Adiposity, immunity, and inflammation: interrelationships in health and disease: a report from 24th Annual Harvard Nutrition Obesity Symposium, June 2023.

The rapidly evolving field of immunometabolism explores how changes in local immune environments may affect key metabolic and cellular processes, including that of adipose tissue. Importantly, these changes may contribute to low-grade systemic inflammation. In turn, chronic low-grade inflammation affecting adipose tissue may exacerbate the outcome of metabolic diseases. Novel advances in our understanding of immunometabolic processes may critically lead to interventions to reduce disease severity and progression. An important example in this regard relates to obesity, which has a multifaceted effect on immunity, activating the proinflammatory pathways such as the inflammasome and disrupting cellular homeostasis. This multifaceted effect of obesity can be investigated through study of downstream conditions using cellular and systemic investigative techniques. To further explore this field, the National Institutes of Health P30 Nutrition Obesity Research Center at Harvard, in partnership with Harvard Medical School, assembled experts to present at its 24th Annual Symposium entitled "Adiposity, Immunity, and Inflammation: Interrelationships in Health and Disease" on 7 June, 2023. This manuscript seeks to synthesize and present key findings from the symposium, highlighting new research and novel disease-specific advances in the field. Better understanding the interaction between metabolism and immunity offers promising preventative and treatment therapies for obesity-related immunometabolic diseases.

Long-term moderate calorie restriction inhibits inflammation without impairing cell-mediated immunity: a randomized controlled trial in non-obese humans.

Calorie restriction (CR) inhibits inflammation and slows aging in many animal species, but in rodents housed in pathogen-free facilities, CR impairs immunity against certain pathogens. However, little is known about the effects of long-term moderate CR on immune function in humans. In this multi-center, randomized clinical trial to determine CR's effect on inflammation and cell-mediated immunity, 218 healthy non-obese adults (20-50 y), were assigned 25% CR (n=143) or an ad-libitum (AL) diet (n=75), and outcomes tested at baseline, 12, and 24 months of CR. CR induced a 10.4% weight loss over the 2-y period. Relative to AL group, CR reduced circulating inflammatory markers, including total WBC and lymphocyte counts, ICAM-1 and leptin. Serum CRP and TNF-α concentrations were about 40% and 50% lower in CR group, respectively. CR had no effect on the delayed-type hypersensitivity skin response or antibody response to vaccines, nor did it cause difference in clinically significant infections. In conclusion, long-term moderate CR without malnutrition induces a significant and persistent inhibition of inflammation without impairing key in vivo indicators of cell-mediated immunity. Given the established role of these pro-inflammatory molecules in the pathogenesis of multiple chronic diseases, these CR-induced adaptations suggest a shift toward a healthy phenotype.

A Role of the Inflammasome in the Low Storage Capacity of the Abdominal Subcutaneous Adipose Tissue in Obese Adolescents.

The innate immune cell sensor leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome controls the activation of caspase-1, and the release of proinflammatory cytokines interleukin (IL)-1ß and IL-18. The NLRP3 inflammasome is implicated in adipose tissue inflammation and the pathogenesis of insulin resistance. Herein, we tested the hypothesis that adipose tissue inflammation and NLRP3 inflammasome are linked to the downregulation of subcutaneous adipose tissue (SAT) adipogenesis/lipogenesis in obese adolescents with altered abdominal fat partitioning. We performed abdominal SAT biopsies on 58 obese adolescents and grouped them by MRI-derived visceral fat to visceral adipose tissue (VAT) plus SAT (VAT/VAT+SAT) ratio (cutoff 0.11). Adolescents with a high VAT/VAT+SAT ratio showed higher SAT macrophage infiltration and higher expression of the NLRP3 inflammasome-related genes (i.e., TLR4, NLRP3, IL1B, and CASP1). The increase in inflammation markers was paralleled by a decrease in genes related to insulin sensitivity (ADIPOQ, GLUT4, PPARG2, and SIRT1) and lipogenesis (SREBP1c, ACC, LPL, and FASN). Furthermore, SAT ceramide concentrations correlated with the expression of CASP1 and IL1B. Infiltration of macrophages and upregulation of the NLRP3 inflammasome together with the associated high ceramide content in the plasma and SAT of obese adolescents with a high VAT/VAT+SAT may contribute to the limited expansion of the subcutaneous abdominal adipose depot and the development of insulin resistance.

Mechanisms of disease: inflammasome activation and the development of type 2 diabetes.

