Single cell RNAseq reveals distinct differentiation traits of COMMD10-deficient Ly6Chi monocytes in the injured liver

Hepatic macrophages play a central role in the initiation, progression and resolution of various liver diseases. Specifically, infiltrating Ly6Chi monocytes and their-derived macrophage (MoMFs) descendants prevail in acute or chronic liver injury, display marked transcriptional variance and provide crucial functional plasticity. A specific example of MoMFs are lipid associated macrophages (LAMs) involved in progression of metabolic liver disease (Remmerie et al., 2020). Recent studies have uncovered binary developmental trajectories of monocytes in the BM with Neutrophil-like (NeuMo) and dendritic cell (DC)-like (DCMo) monocytes (Weinreb et al., 2020; Yanez et al., 2017), hence further challenging our current comprehension of macrophage heterogeneity in the diseased liver. Yet, the molecular factors regulating their inflammatory versus restorative activity remains enigmatic. The COMMD (copper metabolism MURR1 domain) family includes 10 evolutionarily conserved proteins. Functions of COMMD proteins are still being defined, but they seem to play non-redundant roles in regulating transcription and protein trafficking. Utilizing conditional COMMD10 knockout mice we uncovered a role for COMMD10 in limiting inflammasome activation in Ly6Chi monocytes during experimental sepsis and colitis (Mouhadeb et al., 2018), and in supporting phagolysosomal biogenesis and maturation in KCs and BM-derived macrophages infected with Staphylococcus aureus (Ben Shlomo et al., 2019). Hence, these studies mark COMMD10 as a candidate mediator of monocyte and macrophage fate and immune responses. Here we studeis the effect of COMMD10-deficiency on Ly6Chi monocyte differentiation and inflammatory behaviour in the injured liver, using an acute model of acetaminophen-induced liver injury (AILI). Single cell RNAseq analysis of sorted Ly6Chi monocytes, comparing between COMMD10+ and COMMD10-deficient cells, revealed increased differentiation bias of Ly6Chi monocytes towards NeuMo and LAM differentiation fates. Collectively, COMMD10 appears as an important regulator of Ly6Chi monocyte fate decisions and reparative behavior in the diseased liver. Overall design: Here, we have performed single cell RNAseq analysis of sorted Ly6Chi monocytes in acute model of Acetaminophen-induced liver injury (AILI), comparing between COMMD10-deficient and sufficient Ly6Chi monocytes. Ly6Chi monocytes were sorted during the necro-inflammatory phase at 24 hours following AILI from LysMDCommd10 (Sample - LysM-4, 5229 cells) and Commd10fl/fl (two samples C10-1 and C10-3, 4860 and 8149 cells respectively) livers. Cells were counted and diluted to a final concentration in PBS supplemented with 0.04% BSA. Cellular suspension was loaded onto Next GEM Chip G and then ran on a Chromium Controller instrument to generate GEM emulsion (10x Genomics). Single-cell 3' RNA-seq libraries were generated according to the manufacturer's protocol (10x Genomics Chromium Single Cell 3' Reagent Kit User Guide v3/v3.1 Chemistry). Final libraries were quantified using NEBNext Library Quant Kit for Illumina (NEB) and high sensitivity D1000 TapeStation (Agilent). Libraries were pooled according to targeted cell number, aiming for ~50,000 reads per cell. Pooled libraries were sequenced on a NovaSeq 6000 instrument using an SP 100 cycles reagent kit (Illumina).