morbidity.

NIH Research Projects · FY 2025 · 2023-07

This K23 application proposes a research and career development plan for Catherine Hoyt, PhD, OTD to establish herself as an independent rehabilitation scientist focused on the early identification and intervention for developmental deficits among infants and toddlers with sickle cell disease (SCD). SCD is the most common monogenic disorder in humans and is primarily inherited by who identify as Black or of African descent. Complications associated with SCD (e.g., infection, pain, stroke) are common in the first years of life. In our earlier work, we found that developmental deficits were present in > 50% of children with SCD before the age of 3 but are none had been diagnosed or referred to intervention services. Further, children whose caregivers participated in a home-based caregiver education program demonstrated improved test scores on standardized measures. Thus, when developmental deficits are overlooked, children miss a critical opportunity for intervention that could improve their developmental trajectory. The American Society of Hematology (ASH) recommends frequent developmental screening starting in the first years of life for all children with SCD. Yet few, if any, studies have described the incidence and severity of developmental deficit among children with SCD compared to controls. Consistent with the American Academy of Pediatrics (AAP) guidelines, this research will evaluate children with SCD at 9, 18, and 30 months using the best available developmental measure, the Bayley Scales of Infant Development-4 (Bayley), to determine the incidence of developmental deficit over the first 3 years of life compared to demographically match peers (n = 100, Aim 1). If developmental deficits are identified, scores will be shared with the child’s healthcare team so they can be addressed. Based on theory and evidence, the proposed study will also test a multi-component Sickle Cell Collaboration for Child Development (SCCCD) intervention. The SCCCD combines skilled therapeutic intervention to address developmental deficits, the Parents as Teachers® curriculum and specific SCD education. Our innovative SCCCD intervention is adapted from a pilot study and will provide 12 home visits to caregivers and children with SCD over the course of 1 year (n = 25, Aim 2). Interviews with caregivers who participated, as well as those who declined, will identify contextual determinants (i.e., facilitator and barriers) to prepare for future testing and scaling up of the SCCCD intervention (Aim 3). The results from this K23 award will provide data to understand the onset of developmental deficit in this understudied population and identify the next steps to conduct a randomized control trial to test our SCCCD intervention in an R01 level grant submission. These mentored research aims, combined with a career development plan for advanced training in implementation science (Goal A), mixed methods (Goal B), prospective trial design (Goal C) and professional development (Goals D, E) will enable Dr. Hoyt to launch a career as an independent scientist.

NIH Research Projects · FY 2025 · 2023-07

My training goals outlined in this K23 Career Development Award include to: (1) develop the skills to effectively conduct high-quality, clinically-relevant, mixed-methods analyses of barriers and facilitators of hospital-based violence intervention program (HVIP) enrollment; and (2) complete an individually-tailored training program centered in Community Based Participatory Research (CBPR) and Dissemination and Implementation (D&I) Science. This will support my long-term career goal to become an independent investigator of firearm injury prevention by developing, testing, implementing, and continually refining HVIPs. HVIPs share common outcome goals (decreasing reinjury and death rates among participants), and are, by design, individualized programs that provide counseling and social- and case-based services to achieve additional goals that are unique to each participant. For this reason, I will train in mixed methods study design that supports real-world applicability and adaptability of our findings. I will use this award to study CBPR and D&I science to develop the skills necessary to become a leader in HVIP innovation. My career development plan includes a: (1) robust mentoring plan that provides specific details on mentorship roles and development milestones to capitalize on the complementary areas of expertise among mentorship team members; and (2) didactic training plan through the Master of Science in Clinical Investigation program. My research project is a multi-center, mixed-method analysis of barriers and facilitators of enrollment in Life Outside Violence (LOV), the St. Louis region-wide HVIP. Violent injury is an under addressed public health threat. LOV's mission is to facilitate healing and decrease incidences of retaliation, criminal involvement, reinjury, and death after surviving a violent injury. In early analysis of 158 LOV enrolled patients, three (2%) have suffered a recurrent violent reinjury— well below the national rate (up to 77%). However, only 13% of eligible patients enrolled in the LOV program. To address this enrollment gap, I will investigate the following specific aims, to: (1) Apply multilevel regression models to the STL-HVIP-DR to identify sociodemographic, service delivery, organization, and geographic characteristics associated with LOV program enrollment; (2) Assess barriers and facilitators of enrollment in the LOV program through individual and group interviews with a purposive sample (n~45) of LOV mentors, community members, and patients who enrolled in LOV and those who did not; and (3) Use CBPR methods to design and assess an improved engagement pathway to approach violently injured patients for enrollment in the LOV program. My proposed career development and research plan will address actionable HVIP implementation barriers and facilitators in real-world conditions. We will disseminate our findings to other HVIPs through subsequent R01 applications to scale up the new engagement pathway and identify implementation factors that are consistent across communities to decrease mortality among high-risk patients.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT Stroke is the leading cause of long-term disability among adults in the United States, with half of all stroke survivors experiencing moderate to severe impairment in motor, sensory, or cognitive function that require specialty care. Despite advances in the acute (< 24 hour) care of stroke, such as thrombolysis and recanalization, stroke patients still experience progression of brain injury that negatively affects patient outcomes. Cerebroprotection, the mitigation of damage to the entire neurovascular unit of the brain, is an extremely high priority in stroke care research. Torpor, a state of hypothermia and hypometabolism, has long been hypothesized to represent a cerebroprotective state. We have identified a previously under-studied, conserved population of GABAergic neurons expressing the kappa opioid receptor (KOR) in the medial preoptic area (POA) termed POAKOR+. In preliminary studies, we found that chemogenetic activation of POAKOR+ neurons induced a hypothermic and hypometabolic state that we refer to as synthetic torpor. Preliminary data suggest that induction of synthetic torpor immediately after experimental stroke reduces infarct size and decreases mortality in mice at 72 hours post-stroke. The data also suggest that induction of synthetic torpor alters metabolism of nucleotides, lipids, and the citric acid cycle, while altering metabolites such as ceramides and succinate that are associated with the progression of brain injury after stroke. While promising, these preliminary data highlight several key knowledge gaps that will be addressed in the proposed research study. First, we will investigate whether the cerebroprotective effects observed 72 hours after stroke also improve long-term behavioral outcomes (Aim 1). Second, our preliminary data suggests that the hypothermic depth and duration of synthetic torpor predicts stroke size, thus, we will investigate whether the cerebroprotective effects of synthetic torpor following stroke are dependent or independent of hypothermia (Aim 2). Third, while the metabolic pathways that are altered during synthetic torpor overlap with those altered by stroke, it is unknown if these metabolic changes occur independently of hypothermia and if the altered pathways and metabolites are related to cerebroprotection. We will investigate these metabolic changes, identify the pathways and metabolites that are uniquely altered in response to synthetic torpor, and characterize the temporal-spatial dynamics of cerebroprotection (Aim 3). Identified metabolites and metabolic pathways may represent targets for future cerebroprotective interventions. Succesful completion of the proposed research study will characterize the mechanistic underpinnings underlying synthetic torpor-mediated cerebroprotection and address the knowledge gap on whether induction of a torpor-like state through modulation of specific neural circuits represents a novel cerebroprotective strategy for the treatment of ischemic stroke.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT The Department of Otolaryngology-Head & Neck Surgery at Washington University has had continuous T32 funding for research training during the otolaryngology residency program since 1985. This application seeks to transition funding for research training during otolaryngology from the T32 to R25 program in response to RFA-DC-20-002. The Overall Goal of the Otolaryngology R25 Mentored Research Pathway Training Program is to provide outstanding research and educational opportunities to medical students and resident-investigators in otolaryngology to stimulate an interest in research as part of their future career goals. The Specific Aims of the R25 Training Program are to: 1) Provide mentored research experience with research preceptors conducting basic, clinical, translational, or behavioral research in diseases and conditions related to deafness and other communication disorders. 2) Provide core courses related to the fundamentals of clinical and translational research created by Ors. Piccirillo and Kallogjeri and delivered as short courses, tutorials, video modules and elective courses to ensure trainees acquire in-depth knowledge of relevant basic, clinical, translational, and behavioral research techniques directly related to their research experience and/or research project. 3) Provide access to curricula, seminars, workshops, and tutorials that focus on topics related to professional career development skills. 4) Evaluate all research education program components and track trainees' career development over time. 5) Actively disseminate the digital core courses to encourage adoption by other otolaryngology residency training programs. The foundation of the R25 Program is the mentored research experience with one or more of the program faculty and research preceptors. The diverse program faculty and research preceptors have a proven record of providing outstanding mentored research opportunities. Each year, two predoctoral medical students and two post-doctoral otolaryngology residents will enter the research education pathway. The medical students will spend 9 months in the mentored research activity laboratory and the resident-investigators will spend two years at 80% effort after the PGY-2 year of clinical residency training. The R25 will support two medical students and four resident-investigators per year. Robust tracking and evaluation of the TL 1 program, trainees, and alumni will be used to determine the effectiveness of the training methods and identify opportunities to improve the Program.

