University Of Alabama At Birmingham

NIH Research Projects · FY 2025 · 2023-08

Project Summary Despite hundreds of published articles, meta-analyses, and reviews describing the clinical syndrome of COVID-Associated Pulmonary Aspergillosis (CAPA), there have been zero publications to date exploring the mechanism by which individuals with severe SARS-2 infection succumb to 2° mold infection. This proposal seeks to evaluate the novel conceptual advancement that immune responses targeting intracellular viral pathogens, like SARS-2, promote 2° mold infection via release of bioavailable iron, delayed neutrophil (PMN) recruitment, and decreased antifungal effector (αFE) expression. In Aims 1-3, we explore the hypotheses that lytic programmed cell death (PCD) in pulmonary epithelial cells (PEC) and red blood cell hemolysis increase the availability of nutrients including iron/heme which drive siderophore- dependent acceleration in mold growth. Concurrently, SARS-2 and the antiviral cytokine milieu mitigate PMN recruitment and activation resulting in spore germination into large invasive hyphae that overwhelm αFE mechanisms enabling 2° mold infection in an otherwise immunocompetent host. In Aim1, we will quantify iron, heme, divalent cations, host metal sequestration proteins (MSP), antiviral cytokines, and PMN-recruiting chemokines in SARS-2 BALs vs control infection cohorts (IC). We will also use immunofluorescence (IF) and spatial transcriptomics to characterize lytic PCD, viral ORF3a-mediated cell lysis, and αFE expression in FFPE lung tissues and utilize isogenic mutant fungi to identify critical host and microbial factors mediating mold growth in BALs. In Aim2, we will utilize novel Calu-3 knockout (KO) cell lines and 1°normal human bronchiole epithelial (NHBE) air liquid interface cell cultures to study the impact of the lower airway milieu and direct SARS-2 infection on PEC: lytic PCD, IFN synthesis, αFE secretion, metal sequestration, and ability to respond to 2° fungal stimuli. We will also utilize isogenic mutant fungi to identify critical pathways mediating mold growth and an innovative PMN airway transmigration model to determine the impact of Type I and III IFNs, iron/heme toxicity, and SARS-2 uptake on PMN recruitment, activation, and fungal killing. In Aim3, we will utilize novel conditional KO mice, fluorescent viral and fungal reporter strains, IF, flow cytometry, and single cell sequencing to assess the potential of SARS-2 variants to induce lytic PCD, define the role of lytic PCD and IFN signaling in PECs, macrophages (Mφ), and PMNs, and evaluate their impact on lung pathology, metal release, PMN- recruitment, spore viability, fungal growth, and infection outcome. We will also dissect key host MSP and fungal pathways mediating mold growth, evaluate the role of Mφ and PMN hACE2 on viral and fungal clearance and determine if small molecule inhibitors of lytic PCD, iron chelators, and siderophore biosynthesis inhibitors prevent or mitigate the development of 2° mold infection. We believe that the results of the proposed study will shed new light on the fundamental biology mediating CAPA and may enable the development of improved therapeutic regimens that mitigate the risk of acquiring and succumbing to this devastating infection.

NIH Research Projects · FY 2026 · 2023-08

Project Summary/Abstract: Acute optic neuritis (ON) is often the initial presenting manifestation of autoimmune demyelinating disorders such as Multiple Sclerosis (MS). Good recovery of vision after ON is common, but a considerable number of MS patients experience poor outcomes with severe visual impairment and permanent blindness as disease progresses. The increasing prevalence of MS will cause an immense social and financial challenge for health care systems and more studies are needed to improve treatment success in these diseases. Impaired recycling of cholesterol-rich myelin debris and decreased de novo synthesis of cholesterol have been identified as key limiting factors of recovery in demyelinating animal models. We have demonstrated decreased expression of the main cholesterol efflux transporter1, Abca1, and altered cholesterol homeostasis in the retinogeniculate system of animals with Experimental Autoimmune Encephalomyelitis (EAE)-ON. However, the exact mechanism by which changes in Abca1 expression affect cholesterol recycling remain elusive. We hypothesize that this impairment in cholesterol synthesis and transport significantly affects RGC survival, integrity of synaptic plasticity and remyelination and that restoration of cholesterol homeostasis via Abca1 regulatory feedback ameliorates visual impairment. In specific aim 1, we will determine the regulatory influence of ABCA1 expression on sterol synthesis and synaptic plasticity, cholesterol transport, and myelination in vitro. Eye and brain organoids from healthy subjects and MS patients will be exposed to Abca1 inhibitors, TNFα/IFNγ, or the demyelinating agent Lysolecithin. Effects of rescued Abca1 expression on sterol synthesis, cholesterol uptake and transport, synaptogenesis and myelination will be examined using single-cell RNA-sequencing, immunohistochemistry, and cholesterol efflux assays. In specific aim 2, we will determine the molecular, functional, and structural impact of Abca1 expression changes in the retinogeniculate pathway of EAE-ON and MS. EAE-ON will be induced in mice and effects of Abca1 expression changes will be determined using optokinetic response, optical coherence tomography, pattern electroretinography, and visual evoked potentials, followed by postmortem molecular and histopathologic analysis. Also, disruption in cholesterol homeostasis will be determined in MS donor eye tissue. Our proposed experiments will significantly advance the understanding of the role of cholesterol homeostasis in MS-like ON and provide an invaluable resource for future translational and therapeutic studies.

