NIH Award Research Articles

Multi-Functional Role of RECK in Metabolic Dysfunction-Associated Steatotic Liver Disease Development: A Mass Spectrometry-Proteomic Approach

Metabolic dysfunction-associated steatotic liver disease (MASLD, previously referred to as nonalcoholic fatty liver disease) is characterized by increased fat deposition and inflammation in the liver as well as dysregulation of cellular processes that affect the extracellular matrix (ECM). We previously observed that the expression of Reversion Inducing Cysteine Rich Protein with Kazal motifs (RECK), a key ECM regulatory protein, negatively correlates with MASLD-associated fibrosis and inflammation (metabolic dysfunction-associated steatohepatitis, MASH). However, mechanisms governing this relationship remain unclear. Therefore, we hypothesized that overexpression of RECK is hepatoprotective in the setting of MASH by altering several ECM-linked pathways, as analyzed by proteomics. Accordingly, we generated hepatocyte-specific Reck-overexpressing transgenic mice. Male hepatocyte-specific Reck-overexpressing transgenic mice and littermate controls were fed a Western diet high in fat, carbohydrate, and cholesterol for 24 weeks (n=6/group). Whole liver tissue was submitted for mass spectrometry-proteomic analysis. We identified 370 differentially expressed proteins out of a total of 4693 proteins using a q-value cutoff of ≤0.10; of which 90 proteins were significantly upregulated while 280 significantly downregulated. This included an upregulation of Cyp2b (adjusted p≤0.01) and downregulation of Taok1, C/EBPβ, and Vitronectin (all adjusted p≤0.01), all of which have been shown to be important in liver health and metabolism in the context of MASH. Predictive pathway analysis using Ingenuity Pathway Analysis software (Qiagen) identified several significantly altered pathways, including those involved in ECM regulator pathways (integrin signaling, z-score=6.0; mTOR signaling, z-score=5.3; leukocyte extravasation signaling, z-score=5.1), cell-signaling (chemokine signaling, z-score=5.4; CXCR4 signaling chemokine signaling, z-score=5.0), and inflammatory responses (leukocyte extravasation signaling, z-score=5.1; CXCR4 signaling, z-score=5.0). Interestingly, pathway analysis based on significantly altered proteins predicted the downregulation of critical components of hepatic inflammation and fibrosis, including TGF-β, IL-1β, and TNFα. These novel results reveal the regulatory role of RECK in MASH progression, specifically governing the components of ECM remodeling and inflammation. Our goal is to develop RECK inducers and identify additional therapeutic targets in MASH development and progression. This work was funded by the NIH award R01DK130243. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exploring Endocrine Signaling between Adipose Tissues and the Heart using Transfer Learning and Induced Pluripotent Stem Cell Models

Background: Because of the amount of evidence pointed toward obesity as a co-morbidity of heart disease, our goal in this study is to use a data-driven method to discover potential cross talk signals between adipose and cardiac tissues, which may reveal a physiological reason for this association. For this study, we hypothesized that adipose-cardiac crosstalk signaling can be found by unexpected co-expression between adipose tissue genes encoding secreted proteins and cardiac tissue genes and vice versa. By discovering new crosstalk signals between these two tissues, we can find new associations between heart disease and obesity. Methods: Cross-tissue correlations of gene expression have been used to identify candidate endocrine signals that mediate tissue crosstalk, which are otherwise diffcult to detect in circulation. However, the identification of significant cross-tissue gene co-expression is hindered by immense multiple-testing burden. Our goal here is to abstract gene expression patterns of tissues into eigengenes and then test their correlation with secreted protein coding genes across tissues in a pairwise fashion. We first used the NIH Common Fund Genotype-Tissue Expression data set (GTEx v8) which contains RNA seq gene expression data from matched tissues of over 17,000 transcriptomes, to learn the latent structure of gene expression across tissues. To do so, we used PLIER (pathway level information extractor), a matrix factorization algorithm to extract the latent targets as eigengenes that map to specific biological pathways in the target tissue (e.g., pathways in the heart in the case of adipose to heart crosstalk). The learned gene expression structure is then transferred to a smaller data set with matching donor adipose and heart transcript expression data, from which a correlation matrix is constructed to find significant relationships between source tissue crosstalk signals and target tissue eigengenes and nominate novel crosstalk signals. Results: Adipose to heart tissue cross talk correlations using PLIER derived latent eigengene targets revealed many adipokines that had strong correlations with pathways found in the heart tissue through similar gene expression. Among these strong correlations were eigengene targets that were wrapped in metabolism pathways particularly those of fatty acid, phospholipid, and protein metabolism. Adipokines that showed high significant and positive correlation with cardiac latent variable expression included Adiponectin (R = 0.32, p = 4.7e−13), Glutathione Peroxidase 3 (R = 0.34, p = 5e−15) and Galectin 9 (R = 0.37, p < 2.2e−16) among others. Based on these results, we nominated new potential relationships between adipose and cardiac tissue. Work is currently under way to validate these candidate crosstalk signals using human induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Future Directions In conclusion, we have found strong evidence in silico to suggest a relationship between known secreted proteins from adipose tissue (adipokine) and the expression of important pathways (such as fatty acid and protein metabolism) in the heart. Based on the findings of these correlations between adipokines and latent eigengene pathways within the heart, our future directions will involved the understanding of potential downstream functional consequences mediated by the adipose secreted proteins in iPSC derived models. This project was supported in part by NIH award R03-OD032666 and the University of Colorado Translational Research Scholar Program to E.L. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Distinct PD-1 hiCD4+ T helper cell populations promote CD11c+ T-bet+ B cell expansion in liver and spleen during obesity.

