Reduction of SPARC protects mice against NLRP3 inflammasome activation and obesity.

The comprehensive assessment of long-term effects of reducing intake of energy (CALERIE-II; NCT00427193) clinical trial established that caloric restriction (CR) in humans lowers inflammation. The identity and mechanism of endogenous CR-mimetics that can be deployed to control obesity-associated inflammation and diseases are not well understood. Our studies have found that 2 years of 14% sustained CR in humans inhibits the expression of the matricellular protein, secreted protein acidic and rich in cysteine (SPARC), in adipose tissue. In mice, adipose tissue remodeling caused by weight loss through CR and low-protein diet feeding decreased, while high-fat diet-induced (HFD-induced) obesity increased SPARC expression in adipose tissue. Inducible SPARC downregulation in adult mice mimicked CR's effects on lowering adiposity by regulating energy expenditure. Deletion of SPARC in adipocytes was sufficient to protect mice against HFD-induced adiposity, chronic inflammation, and metabolic dysfunction. Mechanistically, SPARC activates the NLRP3 inflammasome at the priming step and downregulation of SPARC lowers macrophage inflammation in adipose tissue, while excess SPARC activated macrophages via JNK signaling. Collectively, reduction of adipocyte-derived SPARC confers CR-like metabolic and antiinflammatory benefits in obesity by serving as an immunometabolic checkpoint of inflammation.

[WITHDRAWN] Activation of transsulfuration pathway to maintain cysteine is a thermogenic checkpoint for the conservation of energy.

The authors have requested that this preprint be removed from Research Square.

When a calorie is not just a calorie: Diet quality and timing as mediators of metabolism and healthy aging.

An epidemic of obesity has affected large portions of the world, increasing the risk of developing many different age-associated diseases, including cancer, cardiovascular disease, and diabetes. In contrast with the prevailing notion that "a calorie is just a calorie," there are clear differences, within and between individuals, in the metabolic response to different macronutrient sources. Recent findings challenge this oversimplification; calories from different macronutrient sources or consumed at different times of day have metabolic effects beyond their value as fuel. Here, we summarize discussions conducted at a recent NIH workshop that brought together experts in calorie restriction, macronutrient composition, and time-restricted feeding to discuss how dietary composition and feeding schedule impact whole-body metabolism, longevity, and healthspan. These discussions may provide insights into the long-sought molecular mechanisms engaged by calorie restriction to extend lifespan, lead to novel therapies, and potentially inform the development of a personalized food-as-medicine approach to healthy aging.

Innate immune cell-intrinsic ketogenesis is dispensable for organismal metabolism and age-related inflammation.

Aging is accompanied by chronic low-grade inflammation, but the mechanisms that allow this to persist are not well understood. Ketone bodies are alternative fuels produced when glucose is limited and improve indicators of healthspan in aging mouse models. Moreover, the most abundant ketone body, ß-hydroxybutyrate, inhibits the NLRP3 inflammasome in myeloid cells, a key potentiator of age-related inflammation. Given that myeloid cells express ketogenic machinery, we hypothesized this pathway may serve as a metabolic checkpoint of inflammation. To test this hypothesis, we conditionally ablated ketogenesis by disrupting expression of the terminal enzyme required for ketogenesis, 3-Hydroxy-3-Methylglutaryl-CoA Lyase (HMGCL). By deleting HMGCL in the liver, we validated the functional targeting and establish that the liver is the only organ that can produce the life-sustaining quantities of ketone bodies required for survival during fasting or ketogenic diet feeding. Conditional ablation of HMGCL in neutrophils and macrophages had modest effects on body weight and glucose tolerance in aging but worsened glucose homeostasis in myeloid cell-specific Hmgcl-deficient mice fed a high-fat diet. Our results suggest that during aging, liver-derived circulating ketone bodies might be more important for deactivating the NLRP3 inflammasome and controlling organismal metabolism.

The matricellular protein SPARC induces inflammatory interferon-response in macrophages during aging.