NIH Research Projects · FY 2025 · 2023-08

Project Summary The purpose of this NIH F31 application is to obtain support for the PI, W. Jay Stone, for mentored research and career development activities within his MD/PhD degree training that will strengthen his potential to become a successful physician scientist. The project goal is to develop skills in neuroscience that will allow the PI to study key proteins involved in the pathogenesis of synucleinopathies, including Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB), using laboratory assays foundational to research in neuroscience. The primary objective of this research proposal is to investigate the role of 14-3-3θ phosphorylation in the context of environmental toxicant exposure and the neuroinflammatory response associated with synucleinopathies. Work from the lab of Dr. Talene Yacoubian, sponsor of the PI, has established 14-3-3θ as a key mediator of pathologic mechanisms by which neurons degenerate in models of PD and DLB, and serves as a potential target for disease modifying therapy. Furthermore, our lab has revealed that phosphorylation of 14-3-3θ at serine residue 232 (pS232) modulates its ability to interact with and prevent the aggregation of α-syn, the critical pathologic protein implicated in PD and DLB. However, the events which lead to aberrant pS232 levels are unclear. This project aims to increase our understanding of the mechanisms that induce pS232 and promote neurodegeneration in two synucleinopathy-associated events: exposure to the environmental toxicant, trichloroethylene (Aim 1) and neuroinflammation (Aim 2). The long-term objective of our research is to better understand the role of 14-3-3θ phosphorylation within synucleinopathies so that subsequent studies can be devised to target 14-3-3θ for developing potential disease-modifying therapeutics. The proposed training plan for Jay Stone is sponsored by his project mentor, Dr. Talene Yacoubian, and co- sponsored by Dr. David Standaert. The overall goal of the training plan is to provide the PI with a solid foundation for a successful career as a physician scientist. A project based both in translational approaches, while focused on a disease-oriented pathogenesis, is the ideal training environment for any aspiring physician scientist. Included in the training plan are experiences that will help Jay develop in three major areas: 1) rigorous neuroscience research in synucleinopathies, which includes developing familiarity with the existing literature, critical evaluation of data, and training in the responsible conduct of research; 2) training in advanced gene therapeutics, and 3) career and professional development, including grant and manuscript writing, scientific communications, and the translation of research findings to clinical applications. This proposal drives the development of skills required to conduct rigorous scientific research in synucleinopathies and advanced neuroscience skills necessary for the PI’s future career as a neurology physician-scientist focused on precision medicine and neurodegenerative diseases.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY A combination of individual-, network-, community-, and structural-level variables impact all steps in the HIV Care Continuum, particularly in the Deep South states of Alabama (AL), Louisiana (LA), and Mississippi (MS), which disproportionately bear the nation's HIV burden while simultaneously having the fewest healthcare resources. While the individual-level correlates of viral suppression (VS) are well-known, few epidemiologic studies, especially those that are digitally-based, have examined VS from a multilevel lens that accounts for the role of geographic and community-level characteristics germane to the Deep South, such as aggregate poverty measures, racial segregation, and healthcare access. Epidemiological studies also typically exclude people with HIV (PWH) with non-sustained VS (non-VS) or without recent HIV labs, as these are among the hardest to identify and reach due to their limited healthcare involvement. Our highly experienced study team will address these research gaps through the proposed UG3-UH3 study, which will use mHealth technology to optimally recruit, screen, enroll, and retain a prospective digital cohort of 1000 PWH in AL, LA, and MS with non-VS or who otherwise lack evidence of having been in care for the previous 12 months, leveraging long-standing relationships with public health departments in each of these three states. We will assemble a community advisory board (CAB), consisting of PWH, health department partners, and HIV care team members in the three states and we will use the Information System Research (ISR) Framework to tailor features of an existing adherence management app for PWH, WiseApp. We will test usability of the Drive to Zero app prototype with PWH and informaticians. Eligible cohort participants will be PWH ages ≥18 years, living in AL, LA, or MS, living with HIV for ≥12 months, and entered in the Enhanced HIV/AIDS Reporting System (eHARS), a standardized document-based surveillance database used by all state health departments to report diagnosis and outcomes, with a recorded VL≥200 c/mL ≤12 months or have missing HIV lab values during that same period. Participants will be followed for three years with iterative annual psychosocial quantitative survey assessments conducted via the Drive to Zero app to assess predictors, mediators, and moderators for non-VS. We will contextualize quantitative data by using geospatial analysis, linking participant residence addresses at time of enrollment to Census tract data. Participants of the digital cohort (n=45) will also be eligible to participate in qualitative in-depth interviews to contextualize quantitative results. The findings from this study will inform efforts to recruit large, digital cohorts of PWH and will be among the first to demonstrate effective methods of retaining digital cohorts for HIV as well as other health conditions. Study findings will also help identify multilevel factors that contribute to non-VS in the Deep South, thereby elucidating future areas for intervention, and will directly lead to a follow- up R-level application.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Human tuberculosis (TB) is caused mainly by Mycobacterium tuberculosis (Mtb) and represents an enormous challenge to global health because of the inadequacy of currently available drugs and vaccines. The most common clinical manifestation is pulmonary TB, and Bacille Calmette Guerin (BCG) is the only licensed vaccine for protection against TB; however, its efficacy is highly variable. Today at least 52 countries have reported multidrug-resistant (MDR) and extensive drug-resistant (XDR) TB cases which cannot be cured or contained by current TB therapy. Thus, there is an urgent need to develop new therapies/vaccines that effectively prevent or cure TB. It is well established that the generation of an adaptive immune response against Mtb occurs inside germinal centers (GCs) in secondary lymphoid organs (SLOs), such as spleen and lesion draining lymph nodes, where antigen‐presenting cells (APCs) and antigen-specific circulating T and B lymphocytes interact, clonally expand, and are disseminated to sites of infection. We found that mice vaccinated with BCG and exposed to Mycobacterium avium [a non-tuberculous mycobacterium (NTM)] via drinking water provide more robust and longer-term protection than BCG alone as determined by reduced Mtb bacterial burden and inflammatory progression of infection. Interestingly, these mice also developed ectopic germinal centers (eGC) in the lungs and have an increased number of B-cells and higher levels of anti-Mtb cell lysate-specific IgA and IgG antibodies. These findings suggest that NTM and B-cells play a critical role in generating protective immunity against pulmonary Mtb infection, and the formation of eGC in these mice is a crucial factor in this improved immunity. Thus, investigating the mechanism of eGC formation and the role of NTM and B-cells in its stimulation is an important question to understand TB pathogenesis and develop effective vaccines and therapies. In this K99/R00 application, we propose three aims: 1) investigating the key differences between eGC in lungs and conventional GCs in lymph nodes, 2) evaluating the role of NTM and B-cells in eGC stimulation, and 3) characterizing the antigen-specificity and affinity of eGC B-cells against Mtb antigens. The results of this proposal will bring us one step closer to understanding the B-cell and antibody-mediated mechanisms of protection from TB.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Streptococcal bacteria contribute to infections of vital organs through their surface proteins which facilitate their adherence, colonization, and biofilm formation. In the oral cavity, S. mutans use its surface adhesins AgI/II and GbpC to adhere to Gp340 for initial adherence, dextran to promote biofilm development, and in some cases other microbes which are incorporated into biofilms. More generally many members of the streptococcal species express an AgI/II-like homolog which they use to adhere to both shared and species-specific targets, implicating them in contributing to the disease state of infections involving these streptococci. At sites of physical damage to gums or gum disease, streptococcal species can infiltrate the blood stream and become systemic opportunistic pathogens. This proposal focuses on characterizing the molecular mechanisms of host-microbe and microbe-microbe interactions involving the AgI/II-family of proteins. In structural studies of SspB the Deivanayagam lab discovered a peptide-binding cleft within the V-region, a shared cleft housing a calcium ion. This peptide has nanomolar adherence with the V-regions of SspB, AgI/II and GbpC; the peptide inhibits the V-regions adherence to SRCR region of Gp340 and reduces biofilm formation. In studies with GbpC, this cleft was shown to also be involved in the V-regions adherence to dextran. Our preliminary studies have shown not only shared adherence targets, but also conserved areas of adherence. Both the apical V-region and cell-wall anchored C-terminal regions of AgI/II-family proteins have been shown to be involved in adherence host surfaces. In this proposal we plan to investigate the hypothesis that the Extracellular matrix (ECM) protein interactions among AgI/II-family proteins would share similar but distinct motifs that contribute to the initiation or progression of infections outside the oral cavity. The specific aims are Aim1: Characterize the interactions between AgI/II-family proteins and ECMs. Aim 2: Determine AgI/II proteins’ ability to interact with other pathogenic microorganisms. The proposed studies will elucidate shared areas of adherence to determine the potential for designing inhibitors to prevent the interaction which would reduce the ability of these streptococci to contribute to infection and disease.

