Human visceral and subcutaneous adipose stem and progenitor cells retain depot-specific adipogenic properties during obesity.

Abdominal obesity associates with cardiometabolic disease and an accumulation of lipids in the visceral adipose depot, whereas lipid accumulation in the subcutaneous depot is more benign. We aimed to further investigate whether the adipogenic properties where cell-intrinsic, or dependent on a depot-specific or obesity-produced microenvironment. We obtained visceral and subcutaneous biopsies from non-obese women (n = 14) or women living with morbid obesity (n = 14) and isolated adipose stem and progenitor cells (ASPCs) from the stromal vascular fraction of non-obese (n = 13) and obese (n = 13). Following in vitro differentiation into mature adipocytes, we observed a contrasting pattern with a lower gene expression of adipogenic markers and a higher gene expression of immunogenic markers in the visceral compared to the subcutaneous adipocytes. We identified the immunogenic factor BST2 as a marker for visceral ASPCs. The effect of obesity and insulin resistance on adipogenic and immunogenic markers in the in vitro differentiated cells was minor. In contrast, differentiation with exogenous Tumor necrosis factor resulted in increased immunogenic signatures, including increased expression of BST2, and decreased adipogenic signatures in cells from both depots. Our data, from 26 women, underscore the intrinsic differences between human visceral and subcutaneous adipose stem and progenitor cells, suggest that dysregulation of adipocytes in obesity mainly occurs at a post-progenitor stage, and highlight an inflammatory microenvironment as a major constraint of human adipogenesis.

Human Brown Adipocyte Thermogenesis Is Driven by ß2-AR Stimulation.

Stimulation of brown adipose tissue (BAT) thermogenesis in humans has emerged as an attractive target to improve metabolic health. Pharmacological stimulations targeting the ß3-adrenergic receptor (ß3-AR), the adrenergic receptor believed to mediate BAT thermogenesis, have historically performed poorly in human clinical trials. Here we report that, in contrast to rodents, human BAT thermogenesis is not mediated by the stimulation of ß3-AR. Oral administration of the ß3-AR agonist mirabegron only elicited increases in BAT thermogenesis when ingested at the maximal allowable dose. This led to off-target binding to ß1-AR and ß2-AR, thereby increasing cardiovascular responses and white adipose tissue lipolysis, respectively. ADRB2 was co-expressed with UCP1 in human brown adipocytes. Pharmacological stimulation and inhibition of the ß2-AR as well as knockdown of ADRB1, ADRB2, or ADRB3 in human brown adipocytes all confirmed that BAT lipolysis and thermogenesis occur through ß2-AR signaling in humans (ClinicalTrials.govNCT02811289).

Proteomics-Based Comparative Mapping of the Secretomes of Human Brown and White Adipocytes Reveals EPDR1 as a Novel Batokine.

Adipokines secreted from white adipose tissue play a role in metabolic crosstalk and homeostasis, whereas the brown adipose secretome is less explored. We performed high-sensitivity mass-spectrometry-based proteomics on the cell media of human adipocytes derived from the supraclavicular brown adipose and from the subcutaneous white adipose depots of adult humans. We identified 471 potentially secreted proteins covering interesting categories such as hormones, growth factors, extracellular matrix proteins, and proteins of the complement system, which were differentially regulated between brown and white adipocytes. A total of 101 proteins were exclusively quantified in brown adipocytes, and among these was ependymin-related protein 1 (EPDR1). EPDR1 was detected in human plasma, and functional studies suggested a role for EPDR1 in thermogenic determination during adipogenesis. In conclusion, we report substantial differences between the secretomes of brown and white human adipocytes and identify novel candidate batokines that can be important regulators of human metabolism.

Major TCR Repertoire Perturbation by Immunodominant HLA-B*44:03-Restricted CMV-Specific T Cells.

