Extra virgin olive oil extract rich in secoiridoids induces an anti-inflammatory profile in peripheral blood mononuclear cells from obese children

Abstract

Obesity represents an important public health challenge of the twenty first century reaching epidemic proportions worldwide; this is especially true for the pediatric population. In this context, bioactive compounds from foods are crucial to counteract chronic inflammation as a typical feature of obesity. In particular, extra virgin olive oil (EVOO) is one of the most important functional foods exerting, among others, an anti-inflammatory activity not only due to its major (monounsaturated fatty acids) but also to its minor (phenolics) components, as reported in the last years. However, only a limited number of studies were performed on pediatric population, and even fewer are those focusing on EVOO phenolics that investigate the correlation of the chemical characterization with the biological function. Thus, starting from our in vitro data identifying an EVOO chemical profile characterized by a high content of secoiridoids correlating with an anti-inflammatory effect, we studied the ability of an EVOO extract with the same chemical profile to retain this function ex vivo. Specifically, peripheral blood mononuclear cells (PBMCs) collected from obese children were treated with EVOO and olive oil extracts, characterized by a low polyphenol content, to study the ability of secoiridoids to dampen the inflammatory response. A reduction of pro-inflammatory CD14⁺CD16⁺ monocytes was detected by cytofluorimetric analysis when PBMCs were treated with EVOO as compared to olive oil extracts. According to this, a down modulation of CCL2 and CCL4 chemokines involved in the recruitment of inflammatory cells, was reported in the supernatants of EVOO relative to olive oil extracts treated PBMCs. Moreover, a high-throughput gene expression analysis revealed that PBMCs molecular profile from obese children is greatly modulated after the treatment with EVOO extract in terms of metabolic and inflammatory pathways. Importantly, some of the significantly modulated genes were involved in the pathways promoting the development of severe obesity. Overall, our ex vivo data demonstrated the ability of EVOO to reduce the inflammatory milieu of PBMCs from obese children both at protein and molecular levels. Of note, a good correlation between the EVOO chemical profile and the biological modulations in terms of anti-inflammatory activity was reported.

Keywords: anti-inflammatory activity; extra virgin olive oil (EVOO); factor analysis; pediatric obesity; peripheral blood mononuclear cells (PBMCs); secoiridoids; tandem mass spectrometry.

FIGURE 1

EVOO extract significantly reduced the expression of CD16 molecule among the pro-inflammatory CD14⁺CD16⁺ monocyte population in PBMCs from obese children. (A) Representative dot plot of CD14 and CD16 expression on monocytes (CD64⁺CD45⁺) from PBMCs, each treated for 24 h with Ctrl, EVOO, and olive oil extracts (6.25 μg/mL each). (B) Histogram represents CD16 cell surface expression calculated on CD14⁺CD16⁺ cell population. Data are expressed as a mean of median fluorescence ± SEM (n = 13). (C) Histogram indicates the percentage of cell viability ± SEM (n = 13) in the experimental groups. a: EVOO vs. Ctrl; b: EVOO vs. olive oil ****p < 0.0001, ***p < 0.001.

FIGURE 2

The molecular profile of PBMCs from obese children is greatly modulated after the treatment with EVOO extract in terms of metabolic and inflammatory pathways. (A) Hierarchical clustering using the differentially expressed genes from PBMCs separately treated with EVOO, olive oil, and Ctrl for 6 h (6.25μg/mL; n = 9). (B) Volcano plot representation of differential expression analysis of genes in EVOO vs. olive oil treated PBMCs (p < 0.05 and fold change threshold ≥ 2.4). (C) Networks analysis of obesity and severe obesity pathways generated by IPA for the differentially expressed genes between PBMCs treated with EVOO as compared to olive oil.

FIGURE 3

Summary of the molecular modulations along with the relative pathways induced by a 6-h treatment of PBMCs with EVOO compared to olive oil extracts. Arrow direction indicates the up- or- down modulation of genes in PBMCs treated with EVOO using those treated with olive oil as reference. Green arrows: down modulated genes; red arrows: up modulated genes.

FIGURE 4

EVOO extract significantly reduced the expression of important chemokines at protein level as early as 6 h after PBMCs treatment relative to olive oil. Histograms represent CCL2 (A) and CCL4 (B) protein expression tested by a high-sensitivity immunoassay using Real-Time PCR as a readout, in the supernatant of PBMCs individually treated with EVOO or olive oil for 6 h (6.25μg/mL). Data are expressed as a mean of triplicate concentration values ± SEM of two independent experiments (n = 10). ****p < 0.0001.

FIGURE 5

Factor analysis indicates a good correlation between the EVOO chemical profile and the biological modulations. Score plots (A,C) and factor loadings (B,D) relative to oil polyphenols and gene expression data at 6 h (A,B), and protein data at 6 and 24 h (C,D) were reported. Olive oil polyphenols: (1) 3-hydroxytyrosol; (2) oleuropein isomers; (3) oleuropein aglycone forms; (4) oleochantalic acid; (5) luteolin; (6) ligstroside aglycone forms; (7) apigenin; (8) methoxyluteolin. mRNAs: (a) NQO1; (b) NR4A2; (c) INFγ; (d) SSP1; (e) PPARγ; (f) CCL2; (g) MMP14; (h) MS4A4A; (i) TLR2; (j) CAT; (k) MMP2; (l) FTO; (m) IL-10; (n) CD14; (o) CCL4; (p) S100A8; (q) CD163; (r) CD36; (s) CASP1; (t) THBS1. Proteins 6 h: (u) CCL2 and (v) CCL4. Proteins 24 h: (z) C14⁺C16⁺ cell population.