Over the recent past, the importance of aberrant immune cell activation as one of the contributing mechanisms to the development of insulin-resistance and type 2 diabetes (T2D) has been recognized. Among the panoply of pro-inflammatory cytokines that are linked to chronic metabolic diseases, new data suggests that interleukin-1ß (IL-1ß) may play an important role in initiating and sustaining inflammation-induced organ dysfunction in T2D. Therefore, factors that control secretion of bioactive IL-1ß have therapeutic implications. In this regard, the identification of multiprotein scaffolding complexes, "inflammasomes," has been a great advance in our understanding of this process. The secretion of bioactive IL-1ß is predominantly controlled by activation of caspase-1 through assembly of a multiprotein scaffold, "inflammasome" that is composed of NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) ASC (apoptosis associated speck-like protein containing a CARD) and procaspase-1. The NLRP3 inflammasome appears to be an important sensor of metabolic dysregulation and controls obesity-associated insulin resistance and pancreatic beta cell dysfunction. Initial clinical "proof of concept" studies suggest that blocking IL-1ß may favorably modulate factors related to development and treatment of T2D. However, this potential therapeutic approach remains to be fully substantiated through phase-II clinical studies. Here, we outline the new immunological mechanisms that link metabolic dysfunction to the emergence of chronic inflammation and discuss the opportunities and challenges of future therapeutic approaches to dampen NLRP3 inflammasome activation or IL-1ß signaling for controlling type 2 diabetes.

Effect of different doses of aerobic exercise on total white blood cell (WBC) and WBC subfraction number in postmenopausal women: results from DREW.

BACKGROUND: Elevated total white blood cell (WBC) count is associated with an increased risk of coronary heart disease and death. Aerobic exercise is associated with lower total WBC, neutrophil, and monocyte counts. However, no studies have evaluated the effect of the amount of aerobic exercise (dose) on total WBC and WBC subfraction counts. PURPOSE: To examine the effects of 3 different doses of aerobic exercise on changes in total WBC and WBC subfraction counts and independent effects of changes in fitness, adiposity, markers of inflammation (IL-6, TNF-α, C-reactive protein), fasting glucose metabolism, and adiponectin. METHODS: Data from 390 sedentary, overweight/obese postmenopausal women from the DREW study were used in these analyses. Women were randomized to a non-exercise control group or one of 3 exercise groups: energy expenditure of 4, 8, or 12 kcal kg(-1)⋅week(-1) (KKW) for 6 months at an intensity of 50% VO2peak. RESULTS: A dose-dependent decrease in total WBC counts (trend P = 0.002) was observed with a significant decrease in the 12KKW group (-163.1±140.0 cells/µL; mean±95%CI) compared with the control (138.6±144.7 cells/µL). A similar response was seen in the neutrophil subfraction (trend P = 0.001) with a significant decrease in the 12KKW group (-152.6±115.1 cells/µL) compared with both the control and 4KKW groups (96.4±119.0 and 21.9±95.3 cells/µL, respectively) and in the 8KKW group (-102.4±125.0 cells/µL) compared with the control. When divided into high/low baseline WBC categories (median split), a dose-dependent decrease in both total WBCs (P = 0.003) and neutrophils (P<0.001) was observed in women with high baseline WBC counts. The effects of exercise dose on total WBC and neutrophil counts persisted after accounting for significant independent effects of change in waist circumference and IL-6. CONCLUSION: Aerobic exercise training reduces total WBC and neutrophil counts, in a dose-dependent manner, in overweight/obese postmenopausal women and is especially beneficial for those with systemic low grade inflammation. CLINICAL TRIALS IDENTIFIER: NCT00011193.

Ablations of ghrelin and ghrelin receptor exhibit differential metabolic phenotypes and thermogenic capacity during aging.

BACKGROUND: Obesity is a hallmark of aging in many Western societies, and is a precursor to numerous serious age-related diseases. Ghrelin (Ghrl), via its receptor (growth hormone secretagogue receptor, GHS-R), is shown to stimulate GH secretion and appetite. Surprisingly, our previous studies showed that Ghrl(-/-) mice have impaired thermoregulatory responses to cold and fasting stresses, while Ghsr(-/-) mice are adaptive. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the mechanism, we analyzed the complete metabolic profiles of younger (3-4 months) and older (10-12 months) Ghrl(-/-) and Ghsr(-/-) mice. Food intake and locomotor activity were comparable for both null mice and their wild-type (WT) counterparts, regardless of age. There was also no difference in body composition between younger null mice and their WT counterparts. As the WT mice aged, as expected, the fat/lean ratio increased and energy expenditure (EE) decreased. Remarkably, however, older Ghsr(-/-) mice exhibited reduced fat/lean ratio and increased EE when compared to older WT mice, thus retaining a youthful lean and high EE phenotype; in comparison, there was no significant difference with EE in Ghrl(-/-) mice. In line with the EE data, the thermogenic regulator, uncoupling protein 1 (UCP1), was significantly up-regulated in brown adipose tissue (BAT) of Ghsr(-/-) mice, but not in Ghrl(-/-) mice. CONCLUSIONS: Our data therefore suggest that GHS-R ablation activates adaptive thermogenic function(s) in BAT and increases EE, thereby enabling the retention of a lean phenotype. This is the first direct evidence that the ghrelin signaling pathway regulates fat-burning BAT to affect energy balance during aging. This regulation is likely mediated through an as-yet-unidentified new ligand of GHS-R.

