Dietary Supplementation with 23-Hydroxy Ursolic Acid Reduces the Severity and Incidence of Acute Experimental Autoimmune Encephalomyelitis (EAE) in a Murine Model of Multiple Sclerosis.

23-Hydroxy ursolic acid (23-OH UA) is a potent atheroprotective and anti-obesogenic phytochemical, with anti-inflammatory and inflammation-resolving properties. In this study, we examined whether dietary 23-OH UA protects mice against the acute onset and progression of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). Female C57BL/6 mice were fed either a defined low-calorie maintenance diet (MD) or an MD supplemented with 0.2% wgt/wgt 23-OH UA for 5 weeks prior to actively inducing EAE and during the 30 days post-immunization. We observed no difference in the onset of EAE between the groups of mice, but ataxia and EAE disease severity were suppressed by 52% and 48%, respectively, and disease incidence was reduced by over 49% in mice that received 23-OH UA in their diet. Furthermore, disease-associated weight loss was strikingly ameliorated in 23-OH UA-fed mice. ELISPOT analysis showed no significant differences in frequencies of T cells producing IL-17 or IFN-γ between 23-OH UA-fed mice and control mice, suggesting that 23-OH UA does not appear to regulate peripheral T cell responses. In summary, our findings in EAE mice strongly suggest that dietary 23-OH UA may represent an effective oral adjunct therapy for the prevention and treatment of relapsing-remitting MS.

Macrophage-restricted overexpression of glutaredoxin 1 protects against atherosclerosis by preventing nutrient stress-induced macrophage dysfunction and reprogramming.

BACKGROUND AND AIMS: Deficiency in the thiol transferase glutaredoxin 1 (Grx1) in aging mice promotes, in a sexually dimorphic manner, dysregulation of macrophages and atherogenesis. However, the underlying mechanisms are not known. Here we tested the hypothesis that macrophage-restricted overexpression of Grx1 protects atherosclerosis-prone mice against macrophage reprogramming and dysfunction induced by a high-calorie diet (HCD) and thereby reduces the severity of atherosclerosis. METHODS: We generated lentiviral vectors carrying cluster of differentiation 68 (CD68) promoter-driven enhanced green fluorescent protein (EGFP) or Grx1 constructs and conducted bone marrow (BM) transplantation studies to overexpress Grx1 in a macrophage-specific manner in male and female atherosclerosis-prone LDLR-/- mice, and fed these mice a HCD to induce atherogenesis. Atherosclerotic lesion size was determined in both the aortic root and the aorta. We isolated BM-derived macrophages (BMDM) to assess protein S-glutathionylation levels and loss of mitogen-activated protein kinase phosphatase 1 (MKP-1) activity as measures of HCD-induced thiol oxidative stress. We also conducted gene profiling on these BMDM to determine the impact of Grx1 activity on HCD-induced macrophage reprogramming. RESULTS: Overexpression of Grx1 protected macrophages against HCD-induced protein S-glutathionylation, reduced monocyte chemotaxis in vivo, limited macrophage recruitment into atherosclerotic lesions, and was sufficient to reduce the severity of atherogenesis in both male and female mice. Gene profiling revealed major sex differences in the transcriptional reprogramming of macrophages induced by HCD feeding, but Grx1 overexpression only partially reversed HCD-induced transcriptional reprogramming of macrophages. CONCLUSIONS: Macrophage Grx1 plays a major role in protecting mice atherosclerosis mainly by maintaining the thiol redox state of the macrophage proteome and preventing macrophage dysfunction.

Glutaredoxin 1 controls monocyte reprogramming during nutrient stress and protects mice against obesity and atherosclerosis in a sex-specific manner.

