Single-cell sequencing of PBMC characterizes the altered transcriptomic landscape of classical monocytes in BNT162b2-induced myocarditis

Figures

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  Figure 1. Time Series Analysis of Single-Cell RNA Sequencing Data of PBMCs of a Patient with BNT162b2-MyoC. (A) Overview of single-cell RNA-seq analysis of the PBMCs from patient in the stage of myocarditis(day16), early(day21), and late(day93) recovery stage after BNT162b2 administration, respectively. The samples were performed single-cell RNA-seq and integrated to one dataset with Harmony. (B) The dimensional reduction is performed with the uniform manifold approximation and projection (UMAP). Each dot represents a cell in each corresponding group, and is colored according to cell type The cells are pooled across all patients and separated by conditions: Myocarditis after BNT62b2-administration (BNT162b2-MyoC, left), early (middle), and late (left) recovery stages. Total number of cells per group: Myocarditis (n = 10,580cells), Early recovery (n = 11,745cells) and Late recovery(n = 13,065cells). (C) Normalized expression levels of marker genes for each immune cell type on UMAP plots. CD14 (Classical monocytes and Intermediated monocytes), MS4A7 and FCGR3A (Intermediated monocytes and Non-classical Monocytes), CD1C (Dendritic cells (cDCs)), IL7R (T cells), NKG7(Natural killer cells (NK…

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  Figure 2. Overall Transcriptomic Profiles of Classical Monocytes in BNT162b2-MyoC Stage. (A) A Bar plot showing the proportion of each cell types derived from the patient in BTN162B2-MyoC, early recovery and late recovery phase, respectively. (B) Numbers of differentially expressed genes (DEGs) within each cell type in the patient in the stage of BTN162B2-MyoC compared to early recovery (p-value< 0.01, log2 fold-change (log2 FC) ≥ 0.15 or ≤ −0.15). Down: down-regulated; Up; up-regulated. (C) Gene expression ratio (log2 FC) of classical monocytes in the patients in BTN162B2-MyoC state versus in other groups (early and late recovery state) (horizontal axis) plotted against –Log adjusted p-value (vertical axis), showing DEGs: highly expressed (red) and lowly expressed in BNT162b2-MyoC (blue). (p-value < 0.01, log2 FC ≥ 0.15 or ≤ −0.15). (D) Gene set enrichment analysis with ‘Enrichr’ for up- regulated DEGs of classical monocytes in PBMCs from the patient in BNT162b2-MyoC state versus in early and late recovery stages, respectively. MSigDB_Hallmark_2020_terms are classified. P-value (color) and gene count (vertical axis) of the 20 most significant GO terms are shown.

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  Figure 3. JUN, FOS are the Most Down-regulated in Classical Monocytes in BNT162b2-MyoC Stage. (A) A schematic of the experimental pipeline. PBMCs were obtained from the BNT162b2 vaccinated patient in the stage of myocarditis (day16), early recovery(day21), and late recovery(day93), respectively. The samples were performed single-cell RNA-seq and integrated to one dataset with Harmony. Publicly available datasets of PBMCs from BNT162b2-vaccinated individuals day7 and day21 after primary vaccination (GSE171964), PBMCs from healthy individuals and patients with COVID-19(GSE158055), and PBMCs from control group, patients with Kawasaki, and patients with COVID-19-induced myocarditis (CoV2-MyoC) were explored to understand the transcriptomic features of classical monocytes in more depth. (B) Log adjusted p-value (vertical axis) of DEGs of Classical monocytes in BNT162b2-MyoC plotted against the rank of average expression levels (vertical axis). The expression of AP-1(composed with JUN, JUNB, JUND, FOS, and FOSB. (C) UMAP showing expression levels of regulons of JUN and FOS in classical monocytes in BNT162b2-MyoC and in early recovery stage. (D) Volcano plots showing genes involved in regulatory…

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  Figure 4. Fatty Acid Metabolism is Highly Up-regulated in Classical Monocytes in BNT162b2-MyoC Stage. (A) Enrichment analysis with cellular metabolism-related Hallmark gene sets for DEGs of classical monocytes in each dataset. (B) A diagram of fatty-acid metabolism-pathway and violin plots showing the expression levels of the pathway-related genes in classical monocytes. *** p < 0.001; two-tailed t test. (C) GSEA analysis for the dataset of DEGs of classical monocytes in BNT162b2-MyoC group was conduct against GOBP_LIPID_METABOLIC_PROCESS, GOBP_OXIDATIVE_PHOSPHORYLATION, and GOBP_FATTY_ACID_BETA_OXIDATION gene sets, respectively. A positive enrichment score on the y axis indicates positive correlation with BNT162b2-MyoC group.

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  Figure 5. CEBPB is the Major Transcription Factor to Mediate Fatty Acid Metabolism in Classical Monocytes in BNT162b2-MyoC Stage. (A) A clustered heat map showing the area under the curve (AUC) scores of expression regulation by transcription factors (TFs), as estimated using SCENIC. (B) Regulons with top 5 cell-type-specific activity in classical monocytes in early recovery stage compared to BNT162b2-MyoC stage. (C) The CEBPB-bound motif, which is the most enriched in classical monocytes in BNT162b2-MyoC (NES score of 5.716 and 23 direct targets genes) predicted by iRegulon. (D) A Network plot showing target genes of CEBPB in up-regulated DEGs of classical monocytes in BNT162b2-MyoC. all target genes as squares. squares fille with yellow color are fatty acid metabolism related genes. CEBPB is drawn as a green ellipse. The interactions are shown with directed edges from the CEBPB to the target genes. (E) Normalized expression levels of CEBPB and CEBPD in classical monocytes in each group of datasets. ** p < 0.01; *** p < 0.001; two-tailed t test.

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  Figure 6. Classical monocytes-mediated IL-16 and CXCL signals were enhanced during BNT162B2-MyoC Stage. (A) Bar graphs showing the ranking of major outgoing signals of classical monocytes in the patients in BNT162B2-MyoC compared to early(left) and late(right) recovery stages, respectively. The rank of signals was based on differences in overall information flow of each group. (B) A dot plot showing signaling enrichment of classical monocytes in other datasets for the classical monocytes-specific outgoing signals specific in BNT162b2-MyoC stage. Grey dots indicate insignificant interactions. (C) Circle plots showing cell-to-cell network for increased outgoing signals of classical monocyte in BNT162b2-MyoC stage. Arrows and edge color indicate direction (source: target). Edge thickness indicates the sum of weight key signals between populations. (D) A dot plot showing the relative significance of each cell type for each signaling pathway based on the average expression of the ligand-receptor pair.

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  Figure 7. Classical Monocytes receives Cytotoxic T cell-mediated Incoming Signals in BNT162b2-myocarditis Stage. (A) Bar graphs showing the ranking of major incoming signals of classical monocytes in the patients in BNT162B2-MyoC stage compared to early(left) and late(right) recovery stages, respectively. The rank of signals was based on differences in overall information flow, which is calculated by the total weights in the cellular network, of each group. (B) A dot plot showing signaling enrichment of classical monocytes in other datasets for the classical monocyte-specific incoming signals in BNT162B2-MyoC stage. Grey dots indicate insignificant interactions. (C) Circle plots showing the intercellular network of increased incoming signals of classical monocytes in BNT162B2-MyoC stage. Arrows and edge color indicate direction (source: target). Edge thickness indicates the sum of weight key signals between populations. (D) A dot plot showing the relative significance of each cell type for each signaling pathway based on the average expression of the ligand-receptor pair.