Thiazolidinedione treatment and constitutive-PPARgamma activation induces ectopic adipogenesis and promotes age-related thymic involution.

Age-related thymic involution is characterized by reduction in T cell production together with ectopic adipocyte development within the hematopoietic and thymic niches. Peroxisome proliferator-activated receptor gamma (PPARgamma) is required for adipocyte development, glucose homeostasis and is a target for several insulin-sensitizing drugs. Our prior studies showed that age-related elevation of PPARgamma expression in thymic stromal cells is associated with thymic involution. Here, using clinically relevant pharmacological and genetic manipulations in mouse models, we provide evidence that activation of PPARgamma leads to reduction in thymopoiesis. Treatment of aged mice with antihyperglycemic PPARgamma-ligand class of thiazolidinedione drug, rosiglitazone caused robust thymic expression of classical pro-adipogenic transcripts. Rosiglitazone reduced thymic cellularity, lowered the naïve T cell number and T cell receptor excision circles (TRECs) indicative of compromised thymopoiesis. To directly investigate whether PPARgamma activation induces thymic involution, we created transgenic mice with constitutive-active PPARgamma (CA-PPARg) fusion protein in cells of adipogenic lineage. Importantly, CA-PPARgamma transgene was expressed in thymus and in fibroblast-specific protein-1/S100A4 (FSP1(+)) cells, a marker of secondary mesenchymal cells. The CAPPARgamma fusion protein mimicked the liganded PPARgamma receptor and the transgenic mice displayed increased ectopic thymic adipogenesis and reduced thymopoiesis. Furthermore, the reduction in thymopoiesis in CA-PPARgamma mice was associated with higher bone marrow adiposity and lower hematopoietic stem cell progenitor pool. Consistent with lower thymic output, CAPPARgamma transgenic mice had restricted T cell receptor repertoire diversity. Collectively, our data suggest that activation of PPARgamma accelerates thymic aging and thymus-specific PPARgamma antagonist may forestall age-related decline in T cell diversity.

Analysis of the therapeutic functions of novel melanocortin receptor agonists in MC3R- and MC4R-deficient C57BL/6J mice.

Melanocortin receptor agonists act in the brain to regulate food intake and body weight and, independently of these actions, affect insulin sensitivity. These experiments investigated the function of novel non-selective melanocortin receptor agonists (BIM-22493, BIM-22511) that cross the blood-brain barrier when administered peripherally. Treatment of diet induced obese C57BL/6J (B6) mice with melanocortin agonists administered peripherally improved obesity, hyperinsulinemia (approximately 50%) and fatty liver disease. Specificity of function was determined using B6 melanocortin-3 and melanocortin-4 receptor knockout mice (MC3RKO, MC4RKO). Chow fed MC4RKO but not MC3RKO used for these tests exhibited obesity, hyperinsulinemia and severe hepatosteatosis associated with increased expression of insulin-stimulated genes involved in lipogenesis. Reduced food intake associated with acute BIM-22493 treatment, and weight loss associated with 14 days of treatment with BIM-22511, required functional MC4R but not MC3R. However, while 14 days of treatment with BIM-22511 did not affect body weight and even increased cumulative food intake in MC4RKO, a significant reduction (approximately 50%) in fasting insulin was still observed. Despite lowering insulin, chronic treatment with BIM-22511 did not improve hepatosteatosis in MC4RKO, and did not affect hepatic lipogenic gene expression. Together, these results demonstrate that peripherally administered melanocortin receptor agonists regulate body weight, liver metabolism and glucose homeostasis through independent pathways. MC4R are necessary for melanocortin agonist-induced weight loss and improvements in liver metabolism, but are not required for improvements in hyperinsulinemia. Agonists with activity at MC4R improve glucose homeostasis at least partially by causing weight loss, however other melanocortin receptors may have potential for treating aberrations in glucose homeostasis associated with obesity.

