Crown-like Structures Research Articles

Crown-like structures in breast adipose tissue of breast cancer patients: associations with CD68 expression, obesity, metabolic factors and prognosis

Crown-like structures of the breast (CLS-B), defined by the clustering of macrophages (identified using CD68 immunohistochemical staining) to surround a dying adipocyte, are a sign of adipose-tissue inflammation. In human cohorts, CLS-B positively correlates with older age, obesity, dyslipidemia and higher levels of glucose, insulin, C-reactive protein and IL-6. In an existing cohort of early-stage breast cancer patients, CLS-B were identified using H&E stained histologic sections (hCLS-B), and by CD68 immunohistochemistry (CD68 + CLS-B). We examined associations of H&E and CD68-detected CLS-B with clinicopathologic features using χ2 tests, with metabolic factors using Wilcoxon rank sum tests and with disease free and overall survival using Cox regression models. hCLS-B were detected in 59 of 163 patients with slides (36.2%) and CD68 + CLS-B in 37 of 119 patients with paraffin blocks (31.1%). hCLS-B were positively correlated with higher weight (p = 0.003), BMI (p = 0.0008) and C-reactive protein (p = 0.045). CD68 + CLS-B were positively correlated with higher weight (p = 0.006), BMI p = 0.001), leptin (p = 0.034), insulin (p = 0.008) and Homeostasis Model Assessment (p = 0.027). CD68 + CLS-B were associated with poor distant disease-free with a hazard ratio (HR) of 2.81, 95% confidence interval (CI) 1.20–6.57, and overall survival with HR 3.97 (1.66–9.48), while hCLS-B were not associated with either: HR for distant recurrence 0.59 (0.26–1.30); HR for death 1.04 (0.50–2.16). The presence of hCLS-B and of CD68 + CLS-B were associated with obesity; CD68 + CLS-B were associated with insulin resistance and adverse prognosis. Similar patterns were not seen for hCLS-B. Research is needed to understand the biologic basis for these differences.

Children's Drawings of Coronavirus.

To understand how children perceive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in relation to public representations and to evaluate their interpretations. Children's perceptions of SARS-CoV-2 were evaluated by asking 103 French children, aged 5 to 17 years old, first to draw a coronavirus and then to identify SARS-CoV-2 in a series of 16 images during summer 2020. One hundred three children were included in the study, either during outpatient visits at the hospital (in Marseille and Paris) or through the authors' social network, and were grouped in terms of age, parents' occupation, mode of recruitment, and recollection of having previously seen a representation of a coronavirus. Half of the children drew the coronavirus as circular in shape, and almost all included a crownlike feature. One-third of the drawings had anthropomorphic features. Although the pictorial representations of the virus were fairly accurate overall, the children's interpretations of the crownlike structure were imaginative. The explanations the children gave for their drawings were in some cases surprising. Among the 16 pictures they were shown, the children correctly identified those of SARS-CoV-2, other than the electron micrograph, in more than two-thirds of cases. Children of all ages, even the youngest, and both sexes had a relatively accurate perception of SARS-CoV-2, as evaluated through their drawings and their ability to recognize it among other pictures. The children's drawings of the coronavirus were colorful and had a less frightening tone than expected in the light of media coverage, suggesting that they had developed coping mechanisms.

Hydrophobic-hydrophilic crown-like structure enables aquatic insects to reside effectively beneath the water surface

Various insects utilise hydrophobic biological surfaces to live on the surface of water, while other organisms possess hydrophilic properties that enable them to live within a water column. Dixidae larvae reside, without being submerged, just below the water surface. However, little is known about how these larvae live in such an ecological niche. Herein, we use larvae of Dixa longistyla (Diptera: Dixidae) as experimental specimens and reveal their characteristics. A complex crown-like structure on the abdomen consists of hydrophobic and hydrophilic elements. The combination of these contrasting features enables the larvae to maintain their position as well as to move unidirectionally. Their hydrophobic region leverages water surface tension to function as an adhesive disc. By using the resistance of water, the hydrophilic region serves as a rudder during locomotion.

Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages

A chronic low-grade inflammation within adipose tissue (AT) seems to be the link between obesity and some of its associated diseases. One hallmark of this AT inflammation is the accumulation of AT macrophages (ATMs) around dead or dying adipocytes, forming so-called crown-like structures (CLS). To investigate the dynamics of CLS and their direct impact on the activation state of ATMs, we established a laser injury model to deplete individual adipocytes in living AT from double reporter mice (GFP-labeled ATMs and tdTomato-labeled adipocytes). Hence, we were able to detect early ATM-adipocyte interactions by live imaging and to determine a precise timeline for CLS formation after adipocyte death. Further, our data indicate metabolic activation and increased lipid metabolism in ATMs upon forming CLS. Most importantly, adipocyte death, even in lean animals under homeostatic conditions, leads to a locally confined inflammation, which is in sharp contrast to other tissues. We identified cell size as cause for the described pro-inflammatory response, as the size of adipocytes is above a critical threshold size for efferocytosis, a process for anti-inflammatory removal of dead cells during tissue homeostasis. Finally, experiments on parabiotic mice verified that adipocyte death leads to a pro-inflammatory response of resident ATMs in vivo, without significant recruitment of blood monocytes. Our data indicate that adipocyte death triggers a unique degradation process and locally induces a metabolically activated ATM phenotype that is globally observed with obesity.

257-OR: Short-Term Hyperglucagonemia Induces Adipose Tissue Insulin Resistance and Inflammation In Vivo

Although the effect of glucagon on glucose metabolism has been well characterized, its effect on adipose tissue and lipid metabolism in humans in vivo has been poorly studied with controversial results. We examined the effect of short-term hyperglucagonemia on adipocyte metabolism in 8 healthy normal glucose tolerant subjects (5M/3F, age=35±5, BMI = 24±1) who received a 12-hour (6PM to 6AM) glucagon infusion (6ng/kg/min) with 14C-glycerol infusion; subcutaneous abdominal adipose tissue biopsy was obtained at 6AM. 8 weeks later subjects returned for a repeat study with 12-hour infusion of normal saline. Plasma glucagon increased from 57±3 to 219±21 pg/ml within one hour and remained elevated throughout the study. Glycerol turnover rate (5.1±0.6 vs. 3.8±0.6 µmol/kg•min, p<0.05) increased by 34%, and the adipose tissue insulin resistance index (fasting plasma FFA x Insulin) more than doubled (5.8±0.8 vs. 2.8±0.7 mM•mU/L, p<0.05) after 12-hour glucagon infusion compared to saline. mRNA expression of genes involved in lipolysis in adipose tissue were upregulated after glucagon infusion (ATGL, 1.14±0.07 vs. 0.77±0.03; HSL, 1.17±0.03 vs. 0.9±0.13; MGL, 1.2±0.04 vs. 0.82±0.2, all p<0.05). Plasma concentration of proinflammatory markers (IL-1β, 0.65±0.05 vs. 0.49±0.05 pg/mL; TNF-α, 2.03±0.23 vs. 1.75±0.17 pg/mL, both p<0.05) and lipidomes involved in inflammatory pathways (lysophosphatidylcholine, 126±11 vs. 51±7 nmol/ml, p<0.005; phosphatidylcholine, 385±24 vs. 306±25 nmol/ml, p<0.01; ceramide, 1.7±0.2 vs. 1.2±0.2 nmol/ml, p<0.01) also increased after 12-hour glucagon infusion. H&E and CD68 immunofluorescence staining in adipose tissue displayed the crown-like structures (CLS) and increased infiltration of macrophages following glucagon infusion. Conclusion: Short-term (12-hour) physiologic hyperglucagonemia stimulates adipose tissue lipolysis, induces adipocyte insulin resistance, and promotes adipose tissue and systemic inflammation. Disclosure X. Chen: None. M. Fourcaudot: None. L. Norton: None. R. A. Defronzo: Other Relationship; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Intarcia Therapeutics, Inc., Janssen Pharmaceuticals, Inc., Novo Nordisk, Research Support; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc., Speaker’s Bureau; Self; AstraZeneca, Novo Nordisk. D. Tripathy: None. Funding Foundation for Advancing Veterans’ Health Research

