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The complications of atherosclerosis are a major cause of death and disability in Type 2 diabetes. Defective clearance of apoptotic cells by macrophages (efferocytosis) is thought to lead to increased necrotic core formation and inflammation in atherosclerotic lesions.
To determine if there is defective efferocytosis in a mouse model of obesity and atherosclerosis.
We quantified efferocytosis in peritoneal macrophages and in atherosclerotic lesions of obese ob/ob or ob/ob;Ldlr−/− mice and littermate controls. Peritoneal macrophages from ob/ob and ob/ob;Ldlr−/− mice showed impaired efferocytosis, reflecting defective PI3K activation during uptake of apoptotic cells. Membrane lipid composition of ob/ob and ob/ob;Ldlr−/− macrophages showed an increased content of saturated fatty acids (FAs), and decreased n-3 FAs ((Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA)) compared to controls. A similar defect in efferocytosis was induced by treating control macrophages with saturated free FA (FFA)/BSA complexes, while the defect in ob/ob macrophages was reversed by treatment with EPA/BSA or by feeding ob/ob mice a fish oil diet rich in n-3 FAs. There was also defective macrophage efferocytosis in atherosclerotic lesions of ob/ob;Ldlr−/− mice and this was reversed by a fish oil-rich diet.
The findings suggest that in obesity and Type 2 diabetes elevated levels of saturated FAs and/or decreased levels of n-3 FAs contribute to decreased macrophage efferocytosis. Beneficial effects of fish oil diets in atherosclerotic cardiovascular disease may involve improvements in macrophage function related to reversal of defective efferocytosis, and could be particularly important in Type 2 diabetes and obesity.
Patients with Type 2 diabetes experience both accelerated atherogenesis and increased atherosclerotic complications such as plaque rupture and athero-thrombosis1, 2. While diabetic dyslipidemia is a major factor underlying accelerated atherogenesis, mechanisms acting at the level of the vessel wall may also be involved in plaque formation and complications. Recent studies have suggested that insulin resistance in macrophage foam cells 3 as well as in endothelial cells4 could also contribute to atherosclerotic plaque formation and complications. In macrophages insulin resistance promotes macrophage apoptosis during the endoplasmic reticulum (ER) stress response, and is associated with increased necrotic core formation in atherosclerotic plaques 3, while in endothelial cells insulin resistance and defective protein kinase B (AKT/PKB) activation lead to impaired nitric oxide (NO) bioavailability, increased inflammatory gene expression and markedly accelerated atherogenesis4.
Apoptosis of macrophages and smooth muscle cells in advanced atherosclerotic plaques is thought to lead to increased necrotic core formation, inflammation, plaque disruption and athero-thrombosis5. Macrophages from ob/ob and insulin receptor knockout (Insr−/−) mice show increased susceptibility to apoptosis during the ER stress response 6 and decreased inflammatory responses 7. Increased apoptosis and decreased inflammatory responses of macrophages would not be expected to lead to an enhancement of lesion formation, since efficient efferocytosis is normally anti-inflammatory 8, 9 and should not produce detrimental effects unless the phagocytic system becomes impaired. In fact, several molecular defects that lead to an enhancement of macrophage apoptosis have been associated with diminished formation of early atherosclerotic lesions 10–12. In contrast, various induced mutations that cause defective efferocytosis (such as in the mer-tyrosine kinase receptor) lead to increased necrotic core formation, increased inflammation and accelerated atherogenesis in mouse models 13–15. In the present study we show defective efferocytosis in macrophages of ob/ob mice both in peritoneal macrophages and within atherosclerotic lesions and relate these changes to alterations in membrane fatty acid (FA) composition.
About 6-week old obese ob/ob or db/db mice together with their lean littermate controls, low density lipoprotein receptor knockout (Ldlr−/−) and ob+/−;Ldlr−/− on C57BL/6J background were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA). For study with the AIN76A semi-synthetic diet (0.02% cholesterol), sections of atherosclerotic lesions from diet-fed ob/ob;Ldlr−/− or Ldlr−/− mice for more than 20 weeks were analyzed for lesion area and efferocytosis. Mice on the diet for 4 weeks were used for in vitro peritoneal macrophage efferocytosis or membrane/plasma lipid composition analysis. For olive or fish oil diet study, sections of atherosclerotic lesions from diet-fed ob/ob;Ldlr−/− or Ldlr−/− mice for 6 weeks16 were analyzed for lesion area and efferocytosis. Ob control and ob/ob mice fed the same diets for 4 weeks were used for in vitro macrophage efferocytosis or membrane lipid analysis. Unless otherwise specified, ob control and ob/ob mice were maintained on chow diet for other studies.
