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NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Myelodysplastic Syndromes (MDS) is a group of heterogenous bone marrow failure syndromes often seen with advancing age. Mutations in splicing factors (SFs) such as SF3B1, U2AF1 and SRSF2 are the driving genetic alterations in over half of all MDS. These mutations have classically been linked to alternative splicing of oncogenes or tumor suppressors, but recent studies suggest broader defects including disruption of co-transcriptional splicing, which is the close functional coupling of transcription and splicing. Our group has recently shown that mutant SF3B1 impairs spliceosome assembly and slows RNA Polymerase II (Pol II) elongation, resulting in transcription-replication conflicts and replication stress. These changes reorganize chromatin, reducing promoter accessibility and histone marks. Notably, this model can explain the mutual exclusivity of SF mutations: cumulative transcriptional stress from multiple mutations is unsustainable for clonal expansion. In this application, we seek to define the role of HTATSF1, a protein with roles in both splicing and transcription, in transcriptional dysregulation in SF-mutant MDS. Our preliminary results show reduced interaction of HTATSF1 with mutant SF3B1. We hypothesize that this reduced binding of HTATSF1 to mutant SF3B1 impairs its recruitment to Pol II, disrupting the coordination between transcription and splicing. Conventional genome-wide assays lack spatial and temporal resolution to study the complexity of highly dynamic complexes such as spliceosome and Pol II. To overcome this, we will use multi-color, single-molecule imaging to resolve HTATSF1 recruitment and interaction kinetics in real time. We have leveraged CRISPR/Cas9 to introduce tags (degron for acute degradation and HaloTag for high resolution live-cell imaging in primary murine embryonic stem cells. Two aims are proposed to determine HTATSF1’s role in SF-mutant MDS. In the first aim, we will study how impairment of transcription noted in SF-mutant MDS is linked to HTATSF1. Total Internal Reflection Fluorescence (TIRF) microscopy will be used to track endogenously tagged HTATSF1 at a single-molecule resolution. In the second aim, we will determine HTATSF1’s role in altered splicing, a feature of SF-mutant We will utilize single-molecule imaging as well as differential phosphoproteomics in these studies. Ultimately, our findings may inform the development of therapies targeting transcriptional dysregulation, replication stress, and chromatin dysregulation in MDS.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract: Stem cells are required for tissue homeostasis and regeneration. Accomplishing these tasks requires intimate association with a niche, a cellular microenvironment that forms in a specialized tissue location with precise morphology to enable communication with stem cells. Niches are formed by cells that must be specified for niche identity, receive signals directing migration to the appropriate tissue compartment, and respond to those cues with changes in gene expression and cytoskeletal behavior. Studying this has proven challenging, as most niches are established during embryogenesis when the tissue is inaccessible to live imaging. Under previous GM funding, my lab work established an in vivo live imaging method to enable exploration of each of these facets in the assembling Drosophila testis niche, a tractable and conserved model. Foundational studies in the adult testis have repeatedly unveiled concepts that apply to other systems, yet before my work, we did not know how this niche formed. Our method permits direct in vivo visualization, revealing discreet steps of morphogenesis. This application will perform lab work to investigate the underlying mechanisms for each step. We ask (1) How are niche cells specified? (2) How do regulators of the cytoskeleton enable niche morphogenesis? and (3) What signals direct the location of niche assembly? My previous work showed that signals from adjacent visceral muscle (Vm) are required to assemble the testis niche during embryogenesis. In response to signals, niche cells express the transcription factor islet (mammalian ortholog, Isl1), which I found polarizes F-actin and regulates anterior niche assembly. An open question is whether F-actin polarization directly enables niche morphogenesis, or if it is polarized in consequence of niche assembly. This application will harness our in vivo imaging protocol along with an incisive optogenetic approach to test direct contributions of cytoskeletal regulators in each step of niche development. Our unpublished work supported by GM R15 funds has further shown that Vm cues induce Tbx1 ortholog org-1 expression to influence niche establishment. This proposal will define genetic regulatory mechanisms through which Tbx1 regulates niche identity and morphogenesis. Finally, our data reveal that a gonad-intrinsic, non-niche cell population is guiding niche morphogenesis in concert with signals from adjacent visceral muscle. This represents a novel mechanism for niche development, which we will uncover in this application. Our work will combine the power of Drosophila genetics with incisive assays in cellular mechanics, including live in vivo imaging, optogenetics for precise temporal manipulation of the niche cortical cytoskeleton, and laser ablation to define underlying forces driving niche and stem cell behavior. Mechanisms we unveil in this model will reveal mechanics of niche establishment required to form a compartmentalized niche with appropriate cellular architecture to enable tissue function.

NHLBI - National Heart Lung and Blood Institute

Project Summary/Abstract: Sarcoidosis is a rare and understudied disease. It is characterized by a strong genetic predisposition, with risk often conferred by Major Histocompatibility (MHC) class II genes and a dysregulated immune response against an unidentified inhaled antigen. Antigenic exposure triggers interactions between antigen-presenting cells (APCs) and naïve CD4+ T-cells within lymph nodes (LNs), leading to a polarized Th1 response and granulomatous inflammation primarily observed in the lungs and LNs. Persistence of this heightened inflammatory immune response results in chronic sarcoidosis, with up to 40% of pulmonary cases progressing to irreversible and possibly end-stage fibrotic disease. Disease progression is associated with reduced quality of life, significant morbidity, higher mortality, and increased healthcare costs. Despite the negative impact, the exact immunological mechanisms driving persistent inflammation in sarcoidosis and progression to end stages have not been fully elucidated and pose a barrier to developing effective therapies. Our recent observations and those of other researchers indicate that up to 50% of sarcoidosis patients exhibit paradoxical peripheral lymphopenia, accompanied by CD4+ T-cell anergy and exhaustion, even in the early stages of the disease. This is associated with increased inflammatory activity, severe organ involvement, and disease progression. Loss of CD4+ effector T-cell function is thought to impair immune surveillance, leading to unmitigated inflammation and persistent granulomatous infiltration of affected tissues. Our prior research utilizing bulk RNA-seq analysis of peripheral immune cells suggests that compromised lymphocyte function and survival stem from aberrant, cell-specific, transcriptomic networks and interactions between lymphocytes and hyperactive innate APCs in lymphopenic sarcoidosis. Utilizing single-cell omic analyses, our novel preliminary data expands on this notion and reveals that peripheral naïve CD4+ T-cells from individuals with lymphopenic sarcoidosis possess a genetically imprinted, aberrant transcriptional program with multiple dysregulated immunoregulatory biological pathways that are involved in cell proliferation and death, predisposing them to impaired function and survival. Furthermore, we find an association between lymphopenia and MHC class II genes, and via single-cell RNA-seq of intrathoracic LNs fine needle aspirates, we find evidence that cDC1s in lymphopenic sarcoidosis have a limited ability to process and present antigens underscoring their crucial role in orchestrating adaptive immune responses. Thus, this research aims to elucidate the underlying mechanisms driving paradoxical peripheral lymphopenia in early sarcoidosis. We propose to utilize high-throughput analyses, such as single-cell RNA-seq and ATAC-seq, along with conventional immunology techniques to investigate how naïve CD4+ T-cells and cDC1s contribute to T-cell dysfunction and impaired survival. Focusing on these immune cell subsets will allow us to uncover intrinsic and extrinsic factors that influence immune dysregulation in lymphopenic sarcoidosis and provide critical insights for developing targeted therapies to mitigate disease progression.

