Single-cell RNA sequencing analyses the mechanism of scar-associated macrophages promoting hepatic stellate cell activation

Abstract

Objective  To establish an experimental protocol for inducing human macrophages into scar-associated macrophages (SAMs) in vitro. To investigate the mechanism underlying SAMs regulating hepatic stellate cell (HSC) activation. Methods  Published single-cell RNA sequencing (scRNA-Seq) data, which were obtained from human or murine fibrotic liver tissues, were used to study the expression of fibrosis-related genes in SAMs. Phorbol-12-myristate-13-acetate (PMA) and plasminogen (PLG) were used to induce the transformation of human monocyte THP-1 into SAMs. SAM supernatant was collected to culture with LX-2. The expressions of SAM markers, fibrosis-related genes and HSC activation markers were detected by real-time quantitative polymerase chain reaction（RT-qPCR）. Results  scRNA-seq revealed that SAMs were detected in both mouse fibrotic livers or human injured liver. SAMs highly expressed fibrosis-related genes such as SPP1, CTSD. PMA and PLG increased the expressions of SAM markers CD9, TRME2, LGALS3, CD63 in THP-1. Fibrosis-related genes SPP1, CTSD were highly expressed by SAMs. The supernatant of SAMs activated LX-2. Conclusion  SAMs are accumulated in mouse or human liver injury/fibrosis caused by different causes, and represent similar characteristics and functions. PMA and PLG induce the transformation of human monocyte THP-1 into SAMs. SAMs promote the activation of HSCs via SPP1 and CTSD, thus promoting the occurrence and development of liver fibrosis.

Keywords

single-cell RNA sequencing / scar-associated macrophage / hepatic stellate cell / liver fibrosis