Cells In Visceral Adipose Tissue Research Articles

Androgens Modulate the Immune Profile in a Mouse Model of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is associated with a low-grade inflammation, but it is unknown how hyperandrogenism, the hallmark of PCOS, affects the immune system. Using a PCOS-like mouse model, it is demonstrated that hyperandrogenism affects immune cell populations in reproductive, metabolic, and immunological tissues differently in a site-specific manner. Co-treatment with an androgen receptor antagonist prevents most of these alterations, demonstrating that these effects are mediated through androgen receptor activation. Dihydrotestosterone (DHT)-exposed mice displayed a drastically reduced eosinophil population in the uterus and visceral adipose tissue (VAT). A higher frequency of natural killer (NK) cells and elevated levels of IFN-γ and TNF-α are seen in uteri of androgen-exposed mice, while NK cells in VAT and spleen displayed a higher expression level of CD69, a marker of activation or tissue residency. Distinct alterations of macrophages in the uterus, ovaries, and VAT are also found in DHT-exposed mice and can potentially be linked to PCOS-like traits of the model. Indeed, androgen-exposed mice are insulin-resistant, albeit unaltered fat mass. Collectively, it is demonstrated that hyperandrogenism causes tissue-specific alterations of immune cells in reproductive organs and VAT, which can have considerable implications on tissue function and contribute to the reduced fertility and metabolic comorbidities associated with PCOS.

Mechanisms of γδ T cell accumulation in visceral adipose tissue with aging.

γδ T cells are resident in visceral adipose tissue (VAT) where they show an age-associated increase in numbers and contribute to local and systemic chronic inflammation. However, regulation of this population and mechanisms for the age-dependent accumulation are not known. In this study, we identified a progressive trend of γδ T cell accumulation in VAT over the lifespan in mice and explored physiological mechanisms contributing to accumulation. Using isochronic parabiotic pairs of wild-type (WT) and T cell receptor delta knockout (TCRδ KO) mice at young and old age, we confirmed that VAT γδ T cells are predominately a tissue-resident population which is sustained in aging. Migration of peripheral γδ T cells into VAT was observed at less than 10%, with a decreasing trend by aging, suggesting a minor contribution of recruitment to γδ T cell accumulation with aging. Since tissue-resident T cell numbers are tightly regulated by a balance between proliferation and programmed cell death, we further explored these processes. Using in vivo EdU incorporation and the proliferation marker Ki67, we found that the absolute number of proliferating γδ T cells in VAT is significantly higher in the aged compared to young and middle-aged mice, despite a decline in the proportion of proliferating to non-proliferating cells by age. Analysis of apoptosis via caspase 3/7 activation revealed that VAT γδ T cells show reduced apoptosis starting at middle age and continuing into old age. Further, induction of apoptosis using pharmacological inhibitors of Bcl2 family proteins revealed that VAT γδ T cells at middle age are uniquely protected from apoptosis via a mechanism independent of traditional anti-apoptotic Bcl2-family proteins. Collectively, these data indicate that protection from apoptosis at middle age increases survival of tissue-resident γδ T cells resulting in an increased number of proliferative cells from middle age onward, and leading to the age-associated accumulation of γδ T cells in VAT. These findings are important to better understand how adipose tissue dysfunction and related changes in the immune profile contribute to inflammaging among the elderly.

Canagliflozin enhances endogenous eliminatiton of senescent cells through downregulation of PD-L1

Abstract Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been developed as a new class of anti-diabetic drug, and shown cardio- and renal-protective effects in patients with diabetes, heart failure, and chronic kidney diseases. Also, there's evidence that canagliflozin, one of the SGLT2 inhibitors has expanded lifespan in aged mice, but their detailed protective machinery is still to be clarified. Here we demonstrate that canagliflozin has a effect to eliminate senescent cells from pathological tissues, so-called "senolysis" in some murine disease models and alleviates their pathology. We treated canagliflozin mixed in food at 0.03% (w/w) to high-fat-diet(HFD)-fed obese mice and found that canagliflozin reduced senescent cells in visceral adipose tissue within 1 week. Also, we found that senolytic effect of canagliflozin was provided through enhancing endogenous senolytic function by NK or T cells. This machinery was mediated by enhanced AMPK signaling, which leads to the downregulation of PD-L1 expression on senescent cells. Furthermore, we treated canagliflozin to ApoE-knockout atherosclerotic mice with western diet feeding, progeroid mice and chronological aged mice. Canagliflozin reduced their senescent cell burden, resulting in alleviation of atherosclerosis, extended lifespan of progeroid, and altered physical function due to chronological aging. Our results would be promising evidence that SGLT2 inhibitors can be used as not only cardio- or renal-protective drugs but also anti-aging drugs.