Thioglycollate-elicited peritoneal macrophages were collected from ob/ob or littermate lean control, Ldlr−/− or ob/ob;Ldlr−/− mice 3 days after thioglycollate injection. Cells were pooled from three mice of each genotype and cultured for 1–2 days in DME medium (DMEM) as described 17. For one day culture, cells were washed 2 hours after plating. For 2-day culture, media were replenished on the second day 17.
Fatty acids, such as lauric (LA)-, myristic (MA)-, palmitic (PA)-, palmitoleic (POA)-, oleic (OA)- or linoleic acid (LOA) were complexed with BSA (Sigma-Aldrich) as described 18. Eicosapentaenoic acid (EPA) was prepared as a stock solution in 100% ethanol and then diluted and mixed with essential fatty acid free BSA at a ratio of 5:1 in DMEM/10% FBS before treatment. To study the effects of FFA/BSA on efferocytosis, FFA/BSA complexes were incubated with macrophages for 6–7 h at 0.5mM in DMEM/10% FBS, unless otherwise specified.
Analysis of in vitro efferocytosis was performed as described 17.
Frozen lesion sections from the proximal aorta from Ldlr−/− or ob/ob;Ldlr−/− mice were analyzed. Lesion and necrotic core areas were measured as before3. For assessment of efferocytosis in the lesions, apoptotic cells were detected as before3, except that the frozen sections were permeabilized with 0.1% triton and sodium citrate on ice for 2 minutes. After TdT-mediated dUTP nick end labeling (TUNEL) staining, samples were blocked in 10% goat serum, and stained with macrophage specific rabbit anti-mouse antibody AIA (Accurate Chemical and Scientific) 13. Genomic DNA was stained with Hoechst dye before the slides were mounted with coverslips. Fluorescent images were captured and macrophage efferocytosis in the lesion areas was quantified as previously described 13.
Results are expressed as mean ± S.E.M. (n is noted in the figure legends or figures), and statistical significance of differences was evaluated with Student’s t test.
Using a fluorescence assay that identifies apoptotic cells within macrophage phago-lysosomes 17, we showed that peritoneal macrophages from ob/ob and db/db mice have impaired efferocytosis (Figure 1A). An in vivo defect in efferocytosis was shown by instilling apoptotic cells into the peritoneal cavity of mice during macrophage elicitation (Figure 1B, in vivo). In addition, ob/ob macrophages showed a defect in Fc-receptor (Fc-R) mediated phagocytosis (Figure 1C). These findings suggest a generalized defect in the phagocytic activities of ob/ob macrophages. The decrease in efferocytosis was not associated with diminished binding of apoptotic cells to the cell surface of ob/ob macrophages (Fig 1D), indicating a defect in the uptake of apoptotic cells. Prolonged cell culture of ob/ob macrophages for more than two days was associated with a gradual normalization of efferocytotic efficiency (data not shown). To further assess if defective efferocytosis of ob/ob macrophages might be cell autonomous, we carried out a bone marrow transplantation experiment. When ob control bone marrow was transplanted into lethally irradiated ob/ob mice, the ob control peritoneal macrophages acquired a similar defect in efferocytosis as seen in ob/ob mice transplanted with ob/ob bone marrow. In contrast, when ob/ob bone marrow was transplanted into ob control mice, efferocytotic efficiency of peritoneal macrophages was not significantly different from ob control to ob control transplanted mice (Figure 1E). These findings indicate that the defect in efferocytosis of ob/ob macrophages is reversible and most likely arises from factors exogenous to the macrophage.
