Grants

Ase Theatre

Our community centers on Black folks of Seattle, connected through the historically Black Central District, a neighborhood of deep significance, even as many have been gentrified out but remain tied to it. In February and March 2025, our Dispersed project will host a workshop series where community members will become performers, guided by teaching artists. By exploring themes of Black identity and storytelling, we will foster discussions about personal and collective experiences, amplifying the stories of Black folks in Seattle as a community and place. The production is a community-driven process, where the audience actively participates. Opening night will feature a community celebration with story circles, and all performances will be free and open to the public. The play serves as a catalyst for community connection through dialogue, promoting healing and unity. It is a community event, created by, for, and within our community.

Labor

The Displaced Homemaker Program serves people who have been displaced from their careers as unpaid homemakers. It offers services to help with barriers to self-sufficiency including: child care, transportation, housing, basic necessities, or employment, emotional challenges, medical needs, and/or legal problems.

NCI - National Cancer Institute

Project Summary/Abstract Glioblastoma, IDH-wildtype (GBM; WHO grade 4), the most malignant primary brain tumor, has well-defined pre-necrotic, necrotic and recurrent growth phases. Transition to the necrotic phase in primary and recurrent states involves a massive restructuring of the tumor microenvironment (TME), in which there is a dramatic increase in tumor- associated macrophages (TAMs) that are polarized to an immunosuppressive state. TAMs emerge from the perivascular niche and aggregate in high density with hypoxic glioma cells within the peri-necrotic niche (PN) with remarkable temporal similarity. We hypothesize that the spatial arrangement of TAMs and glioma cells within the highly hypoxic PN represents a biologic engine that serves as a sustainable source of TAM immunosuppression and is reliant on their synergistic interdependencies. We propose investigations to better understand mechanisms underlying these events using patient- derived, molecularly characterized GBM neurosphere cultures and RCAS-tv-a mouse model that allows study of the evolution of TME events following necrosis in real-time in vivo and with advanced spatial transcriptomics. We focus on specific signaling events within the PN that cause polarization of TAMs and have preliminary data indicating that the hypoxic expression of podoplanin (PDPN) by glioma cells activates CLEC5A on TAMs, causing an immunosuppressive phenotype through Syk/STAT3 signaling. We also suggest that the direct effects of hypoxia and TGFβ1 activate Hippo signaling and PDPN expression within this niche. Pharmacologic inhibition of Syk as a single agent prolongs survival and enhances the presence of CD8+ T cells in the RCAS/tv-a model of GBM. We hypothesize that therapeutic targeting of CLEC5A signaling by using Syk inhibitors in addition to standard of care chemoradiation, with or without checkpoint inhibitors, will diminish immunosuppression, enhance immunity and prolong survival in preclinical models of primary and recurrent GBM.

NIAID - National Institute of Allergy and Infectious Diseases

Project Abstract The global tuberculosis (TB) epidemic is fueled by the airborne transmission of Mycobacterium tuberculosis (Mtb), yet the mechanisms underlying cough-mediated aerosolization and the impact of antibiotic treatment and drug resistance on transmission remain poorly understood. We propose to address these gaps using ex vivo cultured nociceptive neurons and a guinea pig model of human cough to investigate the role of host and bacterial factors in TB transmission and evaluate strategies to disrupt this process. Preliminary data show that Mtb glycolipids, sulfolipid-1 (SL-1) and phenolic glycolipid (PGL), activate nociceptive neurons and drive the cough reflex, a critical driver of Mtb aerosolization and person-to-person transmission. Leveraging our innovative nociceptive neuron activation model and Mtb cough measurement and particle capture systems we developed specifically to study Mtb aerobiology, this project will uncover the drivers of Mtb transmission and explore interventions to mitigate it. In the first aim, we will analyze how biological sex, antibiotic treatment, and drug resistance influence Mtb-induced cough and Mtb aerosol production. By comparing male and female guinea pigs, we will determine the impact of host sex on cough frequency and Mtb aerosol generation. We will also evaluate how standard (rifampin-isoniazide-pyrazinamide; RIP) and newer (bedaquiline-pretomanid-linezolid; BPaL) antibiotic regimens reduce cough and Mtb aerosol production and test whether drug-resistant Mtb strains alter these processes, potentially enhancing transmissibility. The second aim will focus on cough suppression as a host-directed intervention. Using inhibitors of nociceptive neuron pathways, we will evaluate their ability to reduce Mtb-induced neuronal activation, cough and infectious particle production. These studies will test whether blocking the cough reflex has potential to mitigate Mtb transmission.

NIAID - National Institute of Allergy and Infectious Diseases

Project Abstract The emergence of antibiotic resistance in pathogenic bacteria poses an eminent threat to the efficacy of our current antibiotic arsenal. Acinetobacter baumannii (Ab) is the top priority pathogen by the WHO and CDC in the fight against antibiotic resistance. Minocycline (MIN), a tetracycline class antibiotic, is one of the most effective antibiotics for treating Ab infections in patients. Unfortunately, MIN resistance is spreading internationally and has begun to emerge in the United States. The greater utility of MIN over other tetracycline class antibiotics lies in structural differences, which render common tetracycline efflux pumps ineffective. While efflux pumps are correlated with MIN resistant Ab, clinical data suggests alternative mechanisms of MIN resistance. To explore the genetic basis for MIN resistance in Ab we employed a machine learning model to predict genetic resistance correlates from clinical isolates. Mutations in ruvB, a DNA repair protein, were strongly correlated with MIN resistant clinical strains of Ab. Mutations in ruvB have not previously been associated with MIN resistance. Consistent with the prediction, tn26 insertion in the Ab strain AB5075 ruvB gene, and deletion of ruvB in strain ATCC 19606, increased MIN minimum inhibitory concentrations to a level that exceeds the MIN resistance breakpoint. RuvB complexes with RuvA and RuvC to resolve Holliday junctions during recombination. However, only ruvB mutants showed the resistance phenotype; neither ruvA nor ruvC mutants were MIN resistant, suggesting loss of the complex’s activity was not the basis for resistance. We observed ruvB mutants produced increased biomass during planktonic growth relative to the other two ruv mutants. Upon examination, the ruvB::tn26 mutant had a 451% increase in biomass and 360% thicker biofilms relative to wildtype. Prior studies in Escherichia coli, although not examined in relation to antibiotic resistance or relevance to disease, demonstrated RuvA stabilizes extracellular DNA (eDNA) to promote biofilm formation. Since RuvB binds RuvA to initiate formation of the RuvABC complex, we hypothesize ruvB disruption promotes increased RuvA eDNA binding and stabilization leading to enhanced biofilm formation and MIN resistance in Ab. Our preliminary experiments indicate ruvB mutant cultures have higher DNA content compared to wild type, supporting our hypothesis. Within this proposal we outline a detailed strategy to understand how ruvB mutations 1) mechanistically lead to MIN resistance, and 2) impact Ab disease progression and MIN resistance in vivo. The findings from this proposal will alter paradigms for how MIN resistance evolves in Ab and paves the way for future studies focused on understanding the correlations between ruvB mutations and infectious disease progression within patients. The results will also help inform future novel counter-resistance therapeutic strategies which will seek to preserve the clinical utility of essential drugs, like MIN, for use against the most dangerous pathogens.

NHLBI - National Heart Lung and Blood Institute

Progressive telomere shortening, which occurs over humans' natural lifespan, is a primary molecular cause of the functional decline of stem cells in high-turnover tissues, including hematopoietic stem cells (HSCs). However, how telomere damage compromises HSCs’ functions is largely unknown. Here, we propose investigating the molecular mechanisms behind telomere damage–induced functional HSCs’ decline to uncover therapeutic strategies to ameliorate bone marrow (BM) failure disorders. In preliminary studies, we demonstrated that telomere damage does not activate programs of apoptosis or senescence in HSCs but instead induces their aberrant activation and differentiation towards the megakaryocytic lineage through the cell-intrinsic upregulation of Ifi20x/IFI16-mediated innate immune signaling response, which directly compromises HSCs’ self-renewal capabilities and eventually leads to their exhaustion. Given that HSCs’ exhaustion in the context of BM failure disorders predisposes to clonal selection, we evaluated whether telomere shortening–induced DNA damage in patients with germline mutations affecting telomere maintenance genes who developed telomere biology disorders (TBDs) is associated with clonal hematopoiesis (CH). We studied the architecture, trajectories, and impact of CH in a cohort of 207 TBD patients. CH was rare in asymptomatic patients but present in 46% of symptomatic patients and involved chromosome 1q aberrations (mainly chromosome 1q gain [Chr1q+]) and recurrent mutations in PPM1D, POT1, TERT promoter, and U2AF1S34. Compared with age-matched healthy controls, patients with TBDs had a significantly higher CH frequency, which increased with age. Regardless of allele burden, Chr1q+ and mutations in U2AF1S34 or TP53 increased the risk of developing myelodysplastic syndromes and acute myeloid leukemia. Further functional studies demonstrated that the U2AF1S34 mutation compensated for the aberrant activation of the TP53 and interferon pathways, which contribute to HSC exhaustion in patients with TBDs. These results suggest that the acquisition of U2AF1S34 mutations in TBDs compensates for the restricted cell fitness caused by germline mutations in telomere maintenance genes, which underscores the importance of understanding the molecular mechanisms of U2AF1S34 mutation–induced tumorigenesis. In this proposal, we will use innovative technologies, such as organoid and induced pluripotent stem cell systems and the MISTRG mouse model to 1) Dissect the IFI16-mediated signaling pathway under telomere attrition and determine the feasibility of targeting IFI16 in humans to rescue telomere-dysfunctional HSC function and 2) Dissect the mechanisms of U2AF1S34 mutation–induced tumorigenesis under telomere stress. The proposed study will expand our understanding of the contribution of telomere damage to HSCs’ functional decline and CH and provide new opportunities for developing strategies to improve the prevention and treatment of hematological disorders associated with telomere dysfunction.

NSF

Inflorescences are flower-bearing structures that ultimately produce fruits and grains. In cereal crops such as economically important corn, wheat and rice, inflorescences have complex branching patterns. The number, length and position of branches collectively compose architecture, an agronomic trait that impacts grain yield and harvesting ability. Inflorescence architecture in cereals is controlled in part by the timing in which a developing branch terminates in a grain-bearing unit called a spikelet. While spikelet development is generally conserved across cereals, little is known about the mechanisms that control it at the molecular level. This project aims to dissect the molecular underpinnings of spikelet development and identify control points for precision breeding or engineering high-yielding cereal crops. The proposed work leverages the model cereal Setaria viridis, which is closely related to corn but is small and rapid cycling, making it ideal for lab-based hypothesis testing. It also has a unique inflorescence architecture where spikelets can be converted to sterile branches, and vice versa, through applications of growth hormones. This genetic system enables rapid discovery that can be directly translated to corn and other cereal crops, ultimately impacting crop productivity and food security. The project also provides cross-disciplinary training in advanced molecular and microscopy techniques and computational biology for undergraduates and high school students. A curriculum based on this project will be deployed in high schools and community colleges, from rural to city schools, providing research experiences that link molecular lab skills to concepts in food security and impacting future workforce development. Inflorescence architecture is an important agronomic trait, directly related to grain yield and harvesting ability. Architecture is determined by position and fate of differentiating stem cell populations called meristems. In grasses, different meristem types and variations in their determinacies give rise to diverse, complex branching patterns of the inflorescence. This proposal is aimed at understanding the molecular mechanisms controlling cell fate decisions during inflorescence development and how those decisions determine architecture and grain bearing potential. The work takes advantage of the model grass Setaria viridis because of its unique inflorescence architecture and emerging breadth of genetics and genomics tools. In Setaria spp., inflorescence branches terminate in either a grain-producing spikelet or sterile bristle, and these structures are paired. The loss-of-function bristleless1 mutant is defective in brassinosteroid biosynthesis and fails to initiate a bristle identity program, resulting in homeotic conversion of bristles to spikelets. In contrast, mutations in the spikeletless (spkl) gene convert spikelets to bristles. Spkl encodes a panicoid grass-specific transcription factor and functional ortholog of maize ramosa1 (ra1), a domestication gene that impacts harvestability by conferring meristem determinacy in ears. Despite its importance in evolution and development, the molecular mechanisms surrounding RA1 function in maize and other panicoid cereals is largely unknown. This proposal aims to i) leverage the unique spikelet vs bristle program in Setaria to dissect molecular mechanisms controlling this core genetic module involving RA1 and growth hormones in shaping meristem fate and inflorescence architecture, and ii) extend these findings through comparative approaches to closely related cereal crops maize and sorghum. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract Late endosomes (multivesicular bodies, MVBs) are basic endocytic organelles for protein homeostasis. Rapid MVB formation is essential for the emergence of cellular responses such as nutrient sensing, signal desensitization, and synaptic vesicle recycling. Additionally, MVBs serve as a frontline defense against pathogens and toxins. Their dysfunctions contribute to disorders like tumor cell invasion, neurological diseases and chronic infections. Despite their physiological significance in health and disease, the molecular mechanisms underlying rapid MVB formation remain poorly understood. The challenges arise from 1) MVB formation processes occurring on hundred milliseconds to a few seconds time scale, and 2) the spatial coexistence of MVBs with other endocytic organelles within diffraction-limited spaces. Thus, traditional methods struggle to capture the rapid membrane remodeling process and to visualize specific molecular players involved. I will push the boundaries of our mechanistic understanding of rapid MVB formation by developing new experimental paradigms that integrate time-resolved electron and super-resolution microscopy imaging techniques. My long-term goal is to reveal the millisecond-to-second progression of MVB formation during fastest clathrin-independent synaptic recycling, called ultrafast endocytic pathway, and understand how proteins coordinate rapid membrane remodeling at the structural level. Towards this goal, I will focus on the following two aspects in this study: 1) Capturing the rapid membrane dynamics during MVB formation and identifying the molecular components driving this process. 2) Developing approaches to visualize these proteins at nanometer resolution using electron microscopy and super-resolution imaging, and study how they rapidly remodel membranes to form MVBs. These experiments will lay the foundations for future research into the general mechanisms of rapid MVB formation. Furthermore, they may also lead to novel strategies to dissect infectious diseases and neurodegenerative diseases driven by defective rapid membrane trafficking.

