The E3 ubiquitin ligase WWP2 regulates pro-fibrogenic monocyte infiltration and activity in heart fibrosis

Fig. 1. Single-cell transcriptomic profiling and trajectory analysis of cardiac macrophages in a murine model of NICM. a Dynamics of cardiac macrophages (live CD45+ CD64+ CD11b+ F4/80+ Ly6G−) from B6J mice treated with Ang-II (500 ng/kg/min). Cardiac macrophages are shown as a percentage with respect to total number of cardiac cells. Three biological replicates were used at each time point. P-values calculated by Mann–Whitney U test, vs percentage at day 0. *P < 0.05, **P < 0.01. b Cardiac CD45+ cells sorted from left ventricle (LV) from B6J mice treated with Ang-II (500 ng/kg/min, 7 days) and saline (controls) were pooled for scRNA-seq analysis on 5497 cells. Global uniform manifold approximation and projection (UMAP) dimension reduction analysis identified six major cell types. DC dendritic cells, NK cells natural killer cells. c Macrophages were re-clustered using Seurat’s Louvain clustering. d Heatmap of the scaled gene expression of top 10 markers for each macrophage cluster from Fig. 1c, based on average log2 fold change (FC). ISGs Interferon (IFN)-stimulated genes, AP-1 Activator protein 1. e Heatmap of enriched pathways identified in each cardiac macrophage cluster. The top five…

Fig. 2. WWP2 dysfunction regulates macrophage infiltration and expansion in the heart at 7 days post Ang II-infusion. a Schematic representation of the experimental setup. Sorted live CD45+ cells from LV underwent scRNA-seq analysis, and seven clusters are represented in the UMAP space. WT, WWP2wt/wt; Mut/Mut, WWP2Mut/Mut. In each experiment, mice were treated with either saline (control) or Ang-II (500 ng/kg/min, 7 days). b Bar plots represent the proportions of macrophage clusters (Fig. 2a) in WT and Mut/Mut following Ang-II treatment. Two-proportion z test, ***P < 0.001 Mut/Mut vs WT. c Expression of Ly6c gene in cardiac macrophages represented in the UMAP space. d Percentage of Ly6chigh cells in WT and Mut/Mut cardiac monocyte compartments, in control or Ang-II-treated mice. n = 5–8 for each group. e Representative flow cytometry for CD11b+/F4/80+ gate in Ang-II-treated WT and Mut/Mut mice (left). Quantification of cardiac macrophages in Mut/Mut compared with WT mice following Ang-II treatment (right). n = 6–9 for each group. f Chemokine ligand-receptor pairs (Molecule 1–2 pairs) derived by CellPhoneDB analysis in cardiac macrophages, P < 0.01 (left). Mean expression of Molecule 1–2…

Fig. 3. WWP2 regulates macrophage activation and profibrotic function. a Scatter plot of log2fold changes (FC) in mRNA expression from scRNA-seq in cardiac macrophages between Ang-II-treated Mut/Mut and WT mice (y axis) and log2FC between WT Ang-II-treated and WT untreated mice (x axis). Differentially expressed genes (DEGs) in red (n = 237, FDR < 0.05). Ang-II treatment: 500 ng/kg/min, 7 days. b Top downregulated pathways in Mut/Mut macrophages identified by gene set enrichment analysis (GSEA) of DEGs. NES, normalized enrichment score. c Violin plots illustrate the expression score of the GSEA-derived pathways across all cardiac macrophage clusters in Mut/Mut and WT mice after treatment with Ang-II (7 days). d qRT-PCR analysis of selected pro-inflammatory and homeostatic/reparatory genes in macrophages sorted from LV of WT and Mut/Mut mice treated with saline or Ang-II (7 days). n = 5–12 for each group. e Representative immunofluorescence staining of smooth muscle aortic alpha-actin (ACTA2, green) in (myo)fibroblasts co-cultured with CD45 + macrophages (red). Scale bar, 100 μm. f Number of cardiac macrophages moving across the proximity border (left), and ACTA2 expression in…