An important early event during phagocytosis is the recruitment of PI-3-kinase (PI3K) to the phagocytic membrane leading to an increase in generation of phosphatidylinositol phosphates (PIPs) within the phagocytic cup 19, 20. In control macrophages, efferocytosis was associated with a rapid increase in PI3K/AKT phosphorylation (Figure 2A) and accumulation of PIP3 in the phagocytic cup (seen as bright localized staining as indicated by arrows in Figure 2B). Ob/ob macrophages had markedly defective PI3K/AKT signaling during efferocytosis, and impaired accumulation of PIP3 in phagocytic membranes (quantification showed ~ 40% decrease in signal in phagocytic cups in ob/ob macrophages compared to control). Similarly, the synthetic PI3K inhibitor LY 294002 produced a severe defect in efferocytosis associated with decreased accumulation of PIP3 in the phagocytic membrane of control macrophages (Figure 2B and C). Since tyrosine phosphorylation of the regulatory subunit of PI3K (P85) can relieve its inhibitory activity on PI3K 21, we carried out immunoprecipitation with a phosphotyrosine antibody and then SDS-PAGE and western blotting with an antibody against the PI3K regulatory subunit P85. There was a rapid increase in the amount of phosphorylated p85 during efferocytosis in control macrophages, but not in ob/ob macrophages. This suggests that p85 phosphorylation is impaired in ob/ob macrophages during efferocytosis leading to decreased PI3K/AKT activation (Figure 2D). To determine if decreased AKT activation might be directly responsible for defective efferocytosis of ob/ob macrophages, we transfected macrophages with adenovirus expressing a constitutively active form of AKT, myr-AKT 22. However, this did not restore the defect in efferocytosis in ob/ob macrophages, suggesting that defective PIP3 generation resulting from decreased PI3K activation is responsible for defective efferocytosis, rather than decreased AKT signaling (Online Figure I).
The bone marrow transplantation experiments showed that the defect in efferocytosis of ob/ob macrophages arose from interaction of the macrophages with exogenous factors. In an attempt to identify the relevant exogenous factors, we first considered whether there might be a direct effect of leptin on phagocytic efficiency, as previously reported for microbial phagocytosis 23. However, addition of leptin or various other adipocytokines (visfatin, adiponectin) or lipopolysaccharide did not influence phagocytic efficiency of ob/ob macrophages (Online Figure II-A). Similarly, increases in glucose in the medium had no effect on efferocytosis of ob/ob macrophages (Online Figure II-B). Since ob/ob macrophages show defective insulin signaling 18, we also analyzed efferocytosis in macrophages from Insr−/− mice. However, macrophages from Insr−/− mice showed no significant defect in efferocytosis (Online Figure II-C).
In contrast to non-obese Insr−/− mice 3, ob/ob mice show marked elevations in plasma FFA levels, and elevated levels of FFAs especially saturated FAs are thought to have an important role in inducing insulin resistance and inflammatory responses in these mice24, 25. We confirmed an increase in plasma FFA levels in ob/ob mice (Figure 3A) and showed that total membrane lipids of ob/ob macrophages have an increased content of saturated FAs and a decreased content of several unsaturated FAs, including the long chain n-3 FAs, Docosahexaenoic acid (DHA) and EPA (Figure 3B). These changes in membrane FA composition in ob/ob mice were similar to those observed for total plasma lipids, where a decrease in DHA and EPA and an increase in C16:0 and C18:0 fatty acids were also observed (Online Figure III-A).
The changes in plasma and membrane FAs in ob/ob mice led us to examine the effects of adding exogenous FFA/BSA complexes on efferocytosis. Strikingly, incubation of wild type macrophages with saturated FFA/BSA complexes but not unsaturated FFA/BSA complexes induced a marked defect in efferocytosis comparable to that observed in ob/ob macrophages. A time course study showed that pre-incubation of WT macrophages with saturated FAs for more than about 5 h induced a defect in efferocytosis (Figure 3C). Since membranes of ob/ob macrophages showed a decreased content of n-3 FAs, we determined effects of incubating ob control and ob/ob macrophages with EPA/BSA complexes. Whereas incubation of ob/ob macrophages with saturated FFA/BSA complexes worsened the defect in efferocytosis (Figure 3D), incubation with EPA/BSA complexes eliminated the defect in efferocytosis in ob/ob macrophages (Figure 3E). Thus, either a decrease in plasma and macrophage membrane n-3 fatty acids and/or an increase in plasma and membrane saturated FAs could be responsible for the defect in efferocytosis in ob/ob macrophages.