Reduction of T cell-derived ghrelin enhances proinflammatory cytokine expression: implications for age-associated increases in inflammation.

Ghrelin (Grln) is a peptide hormone that is predominantly produced in the stomach and stimulates appetite and induces growth hormone (GH) release. We have previously reported that ghrelin is also expressed in T cells and exerts prothymic and anti-inflammatory effects. However, the biologic relevance of T cell-derived ghrelin remains to be determined. Here, we report that acylated-bioactive ghrelin is expressed in human T cells and preferentially segregates within the lipid raft domains upon TCR ligation. The RNA interference (RNAi)-mediated down-regulation of ghrelin in primary human T cells activates IkB, and increases Th1 cytokines and IL-17 secretion. Ghrelin expression declines with increasing age in spleen and T cells and exogenous ghrelin administration in old mice reduces proinflammatory cytokines. These findings demonstrate that ghrelin functions in an autocrine and paracrine capacity to regulate proinflammatory cytokine expression in human and murine T cells and may contribute in regulating "inflamm-aging."

Reduction in hypophyseal growth hormone and prolactin expression due to deficiency in ghrelin receptor signaling is associated with Pit-1 suppression: relevance to the immune system.

In mice and in rats, reduced levels of the growth hormone secretagogue receptor (GHS-R1a) results in reduced body weight and lower levels of serum insulin-like growth factor I (IGF-I). However, the mechanism leading to these impairments has not been elucidated. Studies in primary cultures of pituitary cells from very young mice have shown that GHS-R1a agonists, including ghrelin, increase expression of the pituitary-specific transcription factor (Pit-1) that is critical for differentiation of pituitary cells into somatotrophs, lactotrophs, and thyrotrophs. Hence, we hypothesized that ablation of Ghsr would reduce Pit-1 expression and as a consequence reduce growth hormone (GH) production explaining the lower body weight of Ghsr-/- mice. Here, we now show that Pit-1 mRNA levels are significantly lower in the pituitary gland of Ghsr-/- mice compared to wild-type littermates and also with advancing age. This Pit-1 loss is associated with reduced GH mRNA and fewer GH producing cells. To determine whether reduced GH is caused by reduced expression of Pit-1 in Ghsr-/- mice, we also measured prolactin (PRL) expression in the pituitary gland and in the circulation. PRL mRNA was significantly reduced in Ghsr-/- mice compared to wild-type littermates and fewer cells expressed PRL. The reduction in expression of both GH and PRL is consistent with a Pit-1 regulated pathway and demonstrates that the GHS-R has an important role in the pituitary gland as a modulator of Pit-1 expression and provides a possible mechanism to explain the lower plasma IGF-1 and modestly reduced body weight exhibited by Ghsr-/- mice. We also believe that lower systemic and lymphoid hormone expression may also account, in part, for the enhanced thymic involution and reduced thymic output in Ghsr-/- mice.

CXCL12-induced partitioning of flotillin-1 with lipid rafts plays a role in CXCR4 function.

Lipid rafts play an important role in signal integration and in the cellular activation of a number of cytokine and growth factor receptors. It has recently been demonstrated that flotillin proteins are recruited to lipid raft microdomains upon cellular activation and play a role in neural cell regeneration, receptor signaling and lymphocyte activation. However, little is known about the relevance of the flotillin proteins during T cell responses to chemoattractant stimulation. To this end, cytoplasmic and lipid raft fractions from human T cells were analyzed for flotillin protein redistribution prior to and after CXCL12 stimulation. Flotillin-1, but not flotillin-2, redistributes to lipid rafts upon CXCR4 ligation. Moreover, in CXCL12-treated T cells, flotillin-1 also associates with several raft proteins including LAT, CD48 and CD11a but not Lck. In addition, an increase in CXCR4 association with flotillin-1 in lipid rafts was observed after chemokine treatment. RNAi technology was also utilized to inhibit the expression of flotillin-1, resulting in an inhibition of CXCL12-mediated signaling, function and CXCR4 recruitment into lipid rafts. Together, these data suggest that the increased association of cellular flotillin-1 with lipid raft microdomains during chemokine exposure may play an important role in chemokine receptor signaling and receptor partitioning with lipid rafts.

Alternate day calorie restriction improves clinical findings and reduces markers of oxidative stress and inflammation in overweight adults with moderate asthma.

