Oleate but not stearate induces the regulatory phenotype of myeloid suppressor cells

Tumor infiltrating myeloid cells play contradictory roles in the tumor development. Dendritic cells and classical activated macrophages support anti- tumor immune activity via antigen presentation and induction of pro- inflammatory immune responses. Myeloid suppressor cells (MSCs), for instance myeloid derived suppressor cells (MDSCs) or tumor associated macrophages play a critical role in tumor growth. Here, treatment with sodium oleate, an unsaturated fatty acid, induced a regulatory phenotype in the myeloid suppressor cell line MSC-2 and resulted in an increased suppression of activated T cells, paralleled by increased intracellular lipid droplets formation. Furthermore, sodium oleate potentiated nitric oxide (NO) production in MSC-2, thereby increasing their suppressive capacity. In primary polarized bone marrow cells, sodium oleate (C18:1) and linoleate (C18:2), but not stearate (C18:0) were identified as potent FFA to induce a regulatory phenotype. This effect was abrogated in MSC-2 as well as primary cells by specific inhibition of droplets formation while the inhibition of de novo FFA synthesis proved ineffective, suggesting a critical role for exogenous FFA in the functional induction of MSCs. Taken together our data introduce a new unsaturated fatty acid-dependent pathway shaping the functional phenotype of MSCs, facilitating the tumor escape from the immune system

Dietary oleic acid increases m2 macrophages in the mesenteric adipose tissue.

Several studies have implicated fatty-acids as inflammatory regulators, suggesting that there may be a direct role for common dietary fatty-acids in regulating innate immune cells. In humans, a single high-fat meal increases systemic cytokines and leukocytes. In mice, short term high-fat feeding increases adipose tissue (AT) leukocytes and alters the inflammatory profile of AT macrophages. We have seen that short term high fat feeding to C57BL/6J male mice increases palmitic and oleic acid within AT depots, but oleic acid increase is highest in the mesenteric AT (MAT). In vitro, oleic acid increases M2 macrophage markers (CD206, MGL1, and ARG1) in a murine macrophage cell line, while addition of palmitic acid is able to inhibit that increase. Three day supplementation of a chow diet, with oleic acid, induced an increase in M2 macrophage markers in the MAT, but not in the epididymal AT. We tested whether increases in M2 macrophages occur during short term ad lib feeding of a high fat diet, containing oleic acid. Experiments revealed two distinct populations of macrophages were altered by a three day high milk-fat diet. One population, phenotypically intermediate for F4/80, showed diet-induced increases in CD206, an anti-inflammatory marker characteristic of M2 macrophages intrinsic to the AT. Evidence for a second population, phenotypically F4/80(HI)CD11b(HI) macrophages, showed increased association with the MAT following short term feeding that is dependent on the adhesion molecule, ICAM-1. Collectively, we have shown that short term feeding of a high-fat diet changes two population of macrophages, and that dietary oleic acid is responsible for increases in M2 macrophage polarization

HMF diet induces peritoneal cell adhesion.

<p>(A) F4/80 and CD206 expression, by flow cytometry, on peritoneal macrophages, and SVF macrophages isolated from unwashed or washed MAT. F4/80^Int CD206⁺ (a) and F4/80^HI CD206⁻ (b) macrophage populations are boxed. (B) Total cell counts (per mouse) of peritoneal cells (PCs) or macrophages (Macs) isolated by peritoneal lavage following three day control or milk-fat feeding. (N = 3) (C) C57BL6, MCP-1−/−, or ICAM-1−/− mice were fed control or HMF diet. F4/80 gene expression from HMF unwashed MAT is expressed as fold change relative to mice on control diet. (N = 5–8) (D) F4/80 and MCP-1 gene expression (qPCR, relative to Gapdh) in the washed MAT of mice fed a three day control or HMF diet. (N = 10,11).</p

Dietary oleic acid increases M2 macrophage markers in the MAT.

<p>(A) Change in body-weight during a three day, twice daily, oral gavage with control or purified OA. (B) F4/80 gene expression (qPCR relative to GAPDH) in mesenteric adipose tissue (MAT) or epididymal adipose tissue (EAT) from mice given control or OA gavage. (C) Gene expression (qPCR relative to GAPDH) analysis of M2 and M1 macrophage markers in MAT from gavaged mice. (N = 10, 12).</p

Metabolic and body composition changes in response to 3 day HMF feeding.

<p>The mouse metabolic research unit was utilized to analyze (A) body-weight changes, (B) respiratory exchange ratio (RER) and (C&D) food-intake in mice fed a three day control or 42% HMF diet. (N = 5) (E) Fat and lean mass were measured using MRI. (N = 5).</p

Oleic acid induces M2 macrophage markers in

<p>Arginase-1 (Arg1) and TNFα gene expression fold change from (A) RAW 264.7 macrophages or (B) bone marrow derived macrophages (BMDM) cultured overnight with oleic (OA) or palmitic (PA) acid (500 µM). (N = 3,4) (C) CD206, MGL1 and KLF4 gene expression fold change in RAW 264.7 macrophages following overnight culture in OA (500 µM). (D) Arg1 and CD206 gene expression fold change from RAW 264.7 macrophages cultured in OA (200 µM) and PA (as labeled). Control cells were cultured in BSA and ethanol. (N = 3).</p

Oleic acid increase is highest in MAT.

<p>Mass spectrometry analysis of (A) fatty-acid composition (expressed as a percentage of total fatty acid) in the EAT and MAT following three control or 60% HMF feeding. (B) Change in the percentage of palmitic (PA) and oleic (OA) acid out of total fatty-acids expressed as the difference between control and HMF-fed mice. (N = 5–6).</p

Diet-induced macrophage changes in MAT and EAT.

<p>(A) F4/80 expression (qPCR relative to GAPDH) in MAT and EAT (n = 4–8). (B) MCP-1 expression (qPCR relative to GAPDH) in MAT and EAT (n = 4–8). (C) Changes in MCP-1 protein detected by ELISA in extracts of adipose tissues and expressed as ratios of HMF diet over control diet adipose tissue levels in EAT and MAT. (n = 4–8) (D) Typical flow cytometry pattern of the SVF of the MAT showing (a) F4/80^Int CD206⁺ and (b) F4/80^HI CD206⁻ macrophage populations. (E) Changes in the F4/80^Int CD206⁺ macrophage population expressed as ratios of HMF diet over control diet adipose tissue cells/gm in EAT and MAT (n = 6). (F) Representative flow cytometry of the F4/80^HI CD206⁻ macrophage population in MAT showing CD11b expression with control and HMF diets; and graph of the percentage of these cells in the total macrophage population within the stromavascular fraction of MAT from control and HMF diets. (G) Changes in the F4/80^HI CD206⁻ CD11b^Hi macrophages expressed as ratios of cells/g in HMF diet over control diet EAT and MAT.</p