The risk of chronic diseases caused by aging is reduced by caloric restriction (CR)-induced immunometabolic adaptation. Here, we found that the matricellular protein, secreted protein acidic and rich in cysteine (SPARC), was inhibited by 2 years of 14% sustained CR in humans and elevated by obesity. SPARC converted anti-inflammatory macrophages into a pro-inflammatory phenotype with induction of interferon-stimulated gene (ISG) expression via the transcription factors IRF3/7. Mechanistically, SPARC-induced ISGs were dependent on toll-like receptor-4 (TLR4)-mediated TBK1, IRF3, IFN-ß, and STAT1 signaling without engaging the Myd88 pathway. Metabolically, SPARC dampened mitochondrial respiration, and inhibition of glycolysis abrogated ISG induction by SPARC in macrophages. Furthermore, the N-terminal acidic domain of SPARC was required for ISG induction, while adipocyte-specific deletion of SPARC reduced inflammation and extended health span during aging. Collectively, SPARC, a CR-mimetic adipokine, is an immunometabolic checkpoint of inflammation and interferon response that may be targeted to delay age-related metabolic and functional decline.

Unified Total Synthesis of the Limonoid Alkaloids: Strategies for the De Novo Synthesis of Highly Substituted Pyridine Scaffolds.

Highly substituted pyridine scaffolds are found in many biologically active natural products and therapeutics. Accordingly, numerous complementary de novo approaches to obtain differentially substituted pyridines have been disclosed. This article delineates the evolution of the synthetic strategies designed to assemble the demanding tetrasubstituted pyridine core present in the limonoid alkaloids isolated from Xylocarpus granatum, including xylogranatopyridine B, granatumine A and related congeners. In addition, NMR calculations suggested structural misassignment of several limonoid alkaloids, and predicted their C3-epimers as the correct structures, which was further validated unequivocally through chemical synthesis. The materials produced in this study were evaluated for cytotoxicity, anti-oxidant effects, anti-inflammatory action, PTP1B and Nlrp3 inflammasome inhibition, which led to compelling anti-inflammatory activity and anti-oxidant effects being discovered.

Desmosterol suppresses macrophage inflammasome activation and protects against vascular inflammation and atherosclerosis.

Cholesterol biosynthetic intermediates, such as lanosterol and desmosterol, are emergent immune regulators of macrophages in response to inflammatory stimuli or lipid overloading, respectively. However, the participation of these sterols in regulating macrophage functions in the physiological context of atherosclerosis, an inflammatory disease driven by the accumulation of cholesterol-laden macrophages in the artery wall, has remained elusive. Here, we report that desmosterol, the most abundant cholesterol biosynthetic intermediate in human coronary artery lesions, plays an essential role during atherogenesis, serving as a key molecule integrating cholesterol homeostasis and immune responses in macrophages. Depletion of desmosterol in myeloid cells by overexpression of 3ß-hydroxysterol Δ24-reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol, promotes the progression of atherosclerosis. Single-cell transcriptomics in isolated CD45+CD11b+ cells from atherosclerotic plaques demonstrate that depletion of desmosterol increases interferon responses and attenuates the expression of antiinflammatory macrophage markers. Lipidomic and transcriptomic analysis of in vivo macrophage foam cells demonstrate that desmosterol is a major endogenous liver X receptor (LXR) ligand involved in LXR/retinoid X receptor (RXR) activation and thus macrophage foam cell formation. Decreased desmosterol accumulation in mitochondria promotes macrophage mitochondrial reactive oxygen species production and NLR family pyrin domain containing 3 (NLRP3)-dependent inflammasome activation. Deficiency of NLRP3 or apoptosis-associated speck-like protein containing a CARD (ASC) rescues the increased inflammasome activity and atherogenesis observed in desmosterol-depleted macrophages. Altogether, these findings underscore the critical function of desmosterol in the atherosclerotic plaque to dampen inflammation by integrating with macrophage cholesterol metabolism and inflammatory activation and protecting from disease progression.

IL-27 signalling promotes adipocyte thermogenesis and energy expenditure.