NHLBI - National Heart Lung and Blood Institute

Desmoplakin cardiomyopathy (DSP-CM), an arrhythmogenic cardiomyopathy often affecting young adults, results from pathogenic loss-of-function variants in DSP, leading to impaired cardiac function, myocardial fibrosis, and sudden cardiac death. Fibrosis in DSP-CM localizes to the subepicardium, a region of high tensile stress. My preliminary data using both human heart tissue and human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) demonstrates that DSP haploinsufficiency, which leads to cell adhesion failure, is the primary pathogenic mechanism. However, the precise consequences of the interaction between DSP level and mechanical load, including with the hemodynamic stress imposed by exercise, remain poorly understood at the tissue level. This proposal aims to define the tissue-level pathology driven by mechanical stress in DSP haploinsufficiency and to test whether therapeutic restoration of DSP levels can prevent this pathology. The central hypothesis is that DSP haploinsufficiency causes cardiac tissue to be sensitized to injury, fibrosis, and arrhythmias specifically with mechanical stress, including high intensity exercise, and that restoring DSP levels can mitigate these effects in vivo. To investigate tissue-level pathology and treatment effects, this project will utilize fiber-aligned engineered heart tissues (fEHTs) and a novel Dsp mouse model that I developed to recapitulate patient-level haploinsufficiency and left ventricular fibrosis. Specific Aim 1 will determine the impact of mechanical load, including exercise, on fibrosis and arrhythmias in DSP haploinsufficient models, utilizing exercise protocols, functional assessments, and spatial transcriptomics in the Dsp mouse model, complemented by mechanistic studies in fEHTs. Specific Aim 2 will evaluate whether AAV-mediated gene therapy strategies can rescue DSP haploinsufficiency and prevent fibrosis in vivo. This will involve testing both CRISPR-based transcriptional activation (CRISPRa) to upregulate endogenous Dsp, building on my in vitro rescue data, and a split-intein approach to deliver the functionally sufficient DspII isoform. Both strategies will be assessed in our Dsp-CM mouse model for Dsp protein restoration, cardiac function, and fibrosis. This project will elucidate how mechanical stress impacts DSP-CM progression and provide crucial in vivo proof-of-concept for novel therapeutic strategies, addressing a critical unmet need. By addressing both disease mechanism and therapy, and providing training in advanced cardiac models, gene therapy, exercise modeling, and in vivo physiology, this K08 award will equip me to launch an independent research career focused on understanding and treating inherited cardiac diseases, potentially improving patient lives by informing exercise recommendations and paving the way for new therapeutics.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Natural killer (NK) cells are innate lymphocytes that regulate multiple components of the innate and adaptive immune systems and have direct cytotoxic function against pathogen-infected cells and cancer cells. NK cells complement T cell immune surveillance and can mediate an important graft-versus-leukemia effect following allogeneic hematopoietic stem cell transplantation for acute leukemia. During pregnancy, maternal NK cells are unique mediators in the carefully orchestrated processes of implantation, placentation, and fetal growth; and they are also poised to rapidly respond to infection. In these and other settings, NK cells rely on their expression of inhibitory and activating cell surface receptors to sense their environment and communicate with other cells. Killer immunoglobulin-like receptors (KIRs) comprise a large family of receptors that bind human leukocyte antigen (HLA) molecules and enable NK cells to distinguish healthy from diseased cells. Despite their central importance to NK cell function, it is not yet known how KIR expression is regulated during NK cell development. For over 20 years we have worked to identify and characterize NK cell developmental intermediates (NKDIs) in human tissues and to determine how their differentiation and maturation are regulated. Although KIR acquisition occurs during late stages of NK cell maturation, we now have preliminary data to indicate that prior to this developmental window, early stage NKDIs require a conducive microenvironment to become primed for subsequent development into KIR+ NK cells. Moreover, our findings have uncovered a paradoxical inhibitory role of the NK cell growth factor, interleukin (IL)-15, during the priming phase of human NK cell development, thus challenging current dogma and raising important clinical implications for NK cell mediated therapies. The central goal of this proposal is to elucidate the mechanisms regulating KIR acquisition during human NK cell development. Our specific aims are: 1) To determine how the microenvironment regulates NKDI priming for KIR acquisition; and 2) To elucidate cell intrinsic mechanisms of NKDI priming and its suppression by IL-15. We hypothesize that specific hematopoietic helper cells support NKDI priming by triggering activating receptors through a critical developmental synapse, in turn leading to MEK/ERK/AP-1 signaling and subsequent induction of sense transcription at KIR gene loci. Further, we hypothesize that these critical priming steps are abrogated in the setting of early-stage exposure to IL-15, at least in part through dysregulated STAT5- and mTOR-dependent sense and antisense expression at KIR promoters. In Aim 1 we propose a series of experiments to determine how soluble factors and cell-cell interactions in the microenvironment mediate NKDI priming. In Aim 2 we will investigate NKDI cell intrinsic mechanisms to determine how priming affects KIR promoter expression and how IL-15 subverts NK cell priming and subsequent KIR acquisition. Our overarching goal is to gain a comprehensive understanding of the processes that regulate KIR acquisition during human NK cell development to best understand how NK cell functions can be enhanced in the face of human disease.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