Abstract 14285: Sodium Glucose Co-Transporter 2 Inhibition Eliminates Senescent Cells Through Downregulation of PD-L1 and Improves Pathological Aging

Introduction: Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been developed as a new class of anti-diabetic drug, and shown cardio- and renal-protective effects in patients with diabetes, heart failure, and chronic kidney diseases. SGLT2 inhibitor also has effect to expand lifespan in aged mice, suggesting anti-aging effect. But the detailed machinery of these protective effects is still to be clarified. Here we demonstrate that canagliflozin has a effect to eliminate senescent cells from pathological tissues, so-called "senolysis" in murine aging or age-related disease models. Methods: Canagliflozin mixed in food at 0.03% (w/w) was administered for up to 4 weeks to high-fat-fed diabetic mice, for 2 weeks to ApoE-knockout atherosclerotic mice model, for 20 weeks for chronological aged mice, and for lifetime to Zmpste-knockout progeroid mice. Burden of senescent cell accumilation as well as pathological or aged phenotype were measured in each mice model. Results: Canagliflozin reduced senescent cells in visceral adipose tissue within 1 week. Also, we found that senolytic effect of canagliflozin was provided through enhancing endogenous senolytic function by immune cells such as CD8+T cells. This machinery was mediated by enhanced AMPK signaling through incrase of endogenous AICAR production, which leads to the downregulation of PD-L1 expression on senescent cells. Oppositely, inhibition of AMPK signaling with Compound C administration (10mg/kg ip daily) resulted in abolishing senolytic effect of canagliflozin. Furthermore, canagliflozin reduced their senescent cell burden and alleviated atherosclerosis in ApoE-knockout mice, extended lifespan in progeroid mice, and altered physical function in aged mice. Conclusions: Our results showed new aspect of protective efficacy of SGLT2 inhibitors and would be promising evidence that SGLT2 inhibitors can be used as not only cardio- or renal-protective drugs but also anti-aging drugs.

Obesity accelerates endothelial-to-mesenchymal transition in adipose tissues of mice and humans.

Vascular dysfunction and chronic inflammation are characteristics of obesity-induced adipose tissue dysfunction. Proinflammatory cytokines can drive an endothelial-to-mesenchymal transition (EndoMT), where endothelial cells undergo a phenotypic switch to mesenchymal-like cells that are pro-inflammatory and pro-fibrotic. In this study, we sought to determine whether obesity can promote EndoMT in adipose tissue. Mice in which endothelial cells are lineage-traced with eYFP were fed a high-fat/high-sucrose (HF/HS) or Control diet for 13, 26, and 52 weeks, and EndoMT was assessed in adipose tissue depots as percentage of CD45-CD31-Acta2+ mesenchymal-like cells that were eYFP +. EndoMT was also assessed in human adipose endothelial cells through cell culture assays and by the analysis of single cell RNA sequencing datasets obtained from the visceral adipose tissues of obese individuals. Quantification by flow cytometry showed that mice fed a HF/HS diet display a time-dependent increase in EndoMT over Control diet in subcutaneous adipose tissue (+3.0%, +2.6-fold at 13 weeks; +10.6%, +3.2-fold at 26 weeks; +11.8%, +2.9-fold at 52 weeks) and visceral adipose tissue (+5.5%, +2.3-fold at 13 weeks; +20.7%, +4.3-fold at 26 weeks; +25.7%, +4.8-fold at 52 weeks). Transcriptomic analysis revealed that EndoMT cells in visceral adipose tissue have enriched expression of genes associated with inflammatory and TGFβ signaling pathways. Human adipose-derived microvascular endothelial cells cultured with TGF-β1, IFN-γ, and TNF-α exhibited a similar upregulation of EndoMT markers and induction of inflammatory response pathways. Analysis of single cell RNA sequencing datasets from visceral adipose tissue of obese patients revealed a nascent EndoMT sub-cluster of endothelial cells with reduced PECAM1 and increased ACTA2 expression, which was also enriched for inflammatory signaling genes and other genes associated with EndoMT. These experimental and clinical findings show that chronic obesity can accelerate EndoMT in adipose tissue. We speculate that EndoMT is a feature of adipose tissue dysfunction that contributes to local inflammation and the systemic metabolic effects of obesity..