Clinical significance of crown-like structures to trastuzumab response in patients with primary invasive HER2+ breast cancer.

e12533 Background: Obesity is associated with breast cancer development and worse survival. Obesity can initiate, promote, and maintain systemic inflammation via metabolic reprogramming of macrophages that encircle adipocytes, termed crown-like structures (CLS). In breast cancer patients, CLS are present in 36-50% of patients and have been associated with anthropometric parameters. Here we focus on HER2+ breast cancer. The role of adiposity in HER2+ breast cancer is conflicting which may be attributed to the tumour heterogeneity. Adiposity has also been shown to affect the local immune environment of solid tumours. However, the prognostic significance of CLS in HER2+ breast cancer is still unknown. Methods: We investigated the prognostic significance of CLS in a cohort of 219 patients with primary HER2+ breast cancer who were diagnosed between 1982 to 2012 in Southampton General Hospital. This cohort includes 76 HER2+ trastuzumab naïve patients and 143 HER2+ patients treated with adjuvant trastuzumab. We stained FFPE tumour samples for the expression of CD68, CD16 and CD32B on CLS and correlated these to clinical outcomes. CLS were defined as CLS within distant adipose tissue, CLS within the adipose-tumour border (B-CLS) and intratumoural CLS. CLS were quantified manually in full face sections by two independent scorers and descriptive and Cox regression analysis was carried out. Results: A total of 201 tumours were suitable for CLS analyses. The median follow-up was 34.74 months (range, 0.43-299.08). In the trastuzumab naive cohort, B-CLS≤1 and B-CLS > 1 were present in 37 (52.11%) and 34 (47.89%), respectively. In the trastuzumab treated cohort, B-CLS≤1 were identified in 69 (53.08%) and B-CLS > 1 were found in 61 (46.92%) of the tumours. CLS were more commonly found in the adipose-tumour border (60.89%) rather than in the distant adipose tissue (36.14%) or intratumorally (14.36%). The presence of any CLS was significantly associated with BMI≥25 kg/m2 (p = 0.018). There was strong evidence of association between CD68+CD32B+ B-CLS and BMI≥25 kg/m2 (p = 0.007). Co-expression of CD16 and CD32B by B-CLS was more frequent in patients with BMI≥25 kg/m2 (p = 0.036). Survival analysis showed shorter time to metastatic disease in patients with CD68+ B-CLS > 1 (p = 0.011) in the trastuzumab treated cohort. Subgroup analysis revealed that in the BMI≥25 kg/m2 group, patients with CD68+ B-CLS > 1 had shorter time to metastatic disease compared to patients with B-CLS≤1 (p = 0.004). Multivariate cox regression showed that B-CLS > 1 is an independent prognostic factor for shorter time to metastatic disease in patients with primary HER2+ breast cancer that received adjuvant trastuzumab (HR 6.81, 95%CI (1.38-33.54), p = 0.018). Conclusions: B-CLS can be potentially used as a predictive biomarker to optimize the stratification and personalisation of treatment in HER2-overexpressed breast cancer patients.

Crown-Like Structures in Breast Adipose Tissue: Early Evidence and Current Issues in Breast Cancer.

