Atherogenic lipids and lipoproteins trigger CD36-TLR2-dependent apoptosis in macrophages undergoing endoplasmic reticulum stress

Abstract

Macrophage apoptosis in advanced atheromata, a key process in plaque necrosis, involves the combination of ER stress with other proapoptotic stimuli. We show here that oxidized phospholipids, oxidized LDL, saturated fatty acids (SFAs), and lipoprotein(a) trigger apoptosis in ER-stressed macrophages through a mechanism requiring both CD36 and Toll-like receptor 2 (TLR2). In vivo, macrophage apoptosis was induced in SFA-fed, ER-stressed wild-type but not Cd36⁻(/)⁻ or Tlr2⁻(/)⁻ mice. For atherosclerosis, we combined TLR2 deficiency with that of TLR4, which can also promote apoptosis in ER-stressed macrophages. Advanced lesions of fat-fed Ldlr⁻(/)⁻ mice transplanted with Tlr4⁻(/)⁻Tlr2⁻(/)⁻ bone marrow were markedly protected from macrophage apoptosis and plaque necrosis compared with WT →Ldlr⁻(/)⁻ lesions. These findings provide insight into how atherogenic lipoproteins trigger macrophage apoptosis in the setting of ER stress and how TLR activation might promote macrophage apoptosis and plaque necrosis in advanced atherosclerosis.

Figure 1. OxPLs, oxLDL, and Lp(a) Induce CD36- and TLR2/6-Dependent Apoptosis in ER-Stressed Macrophages

For all the experiments described below, the experimental endpoint was macrophage apoptosis; the data are expressed as the percent of total cells that stained with annexin V and propidium iodide (mean ± SEM; n=4). Differences between values with symbols and no symbols, or between values with different symbols, are statistically significant (p values ranged from < 0.05 to < 0.001). (A) Macrophages were incubated for 28 h with 0.25 μM thapsigargin (Tg) alone or thapsigargin in combination with the indicated concentrations of ox-LDL. (B) Macrophages were left untreated (Un) or incubated for 30 h with 0.25 μM thapsigargin alone or thapsigargin in combination with 10 μg/ml of KDdia-PC or POV-PC, or the phospholipids alone. (C) Macrophages from WT, Sra^−/−, or Cd36^−/− mice were incubated for 18 h with thapsigargin alone or thapsigargin in combination with 10 μg/ml POV-PC or KDdia-PC or with 50 μg/ml of ox-LDL. (D) Macrophages were left untreated or incubated for 23 h with 50 μg/ml KOdia-PC without (mock) or with (PLA2) pretreatment with phospholipase A2, alone or in combination with 0.5 μM thapsigargin (Tg). (E) Macrophages from WT or Tlr2^−/− mice were incubated for 30 h with 0.25 μM thapsigargin alone or thapsigargin combined with KDdia-PC, POV-PC, ox-LDL, or LTA. (F) Macrophages from WT, Cd36^−/−, or Tlr2^−/− mice were incubated for 24 h with thapsigargin alone or thapsigargin combined with 10 μg/ml LTA. (G) Macrophages from WT, Tlr1^−/−, or Tlr6^−/− mice were incubated for 24 h with thapsigargin with or without KOdia-PC, LTA, or 10ug/ml Pam3CSK4 or Pam2CSK4. (H) Macrophages from WT, Cd36^−/−, or Tlr2^−/− mice were incubated for 18 h with 0.25 μM thapsigargin (Tg) alone; 0.25 mM of the indicated fatty acids in complex with BSA; or thapsigargin plus either the fatty acids or BSA control. The unsaturated fatty acids were linoleic acid (18:2) and oleic acid (18:1), and the saturated fatty acids were stearic acid (18:0) and palmitic acid (16:0).

Figure 2. CD36 Ligand-Induced Apoptosis in ER-stressed Macrophages is Dependent on Reactive Oxygen Species (ROS)

(A–B) Macrophages were pre-incubated for 60 min in the absence or presence of 100 μM N-acetylcysteine (NAC) and then incubated for 24 h in medium alone (Un) or containing 0.25 μM thapsigargin (Tg), 20 μg/ml KDdia-PC or Tg and KDdia-PC, as indicated. The cells were then assayed for ROS, using the DCF fluorescence assay, and apoptosis. (C–D) As above, but here the ER stressor was 5 μg/ml 7-ketocholesterol or 2 mM SIN-1 instead of thapsigargin. Differences between values with symbols and no symbols, or between values with different symbols, are statistically significant (p values ranged from < 0.05 to < 0.01).

Figure 3. Lp(a) Triggers Apoptosis in ER-Stressed Macrophages in a CD36-TLR2-Oxidative Stress-Dependent Manner

(A–B) Macrophages from WT, Cd36^−/−, Tlr2^−/−, Tlr1^−/−, or Tlr6^−/− mice were incubated for 24 h with 0.5 μM thapsigargin alone or in combination with 25 μg/ml LDL or Lp(a), 50 μg/ml KOdia-PC, or 10 μg/ml LTA. (C) Apoptosis data for macrophages that were pre-incubated with or without 500 μM N-acetylcysteine (NAC) and then incubated for 24 h with thapsigargin alone or with 50 μg/ml of Lp(a). (D) Macrophages were incubated for 22 h with thapsigargin alone or in combination with 25 μg/ml apo(a) that was pre-treated in the absence (mock) or presence of trypsin and then assayed for apoptosis. The image shows a Coomassie-stained SDS-polyacrylamide electrophoresis gel of mock and trypsin-treated apo(a); for each condition, three different amounts of proteins were loaded per lane, increasing from left to right. (E) As in D, but here the apo(a) was treated with ± PLA₂ prior to trypsinization. *, p < 0.01 compared to other groups without the asterisk.