NIH Research Projects · FY 2025 · 2023-08

(PLEASE KEEP IN WORD, DO NOT PDF) Significant progress has been made in identification of key components of the adult bone marrow (BM) mesenchymal stromal cell (MSC) niche that are critical for maintaining the function of rare, long-term self-renewing hematopoietic stem cells (LT-HSC). In particular, perivascular MSC expressing high levels of Cxcl12 and stem cell factor (Scf) and cell-surface markers including Lepr, Pdgfrα, and CD51, have been localized in very close proximity to LT-HSC and likely contribute to the maintenance of LT-HSC quiescence during steady-state hematopoiesis. We have shown that clonally-derived, Lepr+CD146+Pdgfrα+CD51+Scf+Cxcl12HI primary murine adult BM MSC lines with in vitro and in vivo multilineage developmental potential can significantly enhance the ability of LT-HSC to self-renew after 10 days of co-culturing 20 FACS-sorted LT-HSC with the MSC lines. Further, we show that LT-HSC self-renewal/survival could be further enhanced by increasing MSC expression of the evolutionarily-related noncanonical Wnt ligands, Wnt11 and Wnt5a, which potently suppressed canonical Wnt signaling. Conversely, ectopic expression of the canonical Wnt ligands, Wnt3a or Wnt10b, in MSC resulted in loss of transplantable LT-HSC after co-culture, rapid upregulation of an osteolineage gene expression profile in MSC, and upregulation of both endogenous Wnt11 and Wnt5a expression, suggesting activation of a negative feedback loop that would suppress further canonical Wnt signaling. In addition, we observed that loss of Wnt11 in Prx1+ MSC in vivo increases trabecular bone number in 3-week-old mice, which supports a function for Wnt11 in suppressing MSC differentiation specifically to the osteoblastic cell lineage. Based on these findings, we hypothesize that noncanonical Wnt signaling maintained by both Wnt11 and Wnt5a preserves self-renewal and multipotency of both LT-HSC and MSC within the BM niche. This hypothesis will be addressed in two Aims. Aim 1 will examine whether Wnt11 and Wnt5a coordinately control both LT-HSC and MSC self-renewal and multilineage developmental potential using LT-HSC/MSC co-cultures and primary MSC sublines where biallelic deletions of Wnt11 and Wnt5a can be induced with Cre recombinase. Aim 2 will assess the function of Wnt11 and Wnt5a in regulating LT-HSC and MSC homeostasis in vivo using Prx1-CreER or Wnt11-CreER mice to conditionally delete floxed alleles of Wnt11 and/or Wnt5a in adult BM MSC. Using Wnt11-CreERT;Rosa26-LSL-tdTomato;a-catulinGFP reporter mice, we will also determine the proximity of Wnt11+ MSC with respect to LT-HSC in BM and define the contribution of Wnt11+ MSC to the osteoblast, adipocyte and chondrocyte cell lineages in unperturbed conditions. Together, these studies will be important for understanding noncanonical Wnt signaling-dependent mechanisms that coordinate the life-long production of blood and bone-forming cells by LT-HSC and MSC, which are likely in direct cell-cell contact in vivo. This knowledge can then be applied to enhance the efficacy of stem cell transplantation, tissue repair, and targeted gene correction approaches that benefit patients in the clinic. SPECIFIC AIMS: Numerous studies have shed light on the cellular and molecular factors that control adult long-term self-renewing hematopoietic stem cell (LT-HSC) self-renewal and differentiation during steady-state and stress hematopoiesis. It is appreciated that a number of cell types including various mesenchymal stromal cell (MSC) subsets, sinusoidal endothelial cells (SEC), arterioles, neuronal cells, macrophage, and megakaryocytes all contribute to regulation of HSC homeostasis within the bone marrow (BM) niche 1-7. Using deep confocal imaging of optically cleared BM and highly specific LT-HSC markers like α-catulin, showed that the vast majority of quiescent and dividing LT-HSC are localized near SEC and MSC expressing leptin receptor (Lepr) and high levels of Cxcl128. Other imaging and cell ablation studies have further highlighted the close approximation of LT-HSC with perivascular MSC that express a Nestin-GFP transgene 9 and other cell-surface markers and secreted factors including CD51, Pdgfrα, CD146, and stem cell factor (Scf)10-17. Deletion of Scf from either perivascular MSC or endothelial cells using Lepr-Cre or Tie2-Cre depleted HSC numbers, which was not observed when Scf was deleted from hematopoietic cells, osteoblasts, or Nestin-Cre+ cells 18. Since membrane-bound Scf is essential for maintaining LT-HSC in vivo 19,20, LT-HSC are likely in direct contact with Scf-expressing perivascular MSC during steady-state hematopoiesis in adult mice. Using clonally-derived, Lepr+CD146+Pdgfrα+CD51+Scf+Cxcl12HI primary murine adult BM MSC lines with in vitro and in vivo multilineage developmental potential, we showed that Wnt signaling modulated the ability of LT-HSC to self-renew in long-term co-cultures of 20 FACS-sorted LT-HSC and the clonal MSC lines 21. These MSC lines express high levels of Wnt ligands that activate noncanonical Wnt signaling, particularly Wnt11 and Wnt5a, which potently suppress the canonical (α-catenin-dependent) pathway. This was consistent with the lack of expression of the canonical Wnt target gene, Axin2, in both LT-HSC and MSC in the co-cultures 21. Wnt11 and Wnt5a are also highly expressed in the most primitive, self-renewing adult BM skeletal stem cell subset (mSSC) that gives rise to bone and cartilage in vivo 22, in Nestin-GFP+ stromal cells 23, in Osterix-expressing osteolineage cells 24, and in adult BM stromal progenitor cells by single-cell RNAseq 25. In preliminary co-culture studies, we show that activation of canonical Wnt signaling in the MSC lines through ectopic expression of either Wnt3a or Wnt10b stimulates loss of LT-HSC self-renewal and/or survival and potently activates an osteolineage gene expression program in MSC. Conversely, overexpression of Wnt11 or Wnt5a in MSC enhanced LT-HSC long-term repopulating activity and blocked activation of an osteolineage gene signature in MSC. Further, deletion of Wnt11 in MSC using Prx1-Cre increased deposition of trabecular bone in newborn mice, which suggests an important role for Wnt11 in suppressing osteolineage differentiation of MSC induced by canonical Wnt signaling. Based on these findings, we hypothesize that noncanonical Wnt signaling in MSC maintained by Wnt11 and Wnt5a is essential to preserve the self-renewal and multilineage differentiation potential of both LT-HSC and MSC in the BM niche in order to sustain life-long blood cell and bone production. This hypothesis will be addressed in the following Specific Aims: Aim 1: Functionally define whether Wnt11 and Wnt5a coordinately control both LT-HSC and MSC self-renewal and multilineage developmental potential using LT-HSC/MSC co-cultures and primary MSC sublines with Cre-inducible, biallelic floxed alleles of both Wnt11 and Wnt5a. Transplantation studies will address whether the absence of either Wnt11 or Wnt5a, or both Wnt11/Wnt5a, in MSC results in loss of LT-HSC reconstitution potential after LT-HSC/MSC co-culture in vitro. The ability of MSC to self-renewal in the absence of Wnt11/Wnt5a will be assessed by CFU-F assays, while biased and irreversible activation of MSC commitment to the osteoblast lineage due to the inability to suppress canonical Wnt signaling in MSC will be assessed using osteogenic, adipogenic and chondrogenic growth conditions in vitro and in vivo. Aim 2: Define the unique and redundant functions of Wnt11 and Wnt5a in sustaining adult BM LT-HSC and MSC self-renewal and multilineage developmental potential in vivo during homeostasis. Here, we will use Prx1-Cre, Prx1-CreERT and Wnt11-CreERT mice to delete Wnt11, Wnt5a, or both Wnt11 and Wnt5a, to determine whether they are both essential for maintaining LT-HSC and MSC self-renewal in vivo. We will use Wnt11-CreERT;Rosa26-LSL-tdTomato;a-catulinGFP mice to determine localization of Wnt11-expressing MSC with respect to LT-HSC in adult BM, and for lineage tracing to determine the contribution of tdTomato+ MSC to the osteoblast, adipocyte and chondrocyte cell lineages in an unperturbed setting in vivo. Defining factors that preserve life-long maintenance of both blood-forming LT-HSC and bone-forming MSC has been a fundamental challenge since loss of factors controlling an essential function like self-renewal of either stem cell population would likely result in embryonic lethality. This study will utilize inducible, floxed alleles of the noncanonical Wnt ligands, Wnt11 and Wnt5a, to elucidate their role in coordinate regulation of both adult LT-HSC and MSC self-renewal. Developing a deeper understanding of factors regulating crosstalk between LT-HSC and MSC, which are likely in direct cell-cell contact in adult BM, will significantly enhance our ability to manipulate LT-HSC and MSC in vitro, with the goal of improving the efficacy of stem cell transplantation, tissue repair, and targeted gene correction approaches that can then be applied in the clinic. Modified

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT: Exposure to early life stress (ELS), including abuse, neglect, and household dysfunction, significantly increases the risk of mental illness, chronic kidney disease, and cardiovascular disease (CVD) later in life. The previously characterized effects ELS and chronic disease development in adults may have their origins in ELS- dependent effects on composition and functions of the gut microbiota. The gut microbiota interact directly with the host’s immune and neurological systems and microbial derived metabolites have been shown to mediation cardiovascular function. My recently published research using a mouse model of ELS has determined that ELS alters the gut microbiota independent of maternal inheritance. This suggests ELS-medicated endogenous factors within the offspring are responsible for the ELS microbial phenotype. However, it remains unknown whether ELS-mediated changes in the gut microbiota play a direct role in the genesis risk factors for CVD. This proposal will address these knowledge gaps by identifying ELS-medicated factors that regulate the gut microbiota and elucidating microbial-mediated pathways that lead to increased CVD risk due to ELS. Adolescents and young adults with ELS have increased arterial stiffness and systemic vascular resistance. Using an established mouse model of ELS involving maternal separation, our novel data indicate that ELS is also associated with increased arterial stiffness in adolescent and adult mice. Furthermore, ELS induces superoxide production and endothelial dysfunction in adult mice. This suggests that vascular dysfunction is an important mediator of ELS-induced CVD risk. Our new data in mice show that ELS leads to reduced gut microbial diversity, lower circulating short-chain-fatty acids (SCFAs), and impaired gut barrier function during adolescence. Gut microbial diversity is negatively associated with arterial stiffness in women and reduced SCFAs are associated with hypertension and impaired gut barrier function. This suggests a role for the gut microbiota in ELS-induced vascular dysfunction, though exact mechanisms remain undefined. Therefore, the overall goal of this proposal is to elucidate mechanisms by which microbial metabolites mediate ELS-induced aortic stiffening and endothelial dysfunction and examine the potential of diet in the early intervention of CVD risk.