Abstract Obesity is associated with increased deposition of fat in ectopic tissues, such as within the liver and visceral adipose depots, including the omentum and mesentery. Increased visceral fat is an accompanying feature of the metabolic syndrome and is associated with increased end-organ damage in NAFLD. Emerging evidence implicates intrahepatic B cells in the progression of NAFLD, and increased number of proinflammatory B cells have been found in the steatotic livers of HFD-fed mice. Our preliminary studies demonstrate accumulation of inflammatory T-bet+ CD11c+ B cells in the subcutaneous adipose tissue of obese humans and perigonadal adipose tissue of mice on HFD. We now extend those findings into other sites of ectopic fat deposition to determine the obesity-related changes in T-bet+ B cell frequencies in the liver, omenta, and MAT. Flow cytometry identified increased frequencies of T-bet+ B cells in the spleen and livers, but not visceral adipose, of HFD mice. Furthermore, HFD-associated expansion of T-bet+ B cells in the spleen and liver correlated with a rise of T FHand T PHfrequencies, respectively. Future studies will determine the mechanism of tissue-specific T and B cell interactions associated with obesity-induced chronic inflammation. B.T.E. is supported by the NIH award (K12GM111726). T.H. is supported by a postdoctoral fellowship from the Swedish Research Council. This study was supported by NIH R01 AI32798-01A1 and the Voelcker Fund Young Investigator Awards (E.A.L).

Oxaloacetate regulates mitochondrial complex II respiration in brown fat: dependence on UCP1 expression

Objective: Oxaloacetate (OAA) is a long-known potent inhibitor of succinate dehydrogenase (SDH), a phenomenon that has, however, received only limited attention. We previously found that skeletal muscle mitochondria incubated at low membrane potential (Δψ) or interscapular brown adipose tissue (IBAT) mitochondria, wherein Δψ is intrinsically low, accumulate OAA in amounts sufficient to inhibit SDH (complex II) respiration. We proposed a mechanism wherein low Δψ reduces reverse electron transport (RET) to complex I causing a low NADH/NAD+ ratio favoring malate conversion to OAA. To further assess the mechanism and its physiologic relevance we carried out studies of mice with inherently different levels of IBAT mitochodnrial inner membrane potential. Hypothesis: Mice with differential expression of UCP1 would manifest Δψ-dependent differences in OAA-regulated mitochondrial respiration. Methodology: We examined several parameters in complex II and complex I energized IBAT mitochondria isolated from obesity prone C57BL/6J compared to obesity resistant 129SVE mice and from UCP1 knock-out (UCP1KO) compared to wildtype (WT) mice. Data were analyzed by unpaired, 2-tailed t-test and expressed as mean ± SE. Data: Isolated complex II (succinate)-energized IBAT mitochondria from 129SVE mice versus C57BL/6J displayed greater UCP1 expression, similar O2 flux despite lower Δψ, similar OAA concentrations, and similar NADH/NAD+. When GDP was added to inhibit UCP1, 129SVE IBAT mitochondria, despite their lower Δψ, exhibited much lower respiration (AUC of respiration versus time, 19963 ± 1764 vs 36015 ± 2527, p = 0.0004, n = 6), greater OAA concentrations (22.9 ± 1.8 vs 12.1 ± 3.3, p = 0.016, n = 6), much lower RET (as marked by ROS), and much lower NADH and NADH/NAD+ ratios compared to the C57BL/6J IBAT mitochondria. UCP1 knock-out abolished OAA accumulation by succinate-energized mitochondria associated with markedly greater Δψ, ROS, and NADH, but equal or greater O2 flux compared to WT mitochondria. GDP addition, compared to no GDP, increased Δψ and complex II respiration in WT mice associated with much less OAA. Respiration on complex I substrates for mitochondria of both strains of mice and for WT and UCP1KO mice followed the more classical dynamics of greater O2 flux at lower Δψ. Summary: These findings support the above-mentioned mechanism for OAA- and Δψ-dependent complex II respiration and support its physiologically relevance. Conclusions: Oxaloacetate regulates mitochondrial complex II dynamics in a manner dependent on inner membrane potential as intrinsically determined by mouse strain or genetic manipulation. NIH award, 1 R01 DK123043-01A1, VA Merit Review Award 2 I01 BX000285-06, and the Iowa Fraternal Order of the Eagles This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tissue engineering human thymus from iPSCs to support human T cell development