Simple SummaryObesity increases the risk of postmenopausal, hormone receptor-positive breast cancer and has been linked to a higher risk of recurrence and mortality. During obesity, adipose tissue can become dysfunctional, resulting in chronic low-grade inflammation. Crown-like structures in breast adipose tissue (CLS-B), composed of macrophages surrounding dead or dying adipocytes in a crown-like pattern, are a new histologic marker of local inflammation. In this review, we aim to evaluate the early evidence of CLS-B in breast cancer. There is consistent evidence that CLS-B are more frequently detected among obese compared to non-obese breast cancer patients. Additionally, several studies have found that CLS-B presence is associated with metabolic and inflammatory factors that contribute to breast cancer development and progression. However, more studies are needed to understand the potential clinical utility of CLS-B as a marker of breast cancer risk or prognosis.Obesity is an established risk factor for postmenopausal breast cancer and has been linked to worse breast cancer prognosis, most clearly for hormone receptor-positive breast cancers. The underlying mechanisms of the obesity–breast cancer association are not fully understood, but growing evidence points to the breast adipose tissue microenvironment playing an important role. Obesity-induced adipose tissue dysfunction can result in a chronic state of low-grade inflammation. Crown-like structures of the breast (CLS-B) were recently identified as a histologic marker of local inflammation. In this review, we evaluate the early evidence of CLS-B in breast cancer. Data from preclinical and clinical studies show that these inflammatory lesions within the breast are associated with local NF-κB activation, increased aromatase activity, and elevation of pro-inflammatory mediators (TNFα, IL-1β, IL-6, and COX-2-derived PGE2)—factors involved in multiple pathways of breast cancer development and progression. There is also substantial evidence from epidemiologic studies that CLS-B are associated with greater adiposity among breast cancer patients. However, there is insufficient evidence that CLS-B impact breast cancer risk or prognosis. Comparisons across studies of prognosis were complicated by differences in CLS-B evaluation and deficiencies in study design, which future studies should take into consideration. Breast adipose tissue inflammation provides a plausible explanation for the obesity–breast cancer association, but further study is needed to establish its role and whether markers such as CLS-B are clinically useful.

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.

Viral pores are everywhere

Jones etal. (2021) and Zhang etal. (2021) reveal by cryo-EM the oligomeric crown-like structure formed by a membrane-associated Chikungunya virus replication protein that gates the export of newly synthesized viral RNA from viral replication organelles.

Differential Effects of Chronic Binge Alcohol and Ovariectomy on Apoptosis in Omental Adipose Tissue of SIV‐infected Macaques

Adipose tissue maladaptations including cell death, inflammation, adipokine disruption, dysfunction of adipocytes and adipose tissue metabolism are central to metabolic alterations in HIV, at-risk alcohol use, and aging. Though adipose tissue dysregulation is a well-accepted mechanism meditating metabolic comorbidities in HIV, the effects of alcohol use and estrogen loss on adipose tissue cell death are not fully understood. Using a relevant preclinical model of HIV-infection, the aim of our study was to investigate the differential effects of chronic binge alcohol (CBA) administration and ovarian hormone loss, simulated by ovariectomy (OVX), on alterations in omental adipose tissue (OmAT) expression of genes involved in cell death. CBA or isovolumetric water (VEH) was administered through an intragastric catheter at a concentration of 30% (w/v) for 30 minutes, 5 days a week, with blood alcohol concentrations reaching 50-60 mM. Three months after initiation of CBA/VEH administration, macaques were infected with SIVMac251 and 2.5 months later initiated on ART. After 1 month of ART, macaques underwent sham surgery or ovariectomy. Unbiased quantitative proteomic analysis of OmAT revealed that proteins implicated in the apoptotic pathway were differentially regulated by CBA, OVX and the combination of both. Specifically, CBA and OVX independently led to proteome changes consistent with activation of apoptosis and necrosis pathways while the combination of CBA and OVX led to proteome changes consistent with inhibition of both apoptosis and necrosis. To confirm these results, OmAT expression of genes involved in apoptosis was determined using quantitative PCR. B-cell lymphoma 2 (BCL2), an antiapoptotic gene was increased by CBA but decreased in the CBA/OVX group. CBA increased the gene expression of BCL-2 homologous antagonist killer (BAK), a proapoptotic gene and increased the ratio of BCL2 associated X protein (BAX) to BCL2, an indicator of cell susceptibility to apoptosis. Our quantitative PCR results confirm and support the results of the OmAT proteomic analysis, suggesting that CBA and OVX differentially regulate the apoptotic pathway. However, in contrast to the results from the proteomic analysis, the preliminary gene expression data suggest that CBA is a major driver of apoptosis in the OmAT of female rhesus macaques. Our ongoing studies using TUNEL staining and analysis of crown-like structures will determine the effects of CBA and OVX on OmAT cell death. Additionally, the functional effects of CBA and OVX on adipose-derived stem cell death will be determined using flow cytometry.

Synthesis and Photophysical Properties of [3]Cyclo-1,8-pyrenes via [4 + 2] Cycloaddition Reaction.

Herein, we report the synthesis, characterization, and photophysical properties of the crown-like structure of [3]cyclo-1,8-pyrenes (compounds 9 and 10). Planar pyrenyl arylene-ethynylene macrocycles are used as the precursors to synthesize these pyrene-based cycloarenes by [4 + 2] cycloaddition reaction with good yields. These molecules are confirmed by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. The structure of 9 was unambiguously determined by single-crystal X-ray diffraction. Their photophysical properties are investigated by steady-state absorption, fluorescence, and time-resolved fluorescence spectroscopies, combined with theoretical calculations.

New Glomeromycotan Taxa, Dominikia glomerocarpica sp. nov. and Epigeocarpum crypticum gen. nov. et sp. nov. From Brazil, and Silvaspora gen. nov. From New Caledonia.

Examination of fungal specimens collected in the Atlantic rain forest ecosystems of Northeast Brazil revealed many potentially new epigeous and semihypogeous glomerocarp-producing species of the phylum Glomeromycota. Among them were two fungi that formed unorganized epigeous glomerocarps with glomoid spores of almost identical morphology. The sole structure that distinguished the two fungi was the laminate layer 2 of their three-layered spore wall, which in spores of the second fungus crushed in PVLG-based mountants contracted and, consequently, transferred into a crown-like structure. Surprisingly, phylogenetic analyses of sequences of the 18S-ITS-28S nuc rDNA and the rpb1 gene indicated that these glomerocarps represent two strongly divergent undescribed species in the family Glomeraceae. The analyses placed the first in the genus Dominikia, and the second in a sister clade to the monospecific generic clade Kamienskia with Kamienskia bistrata. The first species was described here as Dominikia glomerocarpica sp. nov. Because D. glomerocarpica is the first glomerocarp-forming species in Dominikia, the generic description of this genus was emended. The very large phylogenetic distance and the fundamental morphological differences between the second species and K. bistrata suggested us to introduce a new genus, here named as Epigeocarpum gen. nov., and name the new species Epigeocarpum crypticum sp. nov. In addition, our analyses also focused on an arbuscular mycorrhizal fungus originally described as Rhizophagus neocaledonicus, later transferred to the genus Rhizoglomus. The analyses indicated that this species does not belong to any of these two genera but represents a new clade at the rank of genus in the Glomeraceae, here described as Silvaspora gen. nov.

Xylo-Oligosaccharides in Prevention of Hepatic Steatosis and Adipose Tissue Inflammation: Associating Taxonomic and Metabolomic Patterns in Fecal Microbiomes with Biclustering.

We have shown that prebiotic xylo-oligosaccharides (XOS) increased beneficial gut microbiota (GM) and prevented high fat diet-induced hepatic steatosis, but the mechanisms associated with these effects are not clear. We studied whether XOS affects adipose tissue inflammation and insulin signaling, and whether the GM and fecal metabolome explain associated patterns. XOS was supplemented or not with high (HFD) or low (LFD) fat diet for 12 weeks in male Wistar rats (n = 10/group). Previously analyzed GM and fecal metabolites were biclustered to reduce data dimensionality and identify interpretable groups of co-occurring genera and metabolites. Based on our findings, biclustering provides a useful algorithmic method for capturing such joint signatures. On the HFD, XOS-supplemented rats showed lower number of adipose tissue crown-like structures, increased phosphorylation of AKT in liver and adipose tissue as well as lower expression of hepatic miRNAs. XOS-supplemented rats had more fecal glycine and less hypoxanthine, isovalerate, branched chain amino acids and aromatic amino acids. Several bacterial genera were associated with the metabolic signatures. In conclusion, the beneficial effects of XOS on hepatic steatosis involved decreased adipose tissue inflammation and likely improved insulin signaling, which were further associated with fecal metabolites and GM.

Early Childhood Fat Tissue Changes-Adipocyte Morphometry, Collagen Deposition, and Expression of CD163+ Cells in Subcutaneous and Visceral Adipose Tissue of Male Children.

Childhood obesity is a complex health problem, and not many studies have been done on adipose tissue remodeling in early childhood. The aim of this study was to examine extracellular matrix remodeling in the adipose tissue of healthy male children depending on their weight status. Subcutaneous and visceral adipose tissue was obtained from 45 otherwise healthy male children who underwent elective surgery for hernia repairs or orchidopexy. The children were divided into overweight/obese (n = 17) or normal weight groups (n = 28) depending on their body mass index (BMI) z-score. Serum was obtained for glucose, testosterone, triglyceride, total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) measurements. Sections of adipose tissue were stained with hematoxylin and eosin to determine the adipocytes’ surface area, and Masson’s trichrome stain was used to detect the adipocytes’ collagen content. Immunohistochemistry for CD163+ cells was also performed. The results showed that male children in the overweight group had higher serum triglyceride levels, greater adipocyte surface area and collagen content in their subcutaneous adipose tissue, more crown-like structures in fat tissues, and more CD163+ cells in their visceral adipose tissue than males in the normal weight group. In conclusion, in male children, obesity can lead to the hypertrophy of adipocytes, increased collagen deposition in subcutaneous adipose tissues, and changes in the polarization and accumulation of macrophages.

Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice.

Background: Fibrosis is a major grafting-related complication that leads to fat tissue dysfunction. Macrophage-induced inflammation is related to the development of fat tissue fibrosis. Necroptosis is a recently discovered pathway of programmed cell necrosis that results in severe inflammation and subsequent tissue fibrosis. Thus, in this study, we investigated the role of macrophage necroptosis in fat graft fibrosis and the underlying mechanisms.Methods: Fibrosis and necroptosis were investigated in mouse fat tissue before and after grafting. An in vitro “crown-like” structure (CLS) cell culture model was developed by co-culturing RAW 264.7 macrophages with apoptotic adipocytes to reproduce in vivo CLS macrophage-adipocyte interactions. Lipid uptake and necroptosis in CLS macrophages were analyzed using Oil-Red-O staining, western blotting, and immunofluorescence. RAW264.7 macrophages were cultured alone or with apoptotic adipocytes and treated with a necroptosis inhibitor (Nec-1 or GSK872) to explore the paracrine effect of necroptotic CLS macrophages on collagen synthesis in fibroblasts in vitro. Mice were treated with Nec-1 to analyze the effect of blocking necroptosis on fat graft fibrosis.Results: Fibrosis was increased after grafting in fat grafts of mice. Macrophages clustered around apoptotic adipocytes or large oil droplets to form a typical CLS in fibrotic depots. This was accompanied by formation and necroptosis of macrophage foam cells (MFCs) in CLSs. RAW 264.7 macrophages co-cultured with apoptotic adipocytes induced CLS formation in vitro, and lipid accumulation in CLS macrophages resulted in the formation and necroptosis of MFCs. Necroptosis of MFCs altered the expression of collagen I and VI in fibroblasts via a paracrine mechanism involving inflammatory cytokines/chemokines, which was reversed by GSK872 or Nec-1 treatment. Furthermore, treatment with Nec-1 ameliorated fat graft fibrosis in mice.Conclusion: Apoptotic adipocytes induced necroptosis of MFCs, and necroptosis of these cells activated collagen synthesis in fibroblasts via a paracrine mechanism. Inhibition of necroptosis in macrophages is a potential approach to prevent fibrosis in fat grafts.

Generation and characterization of a Meflin-CreERT2 transgenic line for lineage tracing in white adipose tissue.

Meflin (Islr) expression has gained attention as a marker for mesenchymal stem cells, but its function remains largely unexplored. Here, we report the generation of Meflin-CreERT2 mice with CreERT2 inserted under the Meflin gene promoter to label Meflin-expressing cells genetically, thereby enabling their lineages to be traced. We found that in adult mice, Meflin-expressing lineage cells were present in adipose tissue stroma and had differentiated into mature adipocytes. These cells constituted Crown-like structures in the adipose tissue of mice after high-fat diet loading. Cold stimulation led to the differentiation of Meflin-expressing lineage cells into beige adipocytes. Thus, the Meflin-CreERT2 mouse line is a useful new tool for visualizing and tracking the lineage of Meflin-expressing cells.

Lipoprotein receptor SR-B1 deficiency enhances adipose tissue inflammation and reduces susceptibility to hepatic steatosis during diet-induced obesity in mice

Scavenger receptor class B type 1 (SR-B1) is a membrane lipoprotein receptor/lipid transporter involved in the pathogenesis of atherosclerosis, but its role in obesity and fatty liver development is unclear. Here, we determined the effects of SR-B1 deficiency on plasma metabolic and inflammatory parameters as well as fat deposition in adipose tissue and liver during obesity. To induce obesity, we performed high-fat diet (HFD) exposure for 12 weeks in male SR-B1 knock-out (SR-B1−/−, n = 14) and wild-type (WT, n = 12) mice. Compared to HFD-fed WT mice, plasma from HFD-fed SR-B1−/− animals exhibited increased total cholesterol, triglycerides (TG) and tumor necrosis factor-α (TNF-α) levels. In addition, hypertrophied adipocytes and macrophage-containing crown-like structures (CLS) were observed in adipose tissue from HFD-fed SR-B1 deficient mice. Remarkably, liver from obese SR-B1−/− mice showed attenuated TG content, dysregulation in hepatic peroxisome proliferator-activated receptors (PPARs) expression, increased hepatic TG secretion, and altered hepatic fatty acid (FA) composition. In conclusion, we show that SR-B1 deficiency alters the metabolic environment of obese mice through modulation of liver and adipose tissue lipid accumulation. Our findings provide the basis for further elucidation of SR-B1's role in obesity and fatty liver, two major public health issues that increase the risk of advanced chronic diseases and overall mortality.

Precision Nutrition to Activate Thermogenesis as a Complementary Approach to Target Obesity and Associated-Metabolic-Disorders.

Simple SummaryRegarding the pandemic of obesity and chronic diseases associated to metabolic alterations that occur nowadays worldwide, here, we review the most recent studies related to bioactive compounds and diet derived ingredients with potential effects to augment the systemic energy expenditure. We specifically focus in two processes: the activation of thermogenesis in adipose tissue and the enhancement of the mitochondrial oxidative phosphorylation capacity in muscles. This may provide relevant information to develop diets and supplements to conduct nutritional intervention studies with the objective to ameliorate the metabolic and chronic inflammation in the course of obesity and related disorders.Obesity is associated to increased incidence and poorer prognosis in multiple cancers, contributing to up to 20% of cancer related deaths. These associations are mainly driven by metabolic and inflammatory changes in the adipose tissue during obesity, which disrupt the physiologic metabolic homeostasis. The association between obesity and hypercholesterolemia, hypertension, cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM) is well known. Importantly, the retrospective analysis of more than 1000 epidemiological studies have also shown the positive correlation between the excess of fatness with the risk of cancer. In addition, more important than weight, it is the dysfunctional adipose tissue the main driver of insulin resistance, metabolic syndrome and all cause of mortality and cancer deaths, which also explains why normal weight individuals may behave as “metabolically unhealthy obese” individuals. Adipocytes also have direct effects on tumor cells through paracrine signaling. Downregulation of adiponectin and upregulation of leptin in serum correlate with markers of chronic inflammation, and crown like structures (CLS) associated to the adipose tissue disfunction. Nevertheless, obesity is a preventable risk factor in cancer. Lifestyle interventions might contribute to reduce the adverse effects of obesity. Thus, Mediterranean diet interventional studies have been shown to reduce to circulation inflammatory factors, insulin sensitivity and cardiovascular function, with durable responses of up to 2 years in obese patients. Mediterranean diet supplemented with extra-virgin olive oil reduced the incidence of breast cancer compared with a control diet. Physical activity is another important lifestyle factor which may also contribute to reduced systemic biomarkers of metabolic syndrome associated to obesity. In this scenario, precision nutrition may provide complementary approaches to target the metabolic inflammation associated to “unhealthy obesity”. Herein, we first describe the different types of adipose tissue -thermogenic active brown adipose tissue (BAT) versus the energy storing white adipose tissue (WAT). We then move on precision nutrition based strategies, by mean of natural extracts derived from plants and/or diet derived ingredients, which may be useful to normalize the metabolic inflammation associated to “unhealthy obesity”. More specifically, we focus on two axis: (1) the activation of thermogenesis in BAT and browning of WAT; (2) and the potential of augmenting the oxidative capacity of muscles to dissipate energy. These strategies may be particularly relevant as complementary approaches to alleviate obesity associated effects on chronic inflammation, immunosuppression, angiogenesis and chemotherapy resistance in cancer. Finally, we summarize main studies where plant derived extracts, mainly, polyphenols and flavonoids, have been applied to increase the energy expenditure.

3D microscopy and deep learning reveal the heterogeneity of crown-like structure microenvironments in intact adipose tissue.

Crown-like structures (CLSs) are adipose microenvironments of macrophages engulfing adipocytes. Their histological density in visceral adipose tissue (VAT) predicts metabolic disorder progression in obesity and is believed to initiate obesity comorbidities. Here, we use three-dimensional (3D) light sheet microscopy and deep learning to quantify 3D features of VAT CLSs in lean and obese states. Obese CLS densities are significantly higher, composing 3.9% of tissue volume compared with 0.46% in lean tissue. Across the states, individual CLS structural characteristics span similar ranges; however, subpopulations are distinguishable. Obese VAT contains large CLSs absent from lean tissues, located near the tissue center, while lean CLSs have higher volumetric cell densities and prolate shapes. These features are consistent with inefficient adipocyte elimination in obesity that contributes to chronic inflammation, representing histological biomarkers to assess adipose pathogenesis. This tissue processing, imaging, and analysis pipeline can be applied to quantitatively classify 3D microenvironments across diverse tissues.

Abstract SP054: Obesity and Breast Cancer: Body Composition, Metabolic Mediators and Inflammation

Abstract Obesity has been associated with an increased risk of postmenopausal breast cancer (predominantly ER+), as well as triple negative premenopausal breast cancer. It has also been associated with an increased risk of breast cancer distant recurrence and death, regardless or menopausal status or IHC defined breast cancer subtype. Obesity effects on recurrence and death are modest, with HRs for either outcome approximating 1.3. Obesity is linked to metabolic alterations that include insulin resistance (with associated high insulin and reduced glucose tolerance), altered lipids and adipokines, hypertension, central obesity, inflammation and increased risks of type 2 diabetes (T2DM) and cardiovascular disease - this constellation of attributes comprises the insulin resistance or metabolic syndrome. There is growing appreciation that BMI [weight (kg)/height (m)2] ≥ 30 kg/m2 is an imperfect measure of both body composition and underlying metabolic status. Recent research has focused on body composition (e.g. central obesity/sarcopenic obesity) and metabolic health (“healthy” obese, “unhealthy” normal weight) as predictors of breast cancer outcomes. Many of the inter-related physiologic changes associated with obesity (including higher levels of insulin, glucose, altered adipokines and, less consistently, circulating inflammatory factors) have individually been associated with increased breast cancer risk and/or poor breast cancer outcomes. These circulating factors, and obesity per se, have, in turn, been associated with changes in breast tissue and tumor micro-environment, including crown-like structures (dying adipocytes surrounded by inflammatory cells) as well as increased infiltration of inflammatory cells, cytokines and chemokines - all of which have been linked to cancer risk and progression in preclinical and/or clinical research. Evidence linking obesity/body composition, circulating obesity-associated physiologic and inflammatory factors to breast cancer outcomes will be discussed as will interventions (notably weight loss, metformin) targeting these factors in breast cancer patients. Recent findings linking breast inflammation to obesity/obesity-associated physiology and breast cancer outcomes will be reviewed. The inter-relatedness of these factors and the likelihood that the breast cancer-obesity link is complex and multifactorial will be highlighted. Citation Format: P Goodwin. Obesity and Breast Cancer: Body Composition, Metabolic Mediators and Inflammation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP054.