Drastic transformation of visceral adipose tissue and peripheral CD4 T cells in obesity.

Obesity has a pronounced effect on the immune response in systemic organs that results in not only insulin resistance but also altered immune responses to infectious diseases and malignant tumors. Obesity-associated microenvironmental changes alter transcriptional expression and metabolism in T cells, leading to alterations in T-cell differentiation, proliferation, function, and survival. Adipokines, cytokines, and lipids derived from obese visceral adipose tissue (VAT) may also contribute to the systemic T-cell phenotype, resulting in obesity-specific pathogenesis. VAT T cells, which have multiple roles in regulating homeostasis and energy utilization and defending against pathogens, are most susceptible to obesity. In particular, many studies have shown that CD4 T cells are deeply involved in the homeostasis of VAT endocrine and metabolic functions and in obesity-related chronic inflammation. In obesity, macrophages and adipocytes in VAT function as antigen-presenting cells and contribute to the obesity-specific CD4 T-cell response by inducing CD4 T-cell proliferation and differentiation into inflammatory effectors via interactions between major histocompatibility complex class II and T-cell receptors. When obesity persists, prolonged stimulation by leptin and circulating free fatty acids, repetitive antigen stimulation, activating stress responses, and hypoxia induce exhaustion of CD4 T cells in VAT. T-cell exhaustion is characterized by restricted effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from functional effector and memory T cells. Moreover, obesity causes thymic regression, which may result in homeostatic proliferation of obesity-specific T-cell subsets due to changes in T-cell metabolism and gene expression in VAT. In addition to causing T-cell exhaustion, obesity also accelerates cellular senescence of CD4 T cells. Senescent CD4 T cells secrete osteopontin, which causes further VAT inflammation. The obesity-associated transformation of CD4 T cells remains a negative legacy even after weight loss, causing treatment resistance of obesity-related conditions. This review discusses the marked transformation of CD4 T cells in VAT and systemic organs as a consequence of obesity-related microenvironmental changes.

Abstract P3128: Obesogenic Diet Promotes Endothelial-to-Mesenchymal Transition In Adipose Tissue

Vascular dysfunction and chronic inflammation are characteristics of obesity-induced adipose tissue dysfunction. Proinflammatory cytokines can drive Endothelial-to-Mesenchymal Transition (EndoMT), where endothelial cells transition into mesenchymal-like interstitial cells that leave the vascular wall and promote tissue inflammation and fibrosis. In this study, we aimed to determine whether obesogenic diet can promote EndoMT in adipose tissue. EndoMT was assessed in 1) transgenic endothelial cell lineage-tracing mice subjected to High-Fat/High-Sucrose (HF/HS) diet, 2) available single cell RNA sequencing datasets from human obese adipose tissue, and 3) cell culture models using primary human adipose-derived endothelial cells. In lineage-tracing mice, obesogenic diet induced EndoMT in a time-dependent manner at 13, 26, and 52 weeks on HF/HS diet in both subcutaneous and visceral adipose tissue. Interestingly, rate of EndoMT was greater in visceral compared to subcutaneous adipose tissue. After 52-weeks on HF/HS diet, EndoMT cells in visceral adipose tissue upregulate inflammatory response and TGF-Beta signaling transcriptional pathways compared to endothelial cells in the same adipose tissue depot. Initiation of EndoMT was also found in single cell RNA sequencing datasets of stromal cells from human obese adipose tissue, indicated by endothelial cells in these datasets containing sub-clusters with reduced Pecam1 and increased Acta2 expression. Finally, the capacity for EndoMT in primary human adipose-derived endothelial cells was validated by bulk RNA sequencing after treatment with proinflammatory cytokines (TNF-Alpha, IFN-Gamma, TGF-Beta1), confirming decreased expression of endothelial cell genes and increased expression of genes associated with EndoMT, inflammatory response, and TGF-Beta signaling. Together, these clinical and experimental findings indicate that chronic obesity can promote EndoMT in adipose endothelial cells.

