Gut microbiota-activated PLCγ2 monocytes drive atrial fibrillation

Figures

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  Fig. 1. Single-cell transcriptional landscape of AF atrial tissue. A scRNA-seq was performed on atrial tissues from AF patients and controls. UMAP plots show the distribution of cells, with colors representing different specimens. B UMAP plots visualize cell types, with colors indicating distinct cell populations. C Dotplot showing marker genes of cell subtypes. D Feature plot showing FCN1 gene expression levels across all cells. E Feature plot showing CD14 gene expression levels across all cells. F Feature plot showing FCGR3A gene expression levels across all cells. G Dot plots illustrating specific cell-cell communication pathways between monocyte and other cells. H Volcano plots highlight DEGs in monocytes, with PLCG2 annotated. Colors indicate statistical significance based on the following criteria: grey (non-significant), green (significant Log2FC only), blue (significant q-value only), and red (significant for both Log2FC and q-value). I Heatmaps illustrate expression of PLCG2 in monocytes of AF and control groups. J Immunofluorescence staining of phospho-PLCγ2 (red) and CD14 (green) in atrial tissues, with DAPI (blue) staining nuclei. White arrows indicate CD14+phospho-PLCγ2+…

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  Fig. 2. phospho-PLCγ2+ monocytes were recruited in atrium of male cAF mice. A The illustration gate of the CD45+ cells in blood of male cAF mice. B The illustration gate of the CD45+CD11b+Ly6G− cells in blood of male cAF mice. C The illustration gate of the Ly6Chiphospho-PLCγ2+ and Ly6Clophospho-PLCγ2+ cells within CD45+ CD11b+Ly6G− population. D Percentage of CD45+CD11b+Ly6G− cells in blood of male cAF mice (n = 5 mice). E Proportion of CD45+CD11b+Ly6G−Ly6Chi monocytes in blood of male cAF mice (n = 5 mice). F Proportion of CD45+CD11b+Ly6G−Ly6Clo monocytes in blood of male cAF mice (n = 5 mice). G Percentage of CD45+CD11b+Ly6G−Ly6Chiphospho-PLCγ2+ monocytes in blood of male cAF mice (n = 5 mice). H Percentage of CD45+CD11b+Ly6G−Ly6Clo phospho-PLCγ2+ monocytes in blood of male cAF mice (n = 5 mice). I The illustration gate of the CD45+ cells in atrium of male cAF mice. J The illustration gate of the CD45+CD11b+Ly6G− cells in atrium of male cAF mice. K The illustration gate of the Ly6Chiphospho-PLCγ2+ and Ly6Clophospho-PLCγ2+ cells within CD45+CD11b+ Ly6G− population. L Percentage of CD45+CD11b+Ly6G− cells in atrium of male cAF mice (n = 5 mice). M Proportion of CD45+CD11b+Ly6G−Ly6Chi…

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  Fig. 3. phospho-PLCγ2+ monocytes promoted EndMT through the SPP1-α9β1 signaling pathway in male cAF mice. A Immunohistochemical staining of SMA (red) and CD31 (green) in the atrial endocardium of male cAF mice, scale bar: 50 µm. B Quantitation of SMA/CD31 fluorescence intensity ratio (n = 5 mice). C Immunohistochemical staining of TWIST (red) and CD31 (green) in the atrial endocardium of male cAF mice, scale bar: 50 µm. D Quantitation of TWIST/CD31 fluorescence intensity ratio (n = 5 mice). E Western blot analysis of EndMT-related protein expression levels in the atrial tissue of male cAF mice. F Masson’s trichrome staining in the atrial tissue of male cAF mice. Blue indicates collagen deposition, scale bar: 25 μm. G Quantification of collagen deposition in the atrial tissue of male cAF mice (n = 5 mice). H qPCR analysis of TWIST mRNA levels in the atrial tissue of male cAF mice (n = 5 mice). I qPCR analysis of Acta2 mRNA levels in the atrial tissue of male cAF mice (n = 5 mice). J qPCR analysis of VE-cadherin mRNA levels in the atrial tissue of male cAF mice (n = 5 mice). K qPCR analysis of Col1a1 mRNA levels in the atrial tissue of male cAF mice (n = 5 mice). L qPCR analysis of Snail…

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  Fig. 4. Monocyte specific knockout of PLCG2 attenuated AF inducibility in male cAF mice. A Representative atrial electrogram recordings from male M-PLCG2−/− sham and cAF mice. Solid underlining represents burst pacing, and dashed underlining indicates AF. B AF induction success rate (n = 6 mice for Sham+WT group, n = 7 mice for remaining each group). C Total AF duration in male cAF mice (n = 6 mice for Sham+WT group, n = 7 mice for remaining each group). Data points represent the cumulative duration of all AF episodes ≥250 ms for each mouse. D Monocytes were isolated from atrial tissues of male M-PLCG2−/− sham and cAF mice. Immunofluorescence staining of phospho-PLCγ2 in monocytes of male M-PLCG2−/− sham and cAF mice, scale bar: 50 µM. E Quantification of phospho-PLCγ2 monocytes (n = 6 mice for Sham+WT group, n = 7 mice for remaining each group). F, G Intracellular DAG and IP3 concentrations in monocytes of male M-PLCG2−/− sham and cAF mice (n = 6 mice for Sham+WT group, n = 7 mice for remaining each group). H Western blot analysis of of phospho-PLCγ2, PKCδ, CaM in monocytes of male M-PLCG2−/− sham and cAF mice. I Representative fluorescence images of migrated monocytes from male…

