Discussion
Dyslipidemia is an important public health problem in industrialized countries, great importance due to its contribution to the development of cardiovascular diseases . Dyslipidemia can have different presentations, whether there is a high level of triglycerides, a high level of total cholesterol, HDL cholesterol, LDL cholesterol, or any combination of high serum lipid levels . Systemic metabolism, in addition to storing and providing energy to the body, influences cellular metabolism. Immune cells, after stimulation by some antigens, suffer metabolic changes that directly influence their functional spectrum. In innate immune cells, the metabolic changes after activation lead to epigenetic changes that condition cells to respond differently to a second stimuli, either enhancing or attenuating their functional phenotype, a physiological condition known as immune training .
In metabolic diseases such as dyslipidemia, monocyte phenotype may be shaped by high blood lipid levels. We hypothesized that elevated serum levels of cholesterol and triglycerides observed in patients with dyslipidemia, could induce the expression of metabolic markers in circulating monocytes, providing them with a phenotype similar to the recently described metabolic activated macrophages onocytes are cells with a short half-life in circulation of around 1 day; however, studies suggest reprogramming can take place at the level of progenitor cells in the bone marrow trained monocytes have been observed 3 months after priming .
It has been reported that the phenotype of metabolic monocytes was CD36+ ABCA1+ ; however, in that study the phenotype was missing PLIN2 and CD14 markers, and the metabolic markers were evaluated individually, with possible overlap with other subpopulations such as M2. This is the first study reporting circulating monocytes with the complete metabolic phenotype (MoMe) in healthy subjects. This population correspond to approximately 3.4% of total monocytes, with the majority of these cells showing high CD14 expression. This could be related to its pro-inflammatory functional spectrum because its similarities, at least in terms of CD14 expression, to the intermediate monocyte phenotype (CD14high CD16low), to which proinflammatory properties are attributed .
When evaluating the percentages of the different populations of circulating monocytes, we found that healthy subjects showed similar levels of MoMe and M2-like monocytes, which are predominantly CD14+ (dim). Regarding the M1-like monocytes, predominantly CD14++ (high), higher frequencies were found in dyslipidemia subjects. This is in accordance with the literature, as hypercholesterolemia induces loss of hematopoietic stem and progenitor cells quiescence and enhances the propensity to generate pro-inflammatory monocytes . We consider that the levels of MoMe may not be influenced by high levels of lipids since the effect of this metabolic alteration has an impact on epigenetic mark and not necessarily in the expression of metabolic receptors. In innate immune cells that are subjected to a stimulus, whether infectious or sterile, undergo long-term metabolic, transcriptional, and epigenetic reprogramming . It is when these cells are subjected to a secondary stimulus, equal to or different from the first stimulus, when they present a greater response capacity, for example, increased proliferation, receptors expression and cytokines production . We need further studies to evaluate the possible epigenetic modifications, including levels of histone acetylation and methylation, in order to clarify this hypothesis.
It is well known that patients with complicated metabolic diseases such as diabetes mellitus 2 or metabolic syndrome, are characterized by a pro-inflammatory state where molecules as glucose, insulin, and palmitate, are elevated, regulating inflammation and with M1 macrophages playing a key role preserving the inflammatory environment . However, it has been proposed that these metabolites also promote the presence of metabolically activated macrophages (MMe), which display a different phenotype from the classically activated (M1) .
Since monocytes from dyslipidemia subjects are exposed to high triglycerides and cholesterol levels, we hypothesize that such condition serves as a priming stimuli driving macrophages stimulated in vitro with high concentrations of glucose, insulin, and palmitate to a stronger polarization into the MMe phenotype.
In dyslipidemia patients and healthy subjects, metabolic stimuli promote polarization into pro-inflammatory MMe, pro-inflammatory M1 and anti-inflammatory M2 macrophages, being M1 the most abundant polarized subset. This was expected free fatty acids (FFA) such as palmitat (the most prevalent dietary FFA, induce metabolic inflammation via TLR4 through NF-kB, MAPK and PI3K, and induce proinflammatory cytokines production such as IL-6, TNF-α, IL-8, and IL-1β .