Fig. 4. Conditional depletion of WWP2 in macrophages protects from Angiotensin II induced cardiac fibrosis. a Left, representative Sirius red (upper) and Masson’s Trichrome (lower) staining of short-axis sections of LV. Scale bar: 0.5 mm. Right, quantification of the area of fibrosis in transverse histological sections with Sirius red staining at the mid-ventricular level. WWP2Mac, WWP2 conditional knockout in macrophages; Control (Ctrl), WWP2flox/flox mice. b Collagen content in LV by hydroxy-phenyl-propionic acid (HPA) assay. n = 10 per experimental group. c Representative M-mode echocardiograms (middle LV long-axis) (left), and quantification of left ventricular internal dimension at end-diastole (LVIDed)(right). n = 12 per experimental group. d Echocardiogram-based quantification of LV ejection fraction (EF%) and fractional shortening (FS%). n = 8–11 per experimental group. e Representative WB showing fibronectin extracellular domain A (EDA-FN), vimentin (VIM), periostin (POSTN), and ACTA2 in LV tissue from control and WWP2Mac mice. f qRT-PCR analysis of selected ECM genes in LV tissue from control and WWP2Mac mice. n = 6 per experimental group. g Representative flow cytometry for…

Fig. 5. WWP2 regulates IRF7 network activity in macrophages in NICM. a Scatter plot of the relative activity of transcription factor (TF)-regulons in cardiac macrophages from WWP2Mut/Mut (Mut/Mut) and WWP2wt/wt (WT) mice treated with Ang-II (7 days). For each regulon, the difference in expression score between Mut/Mut and WT and its statistical significance score are shown on the x axis and y axis, respectively. The number of downstream target genes in each regulon is proportional to the circle size (more details in Methods). The IRF7 regulon showed the largest and most significant expression score difference between Mut/Mut and WT cardiac macrophages. b Average expression score of the IRF7 regulon across cardiac macrophage clusters from WT and Mut/Mut mice treated with Ang-II (7 days). (P values by two-sided Wilcoxon test). c Breakdown of IRF7 targets enrichment in the profibrotic hECM-network (n = 237 genes) previously identified in LV of dilated cardiomyopathy patients39. 80 (33.8%) of these hECM-network genes are targets of IRF7. d, e qRT-PCR (d) and WB (e) of representative IFN signaling genes (IFNα, IFNβ, and IFNγ) in WT and WWP2−/− (−/−) BMDMs with and without LPS stimulation. n =…

Fig. 6. WWP2 regulates IRF7 transcriptional activity in macrophages through non-degradative ubiquitination. a Representative WB of co-immunoprecipitation experiment with WWP2 (upper) or IRF7 (lower) showing a direct interaction of WWP2 with IRF7 in BMDMs following LPS stimulation. WCL, whole cell lysate. b Expression of WWP2 (green) and IRF7 (red) in BMDMs following LPS stimulation. The intensity profiles of WWP2 and IRF7 were plotted along an ideal straight line (yellow) crossing the nuclei of two representative cells, showing the WWP2 and IRF7 signals overlap (bottom right). c In-cell ubiquitylation analysis of IRF7 in BMDMs from control WT and WWP2−/− mice. Cells were treated with MG132 (10uM, 3 hrs) followed by LPS stimulation. d Representative WB measuring p-IRF7 protein expression in WT and WWP2−/− BMDMs with or without LPS stimulation. Quantification of p-IRF7 protein expression is shown in the bar plot below. e Quantification of the IRF7 Interferon-stimulated response element (ISRE) luciferase activity in WT and WWP2−/− BMDMs following LPS treatment. n = 7–11 for each group. f Representative immunoblots of native Polyacrylamide gel electrophoresis (PAGE) identifying monomeric and…