NIDA - National Institute on Drug Abuse

Abstract: Millions of Americans are current or past cocaine users, with 2% of the US population reporting to have used the drug in the past year. Cocaine is highly addictive, and cocaine addiction is devastating to society and individuals, leading to financial loss, job loss and violence within families. Additionally, cocaine abuse is responsible for 40% of drug-related emergency room visits and 1 in every 5 overdoses. Cocaine acts as a monoamine reuptake inhibitor, preventing the dopamine from being transported back into cells after its synaptic release, which leads to prolonged and enhanced dopamine action. Cocaine use especially affects the limbic system, in particular, nucleus accumbens, as well as hippocampus, the amygdala and the prefrontal cortex, leading to changes in activity and long-term rewiring of the circuitry, which causes cocaine craving and addiction. Previous studies identified the major brain regions associated with cocaine addiction, as well as demonstrating that long-term cell- intrinsic molecular changes, such as in epigenetic signaling, underlie the establishment of cocaine addiction. However, the knowledge of how cell type-specific molecular changes resulting from cocaine use lead to rewiring of circuits relevant to cocaine-seeking behavior is currently limited. Moreover, treatments of cocaine addiction that prevent drug seeking via restoring the normal states of neurons and circuits that are dysregulated as the result of repeated cocaine use are desperately needed. We will utilize mice repeatedly exposed to cocaine in a well-established conditioned place preference paradigm to investigate cell type and circuit-specific epigenetic mechanisms underlying cocaine addiction. Using a combination of a novel approach we developed called inducible barcoded rabies virus with activity-dependent neuronal tagging and single-cell genomics, we propose to identify epigenetic changes in specific limbic system neuronal cell types and circuits active during cocaine- seeking behavior. We will then test the hypothesis that cocaine-seeking behavior can be erased by performing epigenetic editing of hippocampal neurons projecting to the nucleus accumbens that are active during detection of cocaine-conditioned cues. To that end, we will utilize a novel gene editing tool we developed called CRISRP- rabies to modulate epigenetic states of specific neuron types within defined hippocampal-NAc circuits conditioned by cocaine use. We believe that our study will discover conceptually novel mechanisms of cocaine addiction and will build a foundation for new types of therapies of drug addiction by rewiring specific circuits responsible for drug seeking behavior.

NIMH - National Institute of Mental Health

Abstract Despite the high prevalence of major depressive disorder (MDD) and its projected rise as the leading cause of global disease burden by 2030, treatment efficacy remains suboptimal. First-line antidepressants have modest efficacy (~50%), and high placebo response rates (~40%) contribute to the failure of antidepressant trials and hinder new drug development. While research underscores the role of antidepressant expectancies in modulating mood across various brain regions, there is a critical need to elucidate how expectancy-driven neural dynamics interact with downstream mood regulation processes to induce sustained mood improvement. Our recent work provides the first computational account of antidepressant placebo effects, where reinforcement learning (RL) model-predicted expectancies—encoded in the salience network (SN)—trigger mood changes perceived as reward signals, which reinforce antidepressant expectancies through an expectancy-mood loop. Furthermore, we and others have demonstrated that enhanced functional connectivity (FC) between the SN and default mode network (DMN) during expectancy processing and at rest predicts long-term antidepressant placebo effects. This evidence suggests that antidepressant expectancies, originating from contextual treatment cues, are represented in the SN and influence mood regulation through top-down connections with the DMN. To test this hypothesis, this study will investigate the causal roles of the SN, DMN, and SN-DMN FC in antidepressant placebo effects using Theta Burst Stimulation (TBS). In a 2x3 factorial design, 200 patients with MDD will be randomized to three counter-balanced TBS conditions (intermittent, continuous, and sham, within-subject) targeting either the SN or DMN (between-subject). These acute experimental manipulations will modulate trial-by-trial expectancy and mood ratings and the neural encoding of model-based expectancies and mood reward signals during the “antidepressant placebo fMRI task”, which manipulates placebo-associated expectancies using visually cued fast-acting antidepressant infusions and sham visual neurofeedback. Led by experts in placebo effects, reinforcement learning, depression, and neuromodulation, this study combines a robust theoretical framework, state-of-the-art neuroimaging, precision functional mapping for personalized TBS targeting, and accelerated TBS, ensuring scientific rigor. The insights gained from this study will deepen our understanding of the neural mechanisms behind placebo effects, enhancing clinical trial design, advancing neuroimaging predictors of treatment response, and accelerating the development of expectancy-based interventions for MDD.