Accumulation of γδ T cells in visceral fat with aging promotes chronic inflammation

Adipose tissue dysfunction is strongly linked to the development of chronic inflammation and cardiometabolic disorders in aging. While much attention has been given to the role of resident adipose tissue immune cells in the disruption of homeostasis in obesity, age-specific effects remain understudied. Here, we identified and characterized a population of γδ T cells, which show unique age-dependent accumulation in the visceral adipose tissue (VAT) of both mice and humans. Diet-induced obesity likewise increased γδ T cell numbers; however, the effect was greater in the aged where the increase was independent of fat mass. γδ T cells in VAT express a tissue-resident memory T cell phenotype (CD44hiCD62LlowCD69+) and are predominantly IL-17A-producing cells. Transcriptome analyses of immunomagnetically purified γδ T cells identified significant age-associated differences in expression of genes related to inflammation, immune cell composition, and adipocyte differentiation, suggesting age-dependent qualitative changes in addition to the quantitative increase. Genetic deficiency of γδ T cells in old age improved the metabolic phenotype, characterized by increased respiratory exchange ratio, and lowered levels of IL-6 both systemically and locally in VAT. Decreased IL-6 was predominantly due to reduced production by non-immune stromal cells, primarily preadipocytes, and adipose-derived stem cells. Collectively, these findings suggest that an age-dependent increase of tissue-resident γδ T cells in VAT contributes to local and systemic chronic inflammation and metabolic dysfunction in aging.

Cellular Senescence in Diabetes Mellitus: Distinct Senotherapeutic Strategies for Adipose Tissue and Pancreatic β Cells.

Increased insulin resistance and impaired insulin secretion are significant characteristics manifested by patients with type 2 diabetes mellitus (T2DM). The degree and extent of these two features in T2DM vary among races and individuals. Insulin resistance is accelerated by obesity and is accompanied by accumulation of dysfunctional adipose tissues. In addition, dysfunction of pancreatic β-cells impairs insulin secretion. T2DM is significantly affected by aging, as the β-cell mass diminishes with age. Moreover, both obesity and hyperglycemia-related metabolic changes in developing diabetes are associated with accumulation of senescent cells in multiple organs, that is, organismal aging. Cellular senescence is defined as a state of irreversible cell cycle arrest with concomitant functional decline. It is caused by telomere shortening or senescence-inducing stress. Senescent cells secrete proinflammatory cytokines and chemokines, which is designated as the senescence-associated secretory phenotype (SASP), and this has a negative impact on adipose tissues and pancreatic β-cells. Recent advances in aging research have suggested that senolysis, the removal of senescent cells, can be a promising therapeutic approach to prevent or improve aging-related diseases, including diabetes. The attenuation of a SASP may be beneficial, although the pathophysiological involvement of cellular senescence in diabetes is not fully understood. In the clinical application of senotherapy, tissue-context-dependent senescent cells are increasingly being recognized as an issue to be solved. Recent studies have observed highly heterogenic and complex senescent cell populations that serve distinct roles among tissues, various stages of disease, and different ages. For example, in high-fat-diet induced diabetes with obesity, mouse adipose tissues display accumulation of p21 Cip1-highly-expressing (p21 high) cells in the early stage, followed by increases in both p21 high and p16 INK4a-highly-expressing (p16 high) cells in the late stage. Interestingly, elimination of p21 high cells in visceral adipose tissue can prevent or improve insulin resistance in mice with obesity, while p16 high cell clearance is less effective in alleviating insulin resistance. Importantly, in immune-deficient mice transplanted with fat from obese patients, dasatinib plus quercetin, a senolytic cocktail that reduces the number of both p21 high and p16 high cells, improves both glucose tolerance and insulin resistance. On the other hand, in pancreatic β cells, p16 high cells become increasingly predominant with age and development of diabetes. Consistently, elimination of p16 high cells in mice improves both glucose tolerance and glucose-induced insulin secretion. Moreover, a senolytic compound, the anti-Bcl-2 inhibitor ABT263 reduces p16 INK4a expression in islets and restores glucose tolerance in mice when combined with insulin receptor antagonist S961 treatment. In addition, efficacy of senotherapy in targeting mouse pancreatic β cells has been validated not only in T2DM, but also in type 1 diabetes mellitus. Indeed, in non-obese diabetic mice, treatment with anti-Bcl-2 inhibitors, such as ABT199, eliminates senescent pancreatic β cells, resulting in prevention of diabetes mellitus. These findings clearly indicate that features of diabetes are partly determined by which or where senescent cells reside in vivo, as adipose tissues and pancreatic β cells are responsible for insulin resistance and insulin secretion, respectively. In this review, we summarize recent advances in understanding cellular senescence in adipose tissues and pancreatic β cells in diabetes. We review the different potential molecular targets and distinctive senotherapeutic strategies in adipose tissues and pancreatic β cells. We propose the novel concept of a dual-target tailored approach in senotherapy against diabetes.