Figure 4. Sustained Activation of NADPH Oxidase by CD36 Ligands in ER-Stressed Macrophages

(A) Macrophages from WT, Cd36^−/−, Nox2^−/−, or Tlr2^−/− mice were incubated for the indicated times with 0.25 μM thapsigargin (Tg) alone or in combination with 20 μg/ml POV-PC and then assayed for the percent of DCF-positive cells (ROS). (B) Macrophages from WT or Nox2^−/− mice were incubated for 24 h with thapsigargin alone or in combination with KOdia-PC, 10 μg/ml LTA, or 0.25 mM palmitic acid (PA). The cells were then assayed for apoptosis. (C) Macrophages were incubated for 4 h in medium alone or medium containing KOdia-PC, thapsigargin or the two reagents combined and then subjected to immunofluorescence for p47^phox (red) and labeling with LysoTracker® Green and the nuclear stain DAPI (blue). Treatment with LPS for 30 min was used as a positive control. Bar, 5 μm. (D) Quantification of the data at two timepoints for the experiment in C, expressed as the percent of total cells that exhibited p47 clustering (mean ± SEM, n=4). Differences between values with symbols and no symbols, or between values with different symbols, are statistically significant (p < 0.05).

Figure 5. TLR2-Dependent ERK Activation Promotes Apoptosis, ROS Generation, and p47^phox Clustering

(A–C) Macrophages were left untreated (Un) or incubated for 24 h with the ER stressors thapsigargin or 7-ketocholesterol alone or in combination with KOdia-PC or LTA. Some of the samples were pre-treated for 1 h with 250 nM of the conventional or pan PKC inhibitors Go6976 and Go6983, respectively; 10 μM of the Src-like kinase inhibitor PP2; or 10 μM of the MEK1 inhibitor PD98059. The cells were then analyzed for apoptosis (A), DCF positivity (B), or p47^phox clustering (C). (D–E) Macrophages from WT, Nox2^−/−, or Tlr2^−/− mice were incubated with KDdia-PC alone for the indicated times. Cell lysates were then immunoblotted for Thr202/Tyr204-phospho-ERK1/2 and total ERK. Differences between values with symbols and no symbols, or between values with different symbols, are statistically significant (p < 0.05).

Figure 6. SFAs Trigger CD36- and TLR2-Dependent Macrophage Apoptosis In Vivo

(A–B) Mice were fed a regular chow diet or a diet containing saturated fatty acids (SFAs) for two weeks. Concanavalin A was administered i.p., and two days later the mice were given an i.p. injection of 150 mM dextrose (vehicle) or 0.4 mg/kg of thapsigargin (Tg) in dextrose. (A) Macrophages were harvested from the peritoneum 24 h later and stained immediately with alexa-488 annexin-V and propidium iodide. Representative fluorescent and bright-field images are shown. Bar, 20 μm. The dot plot shows the quantified data for each individual mouse, which was obtained from 5 individual fields/mouse. The horizontal line represents the mean in each group (*p < 0.05). (B) Spleens harvested from these mice were sectioned and stained for the macrophage marker Mac3 (green) and for apoptosis by TUNEL (red) (Bar, 10 μm; *p < 0.05). (C–F) WT, Cd36^−/−, and Tlr2^−/− mice were fed a SFA-rich diet for 2 wks and then treated with thapsigargin as described above. Peritoneal macrophages (C–D) and splenic macrophages (E–F) were then assayed for apoptosis. (G) ob/ob mice were fed a diet rich in SFAs or unsaturated fatty acids (UFA) for 2 wks, and then freshly isolated peritoneal macrophages were assayed for apoptosis. The horizontal lines in C, E, and G represent the mean value in each group (*p < 0.05).

Figure 7. TLR2/4 Deficiency in Bone Marrow-Derived Cells Suppress Apoptosis and Plaque Necrosis in Advanced Aortic Root Lesions of Fat-Fed Ldlr−/− Mice

Ldlr^−/− mice were transplanted with Tlr2^+/+Tlr4^+/+ (wild-type, WT) or Tlr2^−/−Tlr4^−/− bone marrow and then fed a Western-type diet for 10 wks starting 4 wks after transplantation. (A) Plasma cholesterol, triacylglycerol, HDL, and FPLC lipoprotein profile of the 2 groups of mice. There were no statistical differences between the 2 groups in any of the values. (B–D) Images and quantification of lesion area and percent necrotic area (n=10 and n=9), percent of TUNEL-positive cells (n=10 and n=7), percent activated caspase-3 positive cells (n=8 and n=9) in the lesions of WT → Ldlr^−/− and Tlr2^−/−Tlr4^−/− → Ldlr^−/− mice respectively. Panel B shows hematoxylin and eosin staining of the sections with the necrotic area outlined (dotted black lines), and panel C shows TUNEL (red) and DAPI (blue) staining. The P values shown in panels B–D were derived from the Mann-Whitney test. Bars in B and C, 20 and 15 μm, respectively.