High-calorie diet-induced nutrient stress promotes thiol oxidative stress and the reprogramming of blood monocytes, giving rise to dysregulated, obesogenic, proatherogenic monocyte-derived macrophages. We report that in chow-fed, reproductively senescent female mice but not in age-matched male mice, deficiency in the thiol transferase glutaredoxin 1 (Grx1) promotes dysregulated macrophage phenotypes as well as rapid weight gain and atherogenesis. Grx1 deficiency derepresses distinct expression patterns of reactive oxygen species and reactive nitrogen species generators in male versus female macrophages, poising female but not male macrophages for increased peroxynitrate production. Hematopoietic Grx1 deficiency recapitulates this sexual dimorphism in high-calorie diet-fed LDLR-/- mice, whereas macrophage-restricted overexpression of Grx1 eliminates the sex differences unmasked by high-calorie diet-feeding and protects both males and females against atherogenesis. We conclude that loss of monocytic Grx1 activity disrupts the immunometabolic balance in mice and derepresses sexually dimorphic oxidative stress responses in macrophages. This mechanism may contribute to the sex differences reported in cardiovascular disease and obesity in humans.

Ursolic Acid and Related Analogues: Triterpenoids with Broad Health Benefits.

Ursolic acid (UA) is a well-studied natural pentacyclic triterpenoid found in herbs, fruit and a number of traditional Chinese medicinal plants. UA has a broad range of biological activities and numerous potential health benefits. In this review, we summarize the current data on the bioavailability and pharmacokinetics of UA and review the literature on the biological activities of UA and its closest analogues in the context of inflammation, metabolic diseases, including liver and kidney diseases, obesity and diabetes, cardiovascular diseases, cancer, and neurological disorders. We end with a brief overview of UA's main analogues with a special focus on a newly discovered naturally occurring analogue with intriguing biological properties and potential health benefits, 23-hydroxy ursolic acid.

Inhibition of myeloid HDAC2 upregulates glutaredoxin 1 expression, improves protein thiol redox state and protects against high-calorie diet-induced monocyte dysfunction and atherosclerosis.

BACKGROUND AND AIMS: The thiol transferase glutaredoxin 1 controls redox signaling and cellular functions by regulating the S-glutathionylation status of critical protein thiols. Here we tested the hypothesis that by derepressing the expression of glutaredoxin 1, inhibition of histone deacetylase 2 prevents nutrient stress-induced protein S-glutathionylation and monocyte dysfunction and protects against atherosclerosis. METHODS: Using both a pharmacological inhibitor and shRNA-mediated knockdown of histone deacetylase 2, we determine the role of this deacetylase on glutaredoxin 1 expression and nutrient stress-induced inactivation of mitogen-activated protein kinase phosphatase 1 activity and monocyte and macrophage dysfunction. To assess whether histone deacetylase 2 inhibition in myeloid cells protects against atherosclerosis, we fed eight-week-old female and male HDAC2-/-MyeloidLDLR-/- mice and age and sex-matched LysMcretg/wtLDLR-/- control mice a high-calorie diet for 12 weeks and assessed monocyte function and atherosclerotic lesion size. RESULTS: Myeloid histone deacetylase 2 deficiency in high-calorie diet-fed LDLR-/- mice reduced atherosclerosis in males by 39% without affecting plasma lipid and lipoprotein profiles or blood glucose levels but had no effect on atherogenesis in female mice. Macrophage content in plaques of male mice was reduced by 31%. Histone deacetylase 2-deficient blood monocytes from male mice showed increased acetylation on histone 3, and increased Grx1 expression, and was associated with increased MKP-1 activity and reduced recruitment of monocyte-derived macrophages, whereas in females, myeloid HDAC2 deficiency had no effect on Grx1 expression, did not prevent nutrient stress-induced loss of MKP-1 activity in monocytes and was not atheroprotective. CONCLUSIONS: Specific histone deacetylase 2 inhibitors may represent a potential novel therapeutic strategy for the prevention and treatment of atherosclerosis, but any benefits may be sexually dimorphic.

Oxidatively modified low-density lipoproteins are potential mediators of proteasome inhibitor resistance in multiple myeloma.