Abstract Thymus is the primary immune organ responsible for the generation of diverse and self-tolerant T cells that are essential for fighting off invading pathogens while maintaining immune homeostasis. Loss of thymus function, due to either genetic mutations or environmental insults, can lead to prolonged immunodeficiency that can be life-threatening. Rejuvenating the function of the thymus is challenging as the organ undergoes irreversible involution at young age, during which thymic epithelial cells (TECs) – the thymic stromal cells key to the education and selection of developing T cells – are progressively replaced by infiltrating adipocytes. Regeneration of thymus from the patient’s own stem cells will have tremendous clinical benefits in treating immunodeficiency and immunodysregulatory disorders. We reported here the tissue-engineering of functional artificial thymus from inducible pluripotent stem cells (iPSCs). Taking advantage of the 3D alginate encapsulation technology, we developed a novel protocol to induce the differentiation and maturation of human iPSCs into TECs (iPSC-TECs) at high efficiency. To bioengineer the thymus, iPSC-TECs were injected into decellularized murine thymus scaffolds, in conjunction with T cell progenitors derived from hematopoietic stem/progenitor cells (HSPCs) harvested from the umbilical cord blood. When transplanted under the kidney capsules of hematopoietic humanized mice (aka immunodeficient mice engrafted with HSPCs), iPSC-TEC thymus can support developed a diverse population of human T cells that could mediate adaptive immune responses. Our findings suggest that functional human thymus can be generated from patient-derived iPSCs for regenerative medicine. Supported in part by the NIH (R01 AI123392 and R21 AI126335), NSF (CBET 1804728), and Highmark Health Award (A023948-HIGHMARK-LANNI-FAN).

StomachSim: An in-silico simulator of gastric biomechanics with application to pyloroplasty

Aim: We have developed StomachSim, an in-silico simulator that is designed to simulate the biomechanics and fluid mechanics of a wide range of gastric conditions as well as gastric surgical procedures. Here we demonstrate its use in quantifying the effects of motility disorders and pyloroplasty on the hydrolysis and emptying of a liquid meal inside the stomach. Methods: A physiologically realistic model of the stomach, including the antral motility, is synthesized based on in-vivo imaging, and the mixing and gastric emptying are simulated using computational fluid dynamics (CFD). The enzymatic biochemistry for protein hydrolysis is also implemented by incorporating the secretion of a proteolytic enzyme from the proximal stomach walls and the subsequent enzymatic reaction with the protein dissolved in the liquid meal. Using this model, we measured the effect of stomach motility and the diameter of the pyloric orifice on the retropulsive and pyloric jets, the rate of hydrolysis in different regions, and the emptying of gastric contents. Results: This study presents a proof-of-concept for the application of StomachSim in gastric disorders and surgeries. The emptying rate of our model for the healthy case agreed with the in-vivo measurements of similar caloric density liquid meals. A stomach with a 50% lower motility amplitude had a 35% lower net emptying rate and a 41% lower emptying rate of the hydrolyzed protein. Furthermore, the emptying rate showed a non-linear variation with respect to the motility of the stomach. For a simulated pyloroplasty procedure where the pyloric orifice diameter is doubled and the orifice stays open permanently, the emptying rate of the lower motility case doubled over its original value. Conclusion: StomachSim offers a novel and potentially effective modality for examining the effect of gastric conditions and gastric surgical procedures on gastric function. Our simulations show that gastroparesis and pyloroplasty not only affect the emptying rate but also alter the mixing of contents thereby changing the rate of hydrolysis. The model helped quantify the changes in the flow of contents in the antro-duodenal region for varying degrees of gastroparesis and pyloroplasty. The authors acknowledge support from NSF award CBET 2019405 and NIH award 1R21GM139073-01. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Prospective assessment of nighttime respiratory disturbance in multiple myeloma patients

Chronic intermittent hypoxia, a feature of sleep apnea, is a potent driver of solid tumor cancers and promotes lethal engraftment of malignant plasma cells in a mouse model. The objective of this study was to investigate if nighttime respiratory disturbances affect clinical outcomes in a multiple myeloma patient population. To do so, we prospectively assessed nighttime respiratory disturbance index (RDI) in 44 patients (27 male, 17 female) diagnosed with multiple myeloma at the University of Iowa Hospitals and Clinics. Following this assessment, we categorized patients by RDI and utilized electronic medical records to explore differences in routine myeloma clinical measures. We found 93% of the patients had an RDI equal to clinical sleep apnea. 39% of patients had a moderate or severe RDI. In comparing these patients to those with low RDI, there were no statistical differences in any clinical performance indicator. Additionally, there were no differences in disease symptoms, multiple myeloma staging, or antibody type between these groups. However, patients with a higher nighttime RDI were less likely to respond to induction chemotherapy (p=0.046), a predictor of poor disease prognosis. These data support the hypothesis that nighttime respiratory disturbances negatively impact multiple myeloma patients, but this impact may not be evident in routine clinical tests of patient performance. This research is supported by NIH award R01CA244271 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

94 Evaluation of a grant writing workshop designed to increase submission and award rates for career development awards

OBJECTIVES/GOALS: To improve early career faculty members’ NIH grant writing skills, Clinical and Translational Science Awards (CTSA) hubs have developed a variety of workshop-style programs. However, few articles have evaluated the impact of grant writing workshops on NIH grant submission and award rates. METHODS/STUDY POPULATION: The K Writing program was developed by the Michigan Institute for Clinical and Health Research (MICHR) at the University of Michigan. Since 2012, 435 scholars have participated in the program. The MICHR K Writing program is a three-part workshop series that prepares scholars by providing them with guidelines to write all sections of a career development grant application. Each session focuses on different sections of the K award proposal. During the workshop sessions, participants break into small groups and exchange drafts of their proposal sections and receive peer critique and feedback from senior faculty facilitators who have experience with NIH study sections. RESULTS/ANTICIPATED RESULTS: Between 2012-2018, 273 scholars participated and 57% were female. Our two primary outcomes of interest are submission rates and success rates (the number of grants awarded divided by the number of applications). We plan to examine the effects of several characteristics, including number of sessions attended, cohort year, and faculty vs. postdoctoral status. We will also examine whether there were differences in submission and success rates between female and male researchers and between underrepresented minority scholars and those who identified as white or Asian. Lastly, we will report submission and success rates for each grant mechanism and compare them to the national averages. DISCUSSION/SIGNIFICANCE: Obtaining external research funding is an important part of a faculty career, especially at its early stages. This research has important implications for the design of similar programs intended to increase submission and success rates for federal grant applications.

