Emily L. Goldberg

The ketone metabolite β-hydroxybutyrate blocks NLRP3 inflammasome–mediated inflammatory disease

The ketone bodies β-hydroxybutyrate (BHB) and acetoacetate (AcAc) support mammalian survival during states of energy deficit by serving as alternative sources of ATP. BHB levels are elevated by starvation, caloric restriction, high-intensity exercise, or the low-carbohydrate ketogenic diet. Prolonged fasting reduces inflammation; however, the impact that ketones and other alternative metabolic fuels produced during energy deficits have on the innate immune response is unknown. We report that BHB, but neither AcAc nor the structurally related short-chain fatty acids butyrate and acetate, suppresses activation of the NLRP3 inflammasome in response to urate crystals, ATP and lipotoxic fatty acids. BHB did not inhibit caspase-1 activation in response to pathogens that activate the NLR family, CARD domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome and did not affect non-canonical caspase-11, inflammasome activation. Mechanistically, BHB inhibits the NLRP3 inflammasome by preventing K+ efflux and reducing ASC oligomerization and speck formation. The inhibitory effects of BHB on NLRP3 are not dependent on chirality or starvation-regulated mechanisms like AMP-activated protein kinase (AMPK), reactive oxygen species (ROS), autophagy or glycolytic inhibition. BHB blocks the NLRP3 inflammasome without undergoing oxidation in the TCA cycle, and independently of uncoupling protein-2 (UCP2), sirtuin-2 (SIRT2), the G protein–coupled receptor GPR109A or hydrocaboxylic acid receptor 2 (HCAR2). BHB reduces NLRP3 inflammasome–mediated interleukin (IL)-1β and IL-18 production in human monocytes. In vivo, BHB or a ketogenic diet attenuates caspase-1 activation and IL-1β secretion in mouse models of NLRP3-mediated diseases such as Muckle–Wells syndrome, familial cold autoinflammatory syndrome and urate crystal–induced peritonitis. Our findings suggest that the anti-inflammatory effects of caloric restriction or ketogenic diets may be linked to BHB-mediated inhibition of the NLRP3 inflammasome.

Drivers of age-related inflammation and strategies for healthspan extension.

Aging is the greatest risk factor for the development of chronic diseases such as arthritis, type 2 diabetes, cardiovascular disease, kidney disease, Alzheimers disease, macular degeneration, frailty, and certain forms of cancers. It is widely regarded that chronic inflammation may be a common link in all these age‐related diseases. This raises the question, can one alter the course of aging and potentially slow the development of all chronic diseases by manipulating the mechanisms that cause age‐related inflammation? Emerging evidence suggests that pro‐inflammatory cytokines interleukin‐1 (IL‐1) and IL‐18 show an age‐dependent regulation implicating inflammasome‐mediated caspase‐1 activation in the aging process. The Nod‐like receptor (NLR) family of innate immune cell sensors, such as the nucleotide‐binding domain, leucine‐rich‐containing family, pyrin domain‐containing‐3 (NLRP3) inflammasome controls the caspase‐1 activation in myeloid‐lineage cells in several organs during aging. The NLRP3 inflammasome is especially relevant to aging as it can get activated in response to structurally diverse damage‐associated molecular patterns (DAMPs) such as extracellular ATP, excess glucose, ceramides, amyloids, urate, and cholesterol crystals, all of which increase with age. Interestingly, reduction in NLRP3‐mediated inflammation prevents age‐related insulin resistance, bone loss, cognitive decline, and frailty. NLRP3 is a major driver of age‐related inflammation and therefore dietary or pharmacological approaches to lower aberrant inflammasome activation holds promise in reducing multiple chronic diseases of age and may enhance healthspan.

