WWP2 regulates Ly6Chigh monocyte infiltration and activity in heart fibrosis

Non-ischemic cardiomyopathy (NICM) can cause left ventricular dysfunction through interstitial fibrosis, which corresponds to the failure of cardiac remodelling. Recent evidence implicates monocytes/macrophages modulating cardiac fibrosis but targeting these is convoluted, giving their tissue heterogeneity and the antagonizing roles of macrophage subtypes in fibrosis. Here we focus on the role of WWP2, an E3 ubiquitin ligase that acts as a positive genetic regulator of human and murine cardiac fibrosis, and show that its myeloid-specific deletion reduces cardiac fibrosis in hypertension-induced NICM. Using the same model, we establish the functional heterogeneity of macrophages and define an early pro-fibrogenic phase driven by Ccl5-expressing Ly6chigh monocytes. Among other cardiac macrophage subtypes, WWP2 dysfunction primarily affects the Ccl5-dependent infiltration and activation of Ly6chigh monocytes, which causes reduced myofibroblast trans-differentiation. WWP2 interacts with IRF7, promoting its non-degradative monoubiquitination, nuclear translocation and transcriptional activity, including upstream Ccl5. Thus, we identify WWP2 as a key regulator of IRF7-mediated Ccl5/Ly6chigh monocyte axis in heart fibrosis. Overall design: Isolated single cell suspensions were converted to barcoded scRNA-seq libraries (WT, WT Ang-II, WWP2Mut/Mut, and WWP2Mut/Mut Ang-II) by using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit, and Chip Kit V3, loading an estimated 7,000-12,000 cells per library/well and following the manufacturer's instructions. Indexed libraries were sequenced using Illumina HiSeq 4000 sequencer, where 150bp pair-end sequences were obtained. Sequencing reads were aligned and quantified to the mouse genome GRCm38 (mm10-3.0.0 provided by 10x Genomics) using Cellranger count (v3.1.0).