NIH Research Projects · FY 2024 · 2023-07

Project Summary This application is for secondary analyses to determine the long-term effect of treatment, clinical and demographic factors on vision-related quality of life (VRQOL) in the Ocular Hypertension Treatment Study (OHTS) cohort. This application is timely because the US Preventative Services Task Force recently concluded there was “Inadequate evidence that treatment of primary open-angle glaucoma improves health outcomes such as reduced visual impairment, vision-related function, and quality of life” (JAMA Ophthal, May 2022). OHTS is the only US study with 20-year clinical and VRQOL outcomes in a cohort of 1,636 participants with ocular hypertension. The 20 year follow-up is meaningful because it approaches the life expectation of participants whose median age was 55 at baseline. Aim 1 will report cross-sectional 20 year data from the National Eye Institute Visual Function Questionnaire (NEI VFQ) and 15 item Glaucoma Quality Life survey (GQL-15). Aim 2 will focus on longitudinal changes in clinical and VRQOL in the inception cohort of participants who developed POAG in OHTS 1 or 2. Pre-POAG VRQOL data collected in the inception cohort will allow statistical adjustment for individual differences on post-POAG VRQOL which will increase precision of post-POAG impact on VRQOL. Accurate dates of POAG diagnosis in the inception cohort will enable a rigorous test of the effect of duration and rate of change in clinical parameters on VRQOL. We will analyze treatment, clinical and demographic factors affecting VRQOL in the cross-sectional sample at 20 years of follow-up (Aim 1) and in the longitudinal cohort sample (Aim 2). Core tests and measures were completed per protocol at 6 month intervals for 13 years and at 1-3 visits after 20 years of follow-up. Tests and measures to be analyzed include indices from Humphrey 30-2 SITA standard visual fields, best corrected acuity, Pelli-Robson contrast sensitivity, and OCT (assessed at 20 years). VRQOL was assessed using the NEI-VFQ administered every 2 years for 13 years and the GQL-15 administered at 20 years.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY The Down Syndrome – Ambulatory Research in Cognition (DS-ARC) study will develop and validate a smartphone-based digital cognitive assessment designed specifically for participants at risk for Down syndrome-associated Alzheimer’s disease (DS-AD). We will partner with sites in London, Barcelona, Munich, and Gothenburg to develop and validate this approach in different cultures and languages. Nearly all individuals with Down syndrome have elevated levels of Alzheimer’s disease (AD) biomarkers by the time they are in their 30s or 40s, though not all show symptoms of AD. It is during the pre-symptomatic and early symptomatic stages of DS-AD that an in-depth understanding of cognitive changes is crucial, as therapeutic interventions to stop, slow, or prevent disease are focused on these critical periods. Typically, prevention trials and observational studies in AD populations assess cognition with standard “table top” paper and pencil tests. But these conventional methods have several drawbacks. First, performance is influenced by day to day fluctuations in stress, fatigue, sleep patterns, and mood. Second, the testing takes place in environments that are fundamentally different from where cognition is relied upon to function in daily life. Finally, by design, cognition is typically assessed in “one-shot” in extended testing sessions on an annual or semiannual basis. The DS-ARC study will address these difficulties by developing a smartphone-based assessment solution adapted specifically for individuals with Down syndrome. This approach uses a measurement “burst” design in which participants complete very brief cognitive tests on their smartphones several times per day for one week while living in their natural environments. Instead of focusing on one of these measurements, tests are averaged across the week to provide a score that captures and normalizes natural variability and dramatically increases reliability. Studies of our original ARC assessments in autosomal dominant and sporadic AD demonstrate extraordinary reliability and strong relationships with AD biomarkers, while reducing costly and burdensome in-clinic evaluations. The study has two phases: a 2-year development phase and a 3-year validation phase. In the development phase, we will adapt an existing smartphone application called the Ambulatory Research in Cognition (ARC) app for use in DS-AD studies. This will include an iterative pilot testing process for psychometrics and task development, focus groups and user experience testing to address accessibility and compatibility concerns for DS-AD populations, and readiness audits to ensure that the DS- ARC app meets strict compliance guidelines for clinical trial applications. In Phase 2, participants complete DS- ARC assessments every 6 months and complete standard clinical, cognitive, and blood tests for AD biomarkers annually. We hypothesize that DS-ARC assessments are accessible, sensitive, and reliable indicators of cognitive change in DS-AD and will exhibit associations with AD biomarkers and disease stage over and above those of conventional in-clinic assessments.

NIH Research Projects · FY 2024 · 2023-07

Project Summary Small bowel intestinal epithelial cells (IECs) are the first line of defense against human enteric viruses, the most common and leading causes of diarrhea and death in infants and young children. How IECs communicate with intraepithelial lymphocytes (IELs) in the small intestine and orchestrate antiviral responses is heavily understudied. Our overall objectives are to better define the host immune signaling pathways in the gastrointestinal tract during infections and to use that information to develop therapeutic interventions to alleviate diarrhea and sequelae. Our preliminary results contrasted our expectation and demonstrated that the numbers of two IEL subsets are significantly reduced during early rotavirus infection in vivo. We also found that rotavirus infection alters the expression of several pro-inflammatory chemokines in infected human and mouse IECs. Based on these data and prior publications, we hypothesize that the IELs are important mediators of host defense against rotavirus infection and that rotavirus-encoded factors antagonize the antiviral activity of IELs in the host small intestine via inhibition of chemokine expression. Testing these hypotheses is currently hampered by the lack of suitable model systems. Accordingly, we have developed a highly tractable murine rotavirus reverse genetics method, a pathologically relevant neonatal mouse model, and several innovative primary human and murine small bowel organoid culturing systems, which will provide an unprecedented resolution of understanding of IEC-IEL crosstalk in the context of enteric viral infections. In Aim 1, we will define a functional antiviral role of IELs in rotavirus infection in vivo using immunological approaches and gene knockout mice. In Aim 2, we will identify the potential mechanism by which viral factors dampen chemokine expression in infected IECs. Collectively, we expect these studies to establish a new paradigm of mucosal antiviral immunity, especially early in life when most enteric infections occur. We also expect to identify novel rotavirus immune evasion strategies, which will inform new strategies to develop host- based broad-spectrum antiviral therapeutics and next-generation vaccine candidates.

