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.