Proteasome inhibitor (PI) therapy has improved the survival of multiple myeloma (MM) patients. However, inevitably, primary or acquired resistance to PIs leads to disease progression; resistance mechanisms are unclear. Obesity is a risk factor for MM mortality. Oxidized LDL (OxLDL), a central mediator of atherosclerosis that is elevated in metabolic syndrome (co-occurrence of obesity, insulin resistance, dyslipidemia and hypertension), has been linked to an increased risk of solid cancers and shown to stimulate pro-oncogenic/survival signaling. We hypothesized that OxLDL is a mediator of chemoresistance and evaluated its effects on MM cell killing by PIs. OxLDL potently suppressed the ability of the boronic acid-based PIs bortezomib (BTZ) and ixazomib, but not the epoxyketone-based PI carfilzomib, to kill human MM cell lines and primary cells. OxLDL suppressed BTZ-induced inhibition of proteasome activity and induction of pro-apoptotic signaling. These cytoprotective effects were abrogated when lipid hydroperoxides (LOOHs) associated with OxLDL were enzymatically reduced. We also demonstrated the presence of OxLDL in the MM bone marrow microenvironment as well as numerous granulocytes and monocytes capable of cell-mediated LDL oxidation through myeloperoxidase. Our findings suggest that OxLDL may be a potent mediator of boronic acid-based PI resistance, particularly for MM patients with metabolic syndrome, given their elevated systemic levels of OxLDL. LDL cholesterol-lowering therapy to reduce circulating OxLDL, and pharmacologic targeting of LOOH levels or resistance pathways induced by the modified lipoprotein, could deepen the response to these important agents and offer clinical benefit to MM patients with metabolic syndrome.

Dietary 23-hydroxy ursolic acid protects against diet-induced weight gain and hyperglycemia by protecting monocytes and macrophages against nutrient stress-triggered reprogramming and dysfunction and preventing adipose tissue inflammation.

The aim of this study was to determine whether the atheroprotective phytochemical 23-hydroxy ursolic acid protects against diet-induced obesity and hyperglycemia by preventing nutrient stress-induced monocyte reprogramming. After a two week run-in period on a defined, phytochemical-free low-fat maintenance diet, 12-week old female C57BL/6J mice were either kept on the maintenance diet for additional 13 weeks or switched to either a high-calorie diet, a high-calorie diet supplemented with either 0.05% 23-hydroxy ursolic acid or a high-calorie diet supplemented with 0.2% 23-hydroxy ursolic acid. Dietary supplementation with 23-hydroxy ursolic acid reduced weight gain and adipose tissue mass, prevented hyperglycemia, hyperleptinemia and adipose tissue inflammation, and preserved glucose tolerance. 23-Hydroxy ursolic acid also preserved blood monocyte mitogen-activated protein kinase phosphatase-1 activity, a biomarker of monocyte health, and reduced macrophage content in the adipose tissue. Targeted gene profiling by qRT-PCR using custom-designed TaqMan® Array Cards revealed that dietary 23-hydroxy ursolic acid converts macrophages into a transcriptionally hyperactive phenotype with enhanced antioxidant defenses and anti-inflammatory potential. In conclusion, our findings show that dietary 23-hydroxy ursolic acid exerts both anti-obesogenic effects through multiple mechanisms. These include improving glucose tolerance, preventing hyperleptinemia, maintaining blood monocyte function, reducing recruitment of monocyte-derived macrophages into adipose tissues during nutrient stress, and converting these macrophages into an anti-inflammatory, potentially inflammation-resolving phenotype, all contributing to reduced adipose tissue inflammation. Our data suggest that 23-hydroxy ursolic acid may serve as an oral therapeutic and dietary supplement suited for patients at risk for obesity, impaired glucose tolerance and cardiovascular disease.

Sexual dimorphism in glutathione metabolism and glutathione-dependent responses.

