Endogenously produced itaconate negatively regulates innate-driven cytokine production and drives global ubiquitination in human macrophages.

A wide variety of electrophilic derivatives of itaconate, the Kreb's cycle-derived metabolite, are immunomodulatory, yet these derivatives have overlapping and sometimes contradictory activities. Therefore, we generated a genetic system to interrogate the immunomodulatory functions of endogenously produced itaconate in human macrophages. Endogenous itaconate is driven by multiple innate signals restraining inflammatory cytokine production. Endogenous itaconate directly targets cysteine 13 in IRAK4 (disrupting IRAK4 autophosphorylation and activation), drives the degradation of nuclear factor κB, and modulates global ubiquitination patterns. As a result, cells unable to make itaconate overproduce inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and IL-1ß in response to these innate activators. In contrast, the production of interferon (IFN)ß, downstream of LPS, requires the production of itaconate. These data demonstrate that itaconate is a critical arbiter of inflammatory cytokine production downstream of multiple innate signaling pathways, laying the groundwork for the development of itaconate mimetics for the treatment of autoimmunity.

SHIP1 modulation and proteome characterization of microglia.

Understanding microglial states in the aging brain has become crucial, especially with the discovery of numerous Alzheimer's disease (AD) risk and protective variants in genes such as INPP5D and TREM2, which are essential to microglia function in AD. Here we present a thorough examination of microglia-like cells and primary mouse microglia at the proteome and transcriptome levels to illuminate the roles these genes and the proteins they encode play in various cell states. First, we compared the proteome profiles of wildtype and INPP5D (SHIP1) knockout primary microglia. Our findings revealed significant proteome alterations only in the homozygous SHIP1 knockout, revealing its impact on the microglial proteome. Additionally, we compared the proteome and transcriptome profiles of commonly used in vitro microglia BV2 and HMC3 cells with primary mouse microglia. Our results demonstrated a substantial similarity between the proteome of BV2 and mouse primary cells, while notable differences were observed between BV2 and human HMC3. Lastly, we conducted targeted lipidomic analysis to quantify different phosphatidylinositols (PIs) species, which are direct SHIP1 targets, in the HMC3 and BV2 cells. This in-depth omics analysis of both mouse and human microglia enhances our systematic understanding of these microglia models. SIGNIFICANCE: Given the growing urgency of comprehending microglial function in the context of neurodegenerative diseases and the substantial therapeutic implications associated with SHIP1 modulation, we firmly believe that our study, through a rigorous and comprehensive proteomics, transcriptomics and targeted lipidomic analysis of microglia, contributes to the systematic understanding of microglial function in the context of neurodegenerative diseases.

Pharmacological Evaluation of a Pegylated Urocortin-1 Peptide in Experimental Autoimmune Disease Models.

Urocortin-1 (UCN1) is a member of the corticotropin releasing hormone (CRH) family of peptides that acts through CRH-receptor 1 (CRHR1) and CRH-receptor 2 (CRHR2). UCN1 can induce the adrenocorticotropin hormone and downstream glucocorticoids through CRHR1 and promote beneficial metabolic effects through CRHR2. UCN1 has a short half-life and has been shown to improve experimental autoimmune disease. A pegylated UCN1 peptide (PEG-hUCN1) was generated to extend half-life and was tested in multiple experimental autoimmune disease models and in healthy mice to determine effects on corticosterone induction, autoimmune disease, and glucocorticoid induced adverse effects. Cardiovascular effects were also assessed by telemetry. PEG-hUCN1 demonstrated a dose dependent 4-6-fold elevation of serum corticosterone and significantly improved autoimmune disease comparable to prednisolone in several experimental models. In healthy mice, PEG-hUCN1 showed less adverse effects compared with corticosterone treatment. PEG-hUCN1 peptide induced an initial 30% reduction in blood pressure that was followed by a gradual and sustained 30% increase in blood pressure at the highest dose. Additionally, an adeno-associated viral 8 (AAV8) UCN1 was used to assess adverse effects of chronic elevation of UCN1 in wild type and CRHR2 knockout mice. Chronic UCN1 expression by an AAV8 approach in wild type and CRHR2 knockout mice demonstrated an important role of CRHR2 in countering the adverse metabolic effects of elevated corticosterone from UCN1. Our findings demonstrate that PEG-hUCN1 shows profound effects in treating autoimmune disease with an improved safety profile relative to corticosterone and that CRHR2 activity is important in metabolic regulation. SIGNIFICANCE STATEMENT: This study reports the generation and characterization of a pegylated UCN1 peptide and the role of CRHR2 in UCN1-induced metabolic effects. The potency/selectivity, pharmacokinetic properties, pharmacodynamic effects, and efficacy in four autoimmune models and safety profiles are presented. This pegylated UCN1 shows potential for treating autoimmune diseases with reduced adverse effects compared to corticosterone treatment. Continuous exposure to UCN1 through an AAV8 approach demonstrates some glucocorticoid mediated adverse metabolic effects that are exacerbated in the absence of the CRHR2 receptor.