PROJECT SUMMARY Fibrosis and fatty infiltration impair skeletal muscle's regenerative properties and function in traumatic injuries and chronic diseases, and thus there is an urgent need for innovative therapies to restore muscle structure and function. Such muscle degenerative hallmarks are driven by muscle-resident mesenchymal stem cells, also known as fibro-adipogenic progenitors (FAPs). While these cells typically contribute to muscle regeneration by supporting myogenesis through proliferation, secretion of pro-myogenic paracrine factors, and deposition of matrix before apoptotic clearance, they can become resistant to apoptosis in chronic injuries, before differentiating into myofibroblasts and adipocytes. However, extrinsic factors controlling FAPs’ response to muscle injury are not well understood. One such factor may be the biophysical cues during muscle healing. While matrix stiffness regulates mesenchymal stromal cell fate, how FAPs sense the changing matrix stiffness and drive their function in muscle regeneration remains unknown. The mechanism of sensation may be through PIEZO1, a mechanosensitive ion channel. PIEZO1 has been shown to be present in many cells across different tissues, including cells found within muscle, and responds to a range of mechanical cues, especially substrate stiffness. However, it has not yet been identified in FAPs, nor the role PIEZO1 plays on activation. This project will elucidate how FAPs sense the mechanical changes in their environment and downstream processes of PIEZO1 activation. To address this gap, the role of PIEZO1 in mechanosensation in FAPs during muscle regeneration in vivo will be identified in Aim 1. Following conditional knockout of PIEZO1 in FAPs, the muscles will then be injured with barium chloride injection. Muscles without PIEZO1 will show worsened muscle regeneration and increased pathology. Furthermore, matrix stiffening will be prevented following muscle injury to assess the role of mechanosensation in activating PIEZO1 in vivo. Here, muscles with impaired matrix stiffening will behave similarly to those without PIEZO1, showing signs of impaired muscle regeneration and increased pathology, further implicating the importance of PIEZO1 in matrix stiffness sensation and healthy muscle regeneration. Aim 2 seeks to isolate the mechanism of PIEZO1 activation in FAPs and identify downstream effects of activation. Using engineered hydrogels to match stiffnesses that are seen during muscle regeneration, I expect stiffer hydrogels to cause increased PIEZO1 activation. Furthermore, any mediators that are downstream of PIEZO1 will be identified, with a focus on mechanotransductive genes. This project will frame the research within a clinical context and provide a multi-disciplinary training in tissue engineering and stem cell biology to build my career as a future physician-scientist as well as providing therapeutic targets to prevent the development and progression of muscle degeneration following injury.

NINDS - National Institute of Neurological Disorders and Stroke

Summary Mutations in the X-linked gene encoding methyl CpG-binding protein 2 (MeCP2) are associated with neurological diseases ranging from impaired intellectual disability, microcephaly, autism, and Rett (RTT) syndrome. MeCP2 is well studied for binding cytosine methylated DNA through its methyl-binding domain (MBD) and interaction with nucleosomes and chromatin remodeling proteins through the NCoR/SMRT interaction domain (NID) and transcriptional repression domain (TRD) to regulate gene transcription. Numerous mutations in these domains are found in patients, highlighting their functional importance. A small highly conserved intervening domain (ID) is directly adjacent to the MBD but the function of the ID is poorly characterized. Basic amino acids in the ID and two other AT-hook domains in the TRD are thought to bind RNA but knowledge is sparse, not quantitative, and the contribution of the ID largely uncharacterized. Our preliminary data provides evidence that the ID is predominant in binding RNA with high nM affinity. As mutations in the ID are mostly classified as variants of unknown significance, we modeled a set of patient mutations and show decreased RNA binding. As the MBD-DNA binding domain and ID-RNA binding domain are directly adjacent we tested their interaction and show that RNA can compete MeCP2 off DNA. To gain in vivo insight into ID function, we deleted the ID in human stem cells and studied the effect of ID deletion on differentiation to neurons and cortical organoids. We observe phenotypic changes in ∆ID cells suggestive of disrupted differentiation. Based on our new findings we propose the following hypothesis: the MeCP2 ID binds RNAs to modulate MeCP2 genome occupancy and thus fine-tunes gene expression to allow robust cell fate transitions during brain development. We will test this hypothesis in three aims. Aim 1 uses biochemical, biophysical and live imaging approaches to examine the affinity and kinetics of the ID in binding RNAs and in displacing MeCP2 from DNA as well as the functionality of patient ID variants in RNA binding and MeCP2-DNA competition. Aim 2 will examine the function of the MeCP2 ID during brain development by creating ID variants in human neurons, organoids and mouse for phenotypic studies and to test the hypothesis that the ID serves to modulate gene expression patterns to allow robust transitions in cell fate. Aim 3 will investigate the ID as a modulator of gene regulation and MeCP2-DNA occupancy in human neurons. Finally, we also examine the functionality of the MeCP2 mini-gene to determine whether it recapitulates normal ID functions of RNA binding and modulation of DNA binding. This is critical and timely as the mini-gene is being used in one-dose clinical trials. Altogether our studies will look beyond the canonical roles of MeCP2 to provide a more in-depth analysis of how the ID and its RNA binding affects MeCP2 molecular and biological functions.

NCI - National Cancer Institute

PROJECT SUMMARY Pediatric allogeneic hematopoietic stem cell transplant (PAHCT) is an intensive and lengthy inpatient treatment followed by an exceedingly complex and frequently changing outpatient medication regimen. Caregivers assume responsibility for this complex medication regimen during the transition from inpatient to outpatient care making adherence extraordinarily challenging. Indeed, non-adherence to critical medications is common in the first months following discharge and places patients at risk for life-threatening graft-versus-host-disease (GVHD). Recognizing the need for better adherence-focused care, our multidisciplinary team of PAHCT experts developed MedMemos, a novel 4-session tailored adherence promotion intervention comprised of 2 inpatient and 2 outpatient sessions. MedMemos will provide caregivers with a combination of tailored and standardized medication and adherence education videos and a “Medication Management Kit” that provides tailored strategies to target identified barriers to successful medication management. Informed by the ORBIT model, the goals of this study are to 1) iteratively refine the content, timing, dosing, and delivery of MedMemos with patients, caregivers, and providers (Phase Ib) and 2) establish behavioral (adherence) proof-of-concept (Phase IIa). To achieve the first goal, MedMemos will be delivered to cohorts of 5 caregiver/patient dyads and refined based on participant and provider feedback using a mixed-methods, rapid cycle testing design (Aim 1). After completing all 4 MedMemos sessions, caregivers, and patients ≥8 years-old will provide feedback via acceptability, feasibility, and usability measures and brief structured interview to inform the refinement of MedMemos content and procedures. Following each revision, 10 providers will provide their perspective of the acceptability, feasibility, and usability of the revised MedMemos. MedMemos will then be delivered to the next cohort and evaluated until no feasible changes are indicated by participants or providers. To achieve the second study goal, the final MedMemos version will be administered to 10 caregiver/patient dyads in a quasi- experimental, within-subjects design (Aim 2). Acceptability will be demonstrated by caregivers rating MedMemos a mean of ≥ 4 as measured by the intervention acceptability questionnaires (H1). Feasibility of the refined MedMemos program will be assessed by calculating enrollment (≥80% enrollment rate), data completion (≥80% completion of all follow-up assessments), and intervention fidelity (≥80% of fidelity) and completion rates (>80% will attend all MedMemos Sessions) (H2). Finally, proof-of-concept will be demonstrated by ≥75% children taking ≥75% of their immunosuppressant doses at all follow-up time points (i.e., 1-, 2-, & 3-months post PAHCT discharge) as measured by electronic monitor (H3). The proposed study will lay important groundwork for a fully powered randomized clinical trial to test the efficacy of MedMemos. MedMemos will be developed to be a clinically integrated adherence promotion intervention that optimizes medication adherence and minimizes preventable adverse outcomes in children who receive a PAHCT.