NCI - National Cancer Institute

ABSTRACT The recalcitrance of high-grade serous ovarian cancer (HGSOC) to available therapies is largely enabled by the disease’s ability to fast disseminate into the peritoneal cavity and its intrinsic molecular heterogeneity. A first-line platinum-taxane regimen, in combination with PARP inhibitors (PARPi) as maintenance therapy has improved patients’ survival and revolutionized HGSOC management, especially for those carrying mutations in BRCA1/2 genes. Recently, the sensitivity to PARPi has been associated not only with BRCA1/2 mutations, but with a broader homologous recombination deficiency (HRD) signature, which accounts for nearly 50% of HGSOCs. Surprisingly, PARPi sensitivity has also been observed in homologous recombination-proficient (HRP) tumors. Despite successful, the emergence of cross-resistance to platinum/PARPi treatment strongly hindered the long- term clinical benefit, posing a significant clinical challenge. The genetic and epigenetic instability of HGSOCs strongly contributes to the development of cross-resistant clones, which undergo lineage expansion, leading to treatment failure and disease relapse. Furthermore, the mechanisms driving the selection of Platinum/PARPi cross-resistant clones, and their evolution remain poorly understood. A critical question is whether recurrent disease is driven by pre-existing resistant clonal populations or by de novo (acquired) molecular scars selected during the treatment. Addressing this question is crucial for understanding HGSOC progression and developing more effective therapies. To investigate these issues, we have developed a novel somatic mosaic genetically-engineered mouse model (smGEMM) of HGSOC combined with a molecular barcoding technology. This approach allows for the high-throughput isolation and detailed characterization of specific cell lineages, including therapy cross-resistant clones, enabling us to track clonal dynamics and to identify molecular drivers of resistance. To assess this hypothesis, we will pursue specific aims that 1) generate longitudinal lineage-traced models of HRP and HRD HGSOCs using smGEMM models, 2) functionally characterize the clonal dynamics of platinum/PARPi resistance and 3) perform cross-species validation of molecular dependencies driving platinum/PARPi resistance. Our findings will be significant because they will 1) uncover the transcriptomic and genomic mechanisms behind platinum/PARPi cross-resistance in HRP and HRD tumors, 2) reconstruct the evolutionary pathways of aggressive HGSOCs, 3) identify the cross-species molecular signatures of platinum/PARPi resistance. Importantly, this research has the potential to extend beyond HGSOC, as Platinum/PARPi combinaiton is used in other HRD cancers, such as breast and pancreatic cancer. Furthermore, our CRT and sm-GEMM platforms are adaptable to other gynecological cancers, hence benefiting the broader scientific community. Ultimately, this study will address urgent clinical needs by identifying molecular signatures of platinum/PARPi resistance and discovering biomarkers for patient stratification and therapeutic targeting.

NIDA - National Institute on Drug Abuse

Project Summary/Abstract Opioid use disorder (OUD) is a devastating public health crisis, characterized by a lack of inhibitory control over drug seeking. Opioid use causes persistent adaptations in the excitatory circuitry governing motivated behavior, enabling drug-paired cues to trigger seeking despite negative consequences. Therefore, understanding how opioids engage and adapt unique glutamatergic circuit elements to promote maladaptive, reward-driven behavior would provide significant insight into habitual heroin use and identify treatment strategies to prevent relapse. This K99/R00 proposal seeks to determine the projection-specific glutamatergic neurons that functionally guide motivated behavior and reveal the pathway-specific circuit adaptations that emerge during heroin use to drive reward seeking. As I begin my independent career, I aim to develop a research program that investigates the spatiotemporal dynamics of drug-naive and drug-experienced glutamatergic networks and causally implicate pathway-specific ensembles in guiding reward-driven behavior and relapse. The Otis laboratory identified that the glutamatergic pathway from the paraventricular thalamus to the nucleus accumbens shell (PVT→NAc) pervasively governs naturalistic reward-seeking behavior, and stimulation of this pathway is sufficient to profoundly inhibit motivated action. Recently, I established that heroin use dampens PVT→NAc projection activity and weakens downstream synaptic efficacy, functionally disinhibiting reward seeking. Using two-photon (2P) calcium imaging in head-fixed, self-administering mice, we found three unique ensembles emerge in the PVT→NAc pathway during taking, with inhibitory neuronal dynamics reliably predicting goal-directed behavior in sucrose- and heroin-seeking tasks. However, it is currently unknown whether this inhibitory ensemble functionally encodes motivated behavior and actively guides reward seeking. During the K99, I will receive world-class training in 2P single-cell optogenetics to selectively photostimulate the inhibitory PVT→NAc ensemble that encodes goal-directed behavior in sucrose-seeking mice, both before and after heroin exposure. I will learn advanced computational analysis to determine the outcome of ensemble photostimulation on the within- projection dynamics guiding inhibitory control and heroin-induced disinhibition of seeking (Aim 1). I will expand my investigations into other key glutamatergic inputs to the NAc and determine the heroin-induced adaptations in hippocampal circuit- and cell-type-specific connectivity that facilitates relapse (Aim 2) and hippocampus-to- NAc-specific projection neurons that guide heroin-motivated behavior (Aim 3). Collectively, this proposal tests the hypothesis that heroin induces functional adaptations in pathway-specific glutamatergic circuit elements to drive maladaptive reward seeking. Results will reveal the network computations that guide motivated behavior in naïve and drug-exposed systems. This K99/R00 will grant me the unparalleled opportunity to receive training in state-of-the-art approaches as I develop my own independent research program in OUD.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT Autoimmunity is a major driver of chronic morbidity in over 15 million Americans, representing significant unmet medical need. Individuals with Down syndrome (DS, trisomy 21) are at especially increased risk (4-100x) of autoimmunity, suggesting a contributory role of genes on chromosome 21. We identified the chromosome 21- encoded kinase DYRK1A (studied almost exclusively in the context of neurodevelopment) as a candidate driver of autoimmunity risk. Better understanding the underlying mechanisms may highlight DYRK1A as a druggable target to treat or prevent autoimmunity in people with DS and subsets of people without DS. We showed that DYRK1 regulates differentiation of pro-autoimmunity Th17 cells. We further uncovered that this works at least in part by a novel regulatory role of DYRK1A on surface expression of the IL-6 receptor subunits gp130 and IL-6R. These findings highlight a much broader role for DYRK1A in autoimmune biology than previously appreciated because (i) IL-6 is a pleiotropic cytokine that drives autoimmunity through multiple mechanisms including but not limited to Th17 differentiation; and (ii) gp130 mediates signaling of many cytokines other than IL-6 including (immune-relevant) IL-11 and IL-27. Therefore, the DYRK1A-gp130 axis has broad potential to impact biology relevant to autoimmunity and beyond. We hypothesize that DYRK1A regulates cellular and functional response to IL-6 and other gp130 family cytokines in people and murine models of DS. We propose key experiments here to advance our understanding of the DYRK1A- gp130 axis (i) in people with and without DS, (ii) in cell types beyond naïve CD4+ T cells and (iii) in murine models of DS. Pilot high-risk high-reward approaches include first-in-kind immune studies of individuals with DYRK1A syndrome. Our specific aims are: Specific Aim 1. Define how DYRK1A regulates human gp130 signaling. These studies will define how the DYRK1A-gp130 axis is functionally altered and lay keystone data to rationally expand the scope of follow-up studies. Notably, we include people with DS and DYRK1A syndrome who naturally over-/under-express DYRK1A respectively. This first-in-kind allelic series allows unprecedented translationally relevant studies of DYRK1A in immunobiology, including novel studies in immune aging and autoimmune-poised state. Specific Aim 2. Validate altered DYRK1A/gp130 axis in murine models of DS. These studies test the hypothesis that the Dp16 model of DS shows similar dysregulation of the DYRK1A-gp130 axis as seen in people with DS. We further demonstrate unique utility of the murine model, including leveraging novel genetic models to demonstrate the causal role of DYRK1A and mechanistically dissecting T cell generation beyond Th17. This will generate first-in-kind data that the Dp16 model can and should be used to study DS- immunodysregulation.