Effects of Tirzepatide, a Dual GIP and GLP-1 RA, on Lipid and Metabolite Profiles in Subjects With Type 2 Diabetes.

CONTEXT: Tirzepatide substantially reduced hemoglobin A1c (HbA1c) and body weight in subjects with type 2 diabetes (T2D) compared with the glucagon-like peptide 1 receptor agonist dulaglutide. Improved glycemic control was associated with lower circulating triglycerides and lipoprotein markers and improved markers of beta-cell function and insulin resistance (IR), effects only partially attributable to weight loss. OBJECTIVE: Assess plasma metabolome changes mediated by tirzepatide. DESIGN: Phase 2b trial participants were randomly assigned to receive weekly subcutaneous tirzepatide, dulaglutide, or placebo for 26 weeks. Post hoc exploratory metabolomics and lipidomics analyses were performed. SETTING: Post hoc analysis. PARTICIPANTS: 259 subjects with T2D. INTERVENTION(S): Tirzepatide (1, 5, 10, 15 mg), dulaglutide (1.5 mg), or placebo. MAIN OUTCOME MEASURE(S): Changes in metabolite levels in response to tirzepatide were assessed against baseline levels, dulaglutide, and placebo using multiplicity correction. RESULTS: At 26 weeks, a higher dose tirzepatide modulated a cluster of metabolites and lipids associated with IR, obesity, and future T2D risk. Branched-chain amino acids, direct catabolic products glutamate, 3-hydroxyisobutyrate, branched-chain ketoacids, and indirect byproducts such as 2-hydroxybutyrate decreased compared to baseline and placebo. Changes were significantly larger with tirzepatide compared with dulaglutide and directly proportional to reductions of HbA1c, homeostatic model assessment 2-IR indices, and proinsulin levels. Proportional to metabolite changes, triglycerides and diglycerides were lowered significantly compared to baseline, dulaglutide, and placebo, with a bias toward shorter and highly saturated species. CONCLUSIONS: Tirzepatide reduces body weight and improves glycemic control and uniquely modulates metabolites associated with T2D risk and metabolic dysregulation in a direction consistent with improved metabolic health.

Sex Differences in Insulin Sensitivity are Related to Muscle Tissue Acylcarnitine But Not Subcellular Lipid Distribution.

OBJECTIVE: Sex differences in insulin sensitivity are present throughout the life-span, with men having a higher prevalence of insulin resistance and diabetes compared with women. Differences in lean mass, fat mass, and fat distribution-particularly ectopic fat-have all been postulated to contribute to the sexual dimorphism in diabetes risk. Emerging data suggest ectopic lipid composition and subcellular localization are most relevant; however, it is not known whether they explain sex differences in obesity-induced insulin resistance. METHODS: To address this gap, this study evaluated insulin sensitivity and subcellular localization of intramuscular triacylglycerol, diacylglycerol, and sphingolipids as well as muscle acylcarnitines and serum lipidomics in people with obesity. RESULTS: Insulin sensitivity was significantly lower in men (P < 0.05); however, no sex differences were found in localization of intramuscular triacylglycerol, diacylglycerol, or sphingolipids in skeletal muscle. In contrast, men had higher total muscle acylcarnitine (P < 0.05) and long-chain muscle acylcarnitine (P < 0.05), which were related to lower insulin sensitivity (r = -0.42, P < 0.05). Men also displayed higher serum ceramide (P = 0.05) and lysophosphatidylcholine (P < 0.01). CONCLUSIONS: These data reveal novel sex-specific associations between lipid species involved in the coupling of mitochondrial fatty acid transport, ß-oxidation, and tricarboxylic acid cycle flux that may provide therapeutic targets to improve insulin sensitivity.