Lack of disease during chronic human cytomegalovirus (CMV) infection depends on the maintenance of a high-frequency CMV-specific T cell response. The composition of the T cell receptor (TCR) repertoire underlying this response remains poorly characterised, especially within African populations in which CMV is endemic from infancy. Here we focus on the immunodominant CD8+ T cell response to the immediate-early 2 (IE-2)-derived epitope NEGVKAAW (NW8) restricted by HLA-B*44:03, a highly prevalent response in African populations, which in some subjects represents >10% of the circulating CD8+ T cells. Using pMHC multimer staining and sorting of NW8-specific T cells, the TCR repertoire raised against NW8 was characterised here using high-throughput sequencing in 20 HLA-B*44:03 subjects. We found that the CD8+ T cell repertoire raised in response to NW8 was highly skewed and featured preferential use of a restricted set of V and J gene segments. Furthermore, as often seen in immunity against ancient viruses like CMV and Epstein-Barr virus (EBV), the response was strongly dominated by identical TCR sequences shared by multiple individuals, or "public" TCRs. Finally, we describe a pair "superdominant" TCR clonotypes, which were germline or nearly germline-encoded and produced at remarkably high frequencies in certain individuals, with a single CMV-specific clonotype representing up to 17% of all CD8+ T cells. Given the magnitude of the NW8 response, we propose that this major skewing of CMV-specific immunity leads to massive perturbations in the overall TCR repertoire in HLA-B*44:03 individuals.

Immunodominant cytomegalovirus-specific CD8+ T-cell responses in sub-Saharan African populations.

More than 90% of children in Africa are infected with cytomegalovirus (CMV) by the age of 12 months. However, the high-frequency, immunodominant CD8+ T-cell responses that control CMV infection have not been well studied in African populations. We therefore sought to define the immunodominant CMV-specific CD8+ T-cell responses within sub-Saharan African study subjects. Among 257 subjects, we determined the CD8+ T-cell responses to overlapping peptides spanning three of the most immunogenic CMV proteins, pp65, IE-1 and IE-2, using IFN-γ ELISpot assays. A bioinformatics tool was used to predict optimal epitopes within overlapping peptides whose recognition was statistically associated with expression of particular HLA class I molecules. Using this approach, we identified 16 predicted novel CMV-specific epitopes within CMV-pp65, IE-1 and IE-2. The immunodominant pp65-specific, IE-1, IE-2 responses were all either previously well characterised or were confirmed using peptide-MHC tetramers. The novel epitopes identified included an IE-2-specific epitope restricted by HLA*B*44:03 that induced high-frequency CD8+ T-cell responses (mean 3.4% of CD8+ T-cells) in 95% of HLA-B*44:03-positive subjects tested, in one individual accounting for 18.8% of all CD8+ T-cells. These predicted novel CMV-specific CD8+ T-cell epitopes identified in an African cohort will facilitate future analyses of immune responses in African populations where CMV infection is almost universal during infancy.

Okadaic acid: a rapid inducer of lamellar bodies in small intestinal enterocytes.

Okadaic acid (OA) is a polyether fatty acid produced by marine dinoflagellates and the causative agent of diarrhetic shellfish poisoning. The effect of OA on apical endocytosis in the small intestine was studied in organ cultured porcine mucosal explants. Within 0.5-1 h of culture, the toxin caused hyper protein phosphorylation, but no detectable loss of cell polarity or cytoskeletal integrity of the enterocytes. Using a fluorescent membrane marker, FM dye, endocytosis from the brush border was affected by the toxin. Although constitutive uptake into subapical terminal web-localized early endosomes (TWEEs) occurred unimpeded in the presence of OA, FM condensed in larger subapical structures by 1 h, implying a perturbed endosomal trafficking/maturation. The fluorescent lysosomotropic agent Lysotracker revealed induction of large lysosomal structures by OA. Endocytosis from the brush border was studied at the electron microscopic level using the membrane-impermeable marker Ruthenium Red (RR). Like FM dye, RR was taken up into TWEEs and multivesicular bodies (MVBs). However, OA induced the formation of a large number of lamellar bodies (LBs), a type of lysosome-related organelles. LBs are the hallmark of phospholipidosis, a pathological condition characterized by lysosomal phospholipid accumulation. Phospholipidosis is observed in acquired lysosomal storage diseases and is induced by a large number of cationic amphiphilic drugs. Unlike the latter, however, OA does not act by accumulating in acidic organelles, implying a different toxic mechanism of action. We propose that rapid induction of LBs, an indicator of phospholipidosis, should be included in the future toxicity profile of OA.