While no difference was observed between the percentages of MoMe comparing control and dyslipidemic subjects, in vitrodifferentiated monocyte-derived MMe percentages are higher in dyslipidemia than in the control group. This finding suggests, accordingly with our hypothesis, that elevated triglycerides and cholesterol levels serve as the first stimulus inducing immune training in monocytes, which after in vitro stimulation with glucose, insulin and palmitate in high concentrations, respond differently to those not exposed to elevated lipids in circulation.
Furthermore, in dyslipidemic conditions, the higher the body mass index (BMI) the higher the MMe percentage. It has been previously reported that macrophages from adipose tissue, show a positive correlation between CD36 expression and BMI . Nevertheless, this is the first work reporting the correlation between monocyte-derived MMe (full phenotype) frequencies and BMI.
ABCA1, which is present in the plasma membranes of cells such as monocytes/ macrophages, participates in cholesterol efflux donating it to ApoA1 to form HDL, inducing anti-inflammatory signaling . In , we did not find a relationship between serum HDL concentrations and ABCA1 expression, but we found a direct association between LDL concentrations and ABCA1 expression. This finding suggests that the differentiation into MMe may be a result of the metabolic impairment and are not associated in a direct manner to the metabolism of lipids. However, we observed that high levels of LDLc are related to a greater expression of ABCA1 in MMe, suggesting a high cholesterol efflux and HDL formation. The above, could indicate a beneficial role of this cell population in late stages of dyslipidemia. It would be interesting to evaluate molecules such as SIRT6, involved in the expression of ABCA1 .
Regarding the MMe function, we confirm that although they produce pro-inflammatory cytokines such as IL-6 , they do so to a lesser extent than classic M1 macrophages in subjects with dyslipidemia. Interestingly, we also found that these MMe produce IL-10, a cytokine with potent anti-inflammatory and regulatory activities , and that their production is higher in MMe compared to M1. Regarding the polarization to classic M1 and alternative M2 macrophages, we found no differences between healthy people and those with dyslipidemia; in this sense, Baardman and colleagues have shown that hypercholesterolemia alters macrophage metabolism and phenotype, attenuating the inflammatory phenotype in M1 macrophages.
Our results support the idea that pre-existing metabolic alterations such as elevated serum lipid levels, condition monocytes to differentiate into a specific phenotype after a second stimulus. This is supported by our results from our in vitro model where the second stimulus corresponds to the combination of high concentrations of glucose, insulin and palmitate. We observed a preferent polarization of monocytes into MMe in dyslipidemia group compared to healthy subjects. Interestingly, we observed that MMe from dyslipidemia patients produce higher levels of the regulatory cytokine IL-10. It has been previously described that MMe have a dynamic role in obesity (18). MMe have an important role in controlling metabolic alterations of high-fat diet fed mice at early stages, however, even when maintaining their metabolic markers expression, their function becomes detrimental at advanced stages of obesity. It is clear that our experimental approach focuses in monocyte derived macrophages and not in adipose tissue associated macrophages. However, the production of IL-10 as a regulatory mechanism by monocyte derived MMe results of great relevance. In this regard, it is important to point out that our study was carried out in young people with dyslipidemia, who did not present other comorbidities. For this reason, we cannot predict whether metabolically activated monocytes/ macrophages would have a different behavior pattern in subjects with a longer time of evolution of the disease, different metabolic alterations or in the presence of other comorbidities. To elucidate if the chronic exposure of circulating metabolic monocytes to high concentrations of metabolites (such as glucose or free fatty acids) leads to a different functional profile, further follow-up studies should be carried out.
In conclusion, this is the first study in which monocytes with metabolic phenotype (expressing CD36, ABCA1 and PLIN2) in systemic circulation are described by multiparametric analysis. Our study highlights the dual role of metabolically activated macrophages, which regulate the pro-inflammatory milieu characteristic of dyslipidemia through the production of IL-6 and IL-10.