Development of ion pairing LC-MS/MS method for itaconate and cis-aconitate in cell extract and cell media.

Accumulation of Immune Responsive Gene 1(IRG1) in macrophage induced by lipopolysaccharide (LPS) and interferon gamma (IFN-γ) leads to production of itaconate by decarboxylation of cis-aconitate. The biology associated with IRG1 and itaconate is not fully understood. A rapid and sensitive method for measurement of itaconate will benefit the study of IRG1 biology. Multiple HPLC and derivatization methods were tested. An ion pairing LC-MS/MS method using tributylamine/formic acid as ion pairing agents and a HypercarbTM guard column we proposed demonstrated better peak shape and better sensitivity for itaconate. The current protocol allows baseline separation of itaconate, citraconate, and cis-aconitate without derivatization and direct analysis of analytes in 80% methanol/water solution to avoid the dry-down step. It provides the limit of quantitation (LOQ) of 30 pg itaconate on column with a 4.5-minute run time. This method is validated for measurement of itaconate and cis-aconitate in RAW264.7 cell extract and cell media in a 96-well plate format. We applied this method to successfully measure the increase of itaconate and the decrease of cis-aconitate in RAW cell extract and cell media after LPS/IFN-γ treatment.

Nucleotides released from palmitate-activated murine macrophages attract neutrophils.

Obesity and elevation of circulating free fatty acids are associated with an accumulation and proinflammatory polarization of macrophages within metabolically active tissues, such as adipose tissue, muscle, liver, and pancreas. Beyond macrophages, neutrophils also accumulate in adipose and muscle tissues during high-fat diets and contribute to a state of local inflammation and insulin resistance. However, the mechanisms by which neutrophils are recruited to these tissues are largely unknown. Here we used a cell culture system as proof of concept to show that, upon exposure to a saturated fatty acid, palmitate, macrophages release nucleotides that attract neutrophils. Moreover, we found that palmitate up-regulates pannexin-1 channels in macrophages that mediate the attraction of neutrophils, shown previously to allow transfer of nucleotides across membranes. These findings suggest that proinflammatory macrophages release nucleotides through pannexin-1, a process that may facilitate neutrophil recruitment into metabolic tissues during obesity.

The Folate Pathway Inhibitor Pemetrexed Pleiotropically Enhances Effects of Cancer Immunotherapy.

PURPOSE: Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non-small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown. EXPERIMENTAL DESIGN: Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis. RESULTS: Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell-intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1. CONCLUSIONS: Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell-intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade.See related commentary by Buque et al., p. 6890.

2-Hydroxy-oleic acid does not activate sphingomyelin synthase activity.

2-Hydroxy-oleic acid (2OHOA) is a potent anticancer drug that induces cancer cell cycle arrest and apoptosis. Previous studies have suggested that 2OHOA's anticancer effect is mediated by SMS activation in cancer cells, including A549 and U118 cells. To confirm this phenomenon, in this study, we treated both A549 and U118 cells with 2OHOA and measured SMS activity. To our surprise, we found neither 2OHOA-mediated SMS activation nor sphingomyelin accumulation in the cells. However, we noted that 2OHOA significantly reduces phosphatidylcholine in these cells. We also did not observe 2OHOA-mediated SMS activation in mouse tissue homogenates. Importantly, 2OHOA inhibited rather than activated recombinant SMS1 (rSMS1) and rSMS2 in a dose-dependent fashion. Intra-gastric treatment of C57BL/6J mice with 2OHOA for 10 days had no effects on liver and small intestine SMS activities and plasma sphingomyelin levels. The treatment inhibited lysophosphatidylcholine acyltransferase (LPCAT) activity, consistent with the aforementioned reduction in plasma phosphatidylcholine. Because total cellular phosphatidylcholine is used as a predictive biomarker for monitoring tumor responses, the previously reported 2OHOA-mediated cancer suppression could be related to this phosphatidylcholine reduction, which may influence cell membrane structure and properties. We conclude that 2OHOA is not a SMS activator and that its anticancer property may be related to an effect on phosphatidylcholine metabolism.