NIH Research Projects · FY 2025 · 2023-06

Modified Project Summary/Abstract Section Due to their clinical and research training and expertise, physician-scientists are poised to provide new unique insights into disease, ultimately leading to advances in treatments but there is shortage of physician-investigators. While combined MD-PhD programs have helped address this shortage, there are substantially more physicians without PhD degrees graduating from medical schools who form a substantial pool of potential investigators to overcome this shortage. Physician-scientists are especially needed to explore infectious diseases and immune-related diseases to derive new therapeutic advances. Here, the applicant and his team propose to meet this challenge by focusing on non-PhD physicians with a new R38 Stimulating Access to Research in Residency (StARR) Program in Infectious Disease and Immunology research (ID/IMM StARR) at Washington University School of Medicine (WUSM). The ID/IMM StARR Program is designed to provide protected time for mentored research, didactic training, and career development that will allow residents to fulfill board requirements and take advantage of the robust environment at WUSM for physician-scientist training and in ID/IMM research. The Specific Aims are to: 1) Provide Dermatology, Medicine, Neurology, Pathology and Pediatric residents high quality, competency based, rigorous training in basic or clinical/translational ID/IMM research; 2) Provide 1-2 years of mentored research training, didactic training and other scientific enrichment activities, and Individual Development Plans for career development to ensure the success of residents in the StARR Program through close interaction with outstanding scientific mentors, career advisory committees and program directors; 3) Expand the number of well-trained residents ultimately performing research, and pursuing subspecialty fellowships to become independent research faculty in infectious disease and immunology; and 4) Perform robust evaluation and tracking to demonstrate the impact of the StARR Program. Thus, R38 residents will be well-prepared for an ultimate career as independent investigators in ID/IMM research.