NIH Research Projects · FY 2025 · 2023-08

Not only do fear-based disorders such as posttraumatic stress disorder (PTSD) quintuple the rate of suicide relative to those without these disorders, but they also increase the magnitude of age-related cognitive decline and double the risk for developing Alzheimer’s disease and other dementias in older adults. Fear-based disorders like PTSD are linked to fear memories. Due to the seemingly indelible and hyperactive properties of such fear memories, fear-based disorders can worsen with age. Although normative aging and Alzheimer’s disease have dissociable trajectories, one factor that negatively impacts cognitive aging and Alzheimer’s disease is the comorbidity of PTSD. Perturbations in the functional circuitry supporting fear memory extinction are also key neural mechanisms of PTSD. A critical anatomical structure within the neural circuitry underlying this dysfunctional processing is the basolateral amygdala (BLA), which is considered an integrative hub as it receives sensory and contextual information from the prefrontal cortex and hippocampus. While much of the current scientific focus on cognitive aging and Alzheimer’s disease centers on the prefrontal cortex and hippocampus, little is known about the underlying mechanisms of BLA dysfunction in aging, Alzheimer’s disease (AD), and the impact of co-occurring PTSD. One pathological process common to these disorders is underlying neuroinflammation. Importantly, ketogenic and ketone ester diets are known to ameliorate hyperactivity, inflammation, fear-based disorders, and show promise as treatments for the contributing factors to cognitive aging and AD. To date, no study has investigated how aging and AD act in concert to further impair the BLA’s role in extinguishing hyperactive fear memories (a central component of PTSD). To address the current gap in knowledge, this proposal will leverage the TgF344AD rat model of AD to understand the contribution of BLA inflammation, cellular dysfunction, and synaptic circuit impairment to underlying mechanisms of PTSD. Recent data from the lab suggests, relative to young wild type rats, aging and AD impairs fear extinction and recall, and furthermore, the BLA in aged and AD rats is hyperactive. As such, the overarching hypothesis of this proposal is that hyperactive fear memory, the core element of PTSD, increases with aging and is accelerated in AD due to progressive inflammatory-driven neurophysiological deficits in the BLA. In Specific Aim 1, this proposal will leverage a rodent fear conditioning protocol that models a critical component of PTSD (i.e., the inability to extinguish hyperactive fear memory) and assess how aging and Alzheimer’s disease contribute to BLA cellular dysfunction (both ex vivo and in vivo during fear extinction) and inflammation. Additionally, in Specific Aim 2, this proposal will determine if a ketone ester dietary intervention (known to have anti-epileptic and anti-inflammatory properties) or BLA inactivation can facilitate fear memory extinction and improve BLA hyperactivity and pathology in age and AD. The outcomes of this proposal will be critical to developing translational strategies to combat poor quality of life outcomes in older adults.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Functional seizures (FS) are a type of functional neurological disorder (FND) characterized by seizure-like symptoms without EEG correlates. FS are severely debilitating to children and families, making this a significant clinical and societal burden. Recent research has revealed sense of control as a potential target for pediatric FS treatment. Our previously published study demonstrated children with FS have decreased sense of control over their actions compared to matched controls. Only one randomized controlled trial (RCT) for treatment of pediatric FS has been conducted to date, which evaluated Retraining and Control Therapy (ReACT). ReACT, developed by the PI, is a novel, adaptive intervention that aims to increase sense of control via mindfulness and principles of habit reversal, a well-established treatment for involuntary tics. Our recently published pilot RCT demonstrated that ReACT resulted in significantly reduced FS frequency compared to supportive therapy, with 82% of children remaining FS-free for ≥2 months after ReACT. Further, our preliminary data suggest sense of control significantly improves after ReACT, and increased sense of control after ReACT is significantly related to reduced FS frequency, indicating sense of control is an effective target for improving FS. The purpose of this study is to conduct a multi-site pilot feasibility clinical trial of ReACT, a mind and body treatment for pediatric FS. In the study, 54 11-18-year-olds diagnosed with FS will be randomized to engage in ReACT or the CATCH-IT web-based intervention (validated for treating mood in children) at three sites: the University of Alabama at Birmingham, the Yale School of Medicine/Yale New Haven Children’s Hospital and Baylor College of Medicine/Texas Children’s Hospital. FS frequency will be measured from 30 days before treatment to 2 months after treatment. Participants and a family member will complete baseline and follow-up assessments 7 days before treatment and 7 days and 2 months after treatment. Feasibility of recruitment and retention will be measured by meeting annual recruitment goals and the percent of participants who complete the 2-month follow-up. Sessions will be videotaped, and 20% of therapy sessions will be coded for treatment fidelity using published guides to assess intervention fidelity in behavioral clinical trials. Participant adherence to ReACT will be assessed by the percent of recommended treatment sessions completed, and participants and parents will report if the patient used the treatment plan for each FS episode using a FS diary.