Glutathione is the most abundant intracellular low molecular weight thiol in cells and tissues, and plays an essential role in numerous cellular processes, including antioxidant defenses, the regulation of protein function, protein localization and stability, DNA synthesis, gene expression, cell proliferation, and cell signaling. Sexual dimorphisms in glutathione biology, metabolism and glutathione-dependent signaling have been reported for a broad range of biological processes, spanning the human lifespan from early development to aging. Sex-depended differences with regard to glutathione and its biology have also been reported for a number of human pathologies and diseases such as neurodegeneration, cardiovascular diseases and metabolic disorders. Here we review the latest literature in this field and discuss the potential impact of these sexual dimorphisms in glutathione biology on human health and diseases.

The Need for Multi-Omics Biomarker Signatures in Precision Medicine.

Recent advances in omics technologies have led to unprecedented efforts characterizing the molecular changes that underlie the development and progression of a wide array of complex human diseases, including cancer. As a result, multi-omics analyses-which take advantage of these technologies in genomics, transcriptomics, epigenomics, proteomics, metabolomics, and other omics areas-have been proposed and heralded as the key to advancing precision medicine in the clinic. In the field of precision oncology, genomics approaches, and, more recently, other omics analyses have helped reveal several key mechanisms in cancer development, treatment resistance, and recurrence risk, and several of these findings have been implemented in clinical oncology to help guide treatment decisions. However, truly integrated multi-omics analyses have not been applied widely, preventing further advances in precision medicine. Additional efforts are needed to develop the analytical infrastructure necessary to generate, analyze, and annotate multi-omics data effectively to inform precision medicine-based decision-making.

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages.

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

The role of glial-neuronal metabolic cooperation in modulating progression of multiple sclerosis and neuropathic pain.

While the etiology of multiple sclerosis (MS) remains unclear, research from the clinic and preclinical models identified the essential role of inflammation and demyelination in the pathogenesis of MS. Current treatments focused on anti-inflammatory processes are effective against acute episodes and relapsing-remitting MS, but patients still move on to develop secondary progressive MS. MS progression is associated with activation of microglia and astrocytes, and importantly, metabolic dysfunction leading to neuronal death. Neuronal death also contributes to chronic neuropathic pain. Metabolic support of neurons by glia may play central roles in preventing progression of MS and chronic neuropathic pain. Here, we review mechanisms of metabolic cooperation between glia and neurons and outline future perspectives exploring metabolic support of neurons by glia.

Dietary 23-hydroxy ursolic acid protects against atherosclerosis and obesity by preventing dyslipidemia-induced monocyte priming and dysfunction.

BACKGROUND AND AIMS: We demonstrated that dietary ursolic acid (UA) reduces atherosclerotic lesion size and improves kidney function in diabetic mice. Based on structure-function analyses of naturally occurring UA analogs, we synthesized 23-hydroxy ursolic acid (23-OHUA), a compound with structural features predicted to enhance its bioavailability and anti-atherogenic properties compared to UA. The goal of this study was to determine the anti-obesogenic and atheroprotective properties of 23-OHUA and its mechanism of action. METHODS: We performed chemotaxis assays to determine IC50 of phytochemicals on primed THP-1 monocytes. We fed 12-week old female LDLR-/- mice a high-fat diet (HFD) or a HFD supplemented with either 0.05% UA or 0.05% 23-OHUA, and measured monocyte priming, weight gain and atherosclerotic lesion size after 6 and 20 weeks. RESULTS: Both dietary UA and 23-OHUA prevented dyslipidemia-induced loss of MKP-1 activity, and hyper-chemotactic activity, hallmarks of blood monocytes priming and dysfunction, but they did not affect plasma lipids or blood glucose levels nor WBC and monocyte counts. After 20 weeks, mice fed 23-OHUA showed 11% less weight gain compared to HFD-fed control mice and a 40% reduction in atherosclerotic plaque size, whereas UA reduced lesion size by only 19% and did not reduce weight gain. CONCLUSIONS: Dietary 23-OHUA reduces weight gain and attenuates atherogenesis in mice by protecting monocytes against metabolic stress-induced priming and dysfunction. Based on its mechanism of action, 23-OHUA may represent a novel therapeutic approach for the prevention and treatment of obesity and atherosclerosis.