Thermogenesis in brown and beige adipose tissue has important roles in maintaining body temperature and countering the development of metabolic disorders such as obesity and type 2 diabetes1,2. Although much is known about commitment and activation of brown and beige adipose tissue, its multiple and abundant immunological factors have not been well characterized3-6. Here we define a critical role of IL-27-IL-27Rα signalling in improving thermogenesis, protecting against diet-induced obesity and ameliorating insulin resistance. Mechanistic studies demonstrate that IL-27 directly targets adipocytes, activating p38 MAPK-PGC-1α signalling and stimulating the production of UCP1. Notably, therapeutic administration of IL-27 ameliorated metabolic morbidities in well-established mouse models of obesity. Consistently, individuals with obesity show significantly decreased levels of serum IL-27, which can be restored after bariatric surgery. Collectively, these findings show that IL-27 has an important role in orchestrating metabolic programs, and is a highly promising target for anti-obesity immunotherapy.

Enhanced epigenetic profiling of classical human monocytes reveals a specific signature of healthy aging in the DNA methylome.

The impact of healthy aging on molecular programming of immune cells is poorly understood. Here, we report comprehensive characterization of healthy aging in human classical monocytes, with a focus on epigenomic, transcriptomic, and proteomic alterations, as well as the corresponding proteomic and metabolomic data for plasma, using healthy cohorts of 20 young and 20 older males (~27 and ~64 years old on average). For each individual, we performed eRRBS-based DNA methylation profiling, which allowed us to identify a set of age-associated differentially methylated regions (DMRs) - a novel, cell-type specific signature of aging in DNA methylome. Hypermethylation events were associated with H3K27me3 in the CpG islands near promoters of lowly-expressed genes, while hypomethylated DMRs were enriched in H3K4me1 marked regions and associated with age-related increase of expression of the corresponding genes, providing a link between DNA methylation and age-associated transcriptional changes in primary human cells.

IL-33 causes thermogenic failure in aging by expanding dysfunctional adipose ILC2.

Aging impairs the integrated immunometabolic responses, which have evolved to maintain core body temperature in homeotherms to survive cold stress, infections, and dietary restriction. Adipose tissue inflammation regulates the thermogenic stress response, but how adipose tissue-resident cells instigate thermogenic failure in the aged are unknown. Here, we define alterations in the adipose-resident immune system and identify that type 2 innate lymphoid cells (ILC2s) are lost in aging. Restoration of ILC2 numbers in aged mice to levels seen in adults through IL-33 supplementation failed to rescue old mice from metabolic impairment and increased cold-induced lethality. Transcriptomic analyses revealed intrinsic defects in aged ILC2, and adoptive transfer of adult ILC2s are sufficient to protect old mice against cold. Thus, the functional defects in adipose ILC2s during aging drive thermogenic failure.

Ketogenic diet restrains aging-induced exacerbation of coronavirus infection in mice.

Increasing age is the strongest predictor of risk of COVID-19 severity and mortality. Immunometabolic switch from glycolysis to ketolysis protects against inflammatory damage and influenza infection in adults. To investigate how age compromises defense against coronavirus infection, and whether a pro-longevity ketogenic diet (KD) impacts immune surveillance, we developed an aging model of natural murine beta coronavirus (mCoV) infection with mouse hepatitis virus strain-A59 (MHV-A59). When inoculated intranasally, mCoV is pneumotropic and recapitulates several clinical hallmarks of COVID-19 infection. Aged mCoV-A59-infected mice have increased mortality and higher systemic inflammation in the heart, adipose tissue, and hypothalamus, including neutrophilia and loss of γδ T cells in lungs. Activation of ketogenesis in aged mice expands tissue protective γδ T cells, deactivates the NLRP3 inflammasome, and decreases pathogenic monocytes in lungs of infected aged mice. These data establish harnessing of the ketogenic immunometabolic checkpoint as a potential treatment against coronavirus infection in the aged.

Adiponectin preserves metabolic fitness during aging.

Adiponectin is essential for the regulation of tissue substrate utilization and systemic insulin sensitivity. Clinical studies have suggested a positive association of circulating adiponectin with healthspan and lifespan. However, the direct effects of adiponectin on promoting healthspan and lifespan remain unexplored. Here, we are using an adiponectin null mouse and a transgenic adiponectin overexpression model. We directly assessed the effects of circulating adiponectin on the aging process and found that adiponectin null mice display exacerbated age-related glucose and lipid metabolism disorders. Moreover, adiponectin null mice have a significantly shortened lifespan on both chow and high-fat diet. In contrast, a transgenic mouse model with elevated circulating adiponectin levels has a dramatically improved systemic insulin sensitivity, reduced age-related tissue inflammation and fibrosis, and a prolonged healthspan and median lifespan. These results support a role of adiponectin as an essential regulator for healthspan and lifespan.