NCI - National Cancer Institute

Ewing sarcoma (EwS) is an aggressive bone tumor that is driven by the oncogenic fusion transcription factor (TF) EWS::FLI1. Despite a high rate of treatment response, a quarter of patients with localized tumors and almost all patients with metastatic disease relapse at distant sites months or years after entering clinical remission. Thus, subclinical disseminated tumor cells (DTCs) are a major source of recurrence and novel treatment strategies are needed to prevent outgrowth of these residual cells into macroscopic disease. The transcriptional regulator and scaffolding protein menin is highly constitutively expressed by EwS, and our preliminary studies have established that depletion of menin from EwS tumor cells inhibits their capacity to successfully colonize distant sites. Our data also indicate that this is due to a reliance on menin to maintain tumor stemness and transcriptionally regulate cell plasticity downstream of TGFβ. The best studied function of menin in transcriptional regulation is as a binding partner of MLL, where it enables epigenetic activation of gene promoters through deposition of H3K4me3. Interactions between menin and MLL-fusion proteins are required for leukemogenesis in MLL-rearranged leukemia and inhibitors of these interactions are showing promise in clinical trials. Unlike in leukemia, our data suggest that in EwS, menin-mediated control of stemness and cell state is achieved, in part, independently of MLL and H3K4me3 and is instead dependent on menin interactions with other TFs at intragenic and intergenic enhancers. Prior studies from our group demonstrated that TGFβ induces EwS cells to activate gene programs that promote acquisition of more mesenchymal states. Significantly, our preliminary data implicate menin in moderating the TGFβ -dependent transcriptional response. It is the goal of this proposal to test the innovative hypothesis that menin is a master regulator of EwS cell stemness, plasticity, and TGFβ - induced cell state transitions. We will also investigate if menin inhibition impedes colonization of EwS DTCs by inducing tumor cell dormancy. We will use a combination of dTag protein degrader technology, transcriptomic and epigenomic profiling, and in vivo studies of EwS colonization to determine: (i) if menin promotes tumor stemness by amplifying transcription of SOX2 and MYC target genes; (ii) how menin regulates TGFβ -dependent cell plasticity; and (iii) if high menin activity prevents EwS cells from entering a dormant state in metastatic niches. Together these studies will define the molecular mechanisms by which menin promotes EwS metastasis. Elucidation of MLL-dependent and -independent functions will illuminate if and how menin:MLL interaction inhibitors could be immediately repurposed, or if menin could be otherwise targeted to benefit patients who are at risk of metastatic relapse.

NIAID - National Institute of Allergy and Infectious Diseases

Bacterial vaginosis (BV) is a highly prevalent polymicrobial vaginal condition characterized by the overgrowth of anaerobic bacteria such as Gardnerella. Affecting millions of people annually, BV is strongly associated with increased risk of sexually transmitted infections and serious reproductive sequelae. Despite its incidence and clinical burden, BV remains notoriously challenging to treat, with standard metronidazole therapy failing in 50% of patients and recurrence rates approaching 80% within a year. However, the current treatment guidelines overlook dynamic shifts in vaginal physiology, particularly those driven by menstruation and host immune activation, highlighting a critical gap in our understanding of therapeutic failure. Menstruation introduces transient but predictable changes to the vaginal environment, including increased bioavailable iron concentrations, epithelial turnover, and immune activation, that collectively reshape host–microbe interactions. Preliminary data demonstrate that total bacterial load decreases and the BV-associated taxa present upregulate redox enzyme genes (e.g., nitroreductase, ferredoxin) required for metronidazole activation during menses. Concurrently, menstrual bleeding results in a proinflammatory immune response, yet the cause and relevance to antibiotic efficacy remain unclear. This proposal will define menstruation as a critical window that can be leveraged to improve BV therapy outcomes. Aim 1a will characterize the temporal transcriptional dynamics of the host and vaginal microbiome across the menstrual cycle using high-resolution, longitudinal metatranscriptomic data. Utilizing the same well-characterized cohort, Aim 1b will investigate host immune responses resulting from changes in the host and microbiome triggered by menses. These data will reveal how host and bacterial function interact across the menstrual cycle, clarify why pro-inflammatory immune markers increase during menstruation, and determine the role of the vaginal microenvironment in BV treatment when it is administered during menstruation. Aim 2 will evaluate the therapeutic efficacy of menstruation-timed metronidazole delivery using a 3D organotypic vaginal epithelial model colonized with clinically relevant microbial consortia. The model will simulate menstrual conditions via heme supplementation and assess bacterial clearance, host immune responses, and host-microbial gene expression under temporally distinct regimens. The significance of this work lies in its potential to transform BV treatment paradigms by introducing a biologically synchronized, temporally informed strategy to enhance antimicrobial efficacy and microbiome restoration. The innovation stems from integrating host physiological rhythms with microbial therapeutics—an intersection largely unexplored in BV research. By repurposing FDA-approved agents and aligning their use with endogenous biological cycles, this project offers a highly translatable framework for clinical implementation. This F32 proposal will provide robust training in human immunobiology, epidemiologic methods, and in vitro tissue modeling, building toward a career focused on translational approaches to improve reproductive health.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

