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. 2012 Apr 6;287(15):11629-41.
doi: 10.1074/jbc.M111.338673. Epub 2012 Feb 22.

Endoplasmic Reticulum Stress Controls M2 Macrophage Differentiation and Foam Cell Formation

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Free PMC article

Endoplasmic Reticulum Stress Controls M2 Macrophage Differentiation and Foam Cell Formation

Jisu Oh et al. J Biol Chem. .
Free PMC article

Abstract

Macrophages are essential in atherosclerosis progression, but regulation of the M1 versus M2 phenotype and their role in cholesterol deposition are unclear. We demonstrate that endoplasmic reticulum (ER) stress is a key regulator of macrophage differentiation and cholesterol deposition. Macrophages from diabetic patients were classically or alternatively stimulated and then exposed to oxidized LDL. Alternative stimulation into M2 macrophages lead to increased foam cell formation by inducing scavenger receptor CD36 and SR-A1 expression. ER stress induced by alternative stimulation was necessary to generate the M2 phenotype through JNK activation and increased PPARγ expression. The absence of CD36 or SR-A1 signaling independently of modified cholesterol uptake decreased ER stress and prevented the M2 differentiation typically induced by alternative stimulation. Moreover, suppression of ER stress shifted differentiated M2 macrophages toward an M1 phenotype and subsequently suppressed foam cell formation by increasing HDL- and apoA-1-induced cholesterol efflux indicating suppression of macrophage ER stress as a potential therapy for atherosclerosis.