NIH Research Projects · FY 2026 · 2023-06

To maintain its status as the worldwide leader in research, the United States must realize the tremendous scientific power inherent within its student age population. The creation of holistic K-12 programs that spark student interest in science, empower student academic pursuits, and provide students with bona fide research experiences is essential to enhance the migration of talented, STEM-focused students into college STEM majors. By leveraging established partnerships with local high schools and K-12 organizations and listening to the needs of our partners, we have sculpted a new program that will provide holistic training and support to high school students to help them succeed in STEM. Aim 1: To create educational programs that address partner-identified STEM education gaps and to determine if student participation in these programs increases science literacy, scholastic success, college matriculation, and motivation to pursue scientific careers relative to non-participating students. We hypothesize that planned activities will have a strong, positive impact on student success and STEM interest because we are addressing specific needs defined by their educational organizations. Aim 2: To determine whether the context in which research experiences are delivered impacts motivation to enter STEM fields. We hypothesize that, in our student population, research experiences with connections to improving common health conditions will be more motivating for long-term scientific engagement compared with similar research projects that lack such a context. We have established partnerships with local high schools and organizations. All students in these schools and organizations are eligible for all planned activities; essentially all of these students lack opportunities to learn about and pursue an interest in STEM fields, especially research. Our program then possesses great potential to positively impact the confidence and college and career choice of impacted students and thus help inspire the next generation of scientists.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY Opioid use disorder (OUD) is an escalating public health concern, and has resulted in over 550,000 overdose deaths between 1999 and 2020. Specifically, initial exposure to prescription opioids, such as oxycodone (OXY), has contributed to an average of 13,850 deaths annually since 2018 (Centers for Disease Control). While many individuals are able to use opioids as prescribed, a subset of individuals transition to compulsive drug use, which is defined as continued drug intake despite negative consequences, and is a hallmark feature of OUD. Similarly, most rodents will readily self-administer opioids, but will suppress their drug consumption when drug intake is paired with punishment such as foot shock (punishment-sensitive); while ~20-25% will persist in drug intake despite this punishment (punishment-resistant). Elucidating the neural mechanisms underlying individual differences in punishment-resistant drug seeking will be critical for understanding susceptibility to and treatment strategies for compulsive drug use. The ventral pallidum (VP) has emerged as a critical brain area for encoding the relative value and motivation for rewards and translating this motivation into reward seeking. The VP is a heterogeneous nucleus, with different populations playing opposing roles in appetitive behavior. Specifically, we and others have established that glutamatergic VP neurons (VPGlu) are crucial for constraining reward seeking in the face of aversive consequences, by modulating activity of downstream brain areas involved in punishment learning, including the lateral habenula (LHb) and rostromedial tegmental nucleus (RMTg). The objective of this proposal is to establish whether OXY- SA decreases the excitability, in vivo activity and synaptic output of VPGlu neurons, and determine if these adaptations are causally related to punishment-resistant OXY intake. Using complementary approaches of electrophysiology, calcium detection with fiber photometry and chemogenetic manipulations, we will establish whether OXY self-administration decreases excitability of VPGlu neurons, and whether this reduced activity is necessary and sufficient for the emergence of punishment-resistant OXY intake (Aim 1). We will next use electrophysiology and bidirectional optogenetic manipulations to determine whether reduced synaptic output from VPGlu neurons to the LHb or RMTg is causally related to punishment-resistant OXY intake (Aim 2). Finally, we will sequence actively translated mRNA from VPGlu neurons to determine gene networks that confer risk and protection against punishment-resistant OXY intake (Aim 3). This will also allow us to identify and validate potential pharmacological targets that could modulate VPGlu neuron activity in vivo as a therapeutic strategy. Our long-term goal is to elucidate the neural circuit basis of punishment-resistant opioid intake, and to leverage this understanding to develop neuromodulation therapies (such as deep brain stimulation with targeted pharmacology) to treat persistent drug intake despite negative consequences in patients with OUD.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT Rotavirus (RV) is the leading cause of diarrhea-associated morbidity and mortality in children younger than five worldwide. While RV vaccines have substantially decreased RV deaths, many children remain at risk of life-threatening RV disease because of inadequate response to vaccination. We recently identified a potential role for Enterovirus B (EV-B) infection in reducing oral RV vaccine (ORV) performance. Specifically, EV-B infection at the time of vaccination was associated with a lack of seroconversion to ORV in a previously studied cohort of infants from Ghana. However, the signaling pathways and mechanisms associated with this interference have not yet been defined. Determination of the mechanisms underlying EV-B-mediated interference with ORV will leverage complementary analyses in ex vivo human intestinal organoids, in vivo mouse models, and fecal analyses from a human clinical trial cohort of infants receiving RV vaccines. Representative EV-B strains which are prevalent in settings with reduced vaccine efficacy have been selected for these studies. We will interrogate the antiviral signaling pathways induced by EV-B infection, as well as test whether they are required to limit ORV or RV replication, in both pediatric human organoids and wild-type, transgenic or knock-out mouse lines. EV-B co-infection effects on development of adaptive immune responses to RV will also be evaluated in mouse models. Additionally, a distinctive collection of fecal samples from a clinical trial evaluating ORV and non-replicating RV vaccine clinical efficacy will be used to confirm EV-B interference with ORV, define EV-B-associated antiviral cytokines, and assess whether EV-B also interferes with non-replicating RV vaccines. Because these samples were already collected from infants in Ghana, where a larger percentage of infants fail to develop strong protection after vaccination compared with infants in the United States, they provide a rare opportunity to directly compare babies who developed protection after vaccination with those who did not. Such studies would not be feasible in other settings like the US, because with lower rates of infants failing to respond to vaccines at these sites, there would not be enough data to draw statistically valid conclusions. The Ghanaian cohort includes many more non-responders, making it possible to more clearly identify the biological factors, like viral interference, that limit vaccine effectiveness and drive poor vaccine responses across settings. Completion of this proposal will provide key insights into EV-B-mediated interference with ORV, as well as broadly into virus-virus interactions and their consequences for development of immune responses. RV continues to cause nearly 3 million cases of diarrheal illness per year in the United States. The outcomes from this study will help to develop evidence-based interventions to help to mitigate these diarrheal illnesses affecting infants in the US as well as inform current RV policy to enhance vaccine performance in the US.