SUMMARY: Gastrulation is a pivotal event in early development, establishing the body plan and shaping future tissues. Even slight alterations in this process can lead to embryonic or fetal lethality, or developmental defects. While traditionally viewed as a passive energy source, recent studies—including our own—reveal that glucose metabolism instructively regulates development. Our findings in mouse embryos show that co-developing epiblast and mesoderm cells rely on distinct branches of glucose metabolism to drive cell fate transitions and subsequent cell movements. Further, we identified specific metabolic intermediates that are selectively required to instruct distinct developmental outcomes, by modulating FGF/ERK signaling. In this proposal, we will combine multi-omics approaches in mouse embryos, embryo-derived tissue explants and in vitro stem cell-based embryo models to uncover how glucose, as a single nutrient, spatially coordinates signaling networks, protein function, and gene expression to drive lineage-specific fate decisions (Aim 1) and morphogenetic behaviors (Aim 2) by generating distinct metabolic intermediates that regulate ERK signaling during mammalian gastrulation. In Aim 1, we perform cell type-resolved isotope tracing and employ 3D high-resolution two-photon live imaging in transgenic reporter mouse embryos to simultaneously track cellular metabolic states and ERK signaling activity in embryonic domains. Building on our preliminary results, we will test the hypothesis that glycosylation via the Hexosamine Biosynthetic Pathway (HBP) acts as a key metabolic mechanism linking glucose flux to ERK activation during the epiblast-to-mesoderm transition. Using proteomics assays and genetic perturbations, we will determine how HBP-driven glycosylation regulates ERK-dependent mesoderm specification. In Aim 2, guided by our preliminary results, we will establish a direct causal relationship between localized lactate production and ERK functionality in mesodermal migration and subsequent developmental progression. We will analyze how glycolysis-driven lactylation regulates key transcription factor and signaling proteins during mesodermal development, employing integrative genomic, proteomic, and functional analyses. These studies will reveal how spatially regulated glucose metabolism shapes developmental trajectories at the intersection of metabolic and signaling networks. By completion of this study, we expect to discover key metabolic mechanisms that instruct local and global embryo morphogenesis and patterning during gastrulation, and the consequences on early developmental patterning when these processes go awry. The advances will provide insights into how progenitor-level defects induced by metabolite availability may cause pregnancy loss and developmental disorders in humans.

Michigan Department of Education

STEM education initiative grants for MI to expand science, technology, engineering, and mathematics programs in K-12 and postsecondary institutions.

NIDCR - National Institute of Dental and Craniofacial Research

SUMMARY Temporomandibular joint (TMJ) osteoarthritis (OA) is characterized by degeneration of the condylar cartilage. No effective therapies exist for restoring the damaged cartilage to accommodate pediatric patients' craniomaxillofacial growth. Regenerative therapies are needed to repair TMJ cartilage. Microfracture is a technique where perforations are created through the subchondral bone to allow for bone marrow mesenchymal stem cell (MSC) infiltration to create fibrocartilaginous repair tissue. However, MSCs are difficult to localize to the damaged area without a scaffold, and their phenotype is prone to calcification. Matrix-autologous chondrocyte implantation (MACI) is the most effective knee repair approach, where autologous knee chondrocytes are seeded on a collagen scaffold and then implanted in the defect. However, MACI is costly, requires two invasive procedures, and may not be applicable to the narrow TMJ for chondrocyte extraction. Additionally, the collagenous scaffolds that are used for knee repairs do not recapitulate the native TMJ extracellular matrix environment. The objectives of this F32 training grant are to develop regenerative approaches to heal damaged TMJ cartilage for pediatric patients and train me in these approaches to complement my tissue engineering background. Using fibro-elastic cartilage of porcine meniscus, we will use our Meniscal Decellularized (MEND) scaffold to help localize and inform progenitor cells, such as ear cartilage progenitor cells (eCPCs) or MSCs. We hypothesize that regenerative therapy adapting microfracture and MACI/matrix-induced chondrogenesis can be used to repair the damaged TMJ condylar cartilage. We will first compare the in vitro chondrogenic potential and phenotypic stability of eCPCs and MSCs in MEND. Outcomes will include biochemical assays, mechanical testing, immunohistochemistry, histology, and gene expression. Then, to assess in vivo phenotypic stability of the constructs, we will subcutaneously implant cell-seeded MEND in immunocompromised mice and analyze outcomes via biochemical assays, immunohistochemistry, histology, and gene expression. Next, we will compare the repair of adapted microfracture (simulated by an MSC injection) and MACI (simulated by empty or eCPC MEND) for porcine and human TMJ condylar cartilage regeneration in vivo using the semi-orthotopic mouse model and analyze outcomes via biochemical assays, immunohistochemistry, histology, and gene expression. These studies will uncover whether microfracture regenerates TMJ condylar cartilage and if a scaffold, potentially cellular, is needed to improve condylar regeneration. Results will inform development of regenerative therapies for degenerated TMJ condylar cartilage and produce preliminary data for a K99 application to support my training as an independent researcher in the TMJ oral and craniofacial research field.