NIA - National Institute on Aging

PROJECT SUMMARY/ABSTRACT Age-related ovarian dysfunction is accompanied by sustained, low-grade inflammation and maladaptive immune activation—characterized by immune cell accumulation, fibrosis, and multinucleated giant cell (MNGC) formation. These pathological features arise before reproductive senescence and precede inflammatory remodeling seen during aging in other tissues. Yet, the cellular drivers, signaling mechanisms, and intercellular communication underlying these inflammatory responses in ovarian tissue remain largely unknown. This proposal focuses on how innate and adaptive immune components contribute to chronic, low-grade inflammation and structural tissue remodeling in the ovary. We aim to define how immune-tissue crosstalk promotes fibrosis, immune cell accumulation and activation, and other aging hallmarks. Our preliminary data identified proinflammatory programs that are activated in a cell-type-specific manner during early ovarian aging, implicating granulosa cells, tissue-resident macrophages (TRMs), and Type 17 T cells in a cascade of non-resolving inflammatory signaling. Our central hypothesis is that granulosa cell–intrinsic NF-κB activation initiates a feed- forward inflammatory loop with TRMs and Type 17 T cells, resulting in tissue fibrosis, MNGC formation, and immune-mediated ovarian dysfunction. We will test this hypothesis through the following mechanistic aims: Aim 1: Determine whether NF-κB signaling in granulosa cells orchestrates paracrine immune activation. We predict that NF-κB activation initiates proinflammatory signaling that promotes the recruitment and activation of TRMs and Type 17 T cells. Conditional ablation of NF-κB in granulosa cells will allow us to isolate its role in initiating immune crosstalk and downstream fibrotic pathology. Aim 2: Define the functional role of TRMs in sustaining ovarian inflammation. We hypothesize that ovarian TRMs act as critical amplifiers of age-related inflammatory responses and tissue remodeling. Using targeted macrophage depletion strategies, we will assess whether TRMs are required for T cell expansion, fibrotic signaling, and MNGC formation. Aim 3: Characterize Type 17 T cells in the aging ovary and their contribution to fibrotic remodeling. Type 17 T cells have been implicated in chronic inflammation across tissues, but their role in ovarian aging is unexplored. We will assess their phenotype, spatial localization, and impact on inflammatory signaling and fibrotic changes following targeted depletion. This research will also determine the temporal hierarchy of immune activation in ovarian aging and how intercellular inflammatory signaling evolves across the tissue. By delineating cell-type-specific contributions to chronic inflammation, this work will also provide a blueprint for understanding immune-mediated aging in other tissues. Rather than focusing on reproductive endpoints, we use the ovary as a model of immune-driven fibrosis and sterile inflammation—processes central to aging in multiple organs. Findings from this work will identify novel, immunological targets for intervention that could be used to delay ovarian aging and benefit overall health.

NCI - National Cancer Institute

Project Summary Uveal melanoma (UM) is a rare cancer of the eye that often spreads to the liver, with few treatment options once metastasis occurs. While genetic features like monosomy 3, BAP1 loss, GEP class 2, and PRAME expression help identify high-risk tumors, we still don’t fully understand how these tumors spread—especially how they interact with and influence other parts of the body before metastases are visible. This project aims to better understand how UM tumors send signals across different biological compartments— within the eye, into the bloodstream, and through circulating cells. We’re building on a prospective, longitudinal cohort of over 100 newly diagnosed patients, collecting samples from the tumor, aqueous humor (the fluid in the eye), plasma, and peripheral blood at multiple timepoints. By analyzing extracellular vesicles, soluble proteins, and rare circulating cell types, we hope to map how signals move and evolve over time and space. In Aim 1, we’ll examine how high-risk tumor features shape the local environment of the eye. Aim 2 will follow changes in plasma to understand how signals originating in the tumor show up systemically. Aim 3 focuses on rare circulating cells, using advanced imaging tools to determine how these cells reflect or respond to cross- compartment signaling. We’ll integrate data from all compartments and timepoints to define communication “states” that may help explain why some tumors spread early. This will be the most detailed study of its kind in UM. By focusing on communication between compartments— not just single markers—we hope to gain new insight into how early spread happens, and how it might be detected or interrupted before metastasis occurs. These findings could lay the groundwork for future strategies to monitor high-risk patients or develop early intervention approaches. Because UM is a rare cancer with few large-scale, mechanistic studies, this work may also inform approaches for other cancers with low mutational burden and limited immune responsiveness. The project aligns with NCI’s priorities for rare cancer research and directly addresses the goals of NIH Notice NOT-CA-25-010, which encourages innovative, liquid biopsy–based studies of tumor–host communication.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Interstitial lung diseases (ILDs) represent a global clinical burden that affects >5,000,000 people. Commonly, ILDs have no clinically determinable cause and are diagnosed as idiopathic pulmonary fibrosis (IPF), which presents a median survival time of only 2-4 years after diagnosis. Given the unclear pathogenesis of many ILDs, there is a strong clinical need to elucidate biological mechanisms that contribute to their onset and progression. Interestingly, ILDs often exhibit a heterogeneous distribution of (1) healthy tissue regions, (2) early-fibrogenic foci, and (3) mature-fibrotic tissue, each of which presents a unique cellular/biochemical milieu, tissue architecture, and response to therapy. Traditional biological analyses of ILDs, including single-cell RNA sequencing (scRNA-seq) and other omic-scale methods, have mostly generated composite molecular profiles, which may mask important distinctions between foci such that pathological vs. ameliorative states cannot be directly inferred. This project aims to disaggregate early-fibrogenic microenvironments in an ILD-specific manner, uncovering highly resolved cellular/molecular drivers specific to early scar progression. Further, therapeutic testing for ILDs has similarly centered on (1) in vitro models devoid of spatial-biological context and/or (2) bulk treatment of murine lungs, which may obscure intra-tissue variability in the effects of treatment based on local niche. The overarching goals of this proposal are therefore to spatially profile and recapitulate disease/foci- specific “neighborhoods” that specifically drive early fibrogenesis using spatial biology and biomaterial modeling. Overall, this project seeks to shift current paradigms by utilizing spatial-biological data as an anatomical “blueprint” for disease- and niche-specific modeling of early fibrogenesis, enabling unprecedented detail in dissecting the contributions of in situ tissue context to the initiation of scarring across multiple ILDs. Specialized fibroblast phenotypes play a critical role in the fibrosis of all major organs. Based on prior studies, I hypothesize that fibroblast-centered neighborhoods differentiate healthy vs. early-fibrogenic vs. mature-fibrotic tissue regions across human ILDs. Specific Aim 1 will profile ILD-specific foci by integrating (1) machine learning (ML)-based tissue architecture analysis with (2) high-plex immunofluorescence. Specific Aim 2 will recapitulate fibrogenic cell-matrix interactions by using light-catalyzed bioconjugation chemistry to generate user-defined extracellular protein patterning in two model systems. Specific Aim 3 will dissect fibrogenic cellular crosstalk by using 3D bioprinting to recapitulate niche-associated cell-cell interactions (i.e. endothelial cells with specialized fibroblasts). Ultimately, this project will integrate ML, spatial biology, and biomaterials modeling to elucidate the roles of localized biological niches to the onset of scarring across ILDs.

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

PROJECT SUMMARY/ABSTRACT Infants exposed in utero to maternal inflammatory disorders are at risk of long-lasting health impacts, such as altered neurodevelopmental outcomes or dysregulated immune responses. When maternal infections are the inflammatory stimulus, these adverse neonatal outcomes can occur in the absence of congenital infection. Our preliminary data show that infants who are HIV-exposed, but uninfected experience high rates of morbidity and mortality that are associated with maternal inflammation in a dose-dependent fashion. Early reports suggest that acute maternal infection with COVID-19 can have profound impacts on fetal immune development. Inflammatory signaling across the maternal-fetal interface during a critical fetal developmental window is likely to be responsible for these adverse neonatal outcomes. However, critical gaps exist in our understanding of how inflammatory signals are transferred across the placenta. The overarching goal of this proposal is to determine the cellular and molecular mechanisms involved in the transfer of inflammatory signals across the maternal-fetal interface in the setting of acute or chronic viral infection. We will capitalize upon access to samples from pregnancies complicated by either HIV, which results in chronic inflammation, or SARS-CoV-2, which causes severe acute inflammation, to address the following Specific Aims: 1) to profile the transcriptomic signatures of heterogenous maternal and fetal cell types in their in situ environment within placentas from pregnancies affected by HIV or SARS-CoV-2 versus healthy pregnancies; 2) to compare concentrations of inflammatory and regulatory biomarkers in maternal and infant peripheral blood, cord blood and placentas from term pregnancies affected by HIV or SARS-CoV-2 versus healthy pregnancies and determine their association with placental gene expression; and 3) to characterize inflammatory signaling across the maternal-fetal interface using a 3- dimensional in vitro model that incorporates key placental cell types. This proposal includes several innovative components, including the use of advanced bioinformatics to directly correlate placental gene expression data from a single-cell spatial transcriptomics assay to the peripheral concentrations of inflammatory biomarkers within the same participants, and the development of a novel Transwell-based model of the human placenta that can be used to mechanistically interrogate the role of each cell type in the transfer or inflammatory signals. Disentangling the mechanisms responsible for the transfer of inflammation across the maternal-fetal interface will ultimately allow for the identification of therapeutic agents to modulate inflammation in pregnancy, thereby preventing adverse neurodevelopmental and/or immunologic consequences to the fetus.

NINDS - National Institute of Neurological Disorders and Stroke

Abstract/Project Summary As an essential component of most behaviors, the sympathetic system regulates many vital physiological processes, such as heart rate, blood pressure, and respiration. Sympathetic activities exhibit an arousal state- dependent basal level (tone), which is subject to behavioral context-dependent regulation (activation and inactivation). In contrast to its importance, much less is known about top-down sympathetic regulation. Our recent studies reveal several populations of spinal projecting neurons (SPNs) with the capacity to regulate sympathetic preganglionic neurons. We hypothesize that medullary and hypothalamic SPNs may form parallel circuits to control state-dependent sympathetic activities with or without accompanying motor engagement. The objective of this proposed study is to test this hypothesis by analyzing the fundamental circuit architectures of sympathetic controlling SPNs, and determining the roles of these SPNs in regulating the basal tone of sympathetic activity, as well as their response to exercise and pain. The outcomes of this study are expected to advance the fundamental knowledge about autonomic regulation and its health impact.

NIMH - National Institute of Mental Health

Project Summary Social affiliation is critical to the physical and emotional health of a wide variety of species. Disruptions in social functioning - a key feature of conditions like autism, social anxiety, and schizophrenia - can severely limit one's capacity to cultivate healthy social relationships and bonds. The neural circuit mechanisms governing affiliative social behaviors are not well understood, which is an important part of the social processes domain of the Research Domain Criteria (RDoC) and relevant to several psychiatric disorders. Allogrooming (grooming behavior directed toward another individual) is a major form of affiliative social contact through which animals may form, maintain, and strengthen social relationships and is conserved in a wide range of social species, such as birds, bats, rodents, canids, cats, equids, and primates. This project aims to understand the neural mechanisms regulating allogrooming in mice by examining the oxytocin receptor (OXTR) system, a critical modulator within the brain's `social behavior network'. The sponsor has previously identified a pathway from the medial amygdala (MeA) to the medial preoptic area (MPOA) as essential for allogrooming in mice. However, the specific neural encoding of oxytocin-responsive neurons in this circuit remains unknown. I will gain exceptional training in powerful genetic approaches that allow us to target oxytocin receptor cells specifically. The outlined proposal will 1) utilize in vivo optogenetics to manipulate oxytocin-sensitive MeA neurons and their projections to the MPOA and BNST, 2) assess the role of OXTR specifically in the MeA on prosocial behavior, and 3) utilize in vivo freely moving calcium imaging to optically record MeA OXTR neurons during allogrooming behavior. The proposed study will provide an excellent training experience for the applicant, and it will reveal how discrete, anatomically defined pathways descending from the MeA are engaged during allogrooming. These results will set the foundation for a more incisive analysis of how OXTR circuits shape social function in both health and disease.