Figures

FIGURE 1.
FIGURE 1.
M2 macrophage differentiation induces foam cell formation. For A–C and E–H, macrophages from diabetic patients were differentiated with IFNγ to promote M1 differentiation or with IL-4, IL-10, or IC to promote M2 differentiation and then stimulated with oxLDL. A, staining with Oil Red O. Top panel, media, IFNγ-treated cells; Bottom panel, IL-4, IL-10, and IC-treated cells. Arrowheads indicate foam cells. Shown are total cholesterol (B) and cholesteryl ester (C) formation in macrophages incubated with media (gray), IFNγ (white), IL-4 (back-slashed), IL-10 (hatched), and IC (front-slashed) (n = 8 per group). *, p < 0.0001 versus media; #, p < 0.001 versus IFNγ by ANOVA. D, immunofluorescent staining of antibodies to M1 or M2 macrophages in the aortic sinus atherosclerotic lesions from ApoE−/− mice after 8 weeks on Western diet (n = 5 per group). Top panel, arrows indicate stain for M1 marker CCR7 (green), ARDP (red), and co-staining for ARDP and CCR7 (yellow). Bottom panel, arrows indicate stain for M2 marker MR (green), ARDP (red), and costaining for ARDP and MR (yellow). Scale bars, 100 μm. E, macrophage cholesterol uptake assessed by confocal microscopy. Red represents labeled cholesterol uptake after 6 h of stimulation with 1,1′-dioctadecyl-3,3, 3′3′-tetramethylindocarbocyanine perchlorate oxLDL; blue represents nuclear counterstain. Shown are quantification of macrophage cholesterol uptake (F) and cholesterol binding (G) by mean fluorescence absorbance after DiI-oxLDL stimulation (n = 8 per group). *, p < 0.0001 versus media; #, p < 0.01 versus IFNγ by ANOVA for both. H, Western blot for CD36 and SR-A1 expression. IF, immunofluorescence.
FIGURE 2.
FIGURE 2.
M2 macrophage differentiation and cholesterol uptake are ER stress-dependent in diabetics. For A–C, macrophages from diabetic patients were differentiated with INFγ to promote M1 differentiation or with IL-4, IL-10, or IC to promote M2 differentiation and co-incubated with or without PBA (ER stress inhibitor). A, ER stress protein activation/expression. B, flow cytometry quantification of membrane receptors. M1 receptors, CCR7 (black), CD86 (gray); M2 receptors, CD163 (grid), MR (back-slashed) (n = 9 per group). t and tt, p < 0.001 versus same receptor in non-PBA-treated. C, quantification of cholesterol uptake after differentiation with INFγ (white), IL-4 (back-slashed), IL-10 (hatched), or IC (front-slashed) and stimulation with oxLDL (n = 9 per group). *, p < 0.01 versus same stimulation in non-PBA-treated. For D–F, cultured macrophages from diabetic patients were treated with or without ER stress inducer thapsigargin. D, ER stress protein activation/expression. E, flow cytometry quantification of membrane receptors (n = 8 per group). *, p < 0.04; **, p < 0.02; t and tt, p < 0.01 versus same receptor in non-thapsigargin-treated. F, quantification of cholesterol uptake after stimulation with oxLDL with (black) or without (white) thapsigargin (n = 6 per group). *, p < 0.02 versus non-thapsigargin-treated. For G and H, peritoneal macrophages from CHOP−/− or WT mice were differentiated to M1 or M2 macrophages as described above for A–C. G, flow cytometry quantification of membrane receptors (n = 8 per group). * and **, p < 0.04; t and tt, p < 0.01 versus same receptor in WT. H, quantification of cholesterol uptake after stimulation with oxLDL (n = 8 per group). *, p < 0.04 versus same stimulation in WT.
FIGURE 3.
FIGURE 3.
Effects of ER stress on M2 macrophage differentiation and cholesterol uptake are JNK- and PPARγ-dependent. Cultured macrophages from diabetic patients were differentiated with INFγ to promote M1 differentiation or with IL-4, IL-10, or IC to promote M2 differentiation and co-incubated with or without PBA (ER stress inhibitor). A, p-JNK and PPARγ expression. For B–F, peritoneal macrophages from JNK2−/−, LysM-Cre PPARγ−/−, and WT mice were differentiated with IFNγ to promote M1 or IL-4, IL-10, or IC to promote M2 macrophages (n = 8 per group). B, ER stress protein activation/expression. C, flow cytometry quantification of membrane receptors from JNK2−/− macrophages. M1 receptors, CCR7 (black), CD86 (gray); M2 receptors, CD163 (grid), MR (back-slashed). * and **, p < 0.001; t, p < 0.02; tt, p < 0.001 versus same receptor in WT. D, quantification of cholesterol uptake after differentiation with IFNγ (white), IL-4 (back-slashed), IL-10 (hatched), or IC (front-slashed) and stimulation with oxLDL. *, p < 0.04 versus same stimulation in WT. E, flow cytometry quantification of membrane receptors from LyzM-Cre PPARγ−/− macrophages. * and **, p < 0.01; t and tt, p < 0.03 versus same receptor in WT. F, quantification of cholesterol uptake after stimulation with oxLDL. *, p < 0.01 versus same stimulation in WT.
FIGURE 4.
FIGURE 4.
Scavenger receptor signaling induces ER stress and regulates M2 macrophage differentiation. Peritoneal macrophages from CD36−/−, SR-A1−/−, and WT mice were cultured with IFNγ to promote M1 differentiation or with IL-4, IL-10, or IC to promote M2 differentiation and stimulated with modified cholesterol (n = 6 per group). A, ER stress protein activation/expression. Flow cytometry quantification of macrophage membrane receptors CCR7 (black), CD86 (gray), CD163 (grid), MR (back-slashed) after differentiation with IFNγ (B), IL-4 (C), IL-10 (D), or IC (E) and stimulation with oxLDL, AcLDL, or neither. t and tt, p < 0.001 versus same receptor/LDL stimulation by ANOVA. F, flow cytometry quantification of membrane receptors from peritoneal macrophages treated with or without ER stress inducer thapsigargin from CD36−/−, SR-A1−/−, and WT mice (n = 6 per group). t and tt, p < 0.01 versus same receptor in thapsigargin-treated.
FIGURE 5.
FIGURE 5.
Suppression of ER stress and JNK reverses M2 macrophage differentiation and cholesterol deposition. For A–E, macrophages from diabetic patients were differentiated into M1 or M2 and then incubated with or without PBA (ER stress inhibitor). A, flow cytometry quantification of membrane receptors. M1 receptors, CCR7 (black), CD86 (gray); M2 receptors, CD163 (grid), MR (back-slashed) (n = 8 per group). t, p < 0.02; tt, p < 0.01 versus same receptor in non-PBA-treated. B, quantification of cholesterol uptake after IFNγ (white), IL-4 (back-slashed), IL-10 (hatched), or IC (front-slashed) and stimulation with oxLDL (n = 12 per group). *, p < 0.02 versus same stimulation in non-PBA-treated. C, cholesteryl ester formation (n = 6 per group). *, p < 0.04 versus same stimulation by ANOVA. Shown are apoA-I-induced (D) and HDL-induced (E) cholesterol efflux (n = 6 per group). *, p < 0.05 versus same stimulation by ANOVA. For F and G, macrophages from diabetic patients were differentiated into M1 or M2 and then incubated with or without SP600125 (JNK inhibitor). F, flow cytometry quantification of membrane receptors (n = 6 per group). t, p < 0.05; tt, p < 0.04 versus same receptor in non-SP600125-treated. G, quantification of cholesterol uptake after stimulation with oxLDL (n = 4 per group). *, p < 0.02 versus same stimulation in non-SP600125-treated.
FIGURE 6.
FIGURE 6.
Influence of ER stress pathways in macrophage differentiation. ER stress pathways include the following: c-Jun N-terminal kinase (JNK), PPARγ, scavenger receptor CD36, and SR-A1. Macrophage M1 receptors include CCR7 and CD86; M2 receptors include MR and CD163.

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