Supplementary Material for: Characterization of Pattern Recognition Receptor Expression and Functionality in Liver Primary Cells and Derived Cell Lines

Different liver cell types are endowed with immunological properties, including cell-intrinsic innate immune functions that are important to initially control pathogen infections. However, