Bisecting GlcNAc Protein N-Glycosylation Is Characteristic of Human Adipogenesis.

Human adipose tissue contains a major source of adipose-derived stem cells (ADSCs) that have the ability to differentiate into various cell types: in vitro, ADSCs can differentiate into mesenchymal lineages including adipocytes, while in vivo, ADSCs become mature adipocytes. Protein glycosylation has been shown to change in stem cell differentiation, and while ADSCs have been acknowledged for their therapeutic potential, little is known about protein glycosylation during human ADSC adipogenic differentiation. In the present study, the global membrane protein glycosylation of native adipocytes was compared to ADSCs from the same individuals as a model of in vivo adipogenesis. For in vitro adipogenesis, ADSCs were adipogenically differentiated in cell culture using an optimized, large-scale differentiation procedure. The membrane glycome of the differentiated ADSCs (dADSCs) was compared with mature adipocytes and the progenitor ADSCs. A total of 137 glycan structures were characterized across the three cell types using PGC-LC coupled with negative-ion electrospray ionization mass spectrometry (ESI-MS)/MS. Significantly higher levels of bisecting GlcNAc-type N-glycans were detected in mature adipocytes (32.1% of total glycans) and in in vitro dADSC progeny (1.9% of total glycans) compared to ADSCs. This was further correlated by the mRNA expression of the MGAT3 gene responsible for the enzymatic synthesis of this structural type. The bisecting GlcNAc structures were found on the majority of human native adipocyte membrane proteins, suggesting an important role in human adipocyte biology. Core fucosylation was also significantly increased during in vivo adipogenesis but did not correlate with an increase in Fut8 gene transcript. Unexpectedly, low abundance structures carrying rare ß-linked Gal-Gal termini were also detected. Overall, the N-glycan profiles of the in vitro differentiated progeny did not reflect native adipocytes, and the results show that bisecting GlcNAc structures are a characteristic feature of human adipocyte membrane protein N-glycosylation. Raw MS files are available on GlycoPOST (ID: GPST000153 https://glycopost.glycosmos.org/).

Red blood cells exposed to cancer cells in culture have altered cytokine profiles and immune function.

It is now accepted that red blood cells (RBCs) from healthy individuals regulate T-cell activity through modulating cytokine interactions, and that stored RBCs or RBCs from inflammatory cohorts are dysfunctional. Our study aimed to investigate how changes in RBCs that have been intentionally modified can affect T-cell activity as a mechanistic test of this modification. Exposure to a cancer cell line in culture was used to alter the cytokine profile of intact RBCs and the effect of these modified RBCs (ccRBCs) on T-cells was evaluated using flow cytometry. We used RBCs from healthy volunteers and quantified cytokines in RBC lysates and conditioned media using Luminex technology. During in vitro cancer cell exposure, RBCs sequestered a variety of cytokines including IL-8, bFGF, and VEGF. Although unmodified RBCs (oRBCs) stimulated proliferation of T-cells (Jurkat cells and peripheral blood mononucleated cells), ccRBCs augmented this proliferative response (3.5-fold and 1.9-fold more respectively). Unlike oRBCs, T-cells stimulated with ccRBCs were no longer protected from phytohemagglutinin-P-driven overexpression of GATA-3 and T-bet and these T-cells were induced to secrete a variety of cytokines including IL-17 and MCP-3. This study supports the hypothesis that RBCs are capable of binding and releasing cytokines in blood, and that modification of these cells can then also affect the T-cell response.

The emerging role of red blood cells in cytokine signalling and modulating immune cells.

For many years red blood cells have been described as inert bystanders rather than participants in intercellular signalling, immune function, and inflammatory processes. However, studies are now reporting that red blood cells from healthy individuals regulate immune cell activity and maturation, and red blood cells from disease cohorts are dysfunctional. These cells have now been shown to bind more than 50 cytokines and have been described as a sink for these molecules, and the loss of this activity has been correlated with disease progression. In this review, we summarise what is currently understood about the role of red blood cells in cytokine signalling and in modulating the activity of immune cells. We also discuss the implications of these findings for transfusion medicine and in furthering our understanding of anaemia of chronic inflammation. By bringing these disparate units of work together, we aim to shine a light on an area that requires significantly more investigation.

Red blood cells are dynamic reservoirs of cytokines.

Red blood cells (RBCs) have been shown to affect immune function and can induce inflammatory responses after transfusion. The transfusion of washed RBCs can significantly reduce adverse effects, however, the soluble factors that may mediate these effects have not been identified. Previous studies have identified, but not quantified, a small number of chemokines associated with RBCs. We isolated RBCs from healthy volunteers and quantified of a panel of 48 cytokines, chemokines, and growth factors in the lysate, cytosol, and conditioned media of these cells using Luminex® technology. This analysis revealed that, after correcting for white blood cell and platelet contamination, 46 cytokines were detected in RBC lysates, and the median concentration in RBCs was 12-fold higher than in the plasma. In addition, extensive washing of RBCs, such as that performed in proteomics analyses or prior to some RBC transfusions, significantly attenuated the release of six cytokines following incubation at 37 °C. This supports the hypothesis that, alongside its gas exchange function, RBCs play a role in cytokine signalling. This discovery may help supplement disease biomarker research and may shed light on adverse inflammatory processes that can follow RBC transfusion.

Red blood cells: The primary reservoir of macrophage migration inhibitory factor in whole blood.

Red blood cells are widely accepted to be inert carriers of oxygen and haemoglobin, but there is growing evidence that they play a much more critical role in immune function. Macrophage migration inhibitory factor (MIF) is a key cytokine in disease with additional oxido-reductase activity, which aids in managing oxidative stress. Although two studies have reported the presence of MIF in red blood cells, no study has quantified the levels of this protein. In this study, freshly isolated plasma, platelets, leukocytes, and red blood cells from healthy individuals were collected and the concentration of MIF was determined using an enzyme linked immunosorbent assay. This analysis demonstrated that MIF in red blood cells was present at 25 µg per millilitre of whole blood, which is greater than99% of the total MIF and 1000-fold higher concentration than plasma. This result was supported by electrophoresis and Western blot analysis, which identified MIF in its monomer structural form following sample processing. Furthermore, by assessing the level of tautomerase activity in red blood cell fractions in the presence of a MIF inhibitor, it was determined that the red blood cell-derived MIF was also functionally active. Together, these findings have implications on the effect of haemolysis during sample preparation and provide some clue into the inflammatory processes that occur following haemolysis in vivo. These results support the hypothesis that red blood cells are a major reservoir of this inflammatory protein and may play a role in inflammation.

Proteomic Analysis of Human Adipose Derived Stem Cells during Small Molecule Chemical Stimulated Pre-neuronal Differentiation.

BACKGROUND: Adipose derived stem cells (ADSCs) are acquired from abdominal liposuction yielding a thousand fold more stem cells per millilitre than those from bone marrow. A large research void exists as to whether ADSCs are capable of transdermal differentiation toward neuronal phenotypes. Previous studies have investigated the use of chemical cocktails with varying inconclusive results. METHODS: Human ADSCs were treated with a chemical stimulant, beta-mercaptoethanol, to direct them toward a neuronal-like lineage within 24 hours. Quantitative proteomics using iTRAQ was then performed to ascertain protein abundance differences between ADSCs, beta-mercaptoethanol treated ADSCs and a glioblastoma cell line. RESULTS: The soluble proteome of ADSCs differentiated for 12 hours and 24 hours was significantly different from basal ADSCs and control cells, expressing a number of remodeling, neuroprotective and neuroproliferative proteins. However toward the later time point presented stress and shock related proteins were observed to be up regulated with a large down regulation of structural proteins. Cytokine profiles support a large cellular remodeling shift as well indicating cellular distress. CONCLUSION: The earlier time point indicates an initiation of differentiation. At the latter time point there is a vast loss of cell population during treatment. At 24 hours drastically decreased cytokine profiles and overexpression of stress proteins reveal that exposure to beta-mercaptoethanol beyond 24 hours may not be suitable for clinical application as our results indicate that the cells are in trauma whilst producing neuronal-like morphologies. The shorter treatment time is promising, indicating a reducing agent has fast acting potential to initiate neuronal differentiation of ADSCs.

Effect of Laparoscopic Sleeve Gastrectomy on Fasting Gastrointestinal, Pancreatic, and Adipose-Derived Hormones and on Non-Esterified Fatty Acids.

BACKGROUND: Alterations in gastrointestinal, pancreatic, and adipose hormone levels may have a greater role in weight loss than initially appreciated. The laparoscopic sleeve gastrectomy (LSG) operation is now the most frequently performed bariatric operation in many countries, but there are relatively few data regarding its molecular effects. We sought to characterize the effect of LSG on fasting plasma levels of selected hormones and on non-esterified fatty acids (NEFA), and to compare these to levels in non-obese control individuals. MATERIALS AND METHODS: The levels of nine plasma hormones were measured using a multiplex bead-based assay at baseline and at 3 months after operation in 11 obese patients undergoing LSG. NEFA levels were also measured. The levels were compared to those for 22 age- and sex-matched non-obese individuals. RESULTS: At baseline, obese patients showed significantly higher expression of C-peptide, insulin, and leptin and significantly lower ghrelin, glucose-dependent insulinotropic peptide (GIP), and resistin compared to non-obese controls (p < 0.05). LSG resulted in a reduction in BMI from 42.5 ± 6.47 kg/m2 at operation to 35.2 ± 5.14 kg/m2 at 3 months (42 % mean excess weight loss, p < 0.001). LSG led to a significant decrease in ghrelin, glucagon-like peptide-1 (GLP-1), glucagon, leptin, plasminogen activator inhibitor-1 (PAI-1), and NEFA. CONCLUSION: LSG induces marked early changes in the fasting levels of factors thought to be important regulators of obesity and metabolic health. These changes differ somewhat from the findings for operations with a malabsorptive component, suggesting that subtle differences exist in the mechanisms of weight loss between LSG and other bariatric operations.

Priming Adipose-Derived Mesenchymal Stem Cells with Hyaluronan Alters Growth Kinetics and Increases Attachment to Articular Cartilage.

Background. Biological therapeutics such as adipose-derived mesenchymal stem cell (MSC) therapy are gaining acceptance for knee-osteoarthritis (OA) treatment. Reports of OA-patients show reductions in cartilage defects and regeneration of hyaline-like-cartilage with MSC-therapy. Suspending MSCs in hyaluronan commonly occurs in animals and humans, usually without supporting data. Objective. To elucidate the effects of different concentrations of hyaluronan on MSC growth kinetics. Methods. Using a range of hyaluronan concentrations, we measured MSC adherence and proliferation on culture plastic surfaces and a novel cartilage-adhesion assay. We employed time-course and dispersion imaging to assess MSC binding to cartilage. Cytokine profiling was also conducted on the MSC-secretome. Results. Hyaluronan had dose-dependent effects on growth kinetics of MSCs at concentrations of entanglement point (1 mg/mL). At higher concentrations, viscosity effects outweighed benefits of additional hyaluronan. The cartilage-adhesion assay highlighted for the first time that hyaluronan-primed MSCs increased cell attachment to cartilage whilst the presence of hyaluronan did not. Our time-course suggested patients undergoing MSC-therapy for OA could benefit from joint-immobilisation for up to 8 hours. Hyaluronan also greatly affected dispersion of MSCs on cartilage. Conclusion. Our results should be considered in future trials with MSC-therapy using hyaluronan as a vehicle, for the treatment of OA.

Alterations in the Secretome of Clinically Relevant Preparations of Adipose-Derived Mesenchymal Stem Cells Cocultured with Hyaluronan.

Osteoarthritis (OA) can be a debilitating degenerative disease and is the most common form of arthritic disease. There is a general consensus that current nonsurgical therapies are insufficient for younger OA sufferers who are not candidates for knee arthroplasties. Adipose-derived mesenchymal stem cells (MSCs) therapy for the treatment of OA can slow disease progression and lead to neocartilage formation. The mechanism of action is secretion driven. Current clinical preparations from adipose tissue for the treatment of OA include autologous stromal vascular fraction (SVF), SVF plus mature adipocytes, and culture-purified MSCs. Herein we have combined these human adipose-derived preparations with Hyaluronan (Hylan G-F 20: Synvisc) in vitro and measured alterations in cytokine profile. SVF plus mature adipocytes showed the greatest decreased in the proinflammatory cytokines IL-1ß, IFN-γ, and VEGF. MCP-1 and MIP-1α decreased substantially in the SVF preparations but not the purified MSCs. The purified MSC preparation was the only one to show increase in MIF. Overall the SVF plus mature adipocytes preparation may be most suited of all the preparations for combination with HA for the treatment of OA, based on the alterations of heavily implicated cytokines in OA disease progression. This will require further validation using in vivo models.

