Bone marrow adipose cells - cellular interactions and changes with obesity.

The bone marrow is a spatially restricted niche, housing cells of the hematopoietic and mesenchymal lineages in various hierarchical commitment states. Although highly localized, cells within this niche are also subject to regulation by environmental and/or circulatory changes through extensive vascularization. Bone marrow adipocytes, derived from mesenchymal stem cells and once known as marrow space fillers, are a heterogeneous population. These cells reside in distinct niches within the bone marrow and interact with proximal cells, such as hematopoietic precursors and lineage-committed cells. In this diverse cellular milieu, bone marrow adipocytes influence commitment decisions and cellular lineage selection by interacting with stem and progenitor cells. In addition, bone marrow adipocytes respond to environmental changes, such as obesity, by undergoing hypertrophy, hyperplasia or adoption of characteristics resembling those of peripheral brown, beige or white adipocytes. Here, we review recent findings and concepts on the influence of bone marrow adipocytes on hematopoietic and other cellular lineages within this niche. We discuss how changes in local, systemic, cellular and secreted signals impact on mesenchymal stem cell expansion, differentiation and lineage commitment. Furthermore, we highlight that bone marrow adipocytes may be intermediaries conveying environmental cues to influence hematopoietic cellular survival, proliferation and preferential differentiation.

Pannexin 3 deletion reduces fat accumulation and inflammation in a sex-specific manner.

BACKGROUND: Pannexin 3 (PANX3) is a channel-forming glycoprotein that enables nutrient-induced inflammation in vitro, and genetic linkage data suggest that it regulates body mass index. Here, we characterized inflammatory and metabolic parameters in global Panx3 knockout (KO) mice in the context of forced treadmill running (FEX) and high-fat diet (HFD). METHODS: C57BL/6N (WT) and KO mice were randomized to either a FEX running protocol or no running (SED) from 24 until 30 weeks of age. Body weight was measured biweekly, and body composition was measured at 24 and 30 weeks of age. Male WT and KO mice were fed a HFD from 12 to 28 weeks of age. Metabolic organs were analyzed for a panel of inflammatory markers and PANX3 expression. RESULTS: In females there were no significant differences in body composition between genotypes, which could be due to the lack of PANX3 expression in female white adipose tissue, while male KOs fed a chow diet had lower body weight and lower fat mass at 24 and 30 weeks of age, which was reduced to the same extent as 6 weeks of FEX in WT mice. In addition, male KO mice exhibited significantly lower expression of multiple pro-inflammatory genes in white adipose tissue compared to WT mice. While on a HFD body weight differences were insignificant, multiple inflammatory genes were significantly different in quadriceps muscle and white adipose tissue resulting in a more anti-inflammatory phenotype in KO mice compared to WT. The lower fat mass in male KO mice may be due to significantly fewer adipocytes in their subcutaneous fat compared to WT mice. Mechanistically, adipose stromal cells (ASCs) cultured from KO mice grow significantly slower than WT ASCs. CONCLUSION: PANX3 is expressed in male adult mouse adipose tissue and may regulate adipocyte numbers, influencing fat accumulation and inflammation.

Nucleotides released from palmitate-activated murine macrophages attract neutrophils.

Obesity and elevation of circulating free fatty acids are associated with an accumulation and proinflammatory polarization of macrophages within metabolically active tissues, such as adipose tissue, muscle, liver, and pancreas. Beyond macrophages, neutrophils also accumulate in adipose and muscle tissues during high-fat diets and contribute to a state of local inflammation and insulin resistance. However, the mechanisms by which neutrophils are recruited to these tissues are largely unknown. Here we used a cell culture system as proof of concept to show that, upon exposure to a saturated fatty acid, palmitate, macrophages release nucleotides that attract neutrophils. Moreover, we found that palmitate up-regulates pannexin-1 channels in macrophages that mediate the attraction of neutrophils, shown previously to allow transfer of nucleotides across membranes. These findings suggest that proinflammatory macrophages release nucleotides through pannexin-1, a process that may facilitate neutrophil recruitment into metabolic tissues during obesity.

Deconstructing metabolic inflammation using cellular systems.

Over the past years, we have embarked in a systematic analysis of the effect of obesity or fatty acids on circulating monocytes, microvascular endothelial cells, macrophages, and skeletal muscle cells. With the use of cell culture strategies, we have deconstructed complex physiological systems and then reconstructed "partial equations" to better understand cell-to-cell communication. Through these approaches, we identified that in high saturated fat environments, cell-autonomous proinflammatory pathways are activated in monocytes and endothelial cells, promoting monocyte adhesion and transmigration. We think of this as a paradigm of the conditions promoting immune cell infiltration into tissues during obesity. In concert, it is possible that muscle and adipose tissue secrete immune cell chemoattractants, and indeed, our tissue culture reconstructions reveal that myotubes treated with the saturated fatty acid palmitate, but not the unsaturated fatty acid palmitoleate, release nucleotides that attract monocytes and other compounds that promote proinflammatory classically activated "(M1)-like" polarization in macrophages. In addition, palmitate directly triggers an M1-like macrophage phenotype, and secretions from these activated macrophages confer insulin resistance to target muscle cells. Together, these studies suggest that in pathophysiological conditions of excess fat, the muscle, endothelial and immune cells engage in a synergistic crosstalk that exacerbates tissue inflammation, leukocyte infiltration, polarization, and consequent insulin resistance.

Bone marrow adipocytes drive the development of tissue invasive Ly6Chigh monocytes during obesity.

During obesity and high fat-diet (HFD) feeding in mice, sustained low-grade inflammation includes not only increased pro-inflammatory macrophages in the expanding adipose tissue, but also bone marrow (BM) production of invasive Ly6Chigh monocytes. As BM adiposity also accrues with HFD, we explored the relationship between the gains in BM white adipocytes and invasive Ly6Chigh monocytes by in vivo and ex vivo paradigms. We find a temporal and causal link between BM adipocyte whitening and the Ly6Chigh monocyte surge, preceding the adipose tissue macrophage rise during HFD in mice. Phenocopying this, ex vivo treatment of BM cells with conditioned media from BM adipocytes or bona fide white adipocytes favoured Ly6Chigh monocyte preponderance. Notably, Ly6Chigh skewing was preceded by monocyte metabolic reprogramming towards glycolysis, reduced oxidative potential and increased mitochondrial fission. In sum, short-term HFD changes BM cellularity, resulting in local adipocyte whitening driving a gradual increase and activation of invasive Ly6Chigh monocytes.

ARPC1B binds WASP to control actin polymerization and curtail tonic signaling in B cells.

Immune cells exhibit low-level, constitutive signaling at rest (tonic signaling). Such tonic signals are required for fundamental processes, including the survival of B lymphocytes, but when they are elevated by genetic or environmental causes, they can lead to autoimmunity. Events that control ongoing signal transduction are, therefore, tightly regulated by submembrane cytoskeletal polymers like F-actin. The actin-binding proteins that underpin the process, however, are poorly described. By investigating patients with ARPC1B deficiency, we report that ARPC1B-containing ARP2/3 complexes are stimulated by Wiskott Aldrich Syndrome protein (WASP) to nucleate the branched actin networks that control tonic signaling from the B cell receptor (BCR). Despite an upregulation of ARPC1A, ARPC1B-deficient cells were not capable of WASP-mediated nucleation by ARP2/3, and this caused the loss of WASP-dependent structures, including podosomes in macrophages and lamellipodia in B cells. In the B cell compartment, ARPC1B deficiency also led to weakening of the cortical F-actin cytoskeleton that normally curtails the diffusion of BCRs and ultimately resulted in increased tonic lipid signaling, oscillatory calcium release from the endoplasmic reticulum (ER), and phosphorylated Akt. These events contributed to skewing the threshold for B cell activation in response to microbial-associated molecular patterns (MAMPs). Thus, ARPC1B is critical for ARP2/3 complexes to control steady-state signaling of immune cells.

Tissue-specific murine neutrophil activation states in health and inflammation.

Neutrophils are quickly recruited to tissues in response to proinflammatory cues; however, little is known about tissue neutrophil phenotypes in health. We employ a multicolor flow cytometric approach to assess surface markers of activation on neutrophils from the bone marrow, blood, peritoneum, spleen, liver, fat, colon, and oral cavity of healthy mice. Cell preparations were promptly fixed to preserve native surface marker expression levels. Peritoneal, colonic, and oral neutrophils were also assessed in the setting of pHrodo-induced peritonitis, dextran sodium sulfate-induced colitis, and ligature-induced periodontal disease, respectively. Our results demonstrate consistent detectable neutrophil populations in various sterile and nonsterile tissues of healthy mice, and these cells had tissue-specific neutrophil immunophenotypes. Neutrophils derived from biofilm-associated mucosal tissues had 2- to 3-fold higher expression of surface markers of activation, including CD66a, CD11b, and CD62L, compared to neutrophils derived from both sterile healthy tissues as well as tissues in animals treated with broad-spectrum antibiotics. Furthermore, the unique cluster of differentiation (CD) marker activation signatures of tissue-specific neutrophils from the peritoneum, colon, and oral cavity were altered to a proinflammatory immunophenotype with the presence of an inflammatory stimulus. Based on our results, we propose a model whereby a hierarchy of tissue neutrophil immunophenotypes, based on the differential expression of CD markers of activation, correlates with sterile, healthy commensal biofilm-associated and inflamed tissue states.

Increased inflammation, oxidative stress and a reduction in antioxidant defense enzymes in perivascular adipose tissue contribute to vascular dysfunction in type 2 diabetes.

BACKGROUND: Perivascular adipose tissue (PVAT) surrounds most large blood vessels and plays an important role in vascular homeostasis. The present study was conducted to investigate the contribution of PVAT to vascular dysfunction in a rat model of type 2 diabetes. MATERIAL AND METHODS: Several in vivo parameters such as lipid profile (total cholesterol and triglyceride systemic levels), fasting glucose levels, glucose tolerance and insulin sensitivity (through glucose and insulin tolerance tests, respectively) were determined in Goto-Kakizaki (GK) diabetic rats and compared with control Wistar rats. At the vascular level, endothelial dependent and independent relaxation and contraction studies were performed in aortic rings in the absence (PVAT-) or in the presence (PVAT+) of thoracic PVAT. We also evaluated vascular oxidative stress and performed western blots, PCR and immunohistochemistry analysis of cytokines and various enzymes in PVAT. RESULTS: Endothelium-dependent relaxation to acetylcholine, assessed by wire myography, was impaired in GK rats and improved by the antioxidant TEMPOL and by the TLR4 inhibitor, CLI-095 suggesting an increase in oxidative stress and inflammation. In addition, vascular superoxide and peroxynitrite production was increased in the vascular wall of diabetic rats, accompanied by reduced nitric oxide bioavailability. The presence of PVAT had an anticontractile effect in response to phenylephrine in Wistar rats that was lost in GK rats. Western blot and immunohistochemistry analysis revealed that PVAT phenotype shifts, under diabetic conditions, towards a proinflammatory (with increment in CRP, CCL2, CD36), pro-oxidant (increased levels of aldose reductase, and reduced levels of antioxidant deference enzymes) and vasoconstriction state. CONCLUSION: Our data suggest that this rat model of type 2 diabetes is associated with perivascular adipose dysfunction that contributes to oxidative stress, inflammation and endothelial dysfunction.

Lipid-gated monovalent ion fluxes regulate endocytic traffic and support immune surveillance.

Despite ongoing (macro)pinocytosis of extracellular fluid, the volume of the endocytic pathway remains unchanged. To investigate the underlying mechanism, we used high-resolution video imaging to analyze the fate of macropinosomes formed by macrophages in vitro and in situ. Na+, the primary cationic osmolyte internalized, exited endocytic vacuoles via two-pore channels, accompanied by parallel efflux of Cl- and osmotically coupled water. The resulting shrinkage caused crenation of the membrane, which fostered recruitment of curvature-sensing proteins. These proteins stabilized tubules and promoted their elongation, driving vacuolar remodeling, receptor recycling, and resolution of the organelles. Failure to resolve internalized fluid impairs the tissue surveillance activity of resident macrophages. Thus, osmotically driven increases in the surface-to-volume ratio of endomembranes promote traffic between compartments and help to ensure tissue homeostasis.

Reporting Guidelines, Review of Methodological Standards, and Challenges Toward Harmonization in Bone Marrow Adiposity Research. Report of the Methodologies Working Group of the International Bone Marrow Adiposity Society.

The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis, diabetes, anorexia, cancer) as well as treatments (corticosteroid, radiation, chemotherapy, thiazolidinediones). However, to advance the field of BMA research, standardization of methods is desirable to increase comparability of study outcomes and foster collaboration. Therefore, at the 2017 annual BMA meeting, the International Bone Marrow Adiposity Society (BMAS) founded a working group to evaluate methodologies in BMA research. All BMAS members could volunteer to participate. The working group members, who are all active preclinical or clinical BMA researchers, searched the literature for articles investigating BMA and discussed the results during personal and telephone conferences. According to the consensus opinion, both based on the review of the literature and on expert opinion, we describe existing methodologies and discuss the challenges and future directions for (1) histomorphometry of bone marrow adipocytes, (2) ex vivo BMA imaging, (3) in vivo BMA imaging, (4) cell isolation, culture, differentiation and in vitro modulation of primary bone marrow adipocytes and bone marrow stromal cell precursors, (5) lineage tracing and in vivo BMA modulation, and (6) BMA biobanking. We identify as accepted standards in BMA research: manual histomorphometry and osmium tetroxide 3D contrast-enhanced µCT for ex vivo quantification, specific MRI sequences (WFI and H-MRS) for in vivo studies, and RT-qPCR with a minimal four gene panel or lipid-based assays for in vitro quantification of bone marrow adipogenesis. Emerging techniques are described which may soon come to complement or substitute these gold standards. Known confounding factors and minimal reporting standards are presented, and their use is encouraged to facilitate comparison across studies. In conclusion, specific BMA methodologies have been developed. However, important challenges remain. In particular, we advocate for the harmonization of methodologies, the precise reporting of known confounding factors, and the identification of methods to modulate BMA independently from other tissues. Wider use of existing animal models with impaired BMA production (e.g., Pfrt-/-, KitW/W-v) and development of specific BMA deletion models would be highly desirable for this purpose.

Circulating NOD1 Activators and Hematopoietic NOD1 Contribute to Metabolic Inflammation and Insulin Resistance.

Insulin resistance is a chronic inflammatory condition accompanying obesity or high fat diets that leads to type 2 diabetes. It is hypothesized that lipids and gut bacterial compounds in particular contribute to metabolic inflammation by activating the immune system; however, the receptors detecting these "instigators" of inflammation remain largely undefined. Here, we show that circulating activators of NOD1, a receptor for bacterial peptidoglycan, increase with high fat feeding in mice, suggesting that NOD1 could be a critical sensor leading to metabolic inflammation. Hematopoietic depletion of NOD1 did not prevent weight gain but protected chimeric mice against diet-induced glucose and insulin intolerance. Mechanistically, while macrophage infiltration of adipose tissue persisted, notably these cells were less pro-inflammatory, had lower CXCL1 production, and consequently, lower neutrophil chemoattraction into the tissue. These findings reveal macrophage NOD1 as a cell-specific target to combat diet-induced inflammation past the step of macrophage infiltration, leading to insulin resistance.