NIH Research Projects · FY 2026 · 2023-06

ABSTRACT Endothelial cells (ECs) form an essential part of the vasculature and are strategically located between blood and tissues, functioning as a fundamental barrier between the tissue and the immune system. As such, ECs can be viewed as an essential and active component regulating immune responses. We propose that EC's angiogenic vs. immune-modulatory function can be linked through the same genetic mechanism. We recently reported that Myct1 (MYC target 1), encoding a plasma membrane protein, is a novel regulator of angiogenesis. Myct1 expression is mainly restricted to ECs and tumor ECs. Global and EC-specific (Cdh5-Cre; Myct1f/f) Myct1 KO mice display decreased tumor angiogenesis and reduced tumor growth. Unexpectedly, defective tumor angiogenesis leads to an anti-tumor immune microenvironment. Particularly, tumors from Myct1 KO mice contain more CD8+ cytotoxic T lymphocytes (CTLs) than tumors from littermate control mice. While Myct1 deficient ECs display defects in EC motility, they support more robust CD8+ T cell trans-endothelial migration (TEM). Importantly, analysis of human cancers has also identified MYCT1 as a modulator of cancer patients' angiogenic and immune outcomes. Inhibition of Myct1 through knockout, siRNA treatment, or blocking monoclonal antibodies, in combination with anti-PD1 antibody, significantly improved complete tumor regression, suggesting that the better control of cancer depends on reduced angiogenesis and enhanced recruitment of CD8+ CTLs. We identified that Myct1 could control the outcome of angiogenesis vs. CTL recruitment to tumors through Rhoa vs. Rac1 Rho GTPase pathways. Moreover, we identified ZO1, also known as tight junction protein 1, to associate with MYCT1. Deeper mechanistic investigations on the Myct1-Rho GTPases and MYCT1-ZO1 interaction will help better understand how ECs control angiogenesis vs. immunity. As Myct1 function is conserved between mice and humans, findings from the proposed studies have high translational potential and can be applied to human anti-cancer therapies. The overall goal is to uncover novel molecular mechanisms and functions of MYCT1 in ECs and anti-tumor immunity. Particularly, we will test the hypothesis that Myct1 can modulate EC angiogenesis vs. immune regulatory outcome. Aim 1 determines how Myct1-Rho GTPase regulates tumor angiogenesis vs. tumor immunity. Aim 2 investigates the functional significance of MYCT1-ZO in EC permeability and vessel integrity. Aim 3 is to further assess the efficacy of anti-MYCT1 antibodies for targeting MYCT1 in tumor angiogenesis and growth. By completing the proposed studies, we will gain a deeper mechanistic understanding of how Myct1 regulates angiogenesis and the consequence of immune output played by ECs. The outcome will significantly impact the basic EC biology and therapeutic modality for cancer treatment.

NIH Research Projects · FY 2024 · 2023-06

ABSTRACT Growing bones contain a circumferential anatomical structure called the groove of Ranvier (GOR) that may contain skeletal stem cells and signaling cells critical to skeletal development. Clinically, the GOR is important as pediatric fractures involving the GOR (Salter-Harris type II) often affect longitudinal bone growth. However, the identity and functional properties of cells within the GOR that regulate longitudinal bone growth are not known. Our preliminary data suggest that Fibroblast Growth Factor Receptors (FGFRs) within the GOR contribute to a “signaling center” that regulates adjacent growth plate chondrocytes and longitudinal bone growth. This observation establishes a functional link between cells in the GOR and the growth plate and has implications for the pathogenesis of pediatric physeal fractures. We refer to these poorly defined cells as the “GOR signaling center”. Conditional inactivation of Fgfr1 and Fgfr2 with the Osx-Cre transgene (Osx-Cre, DCKO mice) results in decreased longitudinal bone growth in postnatal mice. Through an unknown feedback mechanism, this inactivation also results in increased Fgf9 expression in the fibrous capsule and perichondrium, which is in close proximity to the GOR. Experimentally, we showed that expression of FGF9 in the perichondrium can activate FGFR3 in adjacent proliferating chondrocytes to suppress chondrogenesis. By regulating the expression of Osx-Cre, we show that Cre must be active during embryonic development to elicit this postnatal growth phenotype. Significantly, embryonic, but not postnatal, expression of Osx-Cre specifically targets the GOR. These data functionally define the region encompassing the GOR as the location of a critical FGFR1/2 signaling center by showing that inactivation of Fgfr1/2 in an embryonic cell lineage that gives rise to the GOR, rather than in definitive osteoblasts, is responsible for the dramatic reduction in bone growth observed in Osx-Cre, DCKO mice. In specific aim 1, we will use lineage tracing to differentially label the GOR and single cell mRNA sequencing to identify cell sub-populations within the GOR that have signaling center and/or skeletal stem cell properties. Identification of selective markers for these cell sub-populations will be used to reveal their location relative to the anatomical GOR. In specific aim 2, we will characterize the signaling properties of GOR cells for their ability to regulate osteogenesis and chondrogenesis. The studies proposed here will identify unique features of GOR cells as a potential cell signaling center with the unique ability to regulate adjacent growth plate tissue. These studies will provide mechanistic insight into why a large percentage of pediatric physeal fractures result in growth arrest despite theoretically preserving the proliferating chondrocytes and their intact blood supply. These studies will provide new genetic tools to study the GOR in future grant proposals and potential therapeutic insight that could be used to prevent growth arrest associated with physeal fractures.