Adipose invariant NKT cells interact with CD1d-expressing macrophages to regulate obesity-related inflammation.

Obesity is accompanied by and accelerated with chronic inflammation in adipose tissue, especially visceral adipose tissue (VAT). This low-level inflammation predisposes the host to the development of metabolic disease, most notably type 2 diabetes. We have focused on the capacity of glycolipid-reactive, CD1d-restricted natural killer T (NKT) cells to modulate obesity and its associated metabolic sequelae. We previously reported that CD1d knockout (KO) mice are partially protected against the development of obesity-associated insulin resistance, and these findings were recapitulated in mice with an adipocyte-specific CD1d deficiency, suggesting that NKT cell-adipocyte interactions play a critical role in exacerbating disease. However, many other CD1d-expressing cells contribute to the in vivo responses of NKT cells to lipid antigens. In the present study, we examined the role of CD1d expression by macrophages (Mϕ) in the development of obesity-associated metabolic inflammation using LysMcre-cd1d1f/f mice where the CD1d1gene is disrupted in a Mϕ-specific manner. Unexpectedly, these animals contained a higher frequency of T-bet+ CD4+ T cells in VAT with increased production of Th1 cytokines that aggravated VAT inflammation. Mϕ from mutant mice displayed increased production of IL-12p40, suggesting M1 polarization. These findings indicate that interactions of CD1d on Mϕ with NKT cells play a beneficial role in obesity-associated VAT inflammation and insulin resistance with a sharp contrast to an aggravating role of CD1d in another type of antigen-presenting cell, dendritic cells.

Myeloid Cell-Specific IL-4 Receptor Knockout Partially Protects from Adipose Tissue Inflammation.

IL-4 receptor signaling is supposed to play a major role in anti-inflammatory polarization and proliferation of adipose tissue macrophages. In this study, we examined the metabolic and inflammatory phenotype of C57BL/6J mice (IIl4ra) with LysM-dependent knockout (IIl4ra Δmyel) of the IL-4 receptor α-chain (IL-4Rα), the mandatory signaling component of IL-4 and IL-13, on chow and high-fat diet. Lean IIl4ra Δmyel mice showed decreased insulin sensitivity, no divergent adipose tissue macrophage polarization, but an increased percentage of CD8+ T cells in visceral adipose tissue. After 20 wk of a high-fat diet, IIl4ra Δmyel mice exhibited higher glucose tolerance, no changes in the lymphocyte compartment and fewer M1 macrophages in visceral adipose tissue. In vivo adipose tissue macrophage proliferation measured by BrdU incorporation was unaffected by Il4ra knockout. Interestingly, we show that IL-4Rα signaling directly augmented Itgax (Cd11c) gene expression in bone marrow-derived macrophages and increased the amount of CD11c+ macrophages in adipose tissue explants. Myeloid cell-specific knockout of Il4ra deteriorated insulin sensitivity in lean mice but improved parameters of glucose homeostasis and partially protected from adipose tissue inflammation in obese mice. Hence, IL-4Rα signaling probably plays a minor role in maintaining the macrophage M2 population and proliferation rates in vivo. Moreover, our data indicate that IL-4 signaling plays a proinflammatory role in adipose tissue inflammation by directly upregulating CD11c on adipose tissue macrophages.

Abstract 13311: A Novel Senolytic Drug, Seno-7284 Ameliorates Age-Related Cardiometabolic Diseases