U.S. National Science Foundation

NSF-supported science and engineering research increasingly relies on cutting-edge infrastructure. With its Major Research Instrumentation (MRI) program and Major Multi-user Facilities ("Major Facilities") projects, NSF supports infrastructure projects at the lower and higher range of infrastructure project costs, Foundation-wide, across science and engineering research disciplines. The Foundation-wide Mid-scale Research Infrastructure opportunity is intended to provide NSF with an agile, Foundation-wide process to fund experimental research capabilities in the mid-scale range between MRI and Major Multi-user Facilities. NSF defines Research Infrastructure (RI) as any combination of facilities, equipment, instrumentation, or computational hardware or software, and the necessary human capital in support of the same. Major facilities and mid-scale projects are subsets of research infrastructure. The NSF Mid-scale Research Infrastructure-1 Program (Mid-scale RI-1) supports either design activities or implementation of unique and compelling RI projects. Mid-scale implementation projects may include any combination of equipment, instrumentation, cyberinfrastructure, broadly used large scale datasets and the personnel needed to successfully commission the project. Mid-scale RI-1 design activities include the design efforts intended to lead to eventual implementation of a mid-scale class RI project. Mid-scale RI-1 projects should involve the training of a diverse workforce engaged in the design and implementation of STEM research infrastructure. Mid-scale RI-1 projects should directly enable advances in any of the research domains supported by NSF. Projects may also include upgrades to existing research infrastructure. Mid-scale RI-1 emphasizes strong scientific merit, a response to an identified need of the research community and/or fulfillment of a national need to enable U.S. researchers to be competitive in a global research environment. Well-conceived technical and management plans are essential for both design and implementation proposals, as are well-developed plans (e.g., mentoring and professional development) for student training and the involvement of a diverse STEM workforce in all aspects of mid-scale design and/or implementation activities. The inclusion of individual project participants that will lead to a supportive working environment is especially encouraged at all levels of the project team. Within Mid-scale RI-1, proposers may submit two types of projects, Implementation (e.g., acquisition and/or construction) or Design . The Design track is intended to facilitate progress toward readiness for a mid-scale range implementation project. Both Implementation projects and Design activities may involve new or upgraded research infrastructure. Mid-scale RI-1 "Implementation" projects may have a total project cost ranging from $4 million up to but not including $20 million. Mid-scale RI-1 "Design" activities may request less than $4 million, with a minimum request of $400,000 and a maximum request up to but not including $20 million, as appropriate, to prepare for a future mid-scale range implementation project. Note: Successful award of a Mid-scale RI-1 design activity does not imply NSF's commitment to the future implementation of the project being designed, nor is a Mid-scale RI-1 design award required for the submission of an implementation project. The Mid-scale RI-1 Program seeks to broaden the representation of PIs and institutions in its award portfolio, including a geographically diverse set of institutions (especially those in EPSCoR jurisdictions). Proposals submitted by, or involving partnerships between institutions are encouraged. Participation in this opportunity is encouraged for the full spectrum of diverse talent society has to offer to include PIs who are women, early-career researchers, persons with disabilities, or members of other groups underrepresented in STEM. To improve participation in science and engineering research for persons with disabilities, Mid-scale RI-1 encourages PIs to incorporate accessibility as part of Mid-scale RI-1 design activity and implementation projects. Please consult NSF's Research Infrastructure Guide, or RIG (available at https://www.nsf.gov/bfa/lfo/lfo_documents.jsp), for definitions of certain terms used in this solicitation, such as the Project Execution Plan (PEP) and Design and Execution Plan (DEP). The RIG provides guidance specific to Mid-scale Research Infrastructure Projects, including references to other parts of the RIG as needed. Note that PEP or DEP should be appropriately scaled for the complexity of the project and may not require all of the elements described in the RIG. Mid-scale research infrastructure projects with total project costs beyond the Mid-scale RI-1 Program limit are separately solicited through the Mid-scale RI-2 Program. Proposals to the Mid-scale RI-1 Program with total project costs outside of this solicitation's budgetary limits, either during initial submission or after cost analyses/revisions during subsequent review, are subject to return without further review.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

PROJECT SUMMARY The Midwest Zebrafish Meeting (MWZM) is a biennial scientific conference that brings together researchers using the zebrafish model to study fundamental questions in developmental biology, reproduction, genetics, and disease. The meeting serves as a key regional hub for fostering collaboration, technical training, and early- career development in the zebrafish research community across the Midwest, with participation from researchers nationwide. This year, MWZM will be hosted at the Van Andel Research Institute in Grand Rapids, Michigan. Grand Rapids is centrally located in the Midwest, and easily accessible via plane, train, or automobile. The Van Andel Research Institute (VARI) is well equipped to host a meeting of this size (approximately 175 attendees), with an auditorium and break-out rooms, AV specialists, and Events Team support on site. VARI is located within walking distance of hotels and restaurants. We have arranged for discounted hotel blocks at two neighboring hotels. Zebrafish are a powerful vertebrate model organism uniquely suited for real-time imaging, high-throughput genetic manipulation, and modeling of human developmental disorders. The MWZM will highlight zebrafish research in areas such as visualizing development, tissue patterning, neural development and regeneration, germ and stem cell biology, cardiovascular and hematopoietic development, and disease modeling. This R13 application requests support to enhance the MWZM’s ability to advance research and training in the areas of development and disease, especially neurobiology and cardiovascular biology. The meeting will feature keynote lectures, invited and contributed talks, poster sessions, and technical workshops that highlight zebrafish-based discoveries from researchers in the Midwest. Special emphasis will be placed on providing early-career researchers with opportunities to present their work and participate in professional development sessions. R13 support will ensure the continued success and accessibility of the meeting by helping to offset operational costs, providing travel assistance to trainees, and disseminating educational resources beyond the conference itself. Through this support, MWZM will continue to promote scientific excellence, accelerate biomedical discovery, and strengthen the workforce.

Minnesota Department of Education / SciMathMN

STEM education initiative grants for MN to expand science, technology, engineering, and mathematics programs in K-12 and postsecondary institutions.

National Aeronautics and Space Administration

The Minority University Research and Education Project (MUREP) Space Technology Artemis Research (M-STAR) opportunity is a National Aeronautics and Space Administration (NASA) initiative through its Office of STEM Engagement (OSTEM). It is designed to strengthen the role of Historically Black Colleges and Universities (HBCUs), Tribal Colleges and Universities (TCUs) and Minority Serving Institutions (MSIs) in the future of space exploration.

U.S. Mission to Spain

The Public Diplomacy Section (PD Spain) of the U.S. Embassy Madrid and U.S. Consulate General Barcelona welcomes proposals for creative, engaging projects that line up with PD Spain s main objectives. That includes: Promote citizen participation in the fight against the climate crisis and facilitate better knowledge of the policies and actions of the United States in this area. Ensure that Spanish and /or Andorran students know the United States as a destination for their training, for summer work programs and for learning English. Promote security and defense alliances between the United States and Spain and the Atlantic Alliance (NATO). Communicate the importance of Spain being the venue for the 2022 NATO Summit, the role that Spain plays within NATO and the strategic concept of NATO in the near future. Explain the value of initiatives dedicated to women, peace and security. Support the areas of entrepreneurship, innovation and STEM to increase economic opportunities for young people in Spain and / or Andorra. Increase knowledge about how to do business in the US and highlight the role of Spain and Andorra in the global digital economy. Expand the reach of media literacy and support the media education programs of Spanish institutions with useful and accurate tools to increase understanding of false information and other tactics, to render misinformation campaigns targeting Spain ineffective. Communicate about the common values that the United States, Spain and the EU share and about the promotion of respect for human rights, democracy and the need for democratic changes in places like Venezuela, Cuba and Nicaragua, the power of the law against impunity, privacy, international order based on common rules, and a fair playing field. Encourage collaboration between Spanish and /or Andorran and American organizations that share the defense of human rights. Explore topics such as the rights of LGBTQI + community, racism, sexism, and the rights of people with disabilities. Promote the rights and equality of women, ethnic and religious minorities, the LGBTQI + community, refugees and migrants, people with disabilities and other marginalized populations in Spain and / or Andorra. All programs should ensure they promote diversity and inclusion. Please be aware that projects funded through this APS must include an American element. That could involve a connection or partnership between Spanish and/or Andorran and American organizations or institutions. For example, an American expert could take part, in person or virtually, in your project. Activities might highlight or examine shared values between Spain and/or Andorra and the United States, national interests, etc. You may incorporate a U.S. approach or method you have learned about to addressing an issue or challenge facing your community, institution, or profession. Grant activities may take any number of forms, including academic competitions, cross-border exchanges, conferences, workshops, courses, curriculum development, exhibits, hackathons or app development, online projects, mock trials or moot court competitions, simulations and role-playing activities (e.g., Model Congress, Model United Nations), performances, or other activities. Project timelines should start no earlier than December 1, 2021, and start no later than September 30, 2022, with all activities being completed no later than December 2023. All activities and your evaluation or assessment of the project should be completed within 18 months of starting the project.