NIH Research Projects · FY 2025 · 2023-06

Abstract Neutrophils are the most abundant and predominantly-infected cell type in the sputum, bronchoalveolar lavage fluid, and caseum contents from resected lung tissue of active tuberculosis (TB) patients. Studies of TB in mice, non-human primates, and humans have identified a correlation between neutrophil abundance and increased disease severity. Although there is a growing appreciation for the association of increased neutrophil abundance with active TB disease, it was still unknown if the presence of neutrophils in the lungs of active TB patients is consequential, or if the neutrophils are bystanders reacting to an uncontrolled infection. In particular, the details on how specific neutrophil responses and effector functions impact TB disease have remained elusive. In response to Mycobacterium tuberculosis (Mtb) infection, neutrophils deploy a number of defenses including the extrusion of neutrophil extracellular traps (NETs) via a process of cell death termed NETosis. NETosis usually follows the general steps of 1) histone citrullination, 2) chromatin decondensation, and 3) release of web-like chromatin structures decorated with antimicrobial granule proteins with the potential to bind, trap, and kill pathogens. We have recently discovered that NETosis directly promotes Mtb replication, where genetic or chemical inhibition of NETosis mediates better control of Mtb infection in vitro and in vivo, thus validating NETosis as a potential target for host-directed therapies to treat TB. We have used genetic and chemical approaches to mechanistically dissect the process of NETosis during Mtb infection, which has identified a number of regulatory nodes that can be manipulated to lead to better control of Mtb pathogenesis. We find that in response to Mtb infection in neutrophils, protein arginine deiminase 4 (PAD4) citrullinates histones to decondense chromatin that gets packaged into vesicles for release as NETs in a manner that promotes Mtb replication. We discovered that type I interferon (IFN), which has been associated with NETosis in numerous contexts but without a known mechanism, promotes the formation of chromatin-containing vesicles and NET release. In addition, we discovered a new autophagy-independent role for the ATG5 protein in suppressing NETosis by blocking type I IFN-dependent induction of PAD4 activity during Mtb infection, where increased NETosis in the absence of ATG5 expression in neutrophils leads to susceptibility to Mtb. Multiple studies have linked increased levels of type I IFN signaling with TB pathology in mice and humans. Based on our data that NETosis promotes Mtb replication and pathogenesis, NETosis could contribute to the ways that type I IFN signaling impedes control of Mtb infection. In this proposal, we will dissect how NETosis is regulated during Mtb infection (Aim 1), how NETosis contributes to Mtb replication (Aim 2), and how NETosis contributes to loss of control of infection (Aim 3). By pursuing our Aims, we will identify regulatory mechanisms to be targeted by HDTs aimed at blocking NETosis during Mtb infection as well as determine what consequences result from blocking NETosis during Mtb infection.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY More than 80% of ovarian cancer (OC) cases have already metastasized to the peritoneal cavity at diagnosis, and the five-year survival for these patients is 25%. Within the peritoneal cavity, the most common site of metastasis is the omentum, a well-vascularized, specialized adipose tissue that arises off the greater curvature of the stomach. Because the omentum promotes tumor growth, it is removed as a standard of care for patients with peritoneal metastases. In mouse OC models, the omentum is an early metastatic site, and removing the omentum before tumor implantation reduces the pace of tumor expansion. Once OC cells shed from the primary tumor and form spheroids, some bind to the adjacent omentum, which is considered transcoelomic metastasis. Other data show that OC cells can exit the peritoneal cavity and travel back to the omentum via the circulatory system, underscoring a hematogenous peritoneal metastatic pathway. Overall, the mechanism by which the omentum promotes tumor growth is still unknown and is important to identify so that we can develop more specific therapeutic strategies. Studies suggest that omental macrophages, blood vessels, or lymphoid aggregates called milky spots, are key features that support tumor growth. In addition to these omental features, the adipocytes themselves, the cells that define the omentum as a fat pad, are likely important for tumor expansion. Adipocytes can directly provide fatty acids to metabolically support tumor proliferation or may indirectly foster tumor growth by supporting milky spot formation, as my preliminary data suggest. To move beyond these valuable observational studies, I herein propose using a mouse model wherein the mouse lacks white and brown adipocytes, rendering the peritoneal cavity adipocyte-free, to explore the role of the omentum in OC metastasis without mature adipocytes. Due to a total lack of fat, the mice become lipodystrophic. To overcome this systemic metabolic syndrome, the lipodystrophic mice receive subcutaneous fat transplants, referred to as distal adipocyte rescue of lipodystrophy (DARL) mice. Littermate controls also receive the fat transplant, known as DARC mice for distal adipose-receiving control mice. I hypothesize that the metastatic seeding and dissemination of murine OC cells within the adipocyte-free omentum and associated peritoneum will be impaired in the absence of local, mature adipocytes. While testing this hypothesis, I will assess how the loss of omental adipocytes impacts the overall omental architecture in DARL mice. I will analyze the impact of an adipocyte-free versus adipocyte-rich omentum on tumor growth. Lastly, I will explore the role of hematogenous and transcoelomic spread to the adipocyte-rich or adipocyte-free omentum and consider whether omentum cells support tumors as spheroids that disseminate within the peritoneal cavity. I will pursue these aims under the mentorship of an advisory committee that includes physician-scientists. This training will help improve my understanding of metastasis so that I can ultimately identify new treatment strategies in my career as an oncologist-scientist.