Subcutaneous fat transplantation alleviates diet-induced glucose intolerance and inflammation in mice.

AIMS/HYPOTHESIS: Adipose tissue (AT) distribution is a major determinant of mortality and morbidity in obesity. In mice, intra-abdominal transplantation of subcutaneous AT (SAT) protects against glucose intolerance and insulin resistance (IR), but the underlying mechanisms are not well understood. METHODS: We investigated changes in adipokines, tissue-specific glucose uptake, gene expression and systemic inflammation in male C57BL6/J mice implanted intra-abdominally with either inguinal SAT or epididymal visceral AT (VAT) and fed a high-fat diet (HFD) for up to 17 weeks. RESULTS: Glucose tolerance was improved in mice receiving SAT after 6 weeks, and this was not attributable to differences in adiposity, tissue-specific glucose uptake, or plasma leptin or adiponectin concentrations. Instead, SAT transplantation prevented HFD-induced hepatic triacylglycerol accumulation and normalised the expression of hepatic gluconeogenic enzymes. Grafted fat displayed a significant increase in glucose uptake and unexpectedly, an induction of skeletal muscle-specific gene expression. Mice receiving subcutaneous fat also displayed a marked reduction in the plasma concentrations of several proinflammatory cytokines (TNF-α, IL-17, IL-12p70, monocyte chemoattractant protein-1 [MCP-1] and macrophage inflammatory protein-1ß [ΜIP-1ß]), compared with sham-operated mice. Plasma IL-17 and MIP-1ß concentrations were reduced from as early as 4 weeks after transplantation, and differences in plasma TNF-α and IL-17 concentrations predicted glucose tolerance and insulinaemia in the entire cohort of mice (n = 40). In contrast, mice receiving visceral fat transplants were glucose intolerant, with increased hepatic triacylglycerol content and elevated plasma IL-6 concentrations. CONCLUSIONS/INTERPRETATION: Intra-abdominal transplantation of subcutaneous fat reverses HFD-induced glucose intolerance, hepatic triacylglycerol accumulation and systemic inflammation in mice.

Analysis of in vitro secretion profiles from adipose-derived cell populations.

BACKGROUND: Adipose tissue is an attractive source of cells for therapeutic purposes because of the ease of harvest and the high frequency of mesenchymal stem cells (MSCs). Whilst it is clear that MSCs have significant therapeutic potential via their ability to secrete immuno-modulatory and trophic cytokines, the therapeutic use of mixed cell populations from the adipose stromal vascular fraction (SVF) is becoming increasingly common. METHODS: In this study we have measured a panel of 27 cytokines and growth factors secreted by various combinations of human adipose-derived cell populations. These were 1. co-culture of freshly isolated SVF with adipocytes, 2. freshly isolated SVF cultured alone, 3. freshly isolated adipocytes alone and 4. adherent adipose-derived mesenchymal stem cells (ADSCs) at passage 2. In addition, we produced an 'in silico' dataset by combining the individual secretion profiles obtained from culturing the SVF with that of the adipocytes. This was compared to the secretion profile of co-cultured SVF and adipocytes. Two-tailed t-tests were performed on the secretion profiles obtained from the SVF, adipocytes, ADSCs and the 'in silico' dataset and compared to the secretion profiles obtained from the co-culture of the SVF with adipocytes. A p-value of < 0.05 was considered statistically different. To assess the overall changes that may occur as a result of co-culture we compared the proteomes of SVF and SVF co-cultured with adipocytes using iTRAQ quantitative mass spectrometry. RESULTS: A co-culture of SVF and adipocytes results in a distinct secretion profile when compared to all other adipose-derived cell populations studied. This illustrates that cellular crosstalk during co-culture of the SVF with adipocytes modulates the production of cytokines by one or more cell types. No biologically relevant differences were detected in the proteomes of SVF cultured alone or co-cultured with adipocytes. CONCLUSIONS: The use of mixed adipose cell populations does not appear to induce cellular stress and results in enhanced secretion profiles. Given the importance of secreted cytokines in cell therapy, the use of a mixed cell population such as the SVF with adipocytes may be considered as an alternative to MSCs or fresh SVF alone.