Characterization of a novel AICARFT inhibitor which potently elevates ZMP and has anti-tumor activity in murine models.

AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.

Discovery of N-(6-Fluoro-1-oxo-1,2-dihydroisoquinolin-7-yl)-5-[(3R)-3-hydroxypyrrolidin-1-yl]thiophene-2-sulfonamide (LSN 3213128), a Potent and Selective Nonclassical Antifolate Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase (AICARFT) Inhibitor Effective at Tumor Suppression in a Cancer Xenograft Model.

A hallmark of cancer is unbridled proliferation that can result in increased demand for de novo synthesis of purine and pyrimidine bases required for DNA and RNA biosynthesis. These synthetic pathways are frequently upregulated in cancer and involve various folate-dependent enzymes. Antifolates have a proven record as clinically used oncolytic agents. Our recent research efforts have produced LSN 3213128 (compound 28a), a novel, selective, nonclassical, orally bioavailable antifolate with potent and specific inhibitory activity for aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), an enzyme in the purine biosynthetic pathway. Inhibition of AICARFT with compound 28a results in dramatic elevation of 5-aminoimidazole 4-carboxamide ribonucleotide (ZMP) and growth inhibition in NCI-H460 and MDA-MB-231met2 cancer cell lines. Treatment with this inhibitor in a murine based xenograft model of triple negative breast cancer (TNBC) resulted in tumor growth inhibition.

Synthesis of Diverse N-Acryloyl Azetidines and Evaluation of Their Enhanced Thiol Reactivities.

Acyl azetidines exhibit nonplanar hybridization, leading to lower amide-like character of the corresponding (O)C-N bonds. This impacts N-acryloyl azetidines by producing enhanced electrophilicy at appended Michael acceptors. Herein, reactivity data are reported in the presence of glutathione (GSH) in phosphate buffer (pH 7.4) at 37 °C. Wide reactivity ranges are observed by varying substitution at the Michael acceptor or by modulating the electron-withdrawing character of substituents at the C3 position of the azetidine.

Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis, Leads to Altered Carbohydrate Metabolism in Cancer Cells.

Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD(+) biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD(+) biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500-3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed.

Interleukin-3-deficient mice have increased resistance to blood-stage malaria.

The contribution of interleukin-3 (IL-3), a hematopoietic growth factor and immunoregulatory cytokine, to resistance to blood-stage malaria was investigated by infecting IL-3-deficient (knockout [KO]) mice with Plasmodium berghei NK65. Male IL-3 KO mice, but not female mice, were more resistant to infection than wild-type (WT) mice, as evidenced by lower peak parasitemia and prolonged survival. Both male and female IL-3 KO mice had increased splenomegaly and were more anemic than corresponding WT mice. Anemia was compensated for by an increase in bone marrow and splenic erythropoiesis in IL-3 KO mice, as evidenced by higher levels of erythroid progenitors. Plasma levels of gamma interferon (IFN-γ) and CXCL9 (monokine induced by IFN-γ [MIG]) were found to be significantly reduced in IL-3 KO mice during early stages of infection. In contrast, granulocyte colony-stimulating factor (G-CSF) levels were significantly higher, and the percentage of peripheral blood neutrophils lower, in infected IL-3 KO mice than in WT counterparts. Overall, our results indicate that IL-3 plays a critical role in suppressing protective immunity to P. berghei NK65 infection and that it is involved in inhibiting the development of splenomegaly, anemia, and erythropoiesis. IL-3 also influences IFN-γ, CXCL9, and G-CSF production in response to infection. The abnormal responses seen in infected IL-3 KO mice may be due to the lack of IL-3 during development, to the lack of IL-3 in the infected mature mice, or to both.

Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD+ biosynthesis, in human cancer cells: metabolic basis and potential clinical implications.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD(+), essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD(+) for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic.