Single Cell RNA Sequencing Reveals THBS1 + CD14 + Monocyte Modulates Inflammatory Activation via NRLP3‐Inflammasome in Congenital Heart Block

Figures

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  FIGURE 1. Identification of cell types in CHB. (A) Workflow for single‐cell transcriptome profiling of umbilical cord blood. (B, D) Clustering for 48,395 cells from four groups (CHB‐r, CHB‐nr, LQTS and Control) based on established lineage markers and visualized using UMAP. (C) The general patterns of UCBMC populations. (E) Dot plot showing expression levels of known lineage markers in all 48,395 cells. (F) Cell proportion diversity in four groups.

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  FIGURE 2. Differential expressed genes and functional analyses between conditions. (A) Cell type prioritization analysis with comparative strategies by Augur. (B) Numbers of DEGs with comparative strategies. (C) Active transcriptome regulons in different conditions using SCENIC analysis. (D) ISGs expression level in every cell type with the comparison of CHB and LQTS. (E) Cell communication differs from each condition (from top to bottom was CHB, LQTS and Control). (F) GO enrichment results displayed by comparative strategies.

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  FIGURE 3. Diversity of monocytes in groups. (A) Clustering for monocytes from three conditions based on established lineage markers and visualized using UMAP. (B) Cell type proportion diversity in four groups. (C) Expression levels of DEGs in subsets (Classical Mono, Intermediate Mono, Non‐classical Mono) based on the comparison of CHB and LQTS. (D) Expression level of markers used for identifying subsets. Statistical significance was assessed using the two‐sided Wilcoxon rank‐sum test. ****p < 0.0001. The Control group is shown for descriptive purposes only and was not included in the statistical comparison. (E) Proportion diversity of monocyte subsets identified with THBS1, CD14 and CD16. (F) Expression level of NLRP3‐inflammasome related genes. (G) The expression changes of Thbs1 and IL‐1β were confirmed in BMDMs of conditional knockout mice using RT‐qPCR. (H) GO enrichment results of THBS1+CD14+ monocyte subset. (I) KEGG enrichment results of THBS1+CD14+ monocyte subset.

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  FIGURE 4. Cell trajectory analyses among monocytes. (A) Trajectory of monocytes along pseudotime in a two‐dimensional space. Each point corresponds to a single cell. (B) Trajectory of monocytes along condition in a two‐dimensional space. (C) Expression changes of THBS1, IL‐1β and NLRP3 displayed along pseudotime. (D) Heatmap showing the dynamic changes of gene expression along pseudotime. The DEGs were clustered hierarchically into six groups. (E) Trajectory of monocytes along monocyte subsets (Classical Mono, Intermediate Mono, Non‐classical Mono) in a two‐dimensional space. (F) Expression of THBS1 genes in monocyte trajectory. (G) Trajectory of monocyte subset (THBS1− Mono, THBS1+CD14+ Mono, THBS1+CD14+CD16+ Mono) along condition in a two‐dimensional space.

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  FIGURE 5. Comparison of LQTS and Control revealed the activation of apoptosis. (A) Expression levels of DEGs in subsets (Classical Mono, Intermediate Mono, Non‐classical Mono) based on the comparison of LQTS and Control. Statistical significance was assessed using the two‐sided Wilcoxon rank‐sum test. ***p < 0.001, ****p < 0.0001. The CHB group is shown for descriptive purposes only and was not included in the statistical comparison. (B) The expression changes of DEGs were confirmed in THP‐1 with dexamethasone treatment using RT‐qPCR. Statistical significance was assessed using one‐way analysis of variance (ANOVA) followed by Dunnett's post hoc test, comparing each treatment group to the control group (0 μM), ns, not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) Expression levels of top 2 DEGs in each cell subset based on the comparison of LQTS and Control. (D) Flow cytometry revealed the apoptosis level changes of THP‐1 with dexamethasone treatment using Annexin‐V/PI. Statistical significance was assessed using one‐way analysis of variance (ANOVA) followed by Dunnett's post hoc test, comparing each treatment group to the control group (0 μM), *p < 0.1.

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  FIGURE 6. Identification and functional analyses of lymphoid cells and dendritic cells between conditions. (A–H) Integrated UMAP graph and fraction of lymphoid cells and dendritic cells in the sequenced cells among different groups. (I) Heatmap showing the dynamic changes of gene expression along condition in 15 cell subsets. The DEGs were put into GO enrichment analysis and representative enriched pathways of each group were shown.