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. 2019 Jul 1;10(7):507.
doi: 10.1038/s41419-019-1736-5.

The P-selectin and PSGL-1 axis accelerates atherosclerosis via activation of dendritic cells by the TLR4 signaling pathway

Zhishuai Ye  1   2 , Lei Zhong  2 , Shengnan Zhu  2 , Yinuo Wang  2 , Jie Zheng  2 , Shujing Wang  3 , Jianing Zhang  4 , Rongchong Huang  5   6
Affiliations

The P-selectin and PSGL-1 axis accelerates atherosclerosis via activation of dendritic cells by the TLR4 signaling pathway

Zhishuai Ye et al. Cell Death Dis. .

Abstract

P-selectin and dendritic cells (DCs) are associated with atherosclerosis. However, their interactions in this setting are undefined. Herein, we investigated the role of P-selectin and its receptor P-selectin glycoprotein ligand (PSGL)-1 on atherosclerosis via activation of DCs. In the current study, a total of 34 patients with ST elevation myocardial infarction (STEMI) and 34 healthy control subjects were enrolled. Serum concentration of P-selectin was higher and the myeloid DC/plasmacytoid DC (mDC/pDC) ratio was lower in STEMI patients than in normal individuals. Interestingly, in STEMI patients, P-selectin was decreased and the mDC/pDC ratio was increased at 5-7 days after successful percutaneous coronary intervention, as compared with values on admission. Serum P-selectin was inversely correlated with the mDC/pDC ratio. Moreover, ApoE-/-P-/- and ApoE-/-PSGL-1-/- mice developed small atherosclerotic plaques after feeding of a western diet for 12 weeks and DC infiltration was significantly reduced. P-selectin stimulation markedly induced phenotypic maturation, enhanced secretion of inflammatory cytokines, communication with T cells, and the adhesion and migration of DCs. In vivo, DC maturation was significantly attenuated in P-selectin and PSGL1 knockout mice under hypercholesterolemic and inflammatory conditions. These effects were associated with the activation of myeloid differentiation primary response 88 (MYD88)-dependent and MyD88-independent Toll-like receptor 4 (TLR4) signaling pathways. Taken together, binding of P-selectin to PSGL-1 on DCs contributes to atherosclerosis progression via DC activation via the TLR4 signaling pathway.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Serum concentration of P-selectin was increased and the mDC/pDC ratio was decreased in STEMI patients.
Detection of peripheral blood dendritic cells (DCs) by standardized 3-color flow cytometry. Peripheral blood samples were collected and the cells were stained with human phycoerythrin (PE) anti-CD11c, PE anti-CD123, peridinin chlorophyll protein anti-HLA-DR, and fluorescent isothiocyanate (FITC) lineage cocktail 1 (lin1). (Aa): Events excluding debris and dead cells (P1); (Ab): cells were gated on region P1, a dot blot of lineage marker (x-axis) vs. HLA-DR (y-axis) was used to define the region of Lin−cells (P2); (Ac) pDC was defined as Lin1−HLA−DR+CD123+ (P4); (Ad) mDC was defined as Lin1−HLA-DR+ CD11c+ (P5). Flow cytometric analysis of the percentage and absolute numbers of mDCs (Ba and Bb), percentages and numbers of pDCs (Bc), and the mDC/pDC ratio (Bd) in control subjects (n = 34) and STEMI patients (n = 34) on admission. ELISA analysis of circulating P-selectin (Ca) and flow cytometric analysis of the percentage and absolute numbers of mDCs (Cb and Cc), and the mDC/pDC ratio (Cd) of STEMI patients on admission and at 5–7 days after PCI (n = 34). The circulating mDC/pDC ratio was inversely correlated with serum P-selectin levels in STEMI patients on admission (Da), 5–7 days after successful PCI (Db), and in total (Dc), while no such correlation was found in controls (Dd). mDC: myeloid dendritic cell; pDC plasmacytoid dendritic cell
Fig. 2
Fig. 2. Effect of P-selectin-PSGL on the maturation, communication with T cells, adhesion, and migration of DCs.