Ketogenesis restrains aging-induced exacerbation of COVID in a mouse model.

Increasing age is the strongest predictor of risk of COVID-19 severity. Unregulated cytokine storm together with impaired immunometabolic response leads to highest mortality in elderly infected with SARS-CoV-2. To investigate how aging compromises defense against COVID-19, we developed a model of natural murine beta coronavirus (mCoV) infection with mouse hepatitis virus strain MHV-A59 (mCoV-A59) that recapitulated majority of clinical hallmarks of COVID-19. Aged mCoV-A59-infected mice have increased mortality and higher systemic inflammation in the heart, adipose tissue and hypothalamus, including neutrophilia and loss of γδ T cells in lungs. Ketogenic diet increases beta-hydroxybutyrate, expands tissue protective γδ T cells, deactivates the inflammasome and decreases pathogenic monocytes in lungs of infected aged mice. These data underscore the value of mCoV-A59 model to test mechanism and establishes harnessing of the ketogenic immunometabolic checkpoint as a potential treatment against COVID-19 in the elderly. HIGHLIGHTS: - Natural MHV-A59 mouse coronavirus infection mimics COVID-19 in elderly.- Aged infected mice have systemic inflammation and inflammasome activation.- Murine beta coronavirus (mCoV) infection results in loss of pulmonary γδ T cells.- Ketones protect aged mice from infection by reducing inflammation. ETOC BLURB: Elderly have the greatest risk of death from COVID-19. Here, Ryu et al report an aging mouse model of coronavirus infection that recapitulates clinical hallmarks of COVID-19 seen in elderly. The increased severity of infection in aged animals involved increased inflammasome activation and loss of γδ T cells that was corrected by ketogenic diet.

Dietary Regulation of Immunity.

Integrated immunometabolic responses link dietary intake, energy utilization, and storage to immune regulation of tissue function and is therefore essential for the maintenance and restoration of homeostasis. Adipose-resident leukocytes have non-traditional immunological functions that regulate organismal metabolism by controlling insulin action, lipolysis, and mitochondrial respiration to control the usage of substrates for production of heat versus ATP. Energetically expensive vital functions such as immunological responses might have thus evolved to respond accordingly to dietary surplus and deficit of macronutrient intake. Here, we review the interaction of dietary intake of macronutrients and their metabolism with the immune system. We discuss immunometabolic checkpoints that promote healthspan and highlight how dietary fate and regulation of glucose, fat, and protein metabolism might affect immunity.

Ketogenesis activates metabolically protective γδ T cells in visceral adipose tissue.

Ketone bodies are essential alternative fuels that allow humans to survive periods of glucose scarcity induced by starvation and prolonged exercise. A widely used ketogenic diet (KD), which is extremely high in fat with very low carbohydrates, drives the host into using ß-hydroxybutyrate for the production of ATP and lowers NLRP3-mediated inflammation. However, the extremely high fat composition of KD raises the question of how ketogenesis affects adipose tissue to control inflammation and energy homeostasis. Here, by using single-cell RNA sequencing of adipose-tissue-resident immune cells, we show that KD expands metabolically protective γδ T cells that restrain inflammation. Notably, long-term ad libitum KD feeding in mice causes obesity, impairs metabolic health and depletes the adipose-resident γδ T cells. In addition, mice lacking γδ T cells have impaired glucose homeostasis. Our results suggest that γδ T cells are mediators of protective immunometabolic responses that link fatty acid-driven fuel use to reduced adipose tissue inflammation.

Ketogenic diet activates protective γδ T cell responses against influenza virus infection.