Disparities in spatial access to neurological care in Appalachia: a cross-sectional health services analysis

Appalachia is rural and socioeconomically deprived with a heavy burden of neurological disorders and poor access to healthcare providers. Rates of neurological disorders are increasing over time without equal increases in providers, indicating that Appalachian disparities are likely to worsen. Spatial access to neurological care has not been robustly explored for U.S. areas, so we aimed to examine disparities in the vulnerable Appalachian region. Using 2022 CMS Care Compare physician data, we conducted a cross-sectional health services analysis, where we computed spatial accessibility of neurologists for all census tracts in the 13 states with Appalachian counties. We stratified access ratios by state, area deprivation, and rural-urban commuting area (RUCA) codes then utilized Welch two-sample t-tests to compare Appalachian tracts with non-Appalachian tracts. Using stratified results, we identified Appalachian areas where interventions would have the largest impact. Appalachian tracts (n=6169) had neurologist spatial access ratios between 25% and 35% lower than non-Appalachian tracts (n=18,441; p<0.001). When stratified by rurality and deprivation, three-step floating catchment area spatial access ratios for Appalachian tracts remained significantly lower in the most urban (RUCA=1 [p<0.0001) and most rural tracts (RUCA=9 [p=0.0093]; RUCA=10 [p=0.0227]). We identified 937 Appalachian census tracts where interventions can be targeted. After stratifying by rural status and deprivation, significant disparities in spatial access to neurologists remained for Appalachian areas, indicating both poorer access in Appalachia and that neurologist accessibility cannot be determined solely by remoteness and socioeconomic status. These findings and our identified disparity areas have broad implications for policymaking and intervention targeting in Appalachia. R.B.B. was supported by NIH Award Number T32CA094186. M.P.M. was supported by NIH-NCATS Award Number KL2TR002547.

Neutrophil-mediated fibroblast-tumor cell il-6/stat-3 signaling underlies the association between neutrophil-to-lymphocyte ratio dynamics and chemotherapy response in localized pancreatic cancer: A hybrid clinical-preclinical study.

Partial/complete pathologic response following neoadjuvant chemotherapy (NAC) in pancreatic cancer (PDAC) patients undergoing pancreatectomy is associated with improved survival. We sought to determine whether neutrophil-to-lymphocyte ratio (NLR) dynamics predict pathologic response following chemotherapy in PDAC, and if manipulating NLR impacts chemosensitivity in preclinical models and uncovers potential mechanistic underpinnings underlying these effects. Pathologic response in PDAC patients (n=94) undergoing NAC and pancreatectomy (7/2015-12/2019) was dichotomized as partial/complete or poor/absent. Bootstrap-validated multivariable models assessed associations between pre-chemotherapy NLR (%neutrophils÷%lymphocytes) or NLR dynamics during chemotherapy (ΔNLR = pre-surgery-pre-chemotherapy NLR) and pathologic response, disease-free survival (DFS), and overall survival (OS). To preclinically model effects of NLR attenuation on chemosensitivity, Ptf1aCre/+; KrasLSL-G12D/+;Tgfbr2flox/flox (PKT) mice and C57BL/6 mice orthotopically injected with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1Cre(KPC) cells were randomized to vehicle, gemcitabine/paclitaxel alone, and NLR-attenuating anti-Ly6G with/without gemcitabine/paclitaxel treatment. In 94 PDAC patients undergoing NAC (median:4 months), pre-chemotherapy NLR (p<0.001) and ΔNLR attenuation during NAC (p=0.002) were independently associated with partial/complete pathologic response. An NLR score = pre-chemotherapy NLR+ΔNLR correlated with DFS (p=0.006) and OS (p=0.002). Upon preclinical modeling, combining NLR-attenuating anti-Ly6G treatment with gemcitabine/paclitaxel-compared with gemcitabine/paclitaxel or anti-Ly6G alone-not only significantly reduced tumor burden and metastatic outgrowth, but also augmented tumor-infiltrating CD107a+-degranulating CD8+ T-cells (p<0.01) while dampening inflammatory cancer-associated fibroblast (CAF) polarization (p=0.006) and chemoresistant IL-6/STAT-3 signaling in vivo. Neutrophil-derived IL-1β emerged as a novel mediator of stromal inflammation, inducing inflammatory CAF polarization and CAF-tumor cell IL-6/STAT-3 signaling in ex vivo co-cultures. Therapeutic strategies to mitigate neutrophil-CAF-tumor cell IL-1β/IL-6/STAT-3 signaling during NAC may improve pathologic responses and/or survival in PDAC. Supported by KL2 career development grant by Miami CTSI under NIH Award UL1TR002736, Stanley Glaser Foundation, American College of Surgeons Franklin Martin Career Development Award, and Association for Academic Surgery Joel J. Roslyn Faculty Award (to J. Datta); NIH R01 CA161976 (to N.B. Merchant); and NCI/NIH Award P30CA240139 (to J. Datta and N.B. Merchant).