Interactions of ß tubulin isotypes with glutathione in differentiated neuroblastoma cells subject to oxidative stress.

Microtubules are a major component of the neuronal cytoskeleton. Tubulin, the subunit protein of microtubules, is an α/ß heterodimer. Both α and ß exist as families of isotypes, whose members are encoded by different genes and have different amino acid sequences. The ßII and ßIII isotypes are very prominent in the nervous system. Our previous work has suggested that ßII may play a role in neuronal differentiation, but the role of ßIII in neurons is not well understood. In the work reported here, we examined the roles of the different ß-tubulin isotypes in response to glutamate/glycine treatment, and found that both ßII and ßIII bind to glutathione in the presence of ROS, especially ßIII. In contrast, ßI did not bind to glutathione. Our results suggest that ßII and ßIII, but especially ßIII, may play an important role in the response of neuronal cells to stress. In view of the high levels of ßII and ßIII expressed in the nervous system it is conceivable that these tubulin isotypes may use their sulfhydryl groups to scavenge ROS and protect neuronal cells against oxidative stress.

mTOR drives cerebral blood flow and memory deficits in LDLR-/- mice modeling atherosclerosis and vascular cognitive impairment.

We recently showed that mTOR attenuation blocks progression and abrogates established cognitive deficits in Alzheimer's disease (AD) mouse models. These outcomes were associated with the restoration of cerebral blood flow (CBF) and brain vascular density (BVD) resulting from relief of mTOR inhibition of NO release. Recent reports suggested a role of mTOR in atherosclerosis. Because mTOR drives aging and vascular dysfunction is a universal feature of aging, we hypothesized that mTOR may contribute to brain vascular and cognitive dysfunction associated with atherosclerosis. We measured CBF, BVD, cognitive function, markers of inflammation, and parameters of cardiovascular disease in LDLR-/- mice fed maintenance or high-fat diet ± rapamycin. Cardiovascular pathologies were proportional to severity of brain vascular dysfunction. Aortic atheromas were reduced, CBF and BVD were restored, and cognitive dysfunction was attenuated potentially through reduction in systemic and brain inflammation following chronic mTOR attenuation. Our studies suggest that mTOR regulates vascular integrity and function and that mTOR attenuation may restore neurovascular function and cardiovascular health. Together with our previous studies in AD models, our data suggest mTOR-driven vascular damage may be a mechanism shared by age-associated neurological diseases. Therefore, mTOR attenuation may have promise for treatment of cognitive impairment in atherosclerosis.

Acute maternal oxidant exposure causes susceptibility of the fetal brain to inflammation and oxidative stress.

