Macrophage-derived myeloid differentiation protein 2 plays an essential role in ox-LDL-induced inflammation and atherosclerosis

Abstract

Background: Atherosclerosis is a chronic inflammatory disease. Although Toll-like receptor 4 (TLR4) has been involved in inflammatory atherosclerosis, the exact mechanisms by which oxidized-low-density lipoproteins (ox-LDL) activates TLR4 and elicits inflammatory genesis are not fully known. Myeloid differentiation factor 2 (MD2) is an extracellular molecule indispensable for lipopolysaccharide recognition of TLR4.

Method: Apoe^-/-Md2^-/- mice and pharmacological inhibitor of MD2 were used in this study. We also reconstituted Apoe^-/- mice with either Apoe^-/- or Apoe^-/-Md2^-/- marrow-derived cells. Mechanistic studies were performed in primary macrophages, HEK-293T cells, and cell-free system.

Finding: MD2 levels are elevated in atherosclerotic lesion macrophages, and MD2 deficiency or pharmacological inhibition in mice reduces the inflammation and stunts the development of atherosclerotic lesions in Apoe^-/- mice fed with high-fat diet. Transfer of marrow-derived cells from Apoe-Md2 double knockout mice to Apoe knockout mice confirmed the critical role of bone marrow-derived MD2 in inflammatory factor induction and atherosclerosis development. Mechanistically, we show that MD2 does not alter ox-LDL uptake by macrophages but is required for TLR4 activation and inflammation via directly binding to ox-LDL, which triggers MD2/TLR4 complex formation and TLR4-MyD88-NFκB pro-inflammatory cascade.

Interpretation: We provide a mechanistic basis of ox-LDL-induced macrophage inflammation, illustrate the role of macrophage-derived MD2 in atherosclerosis, and support the therapeutic potential of MD2 targeting in atherosclerosis-driven cardiovascular diseases.

Funding: This work was supported by the National Key Research Project of China (2017YFA0506000), National Natural Science Foundation of China (21961142009, 81930108, 81670244, and 81700402), and Natural Science Foundation of Zhejiang Province (LY19H020004).

Keywords: Atherosclerosis; Inflammation; Macrophages; Myeloid differentiation-2; Oxidized-LDL; Toll-like receptor 4.

Fig. 1

MD2 is elevated in atherosclerotic lesion macrophages. (a) MD2 protein levels in aortas of Apoe^−/− mice fed a high-fat diet (HFD) or normal/low-fat diet (LFD) were detected by western blotting. β-actin was used as loading control. Representative immunoblots were shown. (b) mRNA levels of Md2 in aortic sinus of Apoe^−/- mice fed with LFD and HFD [n = 9]. (c, d) Serum levels of soluble MD2 protein (c) and tumor necrosis factor-α (TNF-α; d) in Apoe^−/− mice fed with LFD and HFD [n = 7]. (e, f) Representative immunofluorescence staining of MD2 (red, e and f), macrophage marker CD68 (green, e), and smooth muscle cell marker α-SMA (green, f). Tissues were counterstained with DAPI (blue). White arrows indicate co-location of MD2 and CD68 (e) or α-SMA (f) staining [scale bar = 50 μm]. (g) Western blot analysis of MD2 protein levels in human peripheral blood mononuclear cells (hPBMCs) isolated from patients with atherosclerosis (AS) and without AS (normal peoples, NP). (h) mRNA levels of Md2 in hPBMCs. (i, j) Serum levels of MD2 protein (i) and tumor necrosis factor-α (TNF-α; j) in hPBMCs isolated from AS [n = 40] and NP [n = 15]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