Running Biomechanics Before ACL Injury In Collegiate Athletes Do Not Differ From Healthy, Matched Controls

Biomechanical movement screens are commonly used to identify potential risk factors contributing to a variety of musculoskeletal injuries. Such an approach is challenging for anterior cruciate ligament (ACL) injuries given the low incidence rate. However, resulting time loss from sport and poor long-term health outcomes justify the need to begin to use available data. A comprehensive assessment of lower extremity joint mechanics during running is a useful first step in exploring potential movement-related risk factors for ACL injuries. PURPOSE: To determine if healthy, baseline running mechanics differ between collegiate athletes who did and did not go on to suffer a primary ACL injury. METHODS: Whole body kinematics and ground reaction forces (GRF) during treadmill running were collected as part of pre-season performance testing. Thirteen division I collegiate athletes (6 female; 19.1 ± 1.1 years, 86.8 ± 17.0 kg, 1.79 ± 0.10 m) went on to suffer an ACL injury (PRE). HC (n = 13) were matched to PRE (6 female, 18.9 ± 1.0 years, 86.2 ± 17.1 kg, 1.79 ± 0.08 m, 4.2 ± 0.4 m/s and by sport). Multiple statistical parametric mapping (SPM) paired t-tests (α = 0.05) were used to explore differences between PRE and HC for vertical GRF, ankle, knee, and hip angles, moments, and powers during the stance phase of running for injured and uninjured limbs. HC were randomly assigned an injured limb. RESULTS: No significant difference were detected between PRE and HC athletes for any variable on either limb during the stance phase of running (all SPM{t} values < critical threshold t-values). Figure 1 depicts the SPM results for knee angles, moments, and powers of the injured limb. CONCLUSIONS: Pre-season screening of running biomechanics in collegiate athletes is unlikely to identify those at risk for sustaining an ACL injury. Screening higher risk movement patterns may reveal ACL injury risk factors. NIH award TL1TR002375.

The Effects of Chemotherapy on Circulating Plasma Omega-9, Omega-3 Fatty Acids and Plasmalogen in Breast Cancer Patients

ObjectivesChemotherapy produces long-term cognitive impairment (CICI) in ∼30% of those receiving solid tumor treatment. Our previous results revealed a positive correlation between doxorubicin treatment and murine hippocampal concentrations of omega-9 fatty acids, relative to untreated controls. Our objective was to measure these and other structurally analogous fatty acids in human plasma following breast-cancer treatment, to determine if they might serve as biomarkers and/or nutritional targets during treatment. MethodsSerum samples were collected from patients (n = 51) at ≥ 2 of 3 visits: immediately prior to standard adjuvant and neo-adjuvant chemotherapy for breast cancer (baseline), prior to the third cycle of chemotherapy, and 6 months after chemotherapy termination. Lipophilic extracts of serum were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to quantify eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA), linoleic acid, oleic acid, mead acid, gondoic acid, erucic acid, nervonic acid and PE(P-18:0/22:6) (plasmalogen). Linear models assessed the impact of visit on fatty acid concentrations, with a fixed intercept used for each subject. A P < 0.05 for chemotherapy was considered significant. ResultsChemotherapy significantly increased plasma concentrations of gondoic, erucic, nervonic acids and plasmalogen (with no effect on the other fatty acids tested). Six months following chemotherapy, concentrations of gondoic, erucic, nervonic acids and plasmalogen approached baseline concentrations. ConclusionsBreast cancer chemotherapy significantly increased plasma concentrations of omega-9 fatty acids previously reported as increased in subjects with increasing severity of cognitive impairment and Alzheimer’s disease. Ongoing work includes correlating the concentrations of these fatty acids with measures of memory and cognition. Future investigations will determine if these compounds may serve as nutritional targets for dietary interventions prior to or during chemotherapy treatment, and/or serve as objective biomarkers of CICI. Funding SourcesThe Ohio State University Stefanie Spielman Breast Cancer Center Kroger Fund, Pelotonia, NIH R01CA189947, NIH Award Number Grant P30 CA016058, OSU, and OSUCCC.

CGAS-STING Mediated Inflammation In Diabetic Wound Macrophages Is Regulated By Histone Demethylase JMJD3

