Fig 2: Upregulation of CD169 on human monocytes following ex vivo TLR9 agonist stimulation. (A) Levels of IFN-α in supernatants from human PBMCs stimulated with TLR9 agonist (MGN1703 3μM) for 24 hrs. Bar-graph depicts average IFN-α levels obtained from stimulation of five individual donors (+/- SEM). UT=untreated. (B) Flow plot demonstrating monocyte subset gating strategy with representative data. Classical monocytes CD14+/CD16int, Intermediate monocytes CD14+/CD16+ and the non-classical monocytes CD14int/CD16+. (C) Flow plots depicting the impact of MGN1703 on CD169 expression on classical monocytes after 24 hrs of stimulation for a representative donor (D) Time-dependent upregulation of CD169 on monocytes. Human PBMCs were stimulated with TLR9 agonist (MGN1703 at 3 μM) for 4, 8, 16 or 24 hrs. Levels of CD169 on monocyte subsets were measured using flow cytometry. Depicted in the graph is the average (+/- SEM) from five individual donors. Levels of CD169 on each monocyte subset were analyzed by paired ANOVA and Dunnett’s multiple comparison tests were performed for each time-point compared to baseline.

Fig 3: Platelet-monocyte aggregates improved postoperative outcomes of AAD patients by restoring the monocyte immune responseFlow cytometry was performed on whole blood from AAD patients and matched HDs by staining for CD41 and immune cell markers.(A) Flow cytometry analysis showed the percentage of HLA-DR+, CD86+, CD40+, CCR2+, and CX3CR1+ monocytes in AAD patients compared to HDs.(B) The ratio of HLA-DR+ to CD206+ monocytes in AAD patients compared to HDs.(C and D) Correlation analysis of the percentage of HLA-DR+ monocytes and postoperative indicators of the length of ICU stay (C) and the time to tracheal extubation (D).(E) Flow cytometric analysis showing the percentage of MPAs (CD14+CD41+CD42+), platelet-neutrophile aggregates (CD66b+CD41+CD42+), platelet-T cell aggregates (CD3+CD41+CD42+), and platelet-B cell aggregates (CD19+CD41+CD42+) in the circulation.(F) Flow cytometric analysis showing the percentage of MPAs in classical monocytes (CD14brightCD16-, CM), intermediate monocytes (CD14+CD16+, IM), and nonclassical monocytes (CD14+CD16++, NCM).(G and H) The correlation analysis of the percentage of MPAs and postoperative indicators, including length of ICU (G) and hospital (H) stays.(I) Correlation analysis of the percentage of platelet-intermediate monocyte aggregates and the time to tracheal extubation.(J) Flow cytometry analysis comparing the expression of HLA-DR, CD86, CD16, CD40, and CCR2 in monocytes with or without platelet aggregation.(K–M) PBMCs from HDs were cultured with platelets from AAD patients at a 1:100 ratio for 24 h. CD40 expression (K), percentage of CD16+ monocytes (L) and cytokines TNF-α and interleukin-6 (IL-6) expression (M) were measured using flow cytometry.(N) Monocytes from HDs were isolated using magnetic beads and stimulated with PRs from AAD patients for 24 h. Flow cytometric analysis showing the percentage of CD16+ monocytes changes. Data are expressed as the mean ± SD. Two-tailed Student’s t tests (A-B, E, J-N), one-way ANOVA (F), and Spearman correlation analysis (C-D, G-I) were performed. AAD: acute aortic dissection; HD: healthy donors; CM: classical monocytes; IM: intermediate monocytes; NCM: nonclassical monocytes; MPAs: platelet-monocyte aggregates; NT: no treatment; PLT: platelet from AAD patients; PRs: platelet releasates.

Fig 4: Platelets maintain the immune functions in AAD patients in an MMP-9-dependent manner(A and B) Platelet samples from 6 AAD patients and 3 HDs were subjected to RNA-Seq. Volcano plots for differential genes between HDs and AAD patients (A) and comparing AAD patients with high (AAD-PLT-High) and low (AAD-PLT-Low) platelet (B). The red and green dots indicate upregulated and downregulated DEGs, respectively, with p < 0.05.(C) Venn diagram generated to show an overlap of mapped DEGs from different comparison groups, including between HD and AAD patients (Set A) and between high-platelet and low-platelet patients (Set B). Sets A and B share an intersecting set (Set C) of 9 genes, including MMP-9.(D) MMP-9 mRNA in platelets from AAD and HDs subjects was measured by qPCR.(E) The ratio of MMP-9 contents to total protein in platelets. MMP-9 and total protein levels were measured using ELISA and BCA, respectively.(F and G) Representative western blot images (F) and statistical analysis of the gray value (G) showing MMP-9 protein levels in platelets.(H) ELISA of MMP-9 in PRs from AAD patients and HDs.(I) Pure HD monocytes were cultured for 24 h with PRs from AAD patients, followed by MMP-9 inhibition. The percentage of CD16+ monocytes was measured using flow cytometry.(J–L) RNA sequencing was performed on pure HD monocytes cultured with or without an MMP-9i following stimulation of PR from AAD patients.(J) Heatmap depicting differentially expressed transcripts. Red indicates increased relative expression, and blue indicates decreased relative expression.(K) The volcano plot shows differentially expressed genes. The red and green dots indicate upregulated and downregulated DEGs, respectively, with p-value <0.05.(L) The top 15 GO terms of differential gene expression data from biological processes (BPs), molecular function (MF), and cellular component (CC) are listed. Data are expressed as the mean ± SD. Two-tailed Student’s t tests (D, E, G-I) were performed. AAD: acute aortic dissection; HD: healthy donors; IM: intermediate monocytes; MMP-9i: inhibitor of matrix metalloproteinase-9; NT: no treatment; PLT: platelet from AAD patients; PRs: platelet releasates.

Fig 5: Long-term effects of vaccination on monocyte subsets in the blood.Monocytes were isolated from PBMCs from healthy donors on day –1 (prevaccine), day 14, day 56, and day 83 after vaccination (and prior to the second dose) using a hyperosmotic Percoll gradient. (A) Cells were Fc blocked and stained with fluorochrome-conjugated antibodies specific for CD14, CD68, and CD16. Total monocytes were identified as CD14+CD68–, and CD14+CD16+ monocytes were also examined. Numbers in the dot plots indicate the frequencies of cells (%) present inside the gate or quadrant. (B) The median fluorescent intensity of CD14 in the total ex vivo CD14+ population was assessed over time. (C) The absolute number of CD14+ cells was calculated by multiplying the total cell yield from the hyperosmotic Percoll enrichment by the percentage of CD14+ cells. (D) Monocyte frequency was calculated by dividing the total number of CD14+ cells by the total number of PBMCs. (E) The absolute number of CD14+CD16+ cells was calculated by multiplying the total cell yield from the hyperosmotic Percoll enrichment by the percentage of CD14+ CD16+ cells. (F) CD14+CD16+ monocyte frequency was calculated by dividing the total number of CD14+CD16+ cells by the total number of PBMCs. Each dot represents an individual donor (n = 10), with blue dots denoting male donors and pink dots denoting female donors. Data are graphed as the mean value ± SD. Statistically significant differences between the groups were determined by repeated measures 1-way ANOVA using Dunnett’s multiple comparisons test; *P < 0.05, **P < 0.01.