Circulating Blood Monocyte Subclasses and Lipid-Laden Adipose Tissue Macrophages in Human Obesity

Abstract

Background: Visceral adipose tissue foam cells are increased in human obesity, and were implicated in adipose dysfunction and increased cardio-metabolic risk. In the circulation, non-classical monocytes (NCM) are elevated in obesity and associate with atherosclerosis and type 2 diabetes. We hypothesized that circulating NCM correlate and/or are functionally linked to visceral adipose tissue foam cells in obesity, potentially providing an approach to estimate visceral adipose tissue status in the non-surgical obese patient.

Methods: We preformed ex-vivo functional studies utilizing sorted monocyte subclasses from healthy donors. Moreover, we assessed circulating blood monocyte subclasses and visceral fat adipose tissue macrophage (ATM) lipid content by flow-cytometry in paired blood and omental-fat samples collected from patients (n = 65) undergoing elective abdominal surgery.

Results: Ex-vivo, NCM and NCM-derived macrophages exhibited lower lipid accumulation capacity compared to classical or intermediate monocytes/-derived macrophages. Moreover, of the three subclasses, NCM exhibited the lowest migration towards adipose tissue conditioned-media. In a cohort of n = 65, increased %NCM associated with higher BMI (r = 0.250,p<0.05) and ATM lipid content (r = 0.303,p<0.05). Among patients with BMI≥25Kg/m2, linear regression models adjusted for age, sex or BMI revealed that NCM independently associate with ATM lipid content, particularly in men.

Conclusions: Collectively, although circulating blood NCM are unlikely direct functional precursor cells for adipose tissue foam cells, their increased percentage in the circulation may clinically reflect higher lipid content in visceral ATMs.

Fig 1. Non-classical monocytes and their derived macrophages exhibit lower lipid accumulation.

Monocyte subclasses were sorted from CD14+ enriched PBMC fraction obtained from the blood of healthy male donors. Sorted monocyte subclasses were cultured ex-vivo for A) 24 hours with oleic acid (200 Μm) or C) 7 days with M-CSF (50 ng/ml), supplemented for the last 2 days with 10% human omental adipose tissue conditioned medium, to mimic adipose tissue macrophages. Cells were subsequently fixed and stained with DAPI (blue) and BODIPY (green), to visualize nucleus and lipid droplets, respectively. Representative images for the three subclasses were obtained by Operetta imaging system (A and C). Bar = 20 μm. Summary of results from 6 or 9 independent donors are shown in B and D, respectively. Statistical comparison was obtained by the Wilcoxon matched pairs non-parametric test. *p<0.05.

Fig 2. Non-classical and intermediate monocytes migrate less towards conditioned medium of omental adipose tissue.

A) Following isolation of PBMCs from peripheral blood of healthy male donors, monocytes were enriched by CD14+ magnetic separation and stained for CD14 and CD16. 1–1.5*10⁶ cells were placed in the upper part of a migration chamber, placed on top of RPMI media containing 10% FBS or 10% human omental adipose tissue conditioned media (hOAT-CM). After 2.5 hours in 37°C, cells in the lower chamber were collected, counted, and B) analyzed by FACS, and the percentage of migrating cells of each subclass from the original fraction cultured was calculated. Summary of the results obtained from 6 independent donors are shown in C. Statistical comparison was obtained by the Wilcoxon matched pairs non-parametric test, *p<0.05.

Fig 3. Analyses of circulating blood monocyte subpopulations and adipose tissue macrophage lipid content by FACS.

A) Circulating blood monocytes subclasses: Following RBC lysis monocytes were identified by gating for small and low granular cells (low side scatter (SSC) and forward scatter (FSC), respectively), and cell doublets were excluded by using Area (FSC-A) vs. Width (FSC-W) signal intensity. T-, B- and NK- lymphocytes were excluded using CD3, CD19 and CD56, respectively. Monocyte subclasses were identified by CD14 and CD16 expression. B) Adipose tissue macrophage (ATM) lipid content: SVF was isolated from omental human fat tissues by mechanical and collagenase digestion. Viable ATM were identified as staining negative for propidium iodide (PI), positive for the leucocyte marker CD45, and positive for the macrophage markers CD14 and CD64. ATMs' lipids content was assessed using the neutral lipid fluorescent dye BODIPY readout, gray- unstained sample, black-stained sample.

Fig 4. Monocyte subclasses association with clinical and adipose tissue parameters.

A) Spearman Correlation analysis between clinical parameters and omental adipose tissue parameter and % circulating monocyte subclass (CM-classical monocytes; IM-intermediate monocytes; NCM-non-classical monocytes). B) Distribution of the patients' cohort to %NCM-low and–high according to the median value (13.2%). Omental ATM lipid content was compared between %NCM-low and %NCM-high C) in the whole cohort (n = 32 vs. n = 33, respectively), or separately D) in men (left graph, n = 13 vs. n = 11, respectively) and in women (right graph, n = 19 vs. n = 22, respectively). Adipose tissue chemokine expression was compared between %NCM-low and %NCM-high E) in the whole cohort (n = 11 vs. n = 15, respectively), or separately F) in men (left graph, n = 4 vs. n = 6, respectively) and in women (right graph, n = 7 vs. n = 9, respectively). Statistical comparison was obtained by the Mann-Whitney U test. *p< 0.05, **p<0.01.