Cellular senescence plays a pivotal role in aging and age-related disorders. Recently, it was shown that elimination of senescent cells, so-called "senolysis" has the potential to become a next generation therapy for age-related disorders including cardio-metabolic diseases. However, currently there is no senolytic drug available in clinical settings. The present study was aimed to identify a novel senolytic reagent effective for cardiometabolic diseases in compounds already available in clinical settings. We demonstrate a compound called "Seno-7284" exhibits senolytic effect in murine models of type 2 diabetes, atherosclerosis, progeroid and chronological aged mice. Seno-7284 reduced the accumulation of senescent cells in visceral adipose tissue of diabetic mice as senescence-associated-beta-galactosidase (SA-beta-gal) staining exhibits. This associated with the suppression in systemic glucose intolerance, and adipose tissue inflammation in four weeks after the administration of Seno-7284. Administrating Seno-7284 for two weeks also suppressed the progression of atherosclerosis in ApoE-KO mice. This drug significantly improved the lifespan of Zm-KO mice by administering it from 12 weeks old. Administration of Seno-7284 from 50 week-old for 20 weeks improves physical function in chronological aged mice. Further analysis including single cell RNA-seq suggested that Seno-7284 stimulates endogenous senolytic function of NK cells and CD8+ T cells. Our results indicate Seno-7284 mediates its biological effects by inducing senolysis in murine aging models. Seno-7284 would become promising therapies for age-related disorders.

Innate Immune System Orchestrates Metabolic Homeostasis and Dysfunction in Visceral Adipose Tissue During Obesity.

Arising incidence of metabolic disorders and related diseases caused by obesity is a global health concern. Elucidating the role of the immune system in this process will help to understand the related mechanisms and develop treatment strategies. Here, we have focused on innate immune cells in visceral adipose tissue (VAT) and summarized the roles of these cells in maintaining the homeostasis of VAT. Furthermore, this review reveals the importance of quantitative and functional changes of innate immune cells when the metabolic microenvironment changes due to obesity or excess lipids, and confirms that these changes eventually lead to the occurrence of chronic inflammation and metabolic diseases of VAT. Two perspectives are reviewed, which include sequential changes in various innate immune cells in the steady state of VAT and its imbalance during obesity. Cross-sectional interactions between various innate immune cells at the same time point are also reviewed. Through delineation of a comprehensive perspective of VAT homeostasis in obesity-induced chronic inflammation, and ultimately metabolic dysfunction and disease, we expect to clarify the complex interactive networks among distinct cell populations and propose that these interactions should be taken into account in the development of biotherapeutic strategies.

Lineage‐tracing Reveals Multiple Roles of Tcf21 Lineage Cells in the Development and Expansion of Visceral Adipose Tissue

The specific correlation between visceral adipose tissue (VAT) and insulin resistance makes VAT a potential target for treating type 2 diabetes. Recent studies have shown that Tcf21, a basic helix-loop-helix transcription factor, is expressed in VAT but not in subcutaneous AT (SAT). Thus, we hypothesized that cells expressing Tcf21 played a critical role in VAT. Using tamoxifen-inducible Tcf21 lineage-tracing mouse lines, we identified that Tcf21 was exclusively expressed in progenitor cells in VAT but not those in SAT. Lineage-tracing showed that most Tcf21 lineage progenitor cells also express Pdgfra, a marker of mesenchymal stem cell. Tcf21 expression was abolished upon adipogenic differentiation. Tcf21 lineage-tracing induced at embryonic day 11.2 (E11.5) revealed that progenitor cells of Tcf21 lineage give rise to all visceral adipocytes. Tamoxifen treatment during postnatal days 2-4 (P2-4) lineage-traced ~40% adipocytes in epididymal VAT (eVAT) at weaning, indicating high adipogenic activity in VAT during the neonatal stage. EdU assay showed that Tcf21 lineage-traced cells actively proliferated during the first 2 weeks after birth followed by a phase of robust adipogenic differentiation between P14 and P21. Tcf21 lineage-tracing induced at 6 weeks of age only labeled less than 1% of adipocytes in eVAT of mice after 12 weeks of high-fat diet (HFD) treatment regardless of a significant expansion of Tcf21 lineage-traced cells, indicating that Tcf21 lineage cells in adult mice were less adipogenic in vivo. In addition, it was found Tcf21 lineage cells also gave rise to a small portion of vascular smooth muscle cells (VSMCs). However, these cells lacked the expression of PDGFRα. RNA-seq analysis of Tcf21 lineage-traced PDGFRα+ cells isolated from mice at different developmental and growth stages showed distinct gene expression profiles. Proliferation genes were highly active in Tcf21 lineage cells at P12, while genes associated with adipogenic differentiation were more active at P12 and P21. Interestingly, some inflammatory genes that are commonly expressed in inflammatory cells were highly enriched in Tcf21 lineage cells isolated from mice after long-term HFD treatment. Single-cell RNA-seq of Tcf21 lineage-traced cells isolated from mice of different ages showed that the gene expression of individual cells became increasingly diverse as the age of the mice increased. Single-cell RNA-seq analysis confirmed the presence of a small subpopulation of Tcf21 lineage-traced VSMCs as well as a Tcf21 lineage-traced inflammatory cell subpopulation that was exclusive to HFD-treated mice. IHC staining showed that Tcf21 lineage-traced inflammatory cells were located in crown-like structures and might play a role in inflammation and phagocytosis. Several additional subpopulations of Tcf21 lineage-traced cells were also identified and are currently being investigated. Our study clearly showed the critical roles of Tcf21 lineage cells in the growth and development of VAT as well as in the development of VAT inflammation in obese mice.