Abstract Recent work has shown that the cGAS-STING inflammatory pathway is elevated in conditions that are marked by chronic inflammation, such as obesity and diabetes. Using single cell RNA (scRNA) sequencing from human wounds, we show that the Sting gene is increased in diabetic wound macrophages. Histone demethylase JMJD3 specifically demethylates lysine 27 on histone 3 (H3K27me3) - a repressive mark for gene transcription. Using diabetic mice, we note on ChIP analysis, a decrease in the H3K27me3 repressive mark at the STING promoter in diabetic wound macrophages relative to littermate controls, with subsequently enhanced STING protein levels by western blot. Additionally, scRNA from wound macrophages obtained from a novel murine strain deficient in Jmjd3 in myeloid-cell lineages (Jmjd3f/fLyz2Cre+), revealed significantly decreased Sting gene levels. Further, ChIP analysis of H3K27me3 levels at the STING promoter from Jmjd3f/fLyz2Cre+ wound macrophages reveal increased H3K27me3 levels compared to littermate controls, with a concomitant decrease in STING protein as determined by western blot. Mechanistically, we use mRNA, protein and ChIP studies to reveal that in diabetic wound macrophages, IL6 is responsible for increased JMJD3 leading to increased JAK1,3/STAT3 activity and increased STING production. Wound healing curves from diabetic mice locally treated with macrophage-specific nanoparticles against JMJD3 showed significantly improved healing, and decreased STING protein production in these wounds. Taken together, this data highlights a unique epigenetic regulator of STING production in diabetic wound macrophages and provides a viable cell-specific, nanotherapeutic approach to non-healing diabetic wounds. Supported by NIH (F32DK126471), Vascular and Endovascular Surgical Society Resident Research Award

Monocyte-directed targeting of VEGFA in the melanoma microenvironment

Abstract VEGFA plays a critical role in the immunosuppressive cancer response and related immunotherapy resistance. Combined with immune checkpoint inhibitors (ICI) such as those against CTLA-4 and PD-L1, anti-VEGFA agents have demonstrated early clinical success in metastatic melanoma and renal cell carcinomas. To overcome toxicity limitations, thereby opening a wider therapeutic window, VEGFA should be targeted near its production source. Recently, localized co-targeting of TGFβ, an immunosuppressive factor, near CD4+ T cells through bispecific “trap” antibodies have demonstrated robust pre-clinical success. Thus, we assert the superiority of a bispecific antibody guiding a “trap” for VEGFA to its cellular source over global anti-VEGFA agents combined with ICIs. We set out to identify the cell population responsible for secreting VEGFA in the melanoma microenvironment. Upon re-analysis of two existing, independent single cell RNA Sequencing datasets of patient-derived melanoma treated with ICIs, we identified myeloid populations as the main producers of VEGFA. We specifically found a classical monocyte population (CD14+ FCN1+ S100A8+) to express high levels of VEGFA. Of note, we discovered significantly higher VEGFA expression in monocytes from non-responders versus responders to ICIs. VEGFA-expressing monocytes are localized to the intratumoral environment, as our analysis of PBMC-derived monocytes lacked high VEGFA expression in comparison to those that were tumor-derived. Based on our findings, we propose the design of a bispecific antibody that simultaneously targets VEGFA and CD14 to leverage the synergistic pro-immune effects and toxicity diminishment that result from localized inhibition. Supported by the NIH (R01 CA236910) and Melanoma Research Alliance Young Investigator Award to Willy Hugo.

A Ketogenic Diet Prevents Methylglyoxal-Evoked Nociception by Scavenging Methylglyoxal

