Edited by: Charles Dudley Mills, BioMedical Consultants, USA
Reviewed by: Klaus Ley, La Jolla Institute for Allergy & Immunology, USA; Janos G. Filep, University of Montreal, Canada
This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology.
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It is well recognized that macrophages in many contexts
Atherosclerotic cardiovascular diseases, which include myocardial infraction and stroke, are the most common causes of morbidity and mortality in western society and will soon be the same world-wide. Atherosclerosis represents a failure to resolve the inflammatory response in the arterial wall initiated by the retention of apolipoprotein B (apoB)-containing lipoproteins (
The study of macrophages over the past decade is characterized by a remarkable expansion of knowledge concerning their origin, functional properties, and potential to both protect from and contribute to disease [e.g., see in Ref. (
Polarization toward the M1 state is induced by several stimuli
On the other side of the polarization spectrum, M2 macrophages are induced
In addition to the M1 and M2 macrophages, oxidized phospholipids present in oxidized LDL induce a macrophage phenotype that is distinct from M1 or M2 phenotypes and that has been termed Mox; these macrophages are characterized by the increases in the expression of nuclear factor erythroid 2-related factor 2 (NRF2)-dependent genes and in reactive oxygen species, and are found in the progressing plaques (
With this background, we will now turn to a more complete consideration of the inflammatory states of macrophages in atherosclerosis progression and regression.
As mentioned above, M1 macrophages are thought to have significant roles in progressing and vulnerable plaques (
The most accepted and robust risk factor for atherosclerosis is low-density lipoprotein-cholesterol (LDL-C). Thus, several studies have tried to understand how cholesterol can induce inflammation in general, and specifically to an activated state. The different mechanisms by which cholesterol can drive macrophage activation could be divided into those direct – how cholesterol affects macrophages, and indirect – how cholesterol affects other cell types through which activation could be induced, for example by the secretion of pro-inflammatory cytokines from T cells (Figure
Accumulation of cholesterol leads to the formation of crystals that are both intra- and extracellular. The presence of cholesterol crystals in early lesions in Apoe-/- mice was recently demonstrated (
A second direct mechanism that can explain macrophage activation by cholesterol is mediated through oxidized LDL. Oxidized LDL is present in both human and mouse atheromas. LDL oxidation is thought to be mediated by enzymes (such as 12/15-lipoxygenase and myeloperoxidase) and by free radicals that are abundant in the atherosclerotic plaque (
A third direct mechanism points toward the increase in plasma membrane cholesterol. The change in the microenvironment of TLRs (
Despite the predominance of data that show a direct link between cholesterol and macrophage inflammation, there are a number of studies that show the opposite – an anti-inflammatory phenotype induced by intracellular cholesterol. The basis for this is rooted in the LXRs and PPARs, which are important nuclear receptors. The ligands for these receptors include lipids, and in addition to regulating many steps of their metabolism, LXRs and PPARs can also suppress inflammatory signaling in macrophages (
It has been long known that T cells participate in plaque inflammation (
Another indirect effect on macrophage polarization/activation by cholesterol is related to its being a major structural component of progressing plaques, so its ongoing accumulation will contribute to enlargement of the atheroma. This, in turn, is likely to contribute to a hypoxic environment because as the atheroma grows, the distance between the intimal cells from an oxygen supply will increase, particularly in mice, which have little capacity to form vasa vasorum. Hypoxia triggers a heavy reliance on glycolysis for energy production and it has been recently recognized that M1 cells are more glycolytic and M2 cells are more fatty acid oxidizing, and that factors that promote one pathway of energy generation over the other will promote the polarization state corresponding to the favored pathway (
Recent studies have found that although both M1 and M2 macrophage numbers are increased during human plaque progression, M1 macrophages were the predominant phenotype in rupture-prone shoulder regions, whereas M2 markers were predominant in the adventitia and in stable cell-rich areas the of plaque (
In mouse plaques, M1 macrophages tend to be diffusely distributed in, and characteristic of, progressing plaques at the usual age that atherosclerotic mice are examined in detail (typically after 12–16 of the consumption of a high-fat, high-cholesterol diet, or ~16–20 weeks of age). Khallou-Laschet et al. have found in apoE-deficient mice of a similar age, but with less advanced atherosclerosis because they were maintained on a low-fat, chow diet that these early plaques were infiltrated by M2 macrophages, with M1 macrophages appearing later. Disease progression correlated with the dominance of M1 over the M2 phenotype (