RANK links thymic regulatory T cells to fetal loss and gestational diabetes in pregnancy.

Successful pregnancies rely on adaptations within the mother1, including marked changes within the immune system2. It has long been known that the thymus, the central lymphoid organ, changes markedly during pregnancy3. However, the molecular basis and importance of this process remain largely obscure. Here we show that the osteoclast differentiation receptor RANK4,5 couples female sex hormones to the rewiring of the thymus during pregnancy. Genetic deletion of Rank (also known as Tnfrsf11a) in thymic epithelial cells results in impaired thymic involution and blunted expansion of natural regulatory T (Treg) cells in pregnant female mice. Sex hormones, in particular progesterone, drive the development of thymic Treg cells through RANK in a manner that depends on AIRE+ medullary thymic epithelial cells. The depletion of Rank in the mouse thymic epithelium results in reduced accumulation of natural Treg cells in the placenta, and an increase in the number of miscarriages. Thymic deletion of Rank also results in impaired accumulation of Treg cells in visceral adipose tissue, and is associated with enlarged adipocyte size, tissue inflammation, enhanced maternal glucose intolerance, fetal macrosomia, and a long-lasting transgenerational alteration in glucose homeostasis, which are all key hallmarks of gestational diabetes. Transplantation of Treg cells rescued fetal loss, maternal glucose intolerance and fetal macrosomia. In human pregnancies, we found that gestational diabetes also correlates with a reduced number of Treg cells in the placenta. Our findings show that RANK promotes the hormone-mediated development of thymic Treg cells during pregnancy, and expand the functional role of maternal Treg cells to the development of gestational diabetes and the transgenerational metabolic rewiring of glucose homeostasis.

Defective immunosuppressive function of Treg cells in visceral adipose tissue in MIF deficient mice

Obesity, a global health problem nowadays, is a state of low-grade chronic inflammation of adipose tissue (AT) associated with increased adipocyte growth and proliferation and immune cell polarization towards an inflammatory phenotype within the stromal vascular fraction (SVF). Pro-inflammatory cells in the AT produce mediators of inflammation (IL-1β, TNF, macrophage migration inhibitory factor – MIF), thereby surpassing the anti-inflammatory response mediated by IL-10 and TGF-β, cytokines produced by regulatory T (Treg) cells. In this study we demonstrate that the absence of the pro-inflammatory cytokine MIF led to obesity and inflammation in the visceral AT (VAT) in 6 months old MIF−/− mice. Besides the increment of pro-inflammatory AT macrophages and the enhanced production of TNF and IL-1β, VAT of MIF−/− mice contained increased numbers of Treg cells. In situ proliferation of Treg cells did not differ between MIF−/− and wild type mice, but Treg cells isolated from the VAT of MIF-deficient mice, and not from the cervical lymph nodes, exhibited lower expression and production of IL-10 and TGF-β. Additionally, SVF cells had significantly lower levels of STAT3 and IL-33, altogether indicating that VAT Treg cells in MIF−/− mice, albeit abundantly present, are not fully functional. These results indicate that MIF is a new regulator of VAT Treg cell function, necessary for their immunosuppressive activities.

Abstract 13075: A Novel Senolytic Drug, Seno-7284 Ameliorates Aging and Age-related Cardiometabolic Disorders

