Liver and Gastroenterology Units, Hadassah University Medical Center, Jerusalem, Israel.
Cytometry A. 2018 Apr;93(4):427-435. doi: 10.1002/cyto.a.23202. Epub 2018 Mar 8.
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-γ, apoptosis (Annexin-V) and α-smooth-muscle-actin (αSMA, 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 αSMA expressions. However, on their co-culture with NK cells, the HSCs formed four subpopulations, which were stratified by αSMA intensities and cell size. NK cells isolated from heathy donors did not activate LX2-cells. In contrast, HCV exposed to NK cells from both F1 and F4 fibrosis grade patients, showed elevated CD107a and INF-γ levels and increased αSMA intensities in two of the four cell populations, with fibrosis scoring showing a linear correlation with αSMA intensities and NK phagocytosis. The αSMA /Size HSCs sub-population showed higher proliferation following F4-NK cells with higher phagocytosis ability, suggesting an active/regulatory population. The αSMA /Size 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. © 2017 International Society for Advancement of Cytometry.
肝星状细胞（HSCs）是一种重要的纤维生成细胞类型，在慢性肝病期间有助于胶原积累。来自 HCV 患者的外周血淋巴细胞被 HSCs 吞噬，并诱导其分化。本研究旨在使用纤维化评分的流式细胞术工具来表征 HSCs 的分化。来自健康供体和具有不同纤维化严重程度的慢性 HCV 患者的 NK 细胞与人类 HSC 系（LX2）共培养。LX2 对 NK 细胞的吞噬作用用 NK 细胞（CD45/CD56/CD3）和 NK 激活标志物（CD107a）以及 INF-γ、凋亡（Annexin-V）和α-平滑肌肌动蛋白（αSMA，作为 LX2 激活的标志物）进行染色。此外，在进行流式细胞术分析之前，还分析了活性氧物种（ROS）和衰老标志物 P15。LX2 单核培养根据大小（前向散射；FSC）、颗粒度和αSMA 表达呈现均匀的细胞群体。然而，在与 NK 细胞共培养时，HSCs 形成了四个亚群，这些亚群根据αSMA 强度和细胞大小分层。来自健康供体的 NK 细胞不会激活 LX2 细胞。相比之下，来自 F1 和 F4 纤维化等级患者的 NK 细胞暴露于 HCV 后，在四个细胞群体中的两个中显示出 CD107a 和 INF-γ 水平升高以及αSMA 强度增加，纤维化评分显示与αSMA 强度和 NK 吞噬作用呈线性相关。αSMA/Size HSCs 亚群在具有更高吞噬能力的 F4-NK 细胞作用下表现出更高的增殖，提示为活性/调节性群体。αSMA/Size 亚群表现出低增殖和吞噬能力，并且与更高的凋亡、增加的 ROS 和 P15 强度相关，提示衰老细胞。总之，NK 细胞导致 HSCs 的异质性分化。流式细胞术可能为表征与肝纤维化严重程度相关的 HSCs 提供一种新方法。
