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The ketogenic diet (KD) has demonstrated benefits in numerous clinical studies and animal models of disease in modulating the immune response and promoting a systemic anti-inflammatory state. Here we investigate the effects of a KD on systemic toxicity in mice following SARS-CoV-2 infection. Our data indicate that under KD, SARS-CoV-2 reduces weight loss with overall improved animal survival. Muted multi-organ transcriptional reprogramming and metabolism rewiring suggest that a KD initiates and mitigates systemic changes induced by the virus. We observed reduced metalloproteases and increased inflammatory homeostatic protein transcription in the heart, with decreased serum pro-inflammatory cytokines (i.e., TNF-α, IL-15, IL-22, G-CSF, M-CSF, MCP-1), metabolic markers of inflammation (i.e., kynurenine/tryptophane ratio), and inflammatory prostaglandins, indicative of reduced systemic inflammation in animals infected under a KD. Taken together, these data suggest that a KD can alter the transcriptional and metabolic response in animals following SARS-CoV-2 infection with improved mice health, reduced inflammation, and restored amino acid, nucleotide, lipid, and energy currency metabolism.

Glioblastoma (GBM) is incurable despite aggressive standard of care treatments (maximal safe surgical resection, radiation, chemotherapy). GBM therapeutic resistance is due to multiple factors, including tumor heterogeneity and a highly immunosuppressive environment. Naturally occurring polyamines have been identified as a putative therapeutic target in other cancers based on their increased presence and function in normal conditions; they are critical for cell growth and proliferation and cellular functions including autophagy and apoptosis. While polyamines are increased in GBM patients, little is known about their impact on GBM growth. In syngeneic immune competent mouse glioma models (GL261, SB28), mass spectrometry data revealed that spermidine (SPD) – a member of the polyamine family – is increased in tumor tissue as compared to non-neoplastic control brain tissue (sham implanted animals). To test the impact of SPD on tumor growth, we treated mouse glioma models with exogenous SPD and found treatment significantly decreased survival. However, in immunocompromised host mice, no such difference was observed, indicating the mechanism through which SPD is driving GBM progression likely involves immune system alterations. Depletion of myeloid derived suppressor cells in vivo via anti-Gr-1 antibody rescues the decrease in survival caused by exogenous SPD, indicating that SPD drives GBM by affecting immune-suppressive cell subsets, namely MDSCs. To assess if SPD is associated with more aggressive GBM growth in human patients, we are currently analyzing polyamine levels in human GBM samples of long vs short term survivors. We are also exploring the effect of dietary polyamines on GBM via the gut-brain-microbiome axis, as polyamines are enriched in many foods and produced by a subset of commensal gut microbes. Understanding the interactions between polyamines, the tumor microenvironment, and the immune response provide a new mechanism for GBM regulation and identify opportunities to alter the environment in the body to enhance immunotherapeutic efficacy.

Background: The gut microbiota is a metabolically active endocrine organ critical to the maintenance of cardiovascular health. Dietary sources of choline are metabolized by microbial enzymes to form trimethylamine (TMA). Metabolism by the host hepatic enzyme flavin-containing monooxygenase 3 (FMO3) converts TMA to the pro-inflammatory molecule trimethylamine N-oxide (TMAO). Human clinical trials have correlated high levels of circulating TMAO to an increased risk of cardiometabolic diseases. However, this meta-organismal pathway has not been evaluated in the context of abdominal aortic aneurysm (AAA). The objective of this study was to determine the effects of a high choline diet on the development of AAA. Methods: C57BL/6J male (n=20) and female (n=20) mice were fed either a standard chow control diet (n = 10 each sex) or a choline-rich diet (1%; n = 10 each sex) for 5 weeks. After 1 week of diet, basal abdominal ultrasounds were performed and angiotensin II (AngII; 1,000 ng/kg/min) was infused for 28 days via implantation of osmotic minipumps. Termination ultrasounds were performed on day 27 and mice were sacrificed on day 28. Aortas were harvested for evaluation of aneurysm progression and plasma was analyzed for the metabolites TMA, TMAO, and choline. To determine whether TMAO was elevated in human patients with AAA, plasma samples from participants with fast growing AAAs (n = 85), slow growing AAAs (n = 84), and normal (non-aneurysmal) aortas (n = 115) were analyzed for plasma TMAO levels via liquid chromatography tandem mass spectrometry (LC-MS/MS). Results: Administration of a choline-rich diet augmented the incidence (P < 0.02) and aortic diameter (P < 0.001) of AAAs in both male and female mice versus placebo-fed mice. Plasma levels of TMA, TMAO, and choline were significantly elevated in choline-fed mice versus normal chow (P < 0.05). Importantly, circulating levels of plasma TMAO were significantly elevated in a step-wise fashion with the rate of aneurysm growth versus non-aneurysmal control patients (fast growing > slow growing > normal patients; P < 0.001). Conclusions: Our results indicate increases in circulating TMAO augments the growth status of aneurysms in human patients and the incidence of AAA in a low penetrant C57BL/6J mouse model.