Introduction: Recent studies have shown elimination of senescent cells, so-called "senolysis" has the potential to become a novel therapy for aging and age-related disorders. However, there is no senolytic drug available in clinical settings currently. Purpose: The present study was aimed to identify a novel senolytic reagent effective for cardiometabolic disease among compounds already available in clinical settings. Here we identified a compound called "seno-7284" which exhibits senolytic effect in murine models of aging and age-related cardiometabolic disorders. Methods: We generated diet-induced obese(DIO) mice model by imposing high-fat diet, atherosclerotic mice model by imposing western diet to ApoE knockout mice. We administered seno-7284 mixed in the diet (0.03% w/w) to each mouse model for 1, 2 or 4 weeks. We harvested tissue samples for molecular biological analysis after measuring glucose/insulin tolerance. We also administered seno-7284 to Zmpste24 homozygous knockout mice as a progeroid mice model from 12-week-old to measure lifespan and to wild-type mice from 1-year-old for 20 weeks to measure physical function. Results: Seno-7284 reduced accumulation of senescent cells in visceral adipose tissue of DIO mice as senescence-associated beta-galactosidase (SA-beta-gal) staining exhibits (figure(fig) a), resulting in improvement of glucose metabolism (fig b). We also found seno-7284 reduced accumulation of senescent cells in the aorta of atherosclerotic mice model (fig c) and inhibited advancing atherosclerosis (fig d). Furthermore, Seno-7284 improved lifespan of progeroid mice (fig e) and physical function of aged mice (fig f). Further analysis including RNA-seq speculated seno-7284 stimulates endogenous senolytic function of immune cells. Conclusion: Our results indicate that seno-7284 would become a promising senolytic drug that exhibits novel therapeutic machinery for aging and age-related cardiometabolic diseases.

A novel senolytic drug, seno-7284 ameliorates aging phenotype and age-related cardiometabolic diseases

Abstract Background Cellular senescence occurs as a result of various genotoxic stresses and it plays a pivotal role in aging and age-related disorders. Recently, it was shown that elimination of senescent cells, so-called “senolysis” has the potential to become a promising novel therapy for age-related disorders in several mice models including cardiovascular diseases. However, there is no senolytic drug available in clinical settings currently. Purpose The present study was aimed to identify a novel senolytic reagent effective for cardiometabolic disease among compounds already available in clinical settings. Here we demonstrate that a compound called “seno-7284” exhibits senolytic effect in murine models of type 2 diabetes, atherosclerosis, progeroid and chronological aging. Methods We generated diet-induced obesity/diabetic mice model by imposing high-fat diet from 4-week-old for two months, atherosclerosis mice model by imposing western diet to ApoE homozygous knockout mice (ApoE-KO mice) from 4-week-old for 3 months. We administered seno-7284 mixed in the diet (0.03% w/w) to each mouse model for 1, 2 or 4 weeks. For the analysis, we carried out some physiological examinations including glucose tolerance test (GTT) and insulin tolerance test (ITT), then harvested tissue samples and took them to molecular biological analysis including real-time PCR, western blotting, RNA-sequence, etc. We also generated Zmpste24 homozygous knockout mice (Zmpste24-KO mice) as a progeroid mice model to measure their lifespan. Seno-7284 was administered to Zmpste24-KO mice from 12-week-old to the end of life. We also administrated seno-7284 to chronological aged mice at 1-year-old for 20 weeks and their physical function was examined with rotarod running test and hand-grip test. Results Seno-7284 reduced the accumulation of senescent cells in visceral adipose tissue of dietary obese mice as senescence-associated beta-galactosidase (SA-beta-gal) staining exhibits (Figure 1a). This effect results in ameliorating insulin tolerance (Figure 1b) and adipose tissue inflammation after 4-week administration of seno-7284. We also found administrating Seno-7284 for two weeks also reduced the accumulation of senescent cells in the atherosclerotic lesion in the aorta of ApoE-KO mice (Figure 1c) and inhibited advancing atherosclerosis (Figure 1d). Surprisingly, seno-7284 significantly improved the lifespan of Zmpste24 KO mice (Figure 1e). Seno-7284 also improved the physical function of chronologically aged mice by administrating it from 1-year-old for 20 weeks (Figure 1f). In-vitro studies didn't exhibit seno-7284 kills senescent cells directly, but further analysis including RNA-seq or metabolomic analysis speculated that seno-7284 stimulates endogenous senolytic function of NK-cells and CD8+ T-cells. Conclusion Our results indicate that seno-7284 would become a promising senolytic drug that exhibits novel therapeutic machinery for aging and age-related cardiometabolic diseases. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research (KAKENHI) C, Niigata University Tsukada medical research grant

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 β-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 and adipose tissue inflammation-a simple story? Fat chance!

A new study in mice reveals time-dependent effects of a ketogenic diet on resident immune cells in visceral adipose tissue, which have consequences for whole-body metabolic homeostasis.