Lipid influx enforces liver regeneration through the recruitment of CD36+ macrophages (scRNA-Seq and RNA-Seq)

The liver possesses a remarkable regeneration capacity under a wide variety of injury conditions, like partial hepatectomy (PHx) and over-dose acetaminophen (APAP) treatment. Macrophages, multitask at the wound site by facilitating wound debridement and producing chemokines, metabolites and growth factors, and consequently determine the balance between ongoing damage and repair following tissue injury. CD36, which is universally expressed in a wide variety of cells (i.e., macrophages, adipocytes, myocytes, enterocytes and hepatocytes), and serves as the predominant fatty acid uptake facilitator of the cells to uptake microenvironmentally enriched lipids in various settings. However, the role for macrophage CD36 in mediating lipid accumulation as well as the regeneration of liver post physiological or pathophysiological injury still remains largely unknown. Herein, by utilizing multiple seq-based methods, we found the transient lipid accumulation in injured livers post PHx or APAP treatment exerted fundamental impacts on hepatic immune remodeling and induced the recruitment of macrophages with high level of CD36 expression and excessive lipid accumulation which facilitate the regeneration of livers. CD36-mediated lipid influx in macrophages triggers the activation of IRE1a signaling pathways via intracellular ceramide accumulation to promote high levels of IL6 production to accelerate liver regeneration. Overall design: Our experimental design involved single cell RNA sequencing (scRNA-seq) to analyze liver cell dynamics post-PHx and sham operations, focusing on two groups with three mice each group. Fresh liver tissues were processed using the Miltenyi Liver Digestion Kit to ensure high-quality single-cell suspensions, with cell viability consistently above 90%. This preparation step was crucial for reliable downstream scRNA-seq analysis. We used the 10X Genomics platform from BGI for sequencing. The data analysis included Seurat-guided clustering and UMAP dimensionality reduction to identify and annotate nine distinct cell populations. To explore this, we employed a controlled experimental design to assess the impact of HFD induced lipid accumulation on liver regeneration and transcriptome changes. Our design included two distinct groups of C57BL/6 male mice of 8 week old, each consisting of three individuals. The experimental group was subjected to a high-fat diet (Research diet, #12492i) for one week prior to PHx to induce acute hepatic lipid accumulation. In contrast, the control group was maintained on a standard diet during the same period. All mice underwent PHx, and liver tissue samples were collected 40 hours post-surgery from both groups. This setup was specifically devised to evaluate the effects of HFD on the liver's regenerative capacity and associated transcriptomic alterations following PHx, providing insights into the influence of lipid metabolism on liver regeneration. Our study explored the potential distinct functions of liver macrophage subpopulations in liver regeneration, including Kupffer cells, LY6C+ MDMs, CD36+ MDMs retrived from C57BL/6 wild type mice, as well as CD36+ MDMs retrived MDM-Ern1KO mice. Each group was represented by three samples. Each samples were pooled of the cells from three or more mice. The cells were isolated from the livers using FACS two days post-PHx and subjected to bulk RNA sequencing.