MD2 deficiency reduces atherosclerosis in HFD-fed ApoE^−/− mice. (a) En face Oil Red O staining of aortas from Apoe^−/− and Apoe^−/−Md2^−/− mice fed a HFD for 16 weeks. Oil Red O staining highlighting neutral lipids (red). Lower panel showing quantification of plaque lesion area from Oil Red O staining. Plaque area was defined as percentage of total surface area of the aorta [n = 6]. (b) Oil Red O staining of aortic sinus. Lower panel showing quantification of lesion area highlighted by Oil Red O staining [n = 6; scale bar = 500 μm]. (c) Representative images of α-SMA (red) staining of aortic sinus. Lower panel showing quantification of α-SMA staining area [n = 6; scale bar = 50 μm]. (d) Representative images of Masson's Trichome staining for collagen deposition. Lower panel showing quantification of fibrotic area [n = 6; scale bar = 50 μm]. (e) Serum levels of pro-inflammatory cytokines TNF-α and IL-6 in mice fed a HFD [n = 10]. (f) mRNA analysis of proinflammatory cytokines (Il-1β, Tnf-α, Il-6) and adhesion molecules (Icam-1, Vcam-1) in aortic sinus [n = 6]. (g) Representative immunofluorescence staining images for CD68 (green) in aortic sinus. Tissues were counterstained with DAPI (blue) [scale bar = 50 μm]. (h) Quantification of CD68-positive area in aortic sinus slices [n = 6]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Bone marrow-derived MD2 play a critical role in the development of atherosclerotic lesionsApoe^−/− mice were irradiated and received bone marrow cells from either Apoe^−/− mice or Apoe^−/−Md2^−/− mice. KO→KO: marrow-derived cells from Apoe^−/- (KO) mice were transplanted in irradiated Apoe^−/− (KO) mice; DKO→KO: marrow-derived cells from Apoe^−/−Md2^−/- (DKO) mice were transplanted in irradiated Apoe^−/− (KO) mice. Mice were then fed a HFD for 16 weeks. (a) Oil Red O staining of aortas from mice transplanted with KO or DKO marrow cells. Lower panel showing quantification of plaque lesion area. [n = 6]. (b) Oil Red O staining of aortic sinus. Lower panel showing quantification of lesion area [n = 6; scale bar = 500 μm]. (c) Representative images of α-SMA (red) staining of aortic sinus. Lower panel showing quantification of α-SMA staining area [n = 6; scale bar = 50 μm]. (d) Representative images of Masson's Trichome staining of aortic sinus. Lower panel showing quantification of fibrotic area [n = 6; scale bar = 50 μm]. (e) Serum levels of TNF-α and IL-6 in mice (n = 8). (f) mRNA levels of inflammatory cytokines (Il-1β, Tnf-α, Il-6) and adhesion molecules (Icam-1, Vcam-1) in aortic sinus [n = 6]. (g) Representative immunofluorescence staining images for CD68 (green) in aortic sinus. Tissues were counterstained with DAPI (blue) [scale bar = 50 μm]. (h) Quantification of CD68-positive area in aortic sinus slices [n = 6]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

MD2 mediates ox-LDL-induced proinflammatory cytokine production and NF-κB activation (a) Primary macrophages isolated from Md2^−/− (KO) and wildtype (WT) mice were challenged with 50 μg/mL ox-LDL for 24 h. Levels of TNF-α and IL-6 cytokines in culture media were measured by ELISA and reported as pg/μg protein [n = 6]. (b) mRNA levels of Tnf-α and Il-6 in macrophages isolated from KO and WT mice. Cells were exposed to 50 μg/mL ox-LDL for 6 h [n = 4]. (c) Levels of IκB in the primary macrophages exposed to 50 μg/mL ox-LDL for 30 min. GAPDH was used as loading control. Lower panel showing densitometric quantification [n = 4]. (d) Immunoblot detection of NF-κB p65 subunit in cytosolic and nuclear fractions prepared from cells exposed to 50 μg/mL ox-LDL for 30 min. Lamin B and GAPDH were used as loading control for nuclear and cytosolic proteins, respectively. Lower panel showing densitometric quantification [n = 4]. (e) Immunofluorescence staining for NF-κB p65 subunit (red) in primary macrophages exposed to ox-LDL for 1 h. Cells were counterstained with DAPI (blue) [scale bar = 50 µm]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

