Fig 1.
Immunofluorescence staining for TREM-1, TREM-2, and HLA-DR and co-localization of TREMs with HLA-DR+ cells in carotid plaques.
H&E staining shows the histology of carotid plaques in AS (panels A, K) and S (panels F, P) patients. Immunofluorescence staining for TREM-1 (panels B, G), TREM-2 (panels L, Q), HLA-DR (panels C, H, M, R), DAPI (panels D, I, N, S) was performed and merged for examining co-localization of TREM-1 and HLA-DR (panels E, J) and for TREM-2 and HLA-DR (panels O, T). Arrows shows the co-localization of TREM-1 and TREM-2 with HLA-DR. These are the representative images of 3 randomly selected samples from independent patients in each experimental group. Mean fluorescence intensity of TREM-1 (panel U), TREM-2 (panel W) and HLA-DR (panel Y) and number of positive cells for TREM-1 (panel V), TREM-2 (panel X) and HLA-DR (panel Z). Data are shown as mean ± SD (N = 3); *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.
Fig 2.
Immunofluorescence staining for Fascin and TREM-2, and co-localization of TREMs with Fascin+ cells in carotid plaques.
Immunofluorescence staining for TREM-1 (panels B, F), TREM-2 (panels J, N), fascin (panels A, E, I, M), DAPI (panels C, G, K, O) was performed and merged for examining the co-localization of TREM-1 and fascin (panels D, H) and for TREM-2 and fascin (panels L, P). Arrows shows the co-localization of TREM-1 and TREM-2 with fascin. These are the representative images of 3 randomly selected samples in each experimental group. Mean fluorescence intensity for fascin (panel Q) and number of positive cells for fascin (panel R). Data are shown as mean ± SD (N = 3); *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.
Fig 3.
Immunofluorescence staining for α-SMA+ cells and co-localization of α-SMA+ cells with TREM-1 and TREM-2 in carotid plaques.
Immunofluorescence staining for TREM-1 (panels B, F), TREM-2 (panels J, N), α-SMA (panels A, E, I, M), DAPI (panels C, G, K, O) was performed and merged for examining the co-localization of TREM-1 and α-SMA (panels D, H) and for TREM-2 and α-SMA (panels L, P). Arrows show the co-localization of TREM-1 and TREM-2 with α-SMA. These are the representative images of 3 randomly selected samples in each experimental group. Mean fluorescence intensity for α-SMA (panel Q) and number of positive cells for fascin (panel R). Data are shown as mean ± SD (N = 3); *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.
Fig 4.
TREM-1 and TREM-2 mRNA expression analysis by RT-PCR.
The cDNA prepared from mRNA was subjected to RT-PCR with gene specific primers (A: TREM-1; B: TREM-2) and the fold-change expression relative to GAPDH as housekeeping gene are shown. Results are expressed as fold-change in S compared to AS. Data are shown as mean ± SD (N = 6); *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.
Fig 5.
Dynamics of myeloid and plasmacytoid dendritic cells in S and AS carotid plaques.
Cell characterization was performed using anti-human antibodies for mDC1 (CD1c+), mCD2 (CD141+) and pDC (CD303+). (A) Cells only are without antibody (background) and cells labeled with CD1C antibody for mDC1. After subtracting background, mDC1 cells were 3.5%; (B) CD141+ cells (mDC2) were 8.78% and CD303+ cells (pDCs) were 1.41%; and (C) Cells only are without antibody (background) and cells labeled with CD1C antibody for mDC1. After subtracting background, (D) mDC1 cells were 0.73% and CD141+ mDC2 were 1.58%, and (E) CD303+ cells (pDCs) were 10.7%. The data represent 4 plaques from independent patients in each experimental group.
Table 1.
Dynamics of dendritic cell phenotype in atherosclerotic carotid plaques of asymptomatic and symptomatic carotid stenosis patient.