HDL functions are impaired by myeloperoxidase (MPO), which selectively targets and oxidizes human apoA1. We previously found that the 4WF isoform of human apoA1, in which the four tryptophan residues are substituted with phenylalanine, is resistant to MPO-mediated loss of function. The purpose of this study was to generate 4WF apoA1 transgenic mice and compare functional properties of the 4WF and wild-type human apoA1 isoforms in vivo. Male mice had significantly higher plasma apoA1 levels than females for both isoforms of human apoA1, attributed to different production rates. With matched plasma apoA1 levels, 4WF transgenics had a trend for slightly less HDL-cholesterol versus human apoA1 transgenics. While 4WF transgenics had 31% less reverse cholesterol transport (RCT) to the plasma compartment, equivalent RCT to the liver and feces was observed. Plasma from both strains had similar ability to accept cholesterol and facilitate ex vivo cholesterol efflux from macrophages. Furthermore, we observed that 4WF transgenic HDL was partially (∼50%) protected from MPO-mediated loss of function while human apoA1 transgenic HDL lost all ABCA1-dependent cholesterol acceptor activity. In conclusion, the structure and function of HDL from 4WF transgenic mice was not different than HDL derived from human apoA1 transgenic mice.

Atherosclerosis is lipid-driven, chronic inflammatory disease of the arterial wall. While commensal microbiota is involved in the distal regulation of systemic immune responses, how this distant connection influences the development of atherosclerosis and what are the underlying mechanisms remains largely unknown. In a mouse model of atherosclerosis, we found that disease was augmented when expression of the otherwise inflammatory cytokine IL23 was ablated. IL23 and its immediate downstream target IL22 restrict atherosclerosis by preventing outgrowth of microbiota, as inactivation of IL23/IL22 signaling led to dysbiosis and expansion of bacteria with pro-atherogenic properties, due to defective production of antimicrobial peptides in the intestine. These pro-atherogenic bacteria contributed to elevated serum levels of several pro-atherogenic metabolites, which in turn induced osteopontin (OPN) expression by subsets of myeloid cells including aortic macrophages. Microbiota transfer from IL23 deficient mice accelerated atherosclerosis, while microbial depletion or IL22 administration reduced aortic osteopontin expression and ameliorated the disease. Overall, our work uncovers the innate inflammatory IL23-IL22 cytokine signaling axis as a key regulator of atherosclerosis that controls diet-induced expansion of pro-atherogenic microbiota, and argues for informed usage of cytokine blockers with regard to cardiovascular side effects driven by microbiota and inflammation

Introduction: The ability of a simple self-assessment tool for estimated functional capacity to predict long-term prognosis in patients with established peripheral artery disease (PAD) is unknown. Objective: This study examined whether subjective measurement of functional capacity estimated by using of the Duke Activity Status Index (DASI) questionnaire predicts long-term prognosis in patients with established PAD. Methods: We examined the relationship between DASI scores and all-cause mortality over a 5-year follow-up in 771 stable patients with established PAD. Results: Two hundred ten patients (27%) died over 5-year follow-up . The lowest DASI scores were associated with a 3.2-fold increased risk of 5-year all-cause mortality (HR 3.23, 95%CI 2.19-4.75, p Conclusions: A simple self-assessment tool of functional capacity provides independent and incremental prognostic for long-term adverse clinical events in stable patients with established PAD beyond each PAD diagnostic subtype.