Missouri Department of Elementary and Secondary Education

STEM education initiative grants for MO to expand science, technology, engineering, and mathematics programs in K-12 and postsecondary institutions.

OD - NIH Office of the Director

ABSTRACT The radiation tolerance of the gastrointestinal (GI) tract can be exceeded in radiation accidents or in the case of nuclear warfare, resulting in a lethal GI acute radiation syndrome (GI-ARS). There are no current FDA- approved medical countermeasures to mitigate GI-ARS; thus, there is an urgent need to identify key mechanisms of repair and regeneration in the irradiated intestinal epithelium. We have shown that the regeneration of the intestinal epithelium in response to severe radiation injury is mediated through revival stem cells (revSCs), which are generated through fetal-like reprogramming and identified by a marker gene Clusterin (Clu) (the mouse ortholog of human CLU). Remarkably, whole animal deletion of Clu significantly sensitizes mice to GI-ARS, while GI-ARS in mice is significantly mitigated via treatment with exogenous mouse Clu protein starting 24 hours post-irradiation. In addition, our preliminary data suggest that the cGAS-STING pathway acts as an upstream regulator of Clu in the irradiated intestinal epithelium. Treatment of mice with a STING agonist MSA-2 starting 24 hours after irradiation significantly improves the regeneration of irradiated small intestines. Thus, the overall goal of this proposal is to develop novel mitigators of the GI-ARS that promote the regeneration of irradiated intestinal epithelium by targeting the STING-CLU axis. In Aim 1, we will define the mechanisms by which CLU mitigates the development of GI-ARS. In Aim 2, we will dissect the STING-CLU axis in regulating the development of GI-ARS. Successful completion of the proposed study will demonstrate the key role of CLU in promoting the regeneration of the intestinal epithelium following acute radiation injury in mice and human intestinal organoids. We anticipate that our findings will significantly impact the successful development of GI-ARS mitigators that target the STING-CLU axis.

NEI - National Eye Institute

PROJECT SUMMARY Age-related macular degeneration (AMD) is the leading cause of irreversible blindness involving gradual dysfunction and degeneration of photoreceptors and the retinal pigment epithelium (RPE). Mitochondrial dysfunction represents a hallmark of AMD. Excessive and persistent reactive oxygen species (ROS) damages mitochondrial DNA (mtDNA), resulting in fewer mitochondria. Impairments in respiratory chain activity and reduced oxidative phosphorylation (OXPHOS) impacts ATP synthesis and further drives ROS production. Our prior work has shown that exogenous mitochondrial transplantation to cells can induce a bioenergetic shift towards increased OXPHOS and ATP production, and reduced ROS. Mitochondrial cell-to-cell transfer in response to stress rescues aerobic respiration to reduce deleterious cell dynamics. We aim to create a sustainable source of mitochondria proximal to the RPE in the form of mesenchymal stem cells (MSCs) transfected with nuclear respiratory factor 1 (NRF1), a driver of mitochondrial biogenesis, for enhanced cell-to- cell transfer of mitochondria. Our objectives are to determine how NRF1 transfection changes MSC transcriptional activity, mitochondrial production and trafficking, and extracellular vesicle (EV) contents, and test the strategy in two rodent models of retinal degeneration. We hypothesize that our cell therapy using NRF1- overexpressing MSCs will not only provide the needed energetic boost via mitochondrial mass transfer to retinal cells, but also induce profound cellular reprogramming through the secretion of EV-containing factors, thereby counteracting and potentially reversing the onset of late-stage dry AMD. We will use a combination of molecular and biochemical methods, including transcriptomics, to characterize the potency of mitochondrial transfer to impact AMD. We will first investigate the impact of NRF1 on mitochondrial transfer mechanisms in MSCs. We will elucidate the role of NRF1 on mitochondrial transfer mechanisms, specifically tunneling nanotubes (TNTs) and EVs, between MSCs and human RPE cells from donor eyes afflicted with AMD, shedding light on NRF1- primed MSCs as mitochondria generators, responsive to signals from stressed cells. We will then examine the influence of NRF1 on MSC senescence and EV composition, providing insights into the multifaceted benefits of NRF1 in MSCs, including metabolic adaptation to AMD stress environments and modulation of EV constituents. Lastly, we will evaluate the impact of NRF1 primed MSC transplantation in vivo. Through longitudinal and long- term efficacy studies and tissue transcriptomic analysis, we will identify disease stages most responsive to NRF1 MSCs when transplanted subretinally in a sodium iodate (NaIO3)-induced model of retinal degeneration and senescence accelerated mouse (SAMP8) model, highlighting the potential to rescue RPE cell degeneration and abrogate AMD progression. Findings from this work will provide crucial insights into the potential of NRF1-based therapies for AMD, offering a promising avenue for the treatment and management of this debilitating disease.