Asthma is an increasingly common disorder responsible for considerable morbidity and mortality. Although obesity is a risk factor for asthma and weight loss can improve symptoms, many patients do not adhere to low calorie diets and the impact of dietary restriction on the disease process is unknown. A study was designed to determine if overweight asthma patients would adhere to an alternate day calorie restriction (ADCR) dietary regimen, and to establish the effects of the diet on their symptoms, pulmonary function and markers of oxidative stress, and inflammation. Ten subjects with BMI>30 were maintained for 8 weeks on a dietary regimen in which they ate ad libitum every other day, while consuming less than 20% of their normal calorie intake on the intervening days. At baseline, and at designated time points during the 8-week study, asthma control, symptoms, and Quality of Life questionnaires (ACQ, ASUI, mini-AQLQ) were assessed and blood was collected for analyses of markers of general health, oxidative stress, and inflammation. Peak expiratory flow (PEF) was measured daily on awakening. Pre- and postbronchodilator spirometry was obtained at baseline and 8 weeks. Nine of the subjects adhered to the diet and lost an average of 8% of their initial weight during the study. Their asthma-related symptoms, control, and QOL improved significantly, and PEF increased significantly, within 2 weeks of diet initiation; these changes persisted for the duration of the study. Spirometry was unaffected by ADCR. Levels of serum beta-hydroxybutyrate were increased and levels of leptin were decreased on CR days, indicating a shift in energy metabolism toward utilization of fatty acids and confirming compliance with the diet. The improved clinical findings were associated with decreased levels of serum cholesterol and triglycerides, striking reductions in markers of oxidative stress (8-isoprostane, nitrotyrosine, protein carbonyls, and 4-hydroxynonenal adducts), and increased levels of the antioxidant uric acid. Indicators of inflammation, including serum tumor necrosis factor-alpha and brain-derived neurotrophic factor, were also significantly decreased by ADCR. Compliance with the ADCR diet was high, symptoms and pulmonary function improved, and oxidative stress and inflammation declined in response to the dietary intervention. These findings demonstrate rapid and sustained beneficial effects of ADCR on the underlying disease process in subjects with asthma, suggesting a novel approach for therapeutic intervention in this disorder.

Gamma secretase-mediated Notch signaling worsens brain damage and functional outcome in ischemic stroke.

Mice transgenic for antisense Notch and normal mice treated with inhibitors of the Notch-activating enzyme gamma-secretase showed reduced damage to brain cells and improved functional outcome in a model of focal ischemic stroke. Notch endangers neurons by modulating pathways that increase their vulnerability to apoptosis, and by activating microglial cells and stimulating the infiltration of proinflammatory leukocytes. These findings suggest that Notch signaling may be a therapeutic target for treatment of stroke and related neurodegenerative conditions.

Uric acid and inflammatory markers.

AIMS: The role of uric acid (UA) in the process of atherosclerosis and atherotrombosis is controversial. Epidemiological studies have recently shown that UA may be a risk factor for cardiovascular diseases and a negative prognostic marker for mortality in subjects with pre-existing heart failure. METHODS AND RESULTS: We evaluate a relationship between UA levels and several inflammatory markers in 957 subjects, free of severe renal failure, from a representative Italian cohort of persons aged 65-95. Plasma levels of UA and white blood cell (WBC) and neutrophil count, C-reactive protein, interleukin-1 receptor antagonist (IL-1ra), interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6r), interleukin-18 (IL-18), and tumor necrosis factor-alpha (TNF-alpha) were measured. Complete information on potential confounders was collected using standard methods. WBC (P=0.0001), neutrophils (P<0.0001), C-reactive protein (P<0.0001), IL-1ra (P<0.0001), IL-6 (P=0.0004), sIL-6r (P=0.002), IL-18 (P<0.0001), TNF-alpha (P=0.0008), and the percentage of subjects with abnormally high levels of C-reactive protein (P=0.004) and IL-6 (P=<0.0001) were significantly higher across UA quintiles. After adjustment for age, sex, behaviour- and disease-related confounders, results were virtually unchanged. In subjects with UA within the normal range, UA was significantly and independently associated with neutrophils count, C-reactive protein, IL-6, IL-1ra, IL-18, and TNF-alpha, whereas non-significant trends were observed for WBC (P=0.1) and sIL-6r (P=0.2). CONCLUSION: A positive and significant association between UA and several inflammatory markers was found in a large population-based sample of older persons and in a sub-sample of participants with normal UA. Accordingly, the prevalence of abnormally high levels of C-reactive protein and IL-6 increased significantly across UA quintiles.