Fig 1: DCD is beneficial to AIS recovery and DCD-containing BM-MSC-derived migrasome effectively promotes phagocytosis of macrophages. A–G) Peripheral blood of AIS patients (acute phase, 0–3d after disease onset, n = 16) and healthy controls (HC, n = 8) were collected. (A) Plasma DCD concentration was assessed with ELISA. * p < 0.05, compared with HC by Student's t-test (mean ± standard deviation). (B) Correlation of clinic parameters and plasma DCD concentration was assessed with Spearman correlation analysis and Point-biserial correlations. * p < 0.05. DM, diabetes mellitus, CHD, coronary heart disease. (C) Representative images of the magnetic resonance diffusion weighted imaging (MR-DWI) of AIS patients with low plasma DCD concentration (DCD = 3.33 ng ml-1) or high plasma DCD concentration (DCD > 3.33 ng ml-1). (D) Association between plasma DCD concentration with infarct scale was estimated with Spearman correlation analysis. (E) Association between plasma DCD concentration with delta NIHSS (NIHSS at 7d minus NIHSS at 1d) was estimated with Spearman correlation analysis. (F) Representative images of the chest Computed Tomography (CT) of AIS patients with low plasma DCD concentration (DCD = 3.39 ng ml-1, median of the cohort) or high plasma DCD concentration (DCD > 3.39 ng ml-1, median of the cohort). (G) Pie charts showing the occurrence of post-stroke pneumonia in AIS patients with low and high plasma DCD concentrations. H) DCD (1 ng ml-1), PBS-migrasomes (PBS-M, 50 µg ml-1) or E. Coli-migrasomes (E. Coli-M, 50 µg ml-1) labeled with Dil (red) were treated to BMDM (15 min). Immunostaining of WGA (green) and DCD (withe) in migrasome-treated BMDM was performed. Experiments were repeated for three times. I,J) BMDM were first pre-stimulated with DCD (1 ng ml-1), PBS-M (50 µg ml-1), or E. Coli-M (50 µg ml-1) for overnight then treated with E. Coli (E. Coli : BMDM = 20:1, 1 h). Phagocytic efficiency of BMDM to GFP expressing E. Coli was assessed with flow cytometry (I) and immunostaining (J). Experiments were repeated three times. ** p < 0.01, compared with PBS-treated group by one-way ANOVA (mean ± standard deviation).
Fig 2: BM-MSC-derived migrasome contains dermcidin (DCD) and improves bacterial clearance of macrophages. A) PBS-migrasomes were isolated and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis. Protein coverage and best peptide-spectrum (PSM) of PBS-M were displayed. B,C) BM-MSC were stimulated with E. Coli (E. Coli : MSC = 20:1, 6 h). (B) Immunostaining was used to evaluate the expression and localization of dermcidin (DCD, green) in BM-MSC. Cell membrane was labeled with WGA (red) and migrasomes were labeled with TSPAN4 (white). (C) DCD level in BM-MSC or migrasomes derived from PBS- or E. Coli-treated BM-MSC was assessed with ELISA. Experiments were repeated three times. *** p < 0.001, by one-way ANOVA (mean ± standard deviation). D) The mRNA level of DCD in BM-MSC was assessed with QPCR at 0–24 h after E. Coli stimulation (E. Coli : MSC = 20:1). ** p < 0.01, *** p < 0.001, compared with 0 h group by one-way ANOVA (mean ± standard deviation). E–H) BMDM were first pre-treated with DCD (0–1 ng ml-1, overnight), and treated with E. Coli (E. Coli: BMDM = 20:1, 1 h). Phagocytic efficiency of BMDM to E. Coli-GFP was assessed with flow cytometry (E) and immunostaining (F). The experiment was repeated three times. ** p < 0.01, compared with DCD (0 ng ml-1) group by one-way ANOVA (mean ± standard deviation). (G) Expression of LAP mediators was analyzed by western blot. Experiments were repeated three times. (H) Immunofluorescence staining showed the localization of LAP mediators in BMDM. I,J) DCD knock-down (DCDKD) BM-MSC was constructed by transfection of Lentivirus carrying DCD-shRNA. DCDKD or vector-transfected BM-MSC were stimulated with E. Coli. Migrasomes derived from BM-MSC were isolated and treated to BMDM (50 µg ml-1 overnight). Phagocytic efficiency of BMDM to GFP expressing E. Coli (E. Coli : BMDM = 20:1, 1 h) was assessed with flow cytometry (I) and immunostaining (J). Experimental process was schematically displayed in Figure S7G (Supporting Information). Experiments were repeated three times. # p < 0.05, ## p < 0.01, compared with BMDM treated with vector-transfected E. Coli-stimulated BM-MSC derived migrasomes by one-way ANOVA (mean ± standard deviation), * p < 0.05, ** p < 0.01, *** p < 0.001, compared with PBS-treated BMDM group by one-way ANOVA (mean ± standard deviation).
Supplier Page from Wuhan Fine Biotech Co., Ltd. for Human PIF/DCD(Proteolysis Inducing Factor/Dermcidin) ELISA Kit