Methylglyoxal is a reactive dicarbonyl byproduct of glycolysis that has been implicated in a growing number of chronic pain conditions, including diabetic peripheral neuropathy (DPN). We previously demonstrated the efficacy of a ketogenic diet in reversing sensory symptoms in a rodent model of DPN. The purpose of this study was to determine whether a ketogenic diet modified methylglyoxal-evoked nociception as a potential mechanism of improving sensory symptoms of DPN. We delivered methylglyoxal to C57Bl/6 mice by intraperitoneal or intraplantar injections. Sensory behaviors were quantified for mechanical thresholds as assessed by von Frey filament testing and spontaneous nonreflexive nociceptive behaviors (shaking, biting, lifting, etc. of the injected paw). Intraperitoneal methylglyoxal injection induced lasting mechanical allodynia in standard chow-fed mice, while ketogenic diet-fed mice were protected. Importantly, ketogenic diet-fed mice exhibited significantly decreased methylglyoxylated-protein concentrations following injection relative to standard, chow-fed mice, and methylglyoxylated-protein concentration correlated negatively with blood ketones. A reaction between the ketone body acetoacetate and methylglyoxal was recently demonstrated in vivo as a potential mechanism of methylglyoxal detoxification, consistent with this observation. To assess whether ketone bodies modified methylglyoxal-evoked nociception by direct methylglyoxal detoxification, we incubated methylglyoxal with either β-hydroxybutyrate or acetoacetate overnight prior to intraplantar injection. Mice receiving an injection of methylglyoxal alone exhibited an increase in spontaneous nociceptive behaviors, whereas coincubation of either acetoacetate or β-hydroxybutyrate reduced these behaviors. In summary, a ketogenic diet prevented the onset of methylglyoxal-evoked mechanical allodynia and increased scavenging of circulating methylglyoxal. Ketone bodies were individually capable of detoxifying methylglyoxal to prevent nociception. These findings provide insight to a potential mechanism by which a ketogenic diet improves DPN in mice and provide a rationale for exploring a ketogenic diet as a therapeutic intervention in other chronic pain conditions associated with elevated methylglyoxal. This work was supported by NIH grants RO1 NS043314 (DEW), the Kansas Institutional Development Award (IDeA) P20 GM103418, Kansas University Training Program in Neurological and Rehabilitation Sciences (NIH T32 award) supported by NIH Award Number T32HD057850, and core support from the Kansas IDDRC P30 HD00228. Methylglyoxal is a reactive dicarbonyl byproduct of glycolysis that has been implicated in a growing number of chronic pain conditions, including diabetic peripheral neuropathy (DPN). We previously demonstrated the efficacy of a ketogenic diet in reversing sensory symptoms in a rodent model of DPN. The purpose of this study was to determine whether a ketogenic diet modified methylglyoxal-evoked nociception as a potential mechanism of improving sensory symptoms of DPN. We delivered methylglyoxal to C57Bl/6 mice by intraperitoneal or intraplantar injections. Sensory behaviors were quantified for mechanical thresholds as assessed by von Frey filament testing and spontaneous nonreflexive nociceptive behaviors (shaking, biting, lifting, etc. of the injected paw). Intraperitoneal methylglyoxal injection induced lasting mechanical allodynia in standard chow-fed mice, while ketogenic diet-fed mice were protected. Importantly, ketogenic diet-fed mice exhibited significantly decreased methylglyoxylated-protein concentrations following injection relative to standard, chow-fed mice, and methylglyoxylated-protein concentration correlated negatively with blood ketones. A reaction between the ketone body acetoacetate and methylglyoxal was recently demonstrated in vivo as a potential mechanism of methylglyoxal detoxification, consistent with this observation. To assess whether ketone bodies modified methylglyoxal-evoked nociception by direct methylglyoxal detoxification, we incubated methylglyoxal with either β-hydroxybutyrate or acetoacetate overnight prior to intraplantar injection. Mice receiving an injection of methylglyoxal alone exhibited an increase in spontaneous nociceptive behaviors, whereas coincubation of either acetoacetate or β-hydroxybutyrate reduced these behaviors. In summary, a ketogenic diet prevented the onset of methylglyoxal-evoked mechanical allodynia and increased scavenging of circulating methylglyoxal. Ketone bodies were individually capable of detoxifying methylglyoxal to prevent nociception. These findings provide insight to a potential mechanism by which a ketogenic diet improves DPN in mice and provide a rationale for exploring a ketogenic diet as a therapeutic intervention in other chronic pain conditions associated with elevated methylglyoxal. This work was supported by NIH grants RO1 NS043314 (DEW), the Kansas Institutional Development Award (IDeA) P20 GM103418, Kansas University Training Program in Neurological and Rehabilitation Sciences (NIH T32 award) supported by NIH Award Number T32HD057850, and core support from the Kansas IDDRC P30 HD00228.

Endogenous interferon-lambda signaling restricts virus replication and disease severity in a murine model of SARS-CoV-2 infection

Abstract Interferon-lambda (IFN-λ) is currently being investigated in a phase III clinical trial as a therapeutic against COVID-19. Exogenous IFN-λ restricts SARS-CoV-2 in vitro and in Balb/c and C57Bl/6 (WT) murine models of infection. However, roles of endogenously produced IFN-λ in SARS-CoV-2 pathogenesis are not currently known, and the overall mechanisms by which IFN-λ modulates the induction of protective immune responses in SARS-CoV-2 infections remains to be elucidated. We find that IFN-λ receptor deficient mice (Ifnlr1−/−) infected with mouse-adapted SARS-CoV-2 lose significantly more weight and have increased SARS-CoV-2 viral replication compared to WT through day 5 post infection. Intriguingly, Ifit3 and Ifitm3 are increased in the lungs of Ifnlr1−/− mice compared to WT following infection, despite similar IFN-α/β and IFN-λ mRNA levels, suggesting compensatory increases in type I IFN signaling are not driving the increased weight loss or ISG induction observed in Ifnlr1−/− mice. Global transcriptomics revealed induction of a suppressive immunoregulatory signature with increased IL-10 as a hallmark in Ifnlr1−/− lungs, suggesting IFN-λ is critically involved in regulating appropriate immune activation to limit SARS-CoV-2 pathogenesis. Histological analysis revealed a significant increase in CD45+ cells (but not neutrophils) in the lungs of Ifnlr1−/− mice compared to WT on day 5 post infection, and we identified increases in pathways associated with myeloid cell function and T cell activation in Ifnlr1−/− by comparative transcriptomics. Overall, broadening the understanding of how IFN-λ regulates SARS-CoV-2 infection, pathogenesis, and immunity will inform the utilization of IFN-λ as an immunotherapy and adjuvant. Supported by NIH Award K22 AI146141 to EAH.

Improving Visualization of cAMP Gradients Using Algorithmic Modelling.

A ubiquitous second messenger molecule, cAMP is responsible for orchestrating many different cellular functions through a variety of pathways. Fӧrster resonance energy transfer (FRET) probes have been used to visualize cAMP spatial gradients in pulmonary microvascular endothelial cells (PMVECs). However, FRET probes have inherently low signal-to-noise ratios; multiple sources of noise can obscure accurate visualization of cAMP gradients using a hyperspectral imaging system. FRET probes have also been used to measure cAMP gradients in 3D; however, it can be difficult to differentiate between true FRET signals and noise. To further understand the effects of noise on experimental data, a model was developed to simulate cAMP gradients under experimental conditions. The model uses a theoretical cAMP heatmap generated using finite element analysis. This heatmap was converted to simulate the FRET probe signal that would be detected experimentally with a hyperspectral imaging system. The signal was mapped onto an image of unlabeled PMVECs. The result was a time lapse model of cAMP gradients obscured by autofluorescence, as visualized with FRET probes. Additionally, the model allowed the simulated expression level of FRET signal to be varied. This allowed accurate attribution of signal to FRET and autofluorescence. Comparing experimental data to the model results at different levels of FRET efficiency has allowed improved understanding of FRET signal specificity and how autofluorescence interferes with FRET signal detection. In conclusion, this model can more accurately determine cAMP gradients in PMVECs. This work was supported by NIH award P01HL066299, R01HL58506 and NSF award 1725937.