NIH Research Projects · FY 2026 · 2023-06

Project Summary The Evers laboratory studies the binding interactions of neurosteroids and cholesterol with integral membrane proteins, with the aim of identifying the specific binding events underlying sterol modulation of protein function. Our major focus is on neurosteroid (NS) modulation of -aminobutyric acid type A receptors (GABAA- R). Neurosteroids are important modulators of neuronal excitability and nervous system development with enormous therapeutic potential as anesthetics, anti-depressants and neuro-protectants. We have shown that there are multiple, subunit-specific binding sites for neurosteroids on GABAA receptors, each of which contributes to the functional effects of neurosteroids. In the proposed research, we will use photolabeling techniques to define the precise sites at which the major classes of neurosteroids bind on the most abundant forms of synaptic and extra-synaptic GABAA receptors and determine the functional significance of each identified binding site by assessing the effect of targeted amino acid substitutions on NS modulation of GABAA- R currents. To identify photo-labeled residues we will utilize state-of-the-art protein chemistry and expression techniques in conjunction with cutting edge mass spectrometry (MS) methods, including middle-down and intact protein MS. High-resolution cryogenic-electron microscopy structures will be obtained to identify the atomic details of novel NS binding sites and to investigate binding interactions that appear to stabilize conformations not captured in current structures. Fluorescence-based binding assays will then be used to measure the site-specific affinity of various NS for the identified binding sites. These assays will be adapted to stopped-flow fluorimetry to determine the state-dependence of binding and to a plate reader format to screen for site-specific agonists and antagonists. The long term goal of our NS program is to develop and use site-specific NS ligands to probe the role of specific NS binding sites and GABAA-R subtypes in the behavioral effects of endogenous NS and the mechanisms of action of NS sedatives and anesthetics. We have also used cholesterol-analogue photolabeling to identify specific binding sites that mediate cholesterol inhibition of the lipid scramblase, nhTMEM16, and cholesterol modulation of mTOR1 by the lysosomal membrane protein GPR155. Both nhTMEM16 and GPR155 have two specific cholesterol binding sites per protein monomer and we are using targeted amino acid substitution to understand the functional role of each site. We are also developing fluorescence-based binding assays to measure cholesterol affinity and sterol specificity for these sites. Novel cholesterol binding sites present new targets for small molecule allosteric modulators of membrane protein function and the tools we have developed are widely applicable to identifying binding sites on other cholesterol-modulated proteins and screening for site-specific ligands.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Targeting brown adipose tissue (BAT) function to increase energy expenditure represents an attractive strategy to treat obesity and the associated type 2 diabetes. BAT and the related beige fat express uncoupling protein 1 (UCP1), a mitochondrial membrane protein that uncouples respiration from ATP synthesis and promotes thermogenesis. Paradoxically, UCP1 null mice are only obese when maintained at thermoneutrality, suggesting the existence of alternative mechanisms of thermogenesis. Consistent with this notion, several UCP1- independent ATP-consuming futile cycles have been identified in brown and beige adipocytes. However, the relative contribution of these pathways to the regulation of whole-body energy metabolism remains unclear. Our preliminary studies suggest that peroxisomes, organelles specialized for lipid metabolism, are involved in an alternative mechanism of adipose tissue thermogenesis based on peroxisomal metabolism of branched chain fatty acids (BCFA). Peroxisomes account for up to 20% of total cellular oxygen consumption. Peroxisomal respiration, unlike mitochondrial oxygen consumption, is not linked to ATP synthesis and instead generates heat. Monomethyl (mm) BCFA are synthesized via de novo lipogenesis using a precursor derived from catabolism of branched chain amino acids (BCAA). Our results reveal that cold treatment increases the gene expression of factors involved in mmBCFA synthesis and beta-oxidation in thermogenic fat. A thermogenic stimulus promotes translocation of BCFA synthetic proteins to peroxisomes, the site of BCFA beta-oxidation. Upregulation of BCFA beta oxidation raises the intracellular temperature in brown adipocytes and increases oxygen consumption rate in WT and UCP1 KO brown adipocytes. Together, these results lead us to hypothesize that peroxisomes are involved in a UCP1-independent mechanism of thermogenesis characterized by a futile process of BCFA synthesis and beta-oxidation. Since de novo synthesis of fatty acids is a highly energy-demanding process and peroxisomal beta-oxidation is not linked to ATP production, we further hypothesize that this futile cycle promotes negative energy balance, leading to protection against obesity and insulin resistance. To test this hypothesis, we propose two specific aims. The first aim will use biochemical, cell culture-based, and in vivo approaches to implicate peroxisomes in a futile process of BCFA metabolism. The second aim will use loss-of-function and gain-of-function mouse models of BCFA beta-oxidation to study its role in thermogenesis and whole-body energy metabolism.