BACKGROUND: Maternal exposure to environmental stressors poses a risk to fetal development. Oxidative stress (OS), microglia activation, and inflammation are three tightly linked mechanisms that emerge as a causal factor of neurodevelopmental anomalies associated with prenatal ethanol exposure. Antioxidants such as glutathione (GSH) and CuZnSOD are perturbed, and their manipulation provides evidence for neuroprotection. However, the cellular and molecular effects of GSH alteration in utero on fetal microglia activation and inflammation remain elusive. METHODS: Ethanol (EtOH) (2.5 g/kg) was administered to pregnant mice at gestational days 16-17. One hour prior to ethanol treatment, N-acetylcysteine (NAC) and L-buthionine sulfoximine (BSO) were administered to modulate glutathione (GSH) content in fetal and maternal brain. Twenty-four hours following ethanol exposure, GSH content and OS in brain tissues were analyzed. Cytokines and chemokines were selected based on their association with distinctive microglia phenotype M1-like (IL-1ß, IFN γ, IL-6, CCL3, CCL4, CCL-7, CCL9,) or M2-like (TGF-ß, IL-4, IL-10, CCL2, CCL22, CXCL10, Arg1, Chi1, CCR2 and CXCR2) and measured in the brain by qRT-PCR and ELISA. In addition, Western blot and confocal microscopy techniques in conjunction with EOC13.31 cells exposed to similar ethanol-induced oxidative stress and redox conditions were used to determine the underlying mechanism of microglia activation associated with the observed phenotypic changes. RESULTS: We show that a single episode of mild to moderate OS in the last trimester of gestation causes GSH depletion, increased protein and lipid peroxidation and inflammatory responses inclined towards a M1-like microglial phenotype (IL-1ß, IFN-γ) in fetal brain tissue observed at 6-24 h post exposure. Maternal brain is resistant to many of these marked changes. Using EOC 13.31 cells, we show that GSH homeostasis in microglia is crucial to restore its anti-inflammatory state and modulate inflammation. Microglia under oxidative stress maintain a predominantly M1 activation state. Additionally, GSH depletion prevents the appearance of the M2-like phenotype, while enhancing morphological changes associated with a M1-like phenotype. This observation is also validated by an increased expression of inflammatory signatures (IL-1ß, IFN-γ, IL-6, CCL9, CXCR2). In contrast, conserving intracellular GSH concentrations eliminates OS which precludes the nuclear translocation and more importantly the phosphorylation of the NFkB p105 subunit. These cells show significantly more pronounced elongations, ramifications, and the enhanced expression of M2-like microglial phenotype markers (IL-10, IL-4, TGF-ß, CXCL10, CCL22, Chi, Arg, and CCR2). CONCLUSIONS: Taken together, our data show that maintaining GSH homeostasis is not only important for quenching OS in the developing fetal brain, but equally critical to enhance M2 like microglia phenotype, thus suppressing inflammatory responses elicited by environmental stressors.

Dyslipidemic Diet-Induced Monocyte "Priming" and Dysfunction in Non-Human Primates Is Triggered by Elevated Plasma Cholesterol and Accompanied by Altered Histone Acetylation.

Monocytes and the recruitment of monocyte-derived macrophages into sites of inflammation play a key role in atherogenesis and other chronic inflammatory diseases linked to cardiometabolic syndrome and obesity. Previous studies from our group have shown that metabolic stress promotes monocyte priming, i.e., enhanced adhesion and accelerated chemotaxis of monocytes in response to chemokines, both in vitro and in dyslipidemic LDLR-/- mice. We also showed that metabolic stress-induced monocyte dysfunction is, at least to a large extent caused by the S-glutathionylation, inactivation, and subsequent degradation of mitogen-activated protein kinase phosphatase 1. Here, we analyzed the effects of a Western-style, dyslipidemic diet (DD), which was composed of high levels of saturated fat, cholesterol, and simple sugars, on monocyte (dys)function in non-human primates (NHPs). We found that similar to mice, a DD enhances monocyte chemotaxis in NHP within 4 weeks, occurring concordantly with the onset of hypercholesterolemia but prior to changes in triglycerides, blood glucose, monocytosis, or changes in monocyte subset composition. In addition, we identified transitory decreases in the acetylation of histone H3 at the lysine residues 18 and 23 in metabolically primed monocytes, and we found that monocyte priming was correlated with the acetylation of histone H3 at lysine 27 after an 8-week DD regimen. Our data show that metabolic stress promotes monocyte priming and hyper-chemotactic responses in NHP. The histone modifications accompanying monocyte priming in primates suggest a reprogramming of the epigenetic landscape, which may lead to dysregulated responses and functionalities in macrophages derived from primed monocytes that are recruited to sites of inflammation.