NIH Research Projects · FY 2026 · 2023-08

ABSTRACT Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to an infection. Due to improved acute survival following sepsis, many patients develop chronic critical illness (CCI), defined as prolonged hospitalization with unresolved organ dysfunction. CCI frequently manifests as a persistent inflammation, immunosuppression, and catabolism syndrome (PICS). Sepsis survivors suffering from PICS have highly variable poor long-term outcomes, especially regarding muscle weakness, which manifests as atrophy, delays recovery, and leads to physical disability. Although we have demonstrated that sepsis leads to physical disability, which warrants interventions to prevent it, the majority of exercise training and nutritional clinical trials have been ineffective to prevent functional decline. A key deficiency of trials to date has been a “one size fits all” approach to intervention, ignoring any heterogeneity in patient populations (age, sex, and race/ethnicity). To develop precision medicine (or interventions), we need a better understanding of the contribution of age, sex, and race/ethnicity, and immunologically active peripheral organs to sepsis-induced physical disability. Part of this precision approach includes the gut microbiome. The gut microbiome uniquely affects host immune status, depends on biological variables, and interacts with and controls end-organ function such as skeletal muscle. Gut-microbiome dysbiosis is known to play a role in the overall pathology of sepsis and may be a driver of PICS and sepsis-induced acute and long-term muscle loss/weakness and physical disability. The Principal Investigator (PI) has demonstrated that older age is associated with worse clinical trajectories, long-term muscle-strength and physical-function outcomes, and has revealed that older adults have greater aberrations of the PICS biomarkers compared to younger individuals. The PI has also shown persistent gut-microbiome dysbiosis in sepsis patients and that gut microbiome differs between ages and sexes in a preclinical animal sepsis model. Given the current findings, the overarching goal for this application is to characterize the role of age, sex, race/ethnicity, and gut microbiome in the development of PICS leading to sepsis-induced muscle loss/weakness and physical disability. The goal of the PI laboratory’s research program over the next 5 years is to characterize (1) sepsis-induced chronic muscle loss/weakness and physical disability in sepsis survivors; (2) the role of PICS pathophysiology; (3) the effects of sex, age, and race/ethnicity on PICS and the development of physical disability; and (4) the unique contribution of the gut microbiome to heterogeneity of PICS and physical disability. Characterizing the heterogeneity of chronic sepsis-induced muscle loss/weakness and physical disability and creating patient profiles at risk of developing physical disability will be the key to applying precision medicine to prevent physical disability. Based on the findings of this study, future interventions can be customized to particular patients in terms of appropriate timing, type, and modality of exercise training and/or gut-function- enhancing treatments to prevent physical disability in an expanding population of chronically ill sepsis survivors.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The prevalence of ulcerative colitis (UC) in children continues to increase yearly. Recent evidence in pediatric UC patients showed significant mitochondrial impairment in the colon tissues. This is important as optimal mitochondrial activity is required for the solemn function of colonic stem cells that replenish the physical barrier of the colon epithelium. Since patients are constantly exposed to environmental factors such as diet, it is critical to reveal the dietary factors that influence mitochondrial function in the colon epithelium as they would be vital in the management of UC in children. Sulfites are endogenous products of several sulfur-containing compounds, and they are also ubiquitous in our diets as preservatives. My preliminary data in colon organoids derived from pediatric patients showed a detrimental role of sulfite on mitochondrial metabolism and differentiation, with worse metabolic outcomes in samples from pediatric UC patients. My analysis of transcriptomic data from 206 children with UC showed that the Mocs1 gene required for downstream clearance of sulfites in the mitochondria is downregulated in the colon of UC patients, suggesting a potential for inefficient sulfite detoxification in the colon. In this study, I will use patient-derived colon organoids to define how sulfites regulate mitochondrial metabolism and differentiation in health and in UC (Aim 1), reveal the sulfite-induced and sulfite susceptibility chromatin sites in the pediatric colon that explains these metabolic and differentiation anomalies (Aim 2), and how sulfites and the loss of epithelial Mocs1 shape colon biology in the complex gut environment in vivo using physiological relevant models (Aim 3). This award will advance my training in disease models of IBD, epithelial biology, and epigenomics as I work toward establishing an innovative career in regenerative nutrition with a focus on pediatric digestive diseases and continue efforts to enhance diverse representation in the biomedical sciences.

NIH Research Projects · FY 2023 · 2023-08

PROJECT ABSTRACT The prevalence of optic nerve hypoplasia (ONH) among individuals with Down syndrome (DS) is more than ~100 folds higher than in the general population. ONH is characterized by a thin, underdeveloped optic nerve that often results from secondary loss of retinal ganglion cells (RGCs) and is the leading cause of childhood legal blindness in developed nations. We have developed a murine model of ONH by manipulating the CASK (calcium/calmodulin dependent serine protein kinase) gene. The ONH pathology of CASK mutant mice recapitulates many aspects of human ONH, including the timing of the onset of pathology (after RGC development, i.e., secondary loss) and the non-progressive nature of the pathology. In addition to ONH, DS and pathology associated with CASK mutation share other similarities such as microcephaly. Biochemical experiments suggest that similar to DS, CASK mutation alters mitochondrial function, resulting in aberrant fatty acid metabolism. We have uncovered a deficiency of the ω-6 polyunsaturated fatty acid arachidonic acid (ARA) in the central nervous system (CNS) of CASK mutant mice. A specific reduction of ARA-containing lipids has been also reported in postmortem human DS brains. In both CASK mutant mice and the DS murine model visual contrast sensitivity is reduced. Based on the literature and our observations, we hypothesize that ARA deficiency in the optic nerve (ON) leads to ONH in DS. If this hypothesis is correct, dietary ARA supplementation could ameliorate ONH in DS. In this proposal, we plan to use two independent murine DS models (Ts65Dn and DP16 mice) to investigate retinal circuit and retinogeniculate connectivity using appropriate markers and tracers along with imaging techniques. Three-dimensional ultrastructural morphometry of the ON in DS murine models will be performed using serial block-face scanning electron microscopy (SBFSEM). Behavioral experiments will document visual defects. Visual circuit function will be evaluated with a novel in vivo electrophysiological technique we have named Network Response to Visual Excitation (NeRVE), a method that will be applicable beyond this proposal to better understand rodent vision processing. Mitochondrial metabolism, reactive oxygen species generation, oxidative damage, and fatty acid metabolic defects in the retina, optic nerve and brain of two independent DS mouse models will be measured. We will also quantify the levels of two ω- polyunsaturated fatty acids (docosahexaenoic acid and ARA), as well as phospholipids, in the ON of both types of DS mice. Finally, we will test if ARA supplementation ameliorates known defects in visual acuity and contrast sensitivity in these two DS mouse models. Our study is likely to provide data on DS pathobiology and provide a clear mechanism for the co-occurrence of ONH in DS. Furthermore, promising results from our ARA supplementation study would be immediately translatable into clinical practice for the amelioration of ONH in individuals with DS.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Optic nerve hypoplasia (ONH) is a very common congenital optic nerve (ON) disorder and is the leading cause of childhood blindness in developed nations. ONH incidence has increased ~8-fold over the last two decades. ONH is characterized by a thin, underdeveloped ON that often results from secondary loss of retinal ganglion cells (RGCs). The most common prenatal determinants of ONH are a young primiparous mother, an unhealthy maternal lifestyle including alcohol abuse, and nutritional deprivation. We have developed and published murine models of ONH by manipulating the X-linked gene CASK, since CASK mutations in humans are associated with ONH. The ONH pathology of CASK mutant mice recapitulates human ONH, including the timing of pathology onset (after RGC development; i.e., secondary loss) and the non-progressive nature of the pathology. Biochemical experiments show that CASK interacts with metabolic proteins and modulates mitochondrial function. CASK deficiency leads to increased fatty acid oxidation and a deficit of the ω-6 fatty acid arachidonic acid (ARA) in the central nervous system (CNS). ARA deficiency is also observed in other conditions associated with ONH. We hypothesize that ONH results from an early ARA deficit, thus ONH can be exacerbated by perturbing brain ARA metabolism (via astrocyte dysfunction) and ameliorated by dietary ARA supplementation. During the third trimester, ARA is exclusively obtained from the mother; in neonates, brain ARA is also obtained from the diet until adequate enzymatic activity (conversion of the essential fatty acid linoleic acid into ARA) is reached. This post-neonatal shift in ARA acquisition from diet to synthesis may contribute to ONH’s non-progressive nature. In the CNS, fatty acid metabolism (including ARA uptake and production) occurs predominantly in astrocytes. In this proposal we plan to test our hypothesis in two independent ONH mouse models: 1) CASK(+/-) heterozygous knockout mice, and 2) a previously published fetal alcohol syndrome (FAS) mouse model. With these models, we will examine mitochondrial metabolism, oxidative damage and fatty acid metabolic defects in the retina, ON and brain. We will also quantify levels of two ω-fatty acids (docosahexaenoic acid and ARA), as well as phospholipids in the ON of both types of ONH mice. Next, we will genetically disrupt the function of astrocytes (crucial for brain ARA metabolism) in a CASK hypomorph ONH model by complete deletion of CASK in astrocytes. We will investigate if this manipulation exacerbates the metabolic defect and ONH as assessed both morphologically and functionally, using a visual behavioral assay and an innovative electrophysiological tool called Network Response to Visual Excitation (NeRVE). Finally, we will test if ARA supplementation ameliorates ONH in the two models described above. Our study is likely to identify ARA deficiency as the final common pathway that explains ONH’s association with nutritional deprivation, maternal diabetes, infantile cholestasis and FAS. Positive results from ARA supplementation will be readily translatable.

NIH Research Projects · FY 2025 · 2023-08

SUMMARY Obesity is particularly severe in terms of escalating kidney dysfunction, disrupted body fluid homeostasis, kidney injury. Our diet-induced obesity (DIO) mouse model (20-wk, ad lib 45% high fat diet, HFD) dampened feeding cycles, impaired kidney mitochondrial metabolism, amplified kidney medullary oxidative stress and excretion of reactive oxygen species (ROS), as well as increased medullary interstitial fibrosis compared to mice on a normal diet. Remarkably, restoring feeding-fasting cycles through time-restricted food intake (TRF), without altering total caloric intake during the final 2 weeks of DIO, re-established whole body diurnal energy metabolism, normalized excretion of oxidants and renal injury markers, as well as abolished renal interstitial fibrosis and T cell infiltration. These remarkable findings clearly indicate that feeding-fasting cycles are critical for kidney health in obesity. The goal of the proposed studies is to determine specifically how timing of feeding-fasting cycles impacts kidney mitochondrial respiration, inflammation and fibrosis in obesity. Our central hypothesis states that timed feeding- fasting ameliorates DIO-driven kidney fibrosis by reinstating kidney mitochondrial function and reducing T cell activation and migration to the kidney. Prior studies suggest that the clock gene, Bmal1, regulates mitochondrial function, but whether this is critical for DIO-induced metabolic dysfunction in the kidney is unknown. Our new data reveal significant, time-of-day differences in mitochondrial respiration in renal medulla of Bmal1 knockouts compared to wildtype littermates. In our model of DIO, we observed that circadian rhythms in whole body energy metabolism are lost but restored by TRF. We also observed that DIO causes a phase shift in the circadian molecular clock in the kidney. We posit that the mechanisms responsible for the TRF effects in obesity to restore kidney mitochondrial metabolism is via re-establishing clock activity and suppressing ROS production. Furthermore, TRF abolishes kidney interstitial fibrosis and kidney vasa recta-associated T cell infiltration in obese mice suggesting that timed feeding-fasting promotes kidney health by reducing kidney T cell inflammation. Our new data found that endothelium-derived ET-1 specifically mediates kidney pro-inflammatory CD4+T cell activation. We show that gut pro-inflammatory T cell activation and cytokine production is ETA dependent and that CD4+T cells in the gut migrate to peripheral tissues. Thus, we further propose that TRF mitigates DIO-driven kidney medullary fibrosis by reinstating diurnal rhythms of kidney endothelium-derived ET-1 with T cell clock activity and ETA dependent T cell activation and migration from the gut to the kidney. Studies will address two specific aims: First, to test the hypothesis that TRF reduces kidney fibrosis in DIO through Bmal1 mediated restoration of mitochondrial function and reduced mitochondrial-derived ROS. Second, to test the hypothesis that TRF mitigates DIO-driven kidney fibrosis by reinstating physiological endothelium-derived ET-1 in the kidney with T cell activation.

NIH Research Projects · FY 2026 · 2023-07

People living in the Deep South experience higher rates of health disparities due to multilevel barriers to care and fewer health resources compared to other areas of the country. Further, there are few health research training programs in the Deep South. Our overarching goal is to train students and working professionals with an interest in health research to increase knowledge and skills addressing health in this area of the country. We will achieve that goal through creating and implementing a 4-year program, Deep South Population Health Training Program. We have built a unique community-academic partnership, including Dr. Emma Kay, founding director of the Magic City Research Institute, one of the few research institutions in the Deep South housed within a community-based organization; and Dr. Sarah MacCarthy, inaugural holder of a health studies endowed chair. Our specific aims include: 1) Develop a hybrid community-based education program that addresses core concepts and hyperlocal approaches to population health, with particular attention to health issues affecting women; 2) Increase knowledge of population health via implementation of the program; 3) Disseminate a program toolkit nationally to enable local adaptations, implementation, and evaluation. Year 1 includes development of an online 14-week course covering core concepts in population health, providing a foundational understanding of factors driving health risk and resilience with attention to factors affecting women and those endemic to the Deep South. The course will be complemented by hyperlocal approaches, including an in-person 1- week residency highlighting knowledge and expertise among local partners and a 6-month period of virtual mentoring and networking salon sessions. Years 2 and 3 include recruitment of two, 1-year cohorts (n=5-7 scholars per cohort) and implementation of the program. Year 4 includes the development and national dissemination of a toolkit with guidance on how others can develop, implement, and evaluate their locally adapted version. Our rigorous evaluation plan consists of pre/post surveys to assess scholars’ mastery of topics taught in the online course, as well as interviews with scholars and surveys with program mentors and faculty/community partners to assess opportunities for program improvement. Through national dissemination of the toolkit, our program will be broadly available; supporting tools will enable organizations to create locally-relevant experiences for future scholars to further their understanding of population health research.

NIH Research Projects · FY 2024 · 2023-07

The kynurenine pathway catabolizes over 95% of all tryptophan primarily through the actions of tryptophan 2,3-dioxygenase 2 (TDO2) in hepatocytes and indoleamine 2,3-dioxygenese 1 (IDO1) in myeloid leukocytes. Increased kynurenine synthesis in dendritic cells (DC) secondary to IDO1 upregulation is strongly linked with the generation of a tolerogenic phenotype by promoting anti-inflammatory signaling, regulatory T cell (Treg) polarization and immune tolerance, an attenuated inflammatory response instigated by immune cells that are repeatedly exposed to TLR ligands. However, while the pathophysiologic relevance of this pathway is well- established, the mechanism behind the immunomodulatory effects of kynurenine remain poorly defined. Using semi-targeted metabolomic approaches, we recently published that systemic increases in kynurenine levels secondary to either exogenous supplementation or chronic inflammation are associated with the formation of the novel cysteine-reactive kynurenine-derived electrophile Kyn-CKA. Kyn-CKA promotes Nrf2-depedent signaling, inhibits TLR4-dependent NF-κB pathways and attenuates inflammatory responses in endotoxin-challenged mice in a redox-dependent manner. In addition, Kyn-CKA engages AhR signaling with 20-fold higher potency than its kynurenine precursor, suggesting a potential pro-tolerogenic role in DC and T-cells. Specifically designed state- of-the-art LC-MS/MS assays will enable the quantification of Kyn-CKA in the context of other kynurenine pathway metabolites in activated and non-activated myeloid leukocytes, as well as the elucidation of rate-limiting cellular uptake and export mechanisms. Primary macrophages, dendritic, and T cells derived from pathway-specific knock-out animals will be harnessed in conjunction with novel bio-orthogonal labeling strategies and isotope- tracing metabolic flux analyses to define the mechanistic basis of the anti-inflammatory actions of Kyn-CKA both in terms of the modulation of specific signaling pathways and its effects on inflammation-elicited changes in energy metabolism. The ability of Kyn-CKA to promote tolerogenic responses will be established by assessing its effect on the maturation and activation of conventional DC subpopulations, its impact on T cell polarization, and its ability to modulate endotoxin resistance and tolerance in vivo. Mice expressing or lacking critical mediators of Kyn-CKA formation and action in cell-specific compartments will be used to obtain mechanistic insights in vivo. In summary, the Research Plan addresses a hitherto unappreciated redox-dependent component of the immunomodulatory actions of the kynurenine pathway, mediated by the formation of electrophilic Kyn-CKA. If successful, our work will enable the potential development of novel Kyn-CKA based pharmacological interventions for dysregulated immune responses such as chronic inflammation, autoimmune diseases, cancer, and allograft rejection.

NIH Research Projects · FY 2025 · 2023-07

Project Summary - Toxin secretion and trafficking by Mycobacterium tuberculosis The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Most pathogenic bacteria use toxins to evade and/or subvert immune responses, to survive and replicate in the host. CpnT is an outer membrane protein with an N-terminal channel domain and a C-terminal toxin domain (TNT), which causes necrotic cell death in host cells. Up-to-date TNT is the only known exotoxin of Mycobacterium tuberculosis. We showed that TNT is secreted into the cytosol of macrophages infected with M. tuberculosis where it hydrolyzes NAD+. NAD+ depletion by TNT activates necroptosis, a programmed cell death pathway. Importantly, necroptosis-deficient mice are more resistent to infection with M. tuberculosis, underlining the importance of this pathway for tuberculosis pathogenesis. Permeabilization of the phagosomal membrane is a critical step in the pathogenesis of M. tuberculosis, not only because it required for M. tuberculosis proteins such as the toxin TNT to reach the cytosol of infected cells, but it also enables M. tuberculosis to escape from the phagosome resulting in bacterial dissemination. Thus, TNT secretion and trafficking are an important components of the intracellular survival strategy of M. tuberculosis and for tuberculosis pathogenesis. This research project will reveal novel molecular mechanisms of toxin secretion and trafficking by M. tuberculosis and the role of these mechanisms in virulence and pathogenesis of M. tuberculosis.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Chronic rhinosinusitis (CRS) is a common inflammatory disease that affects a large portion of the U.S. population, resulting in poor quality of life for those affected and utilizing billions of dollars of health care resources. Efforts in my lab have focused on understanding patterns of inflammatory heterogeneity in large CRS populations with a goal of improving disease endotyping and developing personalized care pathways. I am devoted to mentoring the next generation of clinician-scientists in the field of rhinology and chronic rhinosinusitis research. The number of physicians and other surgeons pursuing clinical or translational research is declining, and this is particularly true in the fields of rhinology and otolaryngology. Maintaining a strong pipeline of trainees on research-related career paths is crucial for the development of the subspecialty and scientific advancement of the field. This will be a prospective translational cohort study to identify the clinical implications and longitudinal stability of chronic rhinosinusitis endotypes. We will enroll CRS patients through our well-established biospecimen repository and determine the stability of inflammatory mediator levels and associated endotype assignment over multiple seasons and determine whether these changes are associated with sinonasal microbial community structure. We will also confirm the longitudinal effects of two commonly used immune-modifying interventions (endoscopic sinus surgery and anti-IL4/13R biologic therapy) on inflammatory mediator levels and endotypic assignment. Collectively, these translational studies will substantially advance basic knowledge of a common airway disease that impacts millions of patients, while providing a range of investigative opportunities for mentees interested in CRS and mechanisms of upper airway inflammation. My research program is centered around a patient-oriented approach that incorporates patient-derived specimens, patient-reported outcome measures, and personalized approaches to care. I anticipate that mentee participation in this research program will lead to new and exciting avenues for patient-oriented research to further characterize CRS endotypes and evaluate the effects of targeted therapies; thus, this project will provide a conducive setting for developing the next generation of clinician-scientists in the field. I have a history of continual extramural funding, more than a decade of experience as a mentor to trainees at all levels, and institutional support to enhance my own skills that will augment the support proposed through the K24 mechanism.

NIH Research Projects · FY 2025 · 2023-07

Project Summary T follicular helper (Tfh) cells are essential for germinal center (GC) responses and long-term humoral immunity. However, the complex regulation that determines the differentiation of Tfh cells, in particular, the initial CXCR5– versus CXCR5+ CD4+ T cell differentiation, the developmental progression of CXCR5+CD4+ T cells to become GC-Tfh cells, and the generation of follicular helper-like memory CD4+ T cells expressing CXCR5, are still not fully understood. Our proposal aims to fill in these knowledge gaps with long-term goals to identify novel genes/pathways underlying the CD4+ T cell differentiation. In our preliminary studies, we have discovered a novel network engaging various factors/pathways that fine-tunes CXCR5+ versus CXCR5– CD4+ T cell differentiation and regulates the generation of cytotoxic CD4+ T cells in the early stage of CD4+ T cell response. By combining RNA-seq, ATAC-seq, and single cell RNA-seq, our preliminary data also suggest that the PD-1+CXCR5+ pre-Tfh cells undergo substantial further differentiation to become PD-1hiCXCR5hi GC-Tfh cells. Additionally, we have generated novel “fate-mapping” reporter mice that will allow us to track the varied CXCR5– and CXCR5+ memory CD4+ T cells. Thus, in this application, we aim to dissect the molecular underpinning of the early stage CXCR5+ versus CXCR5– CD4+ T cell differentiation as well as to elucidate the mechanisms underlying the pre- to GC-Tfh differentiation and the generation of diversified CD4+ T cell memory. Our work will have a profound impact on the field of CD4+ T cell differentiation. The research will not only shed new light on our understanding of the mechanisms underlying the multiple steps of Tfh cell differentiation but also establish new model systems for memory CD4+ T cell studies. This proposal has the potential to provide important knowledge on how to control both the humoral and the cellular arms of the CD4+ T cell response to aid vaccine development for new pandemic threats and help the treatment of infectious diseases and autoimmune disorders.

NIH Research Projects · FY 2025 · 2023-07

Project Summary. The long-term objective of this study is to understand signals involved in development and maintenance of the axial skeleton that can inform regenerative and engineering strategies. Members of the Tgfb superfamily are secreted signaling proteins that regulate many aspects of skeletal biology. Polymorphisms and mutations in genes that regulate Tgfb activity have been associated with pathology in the spine. It’s also been shown using genetically engineered mice that Tgfbr2 is required for development and maintenance of the fibrous tissues in the spine including the annulus fibrosus of the intervertebral disc, ligaments, and tendon. Previous results obtained in my laboratory indicate that Tgfb regulates cell fate decisions in the sclerotome, the embryonic progenitor of the connective tissues in the spine. In this application, we propose to address the instructive mechanisms whereby Tgfb regulates embryonic formation of fibrous tissues in the spine. In addition, we propose to address the problem of sclerotome resegmentation, an embryonic process that creates the spatial organization of tissues in the spine. Alterations in resegmentation would be expected to alter the context in which cells differentiate, affecting permissive signals and competence to respond to instructive signals that govern cell fate decisions. Finally, using a mouse model developed in my laboratory, we will start to determine the mechanisms of how Tgfb acts to maintain fibrous character in the postnatal annulus fibrosus. The overall aims of this proposal are to: 1) Determine the signaling pathways used by Tgfb to generate fibrous tissues of the spine during embryonic development; 2) To map the process of resegmentation and determine the mechanism of Tgfb-mediated regulation and 3) To understand how Tgfb maintains annulus fibrosus postnatally. The experiments described here will provide mechanistic information about development and maintenance of the axial skeleton and provide a foundation for future regeneration and engineering strategies in the spine.

NIH Research Projects · FY 2026 · 2023-07

SUMMARY The increase in longevity, reduction in tooth loss, increase in periodontal diseases and coronal and root dental caries with aging are contributing to an increased number of seniors with oral health care needs. Dental caries and periodontal diseases disproportionately affect Alaska Native older adults: they are 3 times more likely to have untreated dental caries and 1.7 times more likely to have periodontal disease than the overall United States population. Prospective studies on the prognosis of these oral disorders are lacking, particularly those deconstructing racial and ethnic differences. The goal of this research project is to understand outcomes of geriatric patients with periodontal diseases and/or dental caries and identify prognostic factors for success in disease control, with a focus on Alaska Native seniors. Our primary aim is to estimate, separately, 36-month success rates for periodontitis and dental caries management for geriatric patients overall, and for Alaska Native/American Indian seniors. Our secondary aims are 1) to ascertain practitioner-, patient-, oral environment- and tooth-level prognostic characteristics associated with 36-month successful disease control strategies; and 2) to describe initial disease diagnosis and management strategies used by practitioners, and associated factors. We will accomplish these aims by conducting a prospective cohort prognosis study within a tribal healthcare organization serving urban and remote rural geographical locations in Southeast Alaska. In this observational study, approximately 600 patients with periodontitis and/or dental caries will be systematically enrolled and followed up for three years. Data on oral health, patient, and provider characteristics as well as diagnosis and disease management strategies will be collected at baseline, and yearly up to 3 years after enrollment. This study is innovative and highly relevant to public health and clinical research because it addresses the lack of rigorous prospective US practice-based geriatric oral health research, and the uncertainty in prognosis and prognostic factors when managing oral disorders in patients suffering the greatest oral health disparities. Through prognosis research, we can gain important knowledge on how to potentially improve clinical decision making for better Alaska Native geriatric oral health.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Cardiovascular disease (CVD) is the leading cause of death in the United States, and obesity is one of the highest risk factors for CVD. Our lab has shown that restricting intake of high fat diet (HFD) to the 12-hour active period for the last 2 weeks in a 20-week diet induced obesity (DIO) model significantly reduces aortic wall thickness and fibrosis and restores aortic endothelial function. We also found that DIO significantly increases aortic Th17 cells, which are an inflammatory CD4+ T cell subset that are known to drive progression of autoimmunity and organ damage. Interestingly, time restricted feeding (TRF) in the final 2 weeks of the DIO protocol reduced aortic Th17 cells. Th17 cells are the main producer of the inflammatory cytokine, IL-17A. IL- 17A has been known to drive CVD risk factors, however, it is unclear if TRF reduces aortic damage via the IL- 17A pathway. Furthermore, we have preliminary data that TRF in DIO is associated with greater circulating propionate and butyrate, which are two important microbial-derived short chain fatty acids (SCFA). SCFA are important for regulating hypertension and promoting anti-inflammatory T cell subsets, however, their role in DIO induced tissue damage is unclear. DIO is associated with decreased SCFA production, which could indicate that propionate and butyrate are necessary for protection against DIO damage. This led us to hypothesize that reduction of IL-17A and increased SCFA with TRF drive aortic protection and improved endothelial function in DIO. We will use C57Bl6/J mice for our 20-week DIO model with TRF intervention in the final two weeks of feeding. During those 2 weeks, mice will receive anti-mouse IL-17A or IgG antibody daily at Zeitgeber Time (ZT) 0, the start of the inactive period. Using these groups, we will assess aortic damage via pulse wave velocity (PWV) and histology. We will also assess endothelial function via vascular reactivity by stimulating the aorta with acetylcholine to assess endothelial dependent vasorelaxation. Sodium nitroprusside is used to assess endothelial independent vasorelaxation. Furthermore, we will use our TRF intervention in DIO to assess pathogenicity of Th17 cells in the aorta via single cell RNA sequencing. Th17 cells that upregulate the IL-23 receptor (IL-23R) are known to have greater pathogenic capabilities. In aim 2, we will use our 20-week DIO model with C57Bl6/J mice. During the final two weeks, the diet will be supplemented with 5% butyrate and propionate by weight in the inactive period as that is where we see increase of circulating SCFA. We will assess how dietary SCFA affects aortic Th17 cells via flow cytometry. Flow cytometry will allow us to immunophenotype the cells to identify if SCFA have an effect specifically on Th17 cells. We will also assess aortic damage and function via histology and PWV measurements at the conclusion of the 20-week DIO protocol. Endothelial function will also be assessed via vascular reactivity experiments. The main goal of this proposal is to identify how TRF in DIO improves aortic damage and endothelial function through regulation of Th17 cell pathogenicity.

NIH Research Projects · FY 2026 · 2023-07

7. PROJECT SUMMARY / ABSTRACT. Gastrointestinal (GI) mucosal damage and destruction of the gut epithelial barrier are the defining features of the pathogenesis of HIV-1 infection. Accumulated evidence indicates that neutrophils play a critical role in the gastrointestinal and liver damage in HIV-1 infection. Neutrophils infiltrate the GI tract in HIV-1- infected individuals at high levels and their presence is associated with damage to the epithelial barrier, elevated epithelial permeability, and increased disease severity in animal models and HIV-1-infected patients. In this application, we propose that microbial translocation and the resulting systemic innate immune dysregulation mediated by changes in neutrophil subpopulations in circulation, gut-associated lymphoid tissue (GALT), and liver plays a fundamental role in HIV-1 disease progression. The overall objectives of this proposal are to define the role of neutrophil subpopulations and NETosis as driving mechanisms of gastrointestinal and liver damage in HIV-1 infection and to identify the mechanisms responsible for chronic neutrophilic activation in HIV-1 infection in order to reveal the specific checkpoints for intervention. Our central hypothesis is that HIV-1 infection is associated with the induction and expansion of specific neutrophilic subpopulations with increased capacity to produce reactive oxygen species (ROS) and undergo NETosis. ROS and NETs released from activated neutrophils promote damage in the GI mucosa and liver and drive the progression of HIV-1 infection. This hypothesis has been formulated on the basis of our preliminary data and recently published reports demonstrating the critical role of neutrophils in HIV-1 infection. In preliminary studies, we optimized methods for detailed neutrophil characterization and demonstrated that neutrophils from HIV-1-infected individuals display an activated phenotype, immunosuppressive properties, specific transcriptional profile, increased rate of degranulation, and a high capacity to undergo NETosis. Specific properties of the newly identified neutrophil subpopulations strongly indicate that they play a critical role in damaging GI mucosa and the pathogenesis of liver disease in HIV-1-infected individuals. We propose to determine the effect of induction of specific neutrophil subpopulations on the progression of liver disease in ART-treated HIV-1-infected individuals, to identify specific properties of neutrophil subpopulations in the GALT and liver of HIV-1-infected individuals, and determine whether the innate immune dysregulation in these tissues is associated with a shift in the ratio of tissue macrophages exhibiting M1 versus M2 phenotype resulting in lowered efferocytosis and accumulation of neutrophils undergoing NETosis. The significance of the proposed studies is that once the role of neutrophils in the progression of HIV-1 infection is defined, neutrophil activation and induction of pathogenic populations can be pharmacologically targeted.

NIH Research Projects · FY 2025 · 2023-07

PROJECT ABSTRACT This grant application is for the F31-Diversity support of Courtney Swain during her MD-PhD training. The research focus of this proposal is to establish a mechanism in which hyperglycemia and Hedgehog (Hh) signaling conspire to modulate CD8+ T cell exhaustion and regulatory T cell (Treg) immunosuppression in triple-negative breast cancer (TNBC). Breast cancer continues to threaten the lives of many women in the U.S. and worldwide as it accounts for more than 30% of all female cancer cases. Additionally, type 2 diabetes mellitus (T2D) is a highly prevalent morbidity and about a quarter of breast cancer patients are diabetic, which can increase treatment complications and limit therapy options. TNBC tumors are immunologically “cold,” characterized by the limited infiltration of cytotoxic populations and increased abundance of immunosuppressive constituents in the primary tumor. Hyperglycemia in T2D pathogenesis has been implicated to impair CD8+ T cells, lymphocytes critical in tumor killing and immunotherapy response, and their exhaustion process. Upon tumor challenge, subsets of immunologically reactive TCF1+ stem-like and TCF1- transitory effector CD8+ T cells are generated. These CD8+ T cell subsets have been found to be highly essential in tumor control despite their phenotype and nomenclature of early exhaustion. Notably, hyperglycemia exacerbates dysregulated Hh signaling in breast cancer. Initial investigations have revealed that hyperglycemia and Hh signaling may be cooperatively driving dysfunctional CD8+ T cell exhaustion in the mammary tumor milieu. Additionally, the presence of Tregs during mammary tumorigenesis correlates with poorer prognoses in TNBC. Supporting evidence in this proposal underscores that both, Hh signaling and hyperglycemia, impair CD8+ T cell exhaustion and promote Treg immunosuppression. However, the mechanisms by which these factors influence these key T cell populations are unknown. Therefore, this proposal will apply unique model systems of Hh signaling and hyperglycemia to delineate their roles in CD8+ T cell and Treg activity in TNBC. Findings will provide more relevance for combination TNBC therapies, especially for the distinct patient population of diabetic breast cancer patients. The proposed training plan for the PI is sponsored by her PhD mentor, Dr. Lalita Shevde-Samant. The goals of the training plan are to provide the PI with: (i) a rigorous research project using distinctive pre-clinical models of diabetes-associated breast cancer, novel genetically engineered mice, and tumor-immune crosstalk; (ii) opportunities in developing immunologic and bioinformatic techniques and in expanding training in responsible conduct of research, rigor, reproducibility, and principles of scientific integrity; and (iii) a scientifically enriching and equipped environment essential for developing a successful career as an oncologist-scientist. Given this project’s focus on the interaction of two major disease challenges of the U.S., breast cancer and diabetes, the PI will have exemplary guidance and a solid foundation to develop into a very competent physician-scientist.