Interestingly, the incubation of wild type macrophages with saturated FFA/BSA complexes led to defective PI3K/AKT signaling (Fig 4A) and decreased tyrosine phosphorylation of the regulatory subunit of PI3K during efferocytosis (Fig 4B), resembling the changes seen in ob/ob macrophages (Fig 2A and D). This indicates a similar underlying defect in PI3K activation in ob/ob and saturated FA-treated control macrophages and suggests that changes in plasma and macrophage membrane fatty acid composition in ob/ob mice could be responsible for the defect in efferocytosis in ob/ob and ob/ob;Ldlr−/− macrophages. We also carried out experiments that ruled out a role of increased signaling via Toll-like receptor 4 (TLR4) or effects mediated via IκB kinase (IKK), c-Jun N-terminal kinase (JNK) or protein kinase C (PKC) in saturated fatty acid treated macrophages efferocytosis (Online Figure IV).
We next sought to determine if there might be defective macrophage efferocytosis in atherosclerotic lesions of ob/ob mice. Since the ob/ob mutation is not sufficient to produce atherosclerosis, we carried out these studies in ob/ob; Ldlr−/− mice. Mice were fed a high cholesterol, semi-synthetic diet to induce atherosclerosis. Peritoneal macrophages from ob/ob;Ldlr−/− mice fed this diet showed defective efferocytosis compared to Ldlr−/− controls (Fig 5A). On this diet ob/ob;Ldlr−/− mice showed an increase in plasma FFA levels compared to Ldlr−/− controls (Fig 5B). In Ldlr−/− mice fed the atherogenic semi-synthetic diet, levels of n-3 FAs (DHA and EPA) appeared to be reduced than ob/ob and ob/control mice fed the chow diet, but the same tendency was observed as noted earlier i.e. there were lower levels of DHA and EPA and C18:2 FAs and increased C16:0 and C18:0 FAs in ob/ob;Ldlr−/− compared to Ldlr−/−mice in both plasma and macrophage membrane lipid composition (Online Figure III-B and III-C). In the ob/ob;Ldlr−/− mice plasma very low density lipoprotein/low density lipoprotein (VLDL/LDL) and high density lipoprotein (HDL) levels were increased compared to Ldlr−/− controls, similar to previous studies26, 27 (Online Figure V-A and V-B show data after 4 weeks on diet, Online Table I shows data after more than 20 weeks on diet). We confirmed increased lesion area in ob/ob:Ldlr−/− mice fed the semi-synthetic atherogenic diet for more than 20 weeks compared to Ldlr−/− controls, similar to previous observations on a different diet 27 and also demonstrated increased necrotic core formation in advanced lesions from the former group of mice (Figure 5C). To assess efferocytosis we used a triple staining procedure for apoptotic cells, macrophages and nuclei 13, 28. This assay is illustrated in Figure 5D. TUNEL positive material that co-localizes with nuclei (Hoechst positive) signifies apoptotic cells. If apoptotic cells are not within macrophages (defined as AIA staining positive material in close proximity to another nucleus), they are scored as non-phagocytosed apoptotic cells. Otherwise, they are scored as associated with or phagocytosed by macrophages. These assays were scored by a blinded observer. There was a non-significant trend to increased numbers of apoptotic cells within lesions of ob/ob;Ldlr−/− mice, and a significant increase in numbers of non-phagocytosed apoptotic cells, indicating defective efferocytosis in advanced ob/ob atherosclerotic lesions (Figure 5D).
As noted above, EPA/albumin complexes produced an improvement in efferocytosis in ob/ob macrophages (Figure 3E). Fish oil-rich diets are beneficial for atherosclerotic cardiovascular disease, and are rich in n-3 FAs such as EPA and DHA 29. To determine if fish oils could enhance efferocytosis in vivo, we fed ob control and ob/ob mice either olive oil- or fish oil-enriched diets (containing 40% fat) for about 4 weeks, and then isolated peritoneal macrophages from these mice. Efficiency of efferocytosis was significantly decreased in olive oil treated ob/ob mice compared to ob control mice and this defect was reversed in fish oil treated ob/ob mice relative to ob control mice, paralleling marked improvements in AKT phosphorylation on the fish oil diet (Figure 6A). Analysis of membrane lipid composition in the mice fed the fish oil diet showed an increased content of n-3 FAs (DHA and EPA) compared to mice receiving the olive oil diet (Figure 6B shows mean results of two separate analyses). However, there was no consistent change in membrane saturated FAs in mice fed the fish oil diet.
We next wished to determine if a fish oil rich diet would also improve efferocytosis in atherosclerotic lesions. We carried out an analysis of atherosclerotic lesions from Ldlr−/− and ob/ob:Ldlr−/− mice fed olive oil or fish oil diets. Studies were performed at an early time point in fatty streak lesions. In contrast to later time-points when mice treated with olive oil displayed larger atherosclerotic lesions than those receiving fish oil (12 weeks) 16, there was no overall difference in lesion area after only 6 weeks on olive oil or fish oil diets (Figure 7A). The fish oil diet has a high content of DHA and EPA, allowing us to determine if there might be an in vivo effect on apoptosis and efferocytosis by n-3 FAs 16. Analysis of plasma lipid FA composition showed a marked increase in DHA and EPA, with decreases in C18:1 and C20:4 FAs in mice on the fish oil diet compared to the olive oil diet (Online Figure VI-A and VI-B). On the olive oil diet the ob/ob:Ldlr−/− mice showed a 2-fold increase in numbers of apoptotic cells and a 3.5-fold increase in the number of unphagocytosed apoptotic cells outside of macrophages, compared to Ldlr−/− controls (Figure 7B). Remarkably, both the increase in apoptosis and the defect in efferocytosis were completely reversed in mice fed the fish oil diet (Figure 7B).
Our studies show that in addition to the known inability of ob/ob macrophages to phagocytose bacteria 23, 30, they show defective efferocytosis and Fc-R mediated phagocytosis. Unlike the previous reports 23, 30, we did not find that leptin treatment could reverse these defects. The underlying defect appeared to be related to an altered macrophage membrane lipid composition with decreased levels of n-3 fatty acids (DHA, EPA) and increased levels of saturated fatty acids (C16:0, C18:0), leading to defective PI3K activation and failure to generate PIP3 in the macrophage phagocytic membrane.
Our data strongly suggest that the defect in efferocytosis of ob/ob macrophages was related to increased concentrations of saturated FAs and/or decreased concentrations of the n-3 FAs, DHA and EPA. Saturated FFA/BSA complexes induced a similar defect in efferocytosis associated with defective PI3K activation in control macrophages, while EPA/BSA complexes ameliorated the defect in ob/ob macrophages. Analysis of membrane lipid composition of ob/ob and ob/ob;Ldlr−/− macrophages showed increased saturated FAs and decreased n-3 FAs compared to controls, paralleling changes in the composition of plasma lipid FAs. Thus, exposure of macrophages in vivo to increased saturated FAs and decreased n-3 FAs leads to parallel changes in the composition of membrane lipids. Interestingly, feeding a diet high in fish oils appeared to increase membrane n-3 FAs without appreciable changes in content of saturated FAs, and led to a reversal of the defect in ob/ob efferocytosis in peritoneal and lesional macrophages. Products of DHA/EPA such as lipoxins/resolvins/maresins are known to increase efferocytotic efficiency in macrophages and to promote the resolution of inflammation 31, 32. DHA and EPA are converted into these anti-inflammatory products by 15-lipoxygenase, probably accounting for the anti-atherogenic activity of this enzyme in certain settings33. Thus, reduced levels of plasma and membrane n-3 FAs are likely to be an important factor contributing to defective efferocytosis in ob/ob macrophages, whereas increased levels of n-3 FAs and products such as lipoxins/resolvins/maresins are likely to explain beneficial effects of fish oil diets on efferocytosis. A recent report showing that n-3 FAs increase resolvins, protectins in ob/ob mice with beneficial effects on insulin resistance supports this hypothesis 34.