LC-MS and 1H-NMR Untargeted Metabolomic Profiling of Probiotic Yogurt Consumption in C56BL6/N Mice

ObjectivesProbiotic yogurt consumption is associated with gut health, including nutrient production and immune regulation, largely via modulation of the gut microbiome. However, metabolites that mediate host-microbiome interactions are largely unknown. We hypothesize consumption of probiotic yogurt results in distinct metabolic signatures in colon, including decreased levels of branched-chain amino acids, triglycerides, and bile acids relative to probiotic-free yogurt, milk, and water controls. We further hypothesize these differences are driven by increased abundances of Lactobacilli and Bifidobacteria following probiotic yogurt consumption. MethodsA juvenile mouse model (3 weeks old, n = 32 male, 32 female) was fed AIN-93G diet for 3 weeks, followed by 3 weeks of daily gavage of 150 μL probiotic yogurt (PY), heat-inactivated yogurt (HY), milk (M), or water (W). Animals were cohoused in the initial phase and singly housed in the gavage phase. Polar and nonpolar colon extracts were analyzed with ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) and 1H NMR metabolomics. Features detected by1H-NMR and C18 LC-MS in positive mode were compared, pairwise, between diet groups. ResultsMetabolite classes detected via UHPLC-HRMS included bile acids, amino acids and phospholipids. NMR yielded the following number of features with an absolute log2-fold change > 2 and unadjusted P-value < 0.05 between dietary groups: 74 (PY v HY), 17 (PY v M), 57 (PY v C), 79 (HY v M), 14 (HY v C), and 47 (M v C). C18 LC-MS in positive mode yielded the following numbers of metabolites with the same cutoffs used in NMR: 220 (PY v HY), 39 (PY v M), 196 (PY v C), 80 (HY v M), 58 (HY v C), and 61 (M v C). ConclusionsAs hypothesized, probiotic yogurt consumption for 3 weeks significantly altered the colon metabolome relative to controls. Further metabolite identification, pathway enrichment analysis, and next generation microbiome sequencing analyses will aid in identifying specific pathways altered upon PY consumption and correlated with alterations in colonic microbial taxa abundances. Ultimately, this work will help us to better understand how PY consumption influences gastrointestinal health in children. Funding SourcesDannon Gut Microbiome, Yogurt and Probiotics Fellowship Grant awarded to HC. Sample analyses partially supported by an NIH Award.

The Effects of Chemotherapy on Circulating Plasma Carotenoids and Fat-Soluble Vitamins in Breast Cancer Patients

Abstract Objectives Chemotherapy upregulates inflammatory processes as measured by circulating concentrations of pro-inflammatory cytokines and their signaling lipids. Our objective was to observe if breast cancer chemotherapy influenced the circulating concentrations of provitamin A and non-provitamin A carotenoids and fat-soluble vitamins (FSVs) in free-living patients. Methods Serum samples were collected from patients (n = 34) immediately prior to standard adjuvant and neo-adjuvant chemotherapy for breast cancer, and 4 months following chemotherapy commencement. Patient multivitamin and non-steroidal anti-inflammatory (NSAID) drug use was noted at both visits. Lipophilic extracts of serum were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to quantify α- and β-carotene, lycopene, lutein, zeaxanthin, β-cryptoxanthin, retinol, α-tocopherol and phylloquinone. Linear mixed models were developed to assess the relationship between the main factors (i.e., chemotherapy, multivitamin, and NSAID use), and their interaction effects, on serum carotenoid and fat-soluble vitamin concentrations. Random effects included a fixed intercept for each subject. Results Chemotherapy was significantly associated with reduced serum concentrations of α-carotene (P = 0.053) and retinol (P = 0.042), with a trend observed for reduced β-carotene (P = 0.076) and phylloquinone (P = 0.082). There was no main effect of multivitamin or NSAID use on any analytes investigated. An interaction effect was observed for chemotherapy * multivitamin use, with increased concentrations of serum retinol (P = 0.004) and lycopene (P = 0.004), and a trend observed for zeaxanthin (P = 0.087) for those who took multivitamins. Chemotherapy * NSAID use was also significantly associated with a trend in increased serum lutein (P = 0.061) for those who consumed NSAIDS. Conclusions Our results suggest randomized, controlled trials of multivitamin use and/or provitamin A carotenoid-rich food consumption merit further investigation in patients undergoing chemotherapy treatment. Funding Sources This research was supported by The Ohio State University Stefanie Spielman Breast Cancer Center Kroger Fund, Pelotonia, NIH R01CA189947, NIH Award Number Grant P30 CA016058, OSU, and OSUCCC.

657 Characteristics and merits of NIH award recipients of dermatology research

657 Characteristics and merits of NIH award recipients of dermatology research