NIGMS - National Institute of General Medical Sciences

Cellular membranes serve as dynamic platforms for cell communication and signaling. On each plasma and organelle membrane, lipid–protein and protein–protein interactions are spatiotemporally organized to regulate key biological processes, such as receptor activation and oncogenic signaling. Despite their critical roles, these membrane-specific interactions remain challenging to study due to their transient and context-dependent nature. This proposal aims to develop a novel membrane-centric proximity labeling approach to systematically map lipid–protein and protein–protein networks in live cells. Proximity labeling is a powerful technology for mapping the protein interactome and spatial proteome in living systems. However, conventional tools, such as TurboID and APEX2, lack the spatiotemporal control required to target and capture the dynamic and heterogeneous membrane microenvironment. To address this limitation, I will engineer conditionally activated proximity labeling enzymes that sense their membrane microenvironment and directly couple it to their activity. The first phase (K99) will focus on developing Antigen-Controlled TurboID (ACTurbo), a proximity labeling enzyme activated upon binding to protein of interest, and applying it to dissect compartment-specific μ-opioid receptor signaling pathways in neurons. The second phase (R00) will develop Lipid-Activated Biotin Ligases (LABL) to enable phosphoinositide-centric mapping of oncogenic lipid signaling. This work will establish an innovative and versatile framework for studying lipid–protein and protein–protein interactions in their physiological context, addressing fundamental gaps in membrane biology. By systematically mapping lipid–protein networks on live membranes, this project will provide insights into how membrane dynamics regulate cellular signaling in both normal and degenerative conditions, with potential implications for therapeutic interventions. To achieve this goal, I will integrate my expertise in lipid biology, membrane biology, chemical biology, and protein engineering, bolstered by guidance from my primary mentor, Dr. Alice Ting. To facilitate my proposed research and training, I have assembled a team of leading experts, including Dr. David Baker (computational protein design), Dr. Ilme Schlichting (structural biology), Dr. Ivan Soltesz (neuroscience), and Dr. Ruth Huttenhain (GPCR proteomics). Upon completing this proposal, I will be fully equipped to establish my independent research program focused on developing molecular tools to decipher and manipulate the complex and dynamic networks of membranes, proteins, and lipids.

