Vsig4+ resident single-Kupffer cells improve hepatic inflammation and fibrosis in NASH

Abstract

Background: The role of macrophages in the pathogenesis of nonalcoholic steatohepatitis (NASH) is complex and unclear.

Methods: Single-cell RNA sequencing was performed on nonparenchymal cells isolated from NASH and control mice. The expression of Vsig4⁺ macrophages was verified by qPCR, flow cytometry and immunohistochemistry. Primary hepatic macrophages were cocultured with primary hepatocytes or hepatic stellate cells (LX2) cells by Transwell to detect immunofluorescence and oil red O staining.

Results: Two main single macrophage subsets were identified that exhibited a significant change in cell percentage when NASH occurred: resident Kupffer cells (KCs; Cluster 2) and lipid-associated macrophages (LAMs; Cluster 13). Nearly 82% of resident single KCs in Cluster 2 specifically expressed Cd163, and an inhibited subgroup of Cd163⁺ resident single-KCs was suggested to be protective against NASH. Similar to Cd163, Vsig4 was both enriched in and specific to Cluster 2. The percentage of Vsig4⁺-KCs was significantly decreased in NASH in vivo and in vitro. Hepatocytes and hepatic stellate cells produced less lipid droplet accumulation, proinflammatory protein (TNF-α) and profibrotic protein (α-SMA) in response to coculture with Vsig4⁺-KCs than in those cocultured with lipotoxic KCs.

Conclusions: A subgroup of Vsig4⁺ resident single-KCs was shown to improve hepatic inflammation and fibrosis in NASH.

Keywords: KCs, Kupffer cells; Macrophages; NASH, Non-alcoholic steatohepatitis; Vsig4, V-set and immunoglobulin domain-containing 4; scRNA-seq, Single-cell RNA sequencing.