MD2 is essential for ox-LDL-induced TLR4 dimerization and activation via direct interaction with ox-LDL (a) Macrophages from wildtype mice were incubated with 50 µg/mL ox-LDL for indicated time periods. TLR4 was immunoprecipitated (IP) and MD2 was detected by immunoblotting (IB). Lower panel showing the densitometric quantification of MD2-TLR4 association [n = 4]. (b) Interaction between MD2 and TLR4 was confirmed by co-immunoprecipitation assay in HEK-293T cells transfected with MD2-His and TLR4-Flag expressing plasmids. Lower panel showing the densitometric quantification [n = 4]. (c) Dimerization of TLR4 was assessed by co-immunoprecipitation assay in HEK-293T cells transfected with Flag- and HA-tagged TLR4 and His-tagged MD2 plasmids. Cells were exposed to 50 µg/mL ox-LDL for 30 min. Samples were immunoprecipitation (IP) with anti-HA followed by immunoblotting (IB) with anti-Flag. Lower panel showing the densitometric quantification. [n = 4]. (d) Primary macrophages from wildtype (WT) mice and MD2^−/− mice (KO) were incubated with 50 µg/mL ox-LDL for 30 min. TLR4 was immunoprecipitated (IP) and MyD88 was detected by immunoblotting (IB). Lower panel showing the densitometric quantification [n = 4]. (e) Interaction between MyD88 and TLR4 was detected by co-immunoprecipitation assay in HEK-293T cells transfected with TLR4-HA, MyD88-Flag, and MD2-His expressing plasmids. Cells were treated with or without 50 µg/mL ox-LDL for 30 min. Lower panel showing the densitometric quantification [n = 3]. (f) Macrophages isolated from wildtype mice were exposed to 50 μg/mL ox-LDL or LDL for 30 min. Interaction between ApoB100, MD2, and TLR4 was assessed by co-immunoprecipitation. Right panel showing the densitometric quantification [n = 3]. (g) Interaction between ApoB100, MD2, and TLR4 was detected by co-immunoprecipitation in HEK-293T cells transfected with TLR4-HA and MD2-His expressing plasmids. Cells were treated with or without 50 µg/mL ox-LDL for 30 min. Right panel showing the densitometric quantification [n = 4]. (h) Primary macrophages were treated with 50 µg/mL ox-LDL or LDL, and then were stained for MD2 (green) and ApoB100 (red). DAPI (blue) was used to counterstain. Lower panels show higher magnification. (i) Macrophages isolated from wildtype (WT) and Md2^−/− (Md2KO) mice were incubated with 50 µg/mL ox-LDL for 30 min. Interaction between TLR4 and ApoB100 was assessed by co-immunoprecipitation. Lower panel showing the densitometric quantification [n = 3] (j) Macrophages isolated from WT and Tlr4^−/− (Tlr4KO) mice were incubated with 50 µg/mL ox-LDL for 30 min. Interaction between MD2 and ApoB100 was assessed by co-immunoprecipitation. Lower panel showing the densitometric quantification [n = 3]. (k) The interaction between MD2 and ox-LDL was determined using a cell-free assay. rhMD2 was immobilized and DiI-labeled ox-LDL was added. Binding was determined by relative fluorescence intensity (RFI), normalized to blanks without rhMD2 immobilization [n = 4]. (l) Representative immunofluorescence staining of MD2 (red) and ApoB100 (green) in aortic sinus of Apoe^−/− mice maintained on HFD. Tissues were counterstained with DAPI (blue) [scale bar = 50 μm]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

MD2 in macrophages is not required for ox-LDL uptake (a) Primary macrophages isolated from widetype (WT) and Md2^−/− (KO) mice were incubated with 50 µg/mL DiI-labeled ox-LDL for indicated time periods. DiI-ox-LDL uptake was detected as mean fluorescence intensity by flow cytometry [n = 4]. (b) Oil Red O staining of WT and KO macrophages incubated with 50 µg/mL ox-LDL for 24 h. Right panel showing quantification of area highlighted by Oil Red O staining [n = 4]. (c) HEK-293T cells transfected with MD2-His-plasmid alone or co-transfected with MD2-His-and TLR4-HA plasmids were cultured with 50 µg/mL DiI-labeled ox-LDL for 30 min. DiI-ox-LDL uptake was detected by flow cytometry. Flow histograms showing mean fluorescence intensity (MFI) values. (d) The interaction between ApoB100, MD2, and TLR4 was assessed by co-immunoprecipitation in macrophages from wildtype mice. Cells were pretreated with or without LDL-uptake inhibitor Dynasore at 80 µM for 1 h prior to exposure of cells to 50 µg/mL ox-LDL for 30 min. (e) Immunofluorescence staining of macrophages from wildtype mice for MD2 (green) and ApoB100 (red). Cells were treated as indicated in panel D. Cells were counterstained with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Pharmacological inhibition of MD2 by L6H9 reduces the development of atherosclerosis and inflammation in mice. (a) En face Oil Red O staining of aortas. Apoe^−/− mice maintained on HFD were treated with 10 mg/kg L6H9 every other day. Oil Red O staining highlighting neutral lipids (red). Lower panel showing quantification of plaque lesion area [n = 6]. (b) Oil Red O staining of lesion area in aortic sinus. Lower panel showing quantification of lesion area highlighted by Oil Red O staining [n = 6; scale bar = 500 μm]. (c) Representative images of α-SMA staining (red) of aortic sinus. Lower panel showing quantification of α-SMA staining area [n = 6; scale bar = 50 μm]. (d) Representative images of Masson's Trichome staining for collagen deposition. Lower panel showing quantification of fibrotic area [n = 6; scale bar = 50 μm]. (e) Serum levels of pro-inflammatory cytokines TNF-α and IL-6 [n = 6]. (f) Real-time qPCR assay shows the levels of mRNA of proinflammatory cytokines (Il-1β, Tnf-α, Il-6) and adhesion molecules (Icam-1, Vcam-1) in aortas [n = 6]. (g) Representative immunofluorescence staining images for CD68 (green) in aortic sinus. Tissues were counterstained with DAPI (blue) [scale bar = 50 μm]. (h) Quantification of CD68-positive area in aortic sinus slices [n = 4]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Schematic illustration of the underlying mechanism of MD2 in ox-LDL-induced inflammatory responses in atherosclerosis.