Influenza A virus (IAV) infection-associated morbidity and mortality are a key global health care concern, necessitating the identification of new therapies capable of reducing the severity of IAV infections. In this study, we show that the consumption of a low-carbohydrate, high-fat ketogenic diet (KD) protects mice from lethal IAV infection and disease. KD feeding resulted in an expansion of γδ T cells in the lung that improved barrier functions, thereby enhancing antiviral resistance. Expansion of these protective γδ T cells required metabolic adaptation to a ketogenic diet because neither feeding mice a high-fat, high-carbohydrate diet nor providing chemical ketone body substrate that bypasses hepatic ketogenesis protected against infection. Therefore, KD-mediated immune-metabolic integration represents a viable avenue toward preventing or alleviating influenza disease.

Aging Induces an Nlrp3 Inflammasome-Dependent Expansion of Adipose B Cells That Impairs Metabolic Homeostasis.

During aging, visceral adiposity is often associated with alterations in adipose tissue (AT) leukocytes, inflammation, and metabolic dysfunction. However, the contribution of AT B cells in immunometabolism during aging is unexplored. Here, we show that aging is associated with an expansion of a unique population of resident non-senescent aged adipose B cells (AABs) found in fat-associated lymphoid clusters (FALCs). AABs are transcriptionally distinct from splenic age-associated B cells (ABCs) and show greater expansion in female mice. Functionally, whole-body B cell depletion restores proper lipolysis and core body temperature maintenance during cold stress. Mechanistically, the age-induced FALC formation, AAB, and splenic ABC expansion is dependent on the Nlrp3 inflammasome. Furthermore, AABs express IL-1R, and inhibition of IL-1 signaling reduces their proliferation and increases lipolysis in aging. These data reveal that inhibiting Nlrp3-dependent B cell accumulation can be targeted to reverse metabolic impairment in aging AT.

Bone Marrow: An Immunometabolic Refuge during Energy Depletion.

An integrated immunometabolic response during negative energy balance is required for host survival. Three new papers by Jordan et al. (2019), Nagai et al. (2019), and Collins et al. (2019) report that monocytes, naive B cells, and memory CD8 T cells use bone marrow as a haven to tide off periods of metabolic adversity to maintain immune-responsiveness.

Anti-inflammatory effects of oestrogen mediate the sexual dimorphic response to lipid-induced insulin resistance.

KEY POINTS: Oestrogen has been shown to play an important role in the regulation of metabolic homeostasis and insulin sensitivity in both human and rodent studies. Insulin sensitivity is greater in premenopausal women compared with age-matched men, and metabolism-related cardiovascular diseases and type 2 diabetes are less frequent in these same women. Both female and male mice treated with oestradiol are protected against obesity-induced insulin resistance. The protection against obesity-induced insulin resistance is associated with reduced ectopic lipid content in liver and skeletal muscle. These results were associated with increased insulin-stimulated suppression of white adipose tissue lipolysis and reduced inflammation. ABSTRACT: Oestrogen has been shown to play an important role in the regulation of metabolic homeostasis and insulin sensitivity in both human and rodent studies. Overall, females are protected against obesity-induced insulin resistance; yet, the mechanisms responsible for this protection are not well understood. Therefore, the aim of the present work was to evaluate the underlying mechanism(s) by which female mice are protected against obesity-induced insulin resistance compared with male mice. We studied male and female mice in age-matched or body weight-matched conditions. They were fed a high-fat diet (HFD) or regular chow for 4 weeks. We also studied HFD male mice treated with oestradiol or vehicle. Both HFD female and HFD male mice treated with oestradiol displayed increased whole-body insulin sensitivity, associated with reduction in ectopic hepatic and muscle lipid content compared to HFD male mice. Reductions in ectopic lipid content in these mice were associated with increased insulin-stimulated suppression of white adipose tissue (WAT) lipolysis. Both HFD female and HFD male mice treated with oestradiol also displayed striking reductions in WAT inflammation, represented by reductions in plasma and adipose tissue tumour necrosis factor α and interleukin 6 concentrations. Taken together these data support the hypothesis that HFD female mice are protected from obesity-induced insulin resistance due to oestradiol-mediated reductions in WAT inflammation, leading to improved insulin-mediated suppression of WAT lipolysis and reduced ectopic lipid content in liver and skeletal muscle.