Inflammasome-driven catecholamine catabolism in macrophages blunts lipolysis during ageing

Catecholamine-induced lipolysis, the first step in the generation of energy substrates by the hydrolysis of triglycerides, declines with age. The defect in the mobilization of free fatty acids in the elderly is accompanied by increased visceral adiposity, lower exercise capacity, failure to maintain core body temperature during cold stress, and reduced ability to survive starvation. Although catecholamine signalling in adipocytes is normal in the elderly, how lipolysis is impaired in ageing remains unknown. Here we show that adipose tissue macrophages regulate the age-related reduction in adipocyte lipolysis in mice by lowering the bioavailability of noradrenaline. Unexpectedly, unbiased whole-transcriptome analyses of adipose macrophages revealed that ageing upregulates genes that control catecholamine degradation in an NLRP3 inflammasome-dependent manner. Deletion of NLRP3 in ageing restored catecholamine-induced lipolysis by downregulating growth differentiation factor-3 (GDF3) and monoamine oxidase A (MAOA) that is known to degrade noradrenaline. Consistent with this, deletion of GDF3 in inflammasome-activated macrophages improved lipolysis by decreasing levels of MAOA and caspase-1. Furthermore, inhibition of MAOA reversed the age-related reduction in noradrenaline concentration in adipose tissue, and restored lipolysis with increased levels of the key lipolytic enzymes adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL). Our study reveals that targeting neuro-immunometabolic signalling between the sympathetic nervous system and macrophages may offer new approaches to mitigate chronic inflammation-induced metabolic impairment and functional decline.

Lifespan-extending caloric restriction or mTOR inhibition impair adaptive immunity of old mice by distinct mechanisms

Aging of the world population and a concomitant increase in age‐related diseases and disabilities mandates the search for strategies to increase healthspan, the length of time an individual lives healthy and productively. Due to the age‐related decline of the immune system, infectious diseases remain among the top 5–10 causes of mortality and morbidity in the elderly, and improving immune function during aging remains an important aspect of healthspan extension. Calorie restriction (CR) and more recently rapamycin (rapa) feeding have both been used to extend lifespan in mice. Preciously few studies have actually investigated the impact of each of these interventions upon in vivo immune defense against relevant microbial challenge in old organisms. We tested how rapa and CR each impacted the immune system in adult and old mice. We report that each intervention differentially altered T‐cell development in the thymus, peripheral T‐cell maintenance, T‐cell function and host survival after West Nile virus infection, inducing distinct but deleterious consequences to the aging immune system. We conclude that neither rapa feeding nor CR, in the current form/administration regimen, may be optimal strategies for extending healthy immune function and, with it, lifespan.

β-Hydroxybutyrate Deactivates Neutrophil NLRP3 Inflammasome to Relieve Gout Flares

Aging and lipotoxicity are two major risk factors for gout that are linked by the activation of the NLRP3 inflammasome. Neutrophil-mediated production of interleukin-1β (IL-1β) drives gouty flares that cause joint destruction, intense pain, and fever. However, metabolites that impact neutrophil inflammasome remain unknown. Here, we identified that ketogenic diet (KD) increases β-hydroxybutyrate (BHB) and alleviates urate crystal-induced gout without impairing immune defense against bacterial infection. BHB inhibited NLRP3 inflammasome in S100A9 fibril-primed and urate crystal-activated macrophages, which serve to recruit inflammatory neutrophils in joints. Consistent with reduced gouty flares in rats fed a ketogenic diet, BHB blocked IL-1β in neutrophils in a NLRP3-dependent manner in mice and humans irrespective of age. Mechanistically, BHB inhibited the NLRP3 inflammasome in neutrophils by reducing priming and assembly steps. Collectively, our studies show that BHB, a known alternate metabolic fuel, is also an anti-inflammatory molecule that may serve as a treatment for gout.

Immune Memory–Boosting Dose of Rapamycin Impairs Macrophage Vesicle Acidification and Curtails Glycolysis in Effector CD8 Cells, Impairing Defense against Acute Infections

Direct mammalian target of rapamycin (Rapa) complex 1 inhibition by short-term low-dose Rapa treatment has recently been shown to improve CD8 T cell immunological memory. Whereas these studies focused on memory development, the impact of low-dose Rapa on the primary immune response, particularly as it relates to functional effector immunity, is far less clear. In this study, we investigated the impact of acute Rapa treatment on immune effector cell function during the primary immune response to several acute infections. We found that functional CD8 T cell and macrophage responses to both viral and intracellular bacterial pathogens were depressed in mice in vivo and in humans to phorbol ester and calcium ionophore stimulation in vitro in the face of low-dose Rapa treatment. Mechanistically, the CD8 defect was linked to impaired glycolytic switch in stimulated naive cells and the reduced formation of short-lived effector cells. Therefore, more than one cell type required for a protective effector immune response is impaired by Rapa in both mice and humans, at the dose shown to improve immune memory and extend lifespan. This urges caution with regard to the relative therapeutic costs and benefits of Rapa treatment as means to improve immune memory.