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  Fig. 5. Monocyte specific knockout of PLCG2 suppressed colonization of CD45+CD11b+Ly6G−Ly6Clo monocytes in atrium of male cAF mice. A The illustration gate of the CD45+CD11b+Ly6G− cells in blood of male M-PLCG2−/− sham and cAF mice. B Percentage of CD45+CD11b+Ly6G− cells in blood of male M-PLCG2−/− sham and cAF mice (n = 6 mice for Sham+WT group, n = 7 mice for remaining each group). C The illustration gate of the Ly6Chiphospho-PLCγ2+ and Ly6Clo phospho-PLCγ2+ cells within CD45+CD11b+Ly6G− population. D Proportion of CD45+CD11b+Ly6G−Ly6Chiphospho-PLCγ2+ monocytes in blood of male M-PLCG2−/− sham and cAF mice (n = 6 mice for Sham+WT group, n = 7 mice for remaining each group). E Proportion of CD45+CD11b+Ly6G−Ly6Chi monocytes in blood of male M-PLCG2−/− sham and cAF mice (n = 6 mice for Sham+WT group, n = 7 mice for remaining each group). F Percentage of CD45+CD11b+Ly6G−Ly6Clo monocytes in blood of male M-PLCG2−/− sham and cAF mice (n = 6 mice for Sham+WT group, n = 7 mice for remaining each group). G Percentage of CD45+CD11b+Ly6G−Ly6Clophospho-PLCγ2+ monocytes in blood of male M-PLCG2−/− sham and cAF mice (n = 6 mice for Sham+WT group, n = 7 mice for remaining each group). H Proportion of…

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  Fig. 6. Gut microbiota are required for phospho-PLCγ2+ monocyte-mediated regulation of AF inducibility in male cAF mice. A Representative atrial electrogram recordings in antibiotics-treated and/or male cAF mice. Solid underlining represents burst pacing, and dashed underlining indicates AF. B AF induction success rate (n = 7 mice). C Total AF duration in male cAF mice (n = 7 mice). Data points represent the cumulative duration of all AF episodes ≥250 ms for each mouse. D Monocytes were isolated from atrial tissues of antibiotics-treated and/or male cAF mice. Immunofluorescence staining of phospho-PLCγ2 in monocytes, scale bar: 50 µM. E Quantification of phospho-PLCγ2 monocytes (n = 7 mice). F, G Intracellular DAG and IP3 concentrations in monocytes of antibiotics-treated and/or male cAF mice (n = 7 mice). H Representative fluorescence images of migrated monocytes from antibiotics-treated and/or male cAF mice toward MCP-1 (5 ng/mL), scale bar: 50 µM. I Quantification of migrated monocytes (n = 7 mice). J Measurement of intracellular Ca²⁺ levels in monocytes from antibiotics-treated and/or male cAF mice using the fluorescent indicator Fluo-3 AM, scale bar: 50 µM. K Quantification of the…

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  Fig. 7. Gut microbiota promoted recruitment and colonization of phospho-PLCγ2+ monocytes in the atrium of male cAF mice. A The illustration gate of the CD45+CD11b+Ly6G− cells in blood of antibiotics-treated and/or male cAF mice. B Proportion of CD45+CD11b+Ly6G− cells in blood of antibiotics-treated and/or male cAF mice (n = 7 mice). C The illustration gate of the Ly6Chiphospho-PLCγ2+ and Ly6Clophospho-PLCγ2+ cells within CD45+CD11b+Ly6G-population. D Percentage of CD45+CD11b+Ly6G−Ly6Chi phospho-PLCγ2+ monocytes in blood of antibiotics-treated and/or male cAF mice (n = 7 mice). E Proportion of CD45+CD11b+Ly6G−Ly6Chi monocytes in blood of antibiotics-treated and/or male cAF mice (n = 7 mice). F Proportion of CD45+CD11b+Ly6G−Ly6Clo monocytes in blood of antibiotics and/or male cAF mice (n = 7 mice). G Percentage of CD45+CD11b+Ly6G−Ly6Clophospho-PLCγ2+ monocytes in blood of antibiotics and/or male cAF mice (n = 7 mice). H Percentage of CD45+CD11b+Ly6G−Ly6Clo monocytes in atrium of antibiotics-treated and/or male cAF mice (n = 7 mice). I Proportion of CD45+CD11b+Ly6G−Ly6Clo phospho-PLCγ2+ monocytes in atrium of antibiotics-treated and/or male cAF mice (n = 7 mice). J The illustration gate of…

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  Fig. 8. Bacterial MVs primed Syk signaling to promote AF inducibility in male cAF mice. A qPCR quantification of bacterial 16S rDNA in feces of antibiotics-treated and/or male cAF mice (n = 7 mice). B Abundance of bacterial phyla in feces of antibiotics-treated and/or male cAF mice. C Western blot analysis of of p-Syk and Syk in monocytes of antibiotics-treated and/or male cAF mice. D TEM of bacterial MVs, scale bars, 100 nm. E Internalization of DiO-labeled MVs (green) in THP-1 cells at 0 h and 12 h post-treatment, scale bar: 100 µM. F Protein levels of p-Syk, Syk, phospho-PLCγ2, and PLCγ2 in THP-1 cells treated with 100 µg/mL MVs ± R406 (1 or 10 µM). G Intracellular DAG concentration in THP-1 cells (n = 4 independent experiments). H Representative fluorescence images of migrated THP-1 cells toward MCP-1 (5 ng/mL) after MV/R406 treatment, scale bar: 50 µM. I Quantification of migrated THP-1 cells (n = 4 independent experiments). J Measurement of intracellular Ca²⁺ levels in THP-1 cells after treatment with 100 µg/mL MVs ± R406 (1 or 10 µM) using the fluorescent indicator Fluo-3 AM, scale bar: 50 µM. K Fluo-3-AM intensity quantification (n = 4 independent experiments). L Representative…