Background: We previously reported that functional iron deficiency, assessed by low mean corpuscular hemoglobin concentration (MCHC), has been associated with adverse outcomes in non-anemic patients with HF. Yet, the effect of change in MCHC levels on outcomes has not been studied before. Methods: We prospectively enrolled 1,579 subjects with HF undergoing coronary angiography, with 5-year follow-up. Blood indices [hemoglobin (Hb), MCHC, MCV, RDW] were assessed on enrollment and after 6 months of follow up by reviewing medical records. Anemia was defined as Hb levels Results: In our population (age 66 ± 10 years, male 65%, CAD 75%), most patients were non-anemic (n=1,161, 73.5%). In non-anemic patients, mean Hb and median (IQR) MCHC were 13.3±1.2 g/dL and 34.2 (33.43-34.9) g/dL, respectively. Non-anemic HF patients with lower MCHC had higher mortality risk [Q1 vs Q4 Hazard ratio (HR) (95%CI) of 2.5(1.9-3.2), p Conclusion: Relative hypochromia is an independent predictor of increase mortality in patients with HF, even in the setting of normal hemoglobin levels.

Rationale: Trimethylamine N-oxide (TMAO), a metabolite formed in the metabolism of dietary phosphatidylcholine, is elevated in the circulation of patients at increased risk for heart attack and adverse prognosis during heart failure. Objective: We investigated the effects of dietary choline and TMAO on the severity of heart failure following transverse aortic constriction (TAC). Methods and Results: Male C57Blk/6J mice were fed either control diet, a diet containing choline (1.2%) or a diet containing TMAO (0.12%) at 3 weeks prior to surgical TAC and were studied for 12 weeks. Left ventricular (LV) structure and function were monitored at 3 week intervals and myocardial tissue was collected at 12 weeks. Plasma TMAO levels were significantly (p < 0.01) increased in the choline (28.64 ± 2.30 μM) and TMAO (28.18 ± 4.27 μM) compared to the control group (1.87 ± 0.26 μM). Left ventricular ejection fraction (LVEF) was significantly (p < 0.05) worse in mice fed TMAO compared to control diet. LV end-diastolic and end-systolic diameters were significantly (p < 0.05) increased in the TMAO group compared to control diet. Myocardial fibrosis as measured with Picrosirius Red staining was also significantly greater (p < 0.01) in the TMAO and choline groups. Circulating BNP levels were significantly (p < 0.05) increased in the TMAO and choline groups. Conclusions: These data demonstrate that heart failure severity is significantly enhanced in mice fed diets containing either TMAO or choline. Our results suggest that consumption of food high in dietary nutrients that increase TMAO levels such as phosphatidylcholine may increase heart failure severity.

Introduction: ApoAI-ABCA1 mediated nascent HDL assembly promotes cholesterol efflux and reverse cholesterol transport, but the mechanism of HDL assembly is unknown. Objective: To determine role of PIP2 in ApoAI-ABCA1 mediated nascent HDL assembly. Methods and Results: To determine which lipid species are directly bound by apoAI, a lipid-protein overlay assay was performed using PIPstrips and sphingostrips from Echelon Biosciences. ApoAI bound directly to phosphatidylinositol phosphates (PIPs) rather than PC, PS, PE, SM or cholesterol. This interaction was not solely electrostatic, as PIP3, the most negative charged PIP had less apoAI binding than various PIP2 species. ApoAI did not bind to other charged lipids such as sphingosine 1-P, PI(3)-P, PI(4)-P, and PI(5)-P. Surface plasmon resonance assays using immobilized apoAI or PIP2 were performed, showing that apoAI interacted reversibly with PI(4,5)P2 in a dose dependent manner at the nM concentration range. The central domain of apoAI (residues 44-185) was sufficient for PIP2 binding. In RAW264.7 macrophages, depletion of cellular PIPs by treatments with PI3K inhibitor or PTEN inhibitor decreased cholesterol efflux by 45±1.4% and 41±6.4% respectively (po C, pH5, but apoAI had little activity using similar liposomes without PIP2 . ABCA1 expression led to 2-fold higher cell surface PIP2, assayed by flow cytometry using a PIP2 specific antibody. RAW cells stably transfected with PIP2 binding domain 2X PH-PLCδ-eGFP showed enrichment of this PIP2 reporter on the plasma membrane, while expression of ABCA1 led to partial redistribution to cytoplasm, consistent with ABCA1 having PIP2 floppase activity. Conclusions: ApoAI has novel PIP2 binding activity, ABCA1 is a novel PIP2 floppase, and cell surface PIP2 is required for optimal cholesterol acceptor activity of apoAI.