Growth Hormone Receptor Deficiency Protects against Age-Related NLRP3 Inflammasome Activation and Immune Senescence

SUMMARY The hallmarks of age-related immune senescence are chronic inflammation, aberrant expansion of effector memory, and loss of naive T lymphocytes due in part to systemic activation of innate immune sensor NLRP3 inflammasome in myeloid lineage cells. The endogenous mechanisms that regulate inflammasome activation during aging are unknown. Here, we present evidence that growth hormone receptor (GH-R)-dependent downregulation of NLRP3 inflammasomein macrophages is linked to pro-longevity effects that maintain immune system homeostasis in aging. Deletion of GH-R prevented the macrophage-driven age-related activation of inflammasome in response to NLRP3 ligands and also increased the preservation of naive T cells, even in advanced age and with higher IFNγ secretion from effector cells. The mechanism of inflammasome inhibition is linked to autocrine somatotropic axis as ablation of IGF1R in macrophages lowered the NLRP3 inflammasome activation. Together, our findings show that functional somatotropic axis in macrophages controls inflammation, thus linking NLRP3-mediated innate immune signaling to health span and longevity.

Acute neonatal infections 'lock-in' a suboptimal CD8+ T cell repertoire with impaired recall responses.

Microbial infection during various stages of human development produces widely different clinical outcomes, yet the links between age-related changes in the immune compartment and functional immunity remain unclear. The ability of the immune system to respond to specific antigens and mediate protection in early life is closely correlated with the level of diversification of lymphocyte antigen receptors. We have previously shown that the neonatal primary CD8+ T cell response to replication competent virus is significantly constricted compared to the adult response. In the present study, we have analyzed the subsequent formation of neonatal memory CD8+ T cells and their response to secondary infectious challenge. In particular, we asked whether the less diverse CD8+ T cell clonotypes that are elicited by neonatal vaccination with replication competent virus are ‘locked-in’ to the adult memory T cell, and thus may compromise the strength of adult immunity. Here we report that neonatal memory CD8+ T cells mediate poor recall responses compared to adults and are comprised of a repertoire of lower avidity T cells. During a later infectious challenge the neonatal memory CD8+ T cells compete poorly with the fully diverse repertoire of naïve adult CD8+ T cells and are outgrown by the adult primary response. This has important implications for the timing of vaccination in early life.

Contrasting effects of chronic, systemic treatment with mTOR inhibitors rapamycin and metformin on adult neural progenitors in mice

The chronic and systemic administration of rapamycin extends life span in mammals. Rapamycin is a pharmacological inhibitor of mTOR. Metformin also inhibits mTOR signaling but by activating the upstream kinase AMPK. Here we report the effects of chronic and systemic administration of the two mTOR inhibitors, rapamycin and metformin, on adult neural stem cells of the subventricular region and the dendate gyrus of the mouse hippocampus. While rapamycin decreased the number of neural progenitors, metformin-mediated inhibition of mTOR had no such effect. Adult-born neurons are considered important for cognitive and behavioral health, and may contribute to improved health span. Our results demonstrate that distinct approaches of inhibiting mTOR signaling can have significantly different effects on organ function. These results underscore the importance of screening individual mTOR inhibitors on different organs and physiological processes for potential adverse effects that may compromise health span.

Editorial: “Crowning” eosinophils in adipose tissue: does location matter?

