A Novel Flow Cytometry Tool for Fibrosis Scoring Through Hepatic Stellate Cell Differentiation

Hepatic stellate cells (HSCs) are a central fibrogenic cell type that contributes to collagen accumulation during chronic liver disease. Peripheral blood lymphocytes from HCV patients are phagocytized by HSCs and induce their differentiation. This study aimed to characterize HSCs differentiation using a flow cytometry tool for fibrosis scoring. NK cells from healthy donors and from patients with chronic HCV with various severities of fibrosis were co-cultured with a human HSC line (LX2). LX2 phagocytosis of NK cells were stained for NK cells (CD45/CD56/CD3) and NK activation marker (CD107a) as well as INF-gamma, apoptosis (Annexin-V) and alpha-smooth-muscle-actin (alpha SMA, as a marker of LX2 activation). In addition, reactive oxygen species (ROS) and the senescence marker P15 were analyzed prior to flow cytometry analysis. LX2 mono-cultures demonstrated a homogenous cell-population according to size (forward-scattered; FSC), granularity and alpha SMA expressions. However, on their co-culture with NK cells, the HSCs formed four subpopulations, which were stratified by alpha SMA intensities and cell size. NK cells isolated from heathy donors did not activate LX2-cells. In contrast, HCVexposed to NK cells from both F1 and F4 fibrosis grade patients, showed elevated CD107a and INF-gamma levels and increased alpha SMA intensities in two of the four cell populations, with fibrosis scoring showing a linear correlation with aSMA intensities and NK phagocytosis. The alpha SMA(intermediate)/Size(Low) HSCs sub-population showed higher proliferation following F4-NK cells with higher phagocytosis ability, suggesting an active/regulatory population. The alpha SMA(high)/Size(high) subpopulations showed low proliferation and phagocytosis capacity, and were correlated with higher apoptosis, increased ROS and P15 intensities, suggesting senescing cells. Taken together, NK cells lead to heterogeneous differentiation of HSCs. Flow-cytometry may provide a novel means of characterizing HSCs in relation to the severity of liver fibrosis. (C) 2018 International Society for Advancement of Cytometry