Mouse models of obesity and diabetes, such as ob/ob and db/db mice, show dramatically accelerated atherosclerosis in apolipoprotein E knockout and Ldlr−/− backgrounds, but also have pro-atherogenic lipoprotein changes, such as increased VLDL/LDL cholesterol levels26, 27, 35,. Thus, the potential contribution of factors acting at the level of the vessel wall may be obscured by increased hyperlipidemia on the ob/ob background. In the current study we have analyzed atherosclerotic lesions in ob/ob;Ldlr−/− mice fed two different diets. In the first study using a semi-synthetic diet(more than 20 weeks), we found that in advanced lesions of ob/ob;Ldlr−/− mice there was increased lesion area, increased necrotic core and defective efferocytosis compared to Ldlr−/− controls. In the second study we analyzed lesions from ob/ob; Ldlr−/− mice or Ldlr−/− controls that had been fed olive oil or fish oil diets for a short period (6 weeks). In these early lesions we documented increased apoptosis and defective efferocytosis in the ob/ob; Ldlr−/− mice compared to Ldlr−/− controls. It is important to note that even though at this stage overall lesion size was similar in the different groups, it was larger in the ob/ob;Ldlr−/− mice fed even the chow diet when they were older (6 months old) or in mice fed with the olive oil diet longer (12 weeks) 16,27. In the latter study, lesion size was decreased by the fish oil diet 16. The ability of fish oils or n-3 FA supplementation to reduce lesion size has also been noted in several other studies 36, 37 While increased apoptosis in early lesions appears to be associated with an overall decrease in lesion size 38, 39, our observations indicate defective efferocytosis in both early and late lesions of ob/ob;Ldlr−/− mice. Defective efferocytosis likely contributes to defective resolution of inflammation and leads to increased lesion size and features of instability such as necrotic core formation when lesions become advanced40, 41.
Apart from the effects of induced or rare mutations 13–15, the causes of defective efferocytosis in atherosclerotic lesions are unknown even though this appears to be a common defect in advanced human atherosclerotic lesions 28. Interestingly, since similar clearance mechanisms are involved, it has been speculated that oxidized phospholipids in oxidized LDL or derived from apoptotic cells may compete with new apoptotic cells for clearance by macrophages 42. The present study suggest that in type 2 diabetes/obesity increased levels of saturated FAs and/or decreased levels of n-3 FAs may give rise to a defect in efferocytosis. Once defective efferocytosis has been initiated by such a mechanism, accumulation of oxidized lipids in lesions may worsen the defect by the proposed competitive mechanism 42.
Fish oil diets or n-3 long chain FA supplementation ameliorate atherosclerotic cardiovascular disease (CVD) in humans, but these effects are not correlated with improvements in traditional risk factors such as plasma LDL or HDL levels 43. The studies of Serhan and colleagues showing that products of n-3 FAs in macrophages help to resolve inflammation and promote efferocytosis have suggested novel mechanisms to explain the beneficial effects of diets enriched in fish oils or supplemented with n-3 FAs in atherosclerotic CVD, i.e. promotion of efferocytosis and resolution of inflammation31, 32,33. Our studies in the ob/ob mouse model extend these important observations by suggesting that in obesity and diabetes increases in the levels of saturated FAs and decreases in n-3 FAs produce defective efferocytosis, and indicate that these defects can be reversed by dietary supplementation with n-3 FAs. Our studies suggest the possibility that fish oil-rich diets currently recommended for treatment of atherosclerotic CVD 43, 44 could be particularly beneficial for CVD in obesity and Type 2 diabetes.
We especially thank Inge Holm Hansen for her technical assistance in lipid GC analysis and Daniel Traum for assistance in Fc-R mediated phagocytosis. Furthermore, we would like to thank Samuel Silverstein, Richard Deckelbaum, Steven Greenberg and Jorge Plutzky for generous discussions.
SOURCES OF FUNDING
This work was supported by NIH grants HL84555 and HL87123 (to A.R. Tall), NIH HL054591 and HL087123, and US Army Medical Research and Materiel Command (USAMRMC) grant W81XWH-06-1-0212 (to I. Tabas), NIH UO1 HL070524 (to J.L. Breslow), NIH HL62887 and HL92969 (to K.E. Bornfeldt) and HL089466 (to A.H. Hasty).