DCs without transfection or transfected with negative control siRNA or GALNT4 siRNA were treated with bovine serum albumin as a control, LPS (20 ng/mL), P-selectin (100 ng/mL), or P-selectin plus KPL1 (5 μg/mL) for 24 h, respectively. a, b The maturation surface molecular markers of DCs were analyzed by flow cytometry and histograms were created to illustrate the expression levels of CD80, CD86, and MHC- II. c, d The MLR assay was performed using CD4 + T cells as responder cells, which were co-cultured with the indicated DCs and proliferation was determined after 3 days using the Cell Counting Kit-8. e, f Representative pictures of the adhesion of fluorescently labeled DCs on HUVEC monolayers. Scale bar: 100 μm. g, h Representative pictures of the migration of DCs. Scale bar: 50 μm. Data are expressed as the mean ± SD (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 3
Fig. 3. P-selectin-PSGL-1 deficiency suppresses DC maturation under hypercholesterolemic and inflammatory conditions in vivo.
ApoE−/−, ApoE−/−P−/−, and ApoE−/−PSGL-1−/− mice were fed a western diet for 12 weeks. a Representative flow cytometric dot plots showing the percentage of CD11c+ DCs expressing the DC maturation marker CD86 or MHC- II in spleen tissue. b ELISA analysis of the indicated cytokine levels in serum. WT and PSGL1−/− mice were subcutaneously infused with P-selectin, NS, or LPS for 1 day. c, e Representative flow cytometric dot plots showed the percentage of CD11c+ DCs expressing the DC maturation marker CD86 or MHC-II in spleen tissue. d, f qRT-PCR analysis of the indicated cytokine mRNA levels in spleen tissue. Data are expressed as the mean ± SD (n ≥ 5). *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 4
Fig. 4. Knockout of P-selectin or PSGL1 attenuated the development of atherosclerosis and DC accumulation.
ApoE−/−, ApoE−/−P−/−, and ApoE−/−PSGL-1−/− mice were fed a western diet for 12 weeks. a Representative images of Oil red O-stained aortae after en face preparation and quantification of lipid content (lipid content/vascular area) in three different groups. b Representative histological analysis results of the aortic sinus stained with H&E, Movat, and Masson’s trichrome stain, and quantification of the plaque area (plaque area/lumen area), necrotic area (necrotic area/ plaque area), and collagen content (collagen content/plaque area) in aortic sinus. Scale bar: 200 μm. c Representative immunofluorescence staining of the aortic sinus of lesions with Alexa Fluor® 647-conjugated anti-CD11c Ab. Scale bars: 200 and 50 μm, respectively. Data are expressed as the mean ± SD (n = 8). *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 5
Fig. 5. P-selectin induced DC activation through MyD88-dependent TLR4 signaling.
DCs were treated as described in the legend of Fig. 2. a, b Representative western blots showing expression levels of total and phospho-IKKα/β, total and phospho- IkBα, total phospho-NF-κB p65, total and phospho-P38, total and phospho-JNK, and total and phospho-IRAK4. The intensities of the protein bands were quantified and presented as the ratio of phosphorylated protein/total protein vs. controls after normalization to GAPDH (n = 3). c, d ELISA analysis of the TNF-α and IL-6 levels in the supernatants of DC culture medium (n = 3). e Immunohistochemical analysis of MyD88, phospho-IRAK4, and phospho-NF-κB p65 in atherosclerotic lesions of mice (n = 8). Scale bar: 200 and 50 μm, respectively. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 6
Fig. 6. P-selectin induced DC activation through TRIF-dependent TLR4 signaling.
DCs were treated as described in the legend of Fig. 2. a, b Representative western blots showing the expression levels of TRIF, as well as total and phospho-IRF3. The intensities of the protein bands were quantified and presented as the ratio of phosphorylated protein/total protein vs. controls after normalization to GAPDH (n = 3). c, d ELISA analysis of IFN-β levels in the supernatants of DC culture medium (n = 3). e Immunohistochemical analysis of TRIF and phospho-IRF3 (n = 8). Scale bars: 200 and 50 μm, respectively. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
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