Introduction It is well known that the incidence of obesity is increasing at an alarming rate, leading to increased prevalence of insulin resistance and metabolic disease. Excess visceral adiposity is a major risk factor for development of obesityassociated disease and directly correlates with elevated levels of inflammatory markers, including TNF-a, IL-6, and IL-1b. In addition to its role in energy storage, adipose tissue is becoming increasingly accepted as an immunologically active organ [1]. In 2003, seminal studies identified increased macrophage accumulation in obese adipose tissue as the primary source of increased TNF-a in mice fed a high-fat diet [2]. Since these initial observations, many significant findings have increased our understanding of the immunologic changes in adipose tissue during obesity [3]. Perhaps the most remarkable change is the switch from an anti-inflammatory environment, characterized by the presence of alternatively activated macrophages and regulatory T cells, toward a proinflammatory environment, rich in classically activated macrophages, cytotoxic CD8 T cells, and Th1 CD4 T cells. Importantly, many of the leukocyte changes that have been described in mouse models have been confirmed in human adipose tissue. More recently, the surprising observation was made that eosinophils serve a protective role maintaining adipose tissue homeostasis during obesity [4]. Eosinophils were found to be the major cellular source of IL-4, which promotes macrophage-alternative activation. Systemic increases in eosinophils protected mice from high-fat diet-induced insulin resistance. Subsequent studies identified ILC2 cells as the primary source of IL-5 in adipose tissue, responsible for the increase in eosinophils [5]. Although the regulation of immune cells in adipose tissue is not entirely understood, the intersection of immune function and metabolic disease has clear, clinical relevance for human health. In this issue of JLB, Bolus et al. [6] used a Ccr2 mouse model on a high-fat diet to study immune cell regulation in obese adipose tissue. CCR2 is a chemokine receptor known to regulate macrophage migration to sites of inflammation. Ccr2 mice were previously shown to have decreased macrophage accumulation in adipose tissue during high-fat diet-induced obesity, correlating with improved insulin and glucose homeostasis [7]. Previous work by the Hasty lab [8] reported the accumulation of a novel but unidentified myeloid cell in the adipose tissue of obese Ccr2 mice. In the current study, this cell population is confirmed to be eosinophils, validated by nuclear morphology and Siglec-F expression, with the latter being an inhibitory receptor that marks murine eosinophils. Interestingly, the increased number of eosinophils in Ccr2 mice was limited to the peritoneal cavity and adipose tissue but was not observed systemically in the bone marrow, blood, spleen, or liver. Furthering the mechanistic understanding of how eosinophils might help maintain adipose tissue homeostasis during obesity, Bolus et al. [6] created 3-dimensional renderings of immunofluorescent confocal microscopy images, which revealed that eosinophils were recruited specifically to CLS in the obese adipose tissue of Ccr2 but not wild-type mice. This raises an intriguing question: does tissue location matter for eosinophil function? Macrophages that localize near CLS are thought to induce increased inflammatory activity compared with macrophages in interstitial spaces. Given the current data by Bolus et al. [6], it seems feasible that a similar, but perhaps opposite, paradigm also exists for eosinophils (Fig. 1). It is noteworthy that whether the location of immune cells in adipose tissue matters is not well understood. It remains to be determined whether tissue location determines the function of the cell or vice versa. It is not clear whether the increase in eosinophils in obese Ccr2 mice was mediated by increased recruitment during obesity or whether local proliferation was the causative factor. In addition to IL-5 discussed above, eotaxins have been reported to increase in the serum and adipose tissue of obese individuals, implicating them in the regulation of eosinophil recruitment to obese adipose tissue. Bolus et al. [6] measured increased Ccl11 and Ccl3 in obese Ccr2 adipose tissue but reduced Ccl24 expression. Therefore, it is likely that IL-5 and eotaxins regulate eosinophil recruitment to obese adipose tissue, but the precise mechanism remains to be fully delineated (Fig. 1). The increase in eosinophil recruitment to CLS in Ccr2 mice suggests that CCR2-positive cells somehow actively inhibit this process in wild-type obese mice. Whether this regulation is via cytokines or direct cell-cell contact is not known. In support of cytokine-mediated regulation, eosinophil secretion of IL-4 has previously been shown to regulate macrophage alternative activation [4]. Furthermore, the role of ILC2 cells, which are known to regulate eosinophils in adipose tissue, as discussed above, was not tested in the obese Ccr2 mice. It is noteworthy that eosinophils and