NINDS - National Institute of Neurological Disorders and Stroke

The overarching objective of the proposed research is to dissect how primary motor cortex (M1) flexibly controls different types of movements in the macaque monkey. In primates, M1 is a necessary structure for the generation of voluntary limb movements, like reaching towards your phone and then grasping it. M1 appears to operate under distinct computational modes in reaching and grasping. During reaching, neural population dynamics, that is the time-varying pattern of firing rates in populations of neurons in M1, are low-dimensional and with a specific structure (“rotational”). In contrast, neural population dynamics during grasping are high-dimensional and not rotational. The goal of this specific proposal is to understand how M1 can exhibit these different computational modes. My overall premise is that these diverse operating modes of M1 emerge due to relative contributions of two key components: autonomous recurrent dynamics within M1, and external inputs from other brain areas. Here, I will test my central hypothesis that M1 activity during reaching is controlled primarily by strong recurrent dynamics, whereas grasping is dominated by inputs in two Aims. In Aim 1, I will train monkeys to perform two different tasks: a delayed reaching task, and delayed grasping of various objects (using a turntable). I will use high-density electrophysiology (Neuropixels) to measure the neural population dynamics in M1 and two areas that provide input to M1 (dorsal premotor cortex (PMd) and primary somatosensory cortex (S1)) during these tasks. I will then use residual dynamics analysis techniques to identify the dynamical landscape and inputs to M1 underlying reaching and grasping. The residual dynamics approach models M1 neural population activity as a dynamical system and uses trial-to-trial variability to distinguish between input-driven (e.g., rotations with minimal inputs) versus recurrence-driven (e.g., moving point attractors) systems. I will test the hypothesis that M1 dynamics during reaching are better explained by models with recurrent dynamics within M1, whereas those during grasping are better explained by models that leverage inputs from other areas. In Aim 2, I will use causal approaches to further test the hypothesis. I will express red-shifted opsins in inhibitory neuron populations of PMd and S1. I will then stimulate these inhibitory neurons one at a time to silence the area, while recording from M1 as the animals perform the reaching or grasping task. I will then assess the impact of this optogenetic silencing on (PMd/S1) on both behavior and M1 neural dynamics. My hypothesis is that silencing input areas will have stronger impact on both behavior and M1 dynamics during grasping than reaching. Collectively, the two aims will dissect the roles of inputs versus recurrence in M1 dynamics across multiple tasks and illuminate how M1 operates under dramatically different modes to flexibly control arm and hand movement behavior. Impact: The proposed work understands how M1 in concert with other clinically relevant brain areas flexibly controls two fundamental behaviors: reaching and grasping. Such an understanding will enable the development of better brain-computer interfaces and circuit-level interventions for stroke, cortical injury, and neurological disorders.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY / ABSTRACT This MPI R21 proposal directly addresses the objectives of the INvestigation of Co-occurring conditions across the Lifespan to Understand Down syndromE (INCLUDE) Project, emphasizing the creation of innovative biological resources to advance Down syndrome (DS) research. DS is linked to widely dysregulated immune responses, including susceptibility to autoimmunity and infections, but most current research has focused on B cell hyperactivity and interferon-driven pathways, leaving the role of inadequate thymic selection—marked by reduced thymic output and premature thymic involution—largely uncharacterized. Our proposal posits that a fundamental gap in DS research is the absence of a comprehensive understanding of how these thymic defects drive peripheral immune dysfunction. To address this, we will build a well-annotated, deeply profiled biorepository of thymic samples from individuals with trisomy 21 and age-matched disomic controls, enabling a systematic examination of thymic education processes that may underlie the broader immunopathology seen in DS. Although murine models of DS have offered important insights into the genetics and pathology of trisomy 21, they are inherently limited by the fact that the extra copy of human chromosome 21 cannot be fully and precisely replicated in mice. Thymic development, in particular, depends heavily on self-antigens specific to each species, as immature thymocytes are “educated” through interactions with the host’s unique set of self-genes. By establishing a biorepository of human DS thymic tissue, we will circumvent these issues, enabling precise investigations into how an extra copy of chromosome 21 disrupts thymic selection and predisposes individuals with DS to immune dysfunction. To this end, we propose the following two goals. First, through a close collaboration with the Primary Children’s Hospital in Salt Lake City, we will create a comprehensive repository of thymic samples from infants with trisomy 21 and age-matched controls. All samples will undergo HLA genotyping, biological sex determination, and a thorough phenotypic analysis of both T-lineage and non–T- lineage cells, evaluating each thymocyte subset’s responsiveness to T cell receptor (TCR) stimuli at critical developmental checkpoints. Second, we will sequence the TCR repertoire at each developmental stage— evaluating both diversity and amino acid composition—and then compare these results with our existing in- house TCR datasets from individuals with autoimmune diseases and carriers of polymorphisms in genes critical for TCR repertoire formation. This approach will help us pinpoint meaningful similarities or differences among these groups. By filling critical knowledge gaps related to T-lineage ontogeny, our findings may reveal whether this underexplored aspect of thymic development in DS contributes to the documented immune dysfunction.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Project summary/ Abstract The overarching goal of this K99/R00 proposal is to explore aggregate genetic liability and neurocognitive mechanisms in the co-occurrence of alcohol use/problems and suicidal thoughts and behaviors (STB). Excessive alcohol use is prevalent worldwide and represents a significant burden to human health; it is associated with medical and psychopathological problems such as STB. STB are a critical public health concern, with a continuing increase in suicide attempts and deaths every year in the US. The prevalence of STB is particularly high among individuals with alcohol problems and studies have tackled this question from different perspectives. Research indicates that alcohol problems could causally impact risk of STB; there is also empirical support for a shared genetic liability between alcohol use/problems and STB. This shared genetic liability underscores the existence of possible common mechanisms that would be involved in both alcohol use/problems and STB. Decision-making (DM) has been observed in relation with adolescent drinking behavior and alcohol use disorder, while both have been associated with STB. In STB research, DM difficulties have been described and may be characteristic of impulsive suicide attempts, but current findings largely rely on self-reports and lack objective evaluations. Additional knowledge could be gained by relying on a theoretical conceptualization of DM, a systematic evaluation of its underlying neurocognitive mechanisms, and a consideration of the role of genetic factors. Capitalizing on genetics and neuropsychology, we will explore which DM mechanisms play a role in alcohol use/problems and STB co-occurrence, how genetic liability is involved in this association, and whether environmental factors may influence the development of DM and its relation with alcohol use/problems and STB. An improved understanding of these processes will contribute to prevention and intervention efforts by advancing our ability to target potentially modifiable mechanisms according to the influence of genes and environment. This proposal delineates a series of training aims to advance our understanding of the co-occurrence between alcohol use/problems and STB: 1) the candidate will establish expertise in the assessment of aggregate genetic risks and other genetic models, and in advanced statistical methods that will lay the foundation of her independent career; 2) different suicide phenotypes will be used and distinguished according to their association with alcohol use, neurocognitive, and genetic characteristics; 3) the K99/R00 proposal gathers experts in the disciplines of genetics and neuropsychology that will support the development of the scientific project and the pathway to independence. The environment at the Virginia Institute for Psychiatric and Behavioral Genetics is ideal for the candidate’s goal of developing a comprehensive program in genetics, alcohol, and STB research, and the proposed project represents an important contribution toward advancing the understanding of alcohol use/problems and STB through a combination of genetics and neuropsychological methods, consistent with NIAAA’s missions.

NIAID - National Institute of Allergy and Infectious Diseases

Diabetes mellitus (DM) is a group of metabolic diseases that affect how the body utilizes glucose and result in elevated levels of glucose in the blood and urine. DM increases the risk of many infections and their complications, but comorbidity with tuberculosis (TB) stands out due to its tremendous global impact. DM and TB each individually rank in the top ten global causes of death. In 2020, there were approximately 537 million adults with DM and 10 million new cases of active TB. TB patients with DM are twice as likely to die from TB and 1.5 times more likely to experience TB recurrence than non-DM patients. To better protect DM patients from TB, we need to understand how DM alters immune function to favor developing active TB disease. The impact of the metabolic perturbations in DM patients on immune cell function have largely been explored in the context of lymphocytes and macrophages. However, a systems biology comparison of DM patients, TB patients, comorbid patients (DM/TB), and healthy individuals was recently performed and identified neutrophilic inflammation as a central feature of DM/TB. Dysfunctional neutrophils have been implicated in DM vascular complications and impaired wound healing, but it is unclear how they contribute to disease outcomes in Mycobacterium tuberculosis (Mtb)-infected DM patients. Neutrophils are the most abundant and predominantly-infected cell type in the airways and lung tissue of active TB patients, but are unable to control Mtb replication. In addition, we have recently demonstrated that specific neutrophil effector functions can directly promote Mtb replication and pathogenesis. It is currently unclear how neutrophils contribute to disease outcomes in DM/TB patients, particularly because neutrophils are typically discarded from blood cell isolations and must be analyzed immediately without storing. We hypothesize that altered neutrophil function during DM contributes to increased susceptibility to Mtb infection. The proposed experiments will for the first time directly dissect the neutrophil responses associated with the DM/TB comorbidity and determine how neutrophils impact the outcome of Mtb infection in the context of DM. In Aim 1 we will define the neutrophil responses in DM patients that are associated with susceptibility to developing active TB disease. In Aim 2, we will determine how DM neutrophils promote Mtb pathogenesis. The results from the proposed studies will provide the critical foundation for future translational studies aimed at modulating neutrophil responses in DM/TB comorbid human patients as new strategies to treat TB in individuals with DM. Host directed therapies will work to target drug sensitive and resistant infections. Drugs already being developed that target NET release and other neutrophil effector functions, we can exploit these.