The secretion of a wide range of cytokines and chemokines (e.g., IL-1, TNFalpha, MCP-1) by M1 macrophages serves to further activate macrophages, as well as other cell types in the atheroma, such as endothelial and smooth muscle cells. There are also effects on cellular lipid metabolism. In one study of human plaques, macrophages with a marker of M2 macrophages had small lipid droplets and in studies
M1 macrophages also secrete chemokines (such as MCP-1) and cytokines (such as IL-12) that induce chemotaxis of other white blood cells (
Fatty streaks, the initial phase in the development of plaques, were found in children as young as 3 years of age (
Plaque regression can be defined in various ways, such as a reduction in plaque size, plaque cholesterol content, plaque macrophage number/percentage, or a decreased inflammatory state. Of course, multiple changes can occur simultaneously, but not in every case. For example, if the plaque macrophage content decreases, while collagen content increases, as we have observed experimentally in some models of regression [e.g., Ref. (
Some of the currently available regression models include aortic arch transplantation model (
In the transplantation model, the plaque-containing aortic arch from a donor a
As noted above, HDL and its major protein, apoA1, can be increased by injections of apoA1, apoA1 mimetics, or an adenoviral vector expressing apoA1 (
Another example for plaque regression induced by an increase in HDL was shown with our collaborators using an inhibitor of microRNA-33 (miR-33). miR-33 suppresses HDL formation in the liver and its ability to efflux cholesterol from macrophages by suppressing the expression of cholesterol transporter ATP-binding cassette transporter 1 (
The Reversa (
The hepatic overexpression of
Inducing regression just by a diet change, from western diet to chow, in the
There are two major questions we will consider in this section – the origin of the M2 macrophages in regressing plaques and the mechanisms for their increase.
One possibility is that the re-balancing from enrichment in M1 to M2 markers in regressing plaques represents either a change in an individual cell, as can be accomplished
It is tempting to speculate that the pro-activating direct and indirect effects of cholesterol described above are reversed under conditions of regression, under which the plaque content of cellular and extracellular cholesterol is typically reduced. Little experimental evidence, however, is available
Turning to the issue of what regulates the enrichment in M2 macrophages in regressing plaques, there are no results to discuss at this time, but there are a number of possibilities. As mentioned earlier, potent cytokines that polarize macrophages are IL-4 and IL-13. These can be derived from a variety of leukocytes, namely Th2 lymphocytes, eosinophils, and basophils. Even if one or more of these types of leukocytes were the source of polarizing signals, there is still the mystery of how the change in the lipoprotein/lipid environment causes either the recruitment of the cells to the plaque or the stimulation of secretion from either pre-existing or newly recruited cells.
Another major and incompletely understood area is the requirement for, and the function of, the enrichment in M2 macrophages in atherosclerosis regression. Because their properties include tissue remodeling and inflammation resolution, it is tempting to attribute such changes in regressing plaques to M2 macrophages. This would be consistent with studies in which treatment of
The promotion of the resolution of inflammation by M2 macrophages in regressing plaques is likely a consequence of their secretion of IL-10. The plaque remodeling may represent at least two other properties of M2 macrophages, namely their secretion of collagen (
During both the progression and the regression of atherosclerosis, macrophages have central roles. While macrophage phenotypes are diverse and form a continuum (
As noted throughout this review, there are many areas in which our knowledge of macrophage biology in plaques is inadequate. Yet, it is already clear that the inflammatory state of these cells is dynamically influenced by multiple metabolic, genetic, and pharmacologic factors. Deeper understanding of how these factors effect changes in plaque macrophages will likely advance the development of new strategies to reduce the huge burden of cardiovascular morbidity and mortality that persists with existing therapies.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank all of our lab members, past and present, as well as our collaborators, who have contributed to our studies cited in this review. Support for these studies comes from the following NIH grants: HL098055, HL084312, DK95684, and HL117226.