NINDS - National Institute of Neurological Disorders and Stroke

Repeat-expansion disorders myotonic dystrophy 1 and 2 (DM1, DM2) produce disabling cognitive and behavioral deficits, yet the molecular drivers of central-nervous-system (CNS) pathology remain undefined. DM1 is caused by expanded CTG repeats in the dystrophia-myotonica protein kinase (DMPK) gene that produces toxic CUG RNAs, whereas DM2 is caused by expanded CCTG repeats in the cellular nucleic-acid–binding protein (CNBP) gene that yield toxic CCUG RNAs. These expanded RNAs sequester the splicing regulator muscleblind-like protein 1 (MBNL1), disrupt microtubule-associated protein tau (MAPT) processing, and may provoke excitotoxicity via hyperactive CUG-binding protein ELAV-like family 2 (CELF2). The long-term objective is to enable CNS-directed therapies for myotonic dystrophy. The central hypothesis is that repeat-expansion RNA toxicity mis-splices MAPT and activates CELF2-mediated glutamatergic hyperexcitability, jointly driving tauopathy and neuronal loss. This project will delineate convergent and divergent mechanisms of cortical dysfunction in DM1 and DM2 using human induced-pluripotent-stem-cell (iPSC) cortical organoids produced by an optimized protocol validated in more than sixty lines. Organoids derived from this protocol retain physiological DMPK1, CNBP, and MBNL1 expression overcoming the low expression of these genes observed in other organoid models. The optimized protocol will be applied to DM1 iPSC lines (238–1,600 CTG repeats), DM2 lines (8.8–11.9 kb CCTG repeats), and healthy control lines to generate side-by-side disease and reference cortical organoids. Aim 1 will track RNA- foci formation, splice defects, and tau aggregation at 2, 4, and 6 months in DM1, DM2, and control organoids, combining long-read RNA-seq with quantitative neuropathology. Aim 2 will test whether hyper-phosphorylated CELF2 drives glutamatergic mis-splicing, network hyperexcitability, and neuronal loss across DM1 and DM2 organoids, and whether inducible shRNA knock-down of CELF2 restores receptor splicing, electrophysiological balance, and tau status relative to controls. High-density multielectrode-array recordings, long-read transcriptomics, and tau biochemistry will integrate molecular and functional endpoints throughout the project. The study is expected to (i) establish mechanistic links between repeat RNA toxicity and tauopathy, (ii) identify CELF2 as a modifiable driver of excitotoxicity, and (iii) deliver a scalable, biomarker-rich organoid platform for therapeutic screening. By clarifying disease pathways and providing human assay systems, the work will accelerate the development of tau-lowering and synapse-stabilizing strategies, directly advancing NIH priorities to translate mechanistic insight into treatments for rare neurodegenerative diseases.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT African American individuals face a disproportionately higher risk of tobacco-related cardiovascular diseases (CVD) than other races, a disparity not fully explained by traditional and socioeconomic risk factors. Despite lacking approval from the U.S. Food and Drug Administration (FDA) for their safety, e-cigarettes (e-cigs) have become increasingly popular, particularly among youth, and are now among the most commonly used tobacco products alongside traditional cigarettes. Approximately half of African American individuals carry at least one of two genetic variants (G1 and G2) of the apolipoprotein L1 (APOL1) gene, which are exceedingly rare in other populations. APOL1 is widely expressed, particularly in the vasculature. We have shown that carriers of APOL1 G1 and G2 variants have increased susceptibility to tobacco-related CVD, including stroke and coronary heart disease. Dysfunction of vascular endothelial cells (ECs) is a critical precursor to CVD. EC dysfunction also plays a key role in APOL1-associated pathology, including exacerbated renal issues and increased susceptibility to sepsis and severe COVID-19. Recent research indicates that, similar to cigarettes, both e-cigs and menthol—a flavor popular in the African American community—independently impair endothelial function. While studies, including those using induced pluripotent stem cell (iPSC)-derived ECs, demonstrate these effects, the specific impact of APOL1 risk variants on vascular health in African American tobacco product users remains unknown. The goal of the proposed research is to determine the effects of e-cigs, with and without menthol, on endothelial health, compare them to the effects of cigarettes, and identify potential molecular markers and pathways associated with CVD in African American users, with a focus on the APOL1 genotype. As such, this application aims to expand my background and expertise in modeling the CVD risk from tobacco products and to provide specific training in tobacco product-related in vitro assays, iPSC methodology, gene editing, and computational techniques. Building on my prior work in human studies, this research extends to the cellular level to address significant gaps in knowledge regarding the adverse effects of the most popular tobacco products, with and without menthol, among the African American population—a demographic long targeted by the tobacco industry marketing. To achieve this goal, I will use a robust in vitro platform of human iPSC-ECs to address the following aims: Aim K1) to determine the effect of e-cigs and cigarettes on markers of EC dysfunction in G1/G1 iPSC-ECs, Aim R1) to determine the effect of e-cigs and cigarettes on endothelial function in G2/G2 iPSC-ECs, and Aim R2) to determine the effect of e-cigs and cigarettes on inflammatory markers and lipid mediators of inflammation in G1/G1 and G2/G2 iPSC-ECs. This project will deepen our understanding of the adverse effects of widely used tobacco products on vascular health in the CVD-burdened African American population. It also aims to identify molecular markers of cardiovascular injury in this high-risk group, providing insights into the mechanisms of tobacco-related cardiovascular damage and supporting the development of targeted interventions.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary The goal of this project is to understand the mechanisms and potential therapeutic targets of a novel inborn error of immunity (IEI) caused by variants in toll like receptor 8 (TLR8). We recently identified genetic variants in TLR8 leading to gain-of-function (GOF) of the encoded protein as the cause of a new IEI presenting with profound neutropenia with recurrent infections, lymphoproliferation, T and B cell abnormalities, and bone marrow failure. Toll-like receptor 8 (TLR8) is an endosomal TLR encoded on the X chromosome that recognizes single-stranded RNA (ssRNA) and is expressed in neutrophils and other myeloid cells, including myeloid progenitors. Relatively little is known about human TLR8, largely because the murine equivalent differs in structure and ability to sense ssRNA. There is no available targeted therapy for patients with TLR8 GOF; however, patients have benefited from hematopoietic stem cell therapy, confirming that disease is driven by the immune system. There is a gap in our understanding of how GOF in TLR8 protein function leads to the clinical features of this newly recognized disease. As TLR8 expression is myeloid-specific and, in most cases of TLR8 GOF disease, expressed in a mosaic fashion (with approximately 10-20% of cells harboring TLR8 variants), we hypothesize that myeloid cells expressing mutant TLR8 produce inflammatory cytokines leading to T cell activation, and this inflammatory state and dysfunctional T cells contribute to the disease in patients including lymphoproliferation and bone marrow failure. Given the differences between mouse and human TLR8, we recently generated transgenic mice that conditionally express either WT or GOF human TLR8. Herein, we will use these novel models to address several gaps in our knowledge of TLR8 biology and TLR8 GOF disease, as well as to test potential therapies. In Aim 1, we will identify mechanisms of TLR8 GOF disease, using our transgenic mouse models and human cell xenografts to determine how TLR8 GOF myeloid cells influence hematopoiesis and T cell development and identify putative disease-driving cytokines. In addition, we will formally test the role of T cells in the disease pathogenesis by crossing TLR8 GOF mice to T-cell deficient mice and determining the effects on hematopoiesis, cytokine production and disease progression. In Aim 2, we will identify therapeutic targets of TLR8 GOF disease using inhibitors of TLR8 signaling and blocking candidate cytokines in our transgenic mice. Together, these studies will lead to an increased understanding of the pathology of TLR8 GOF and will identify therapeutic targets. The long-term goal of this application and our work is to develop a mechanistic understanding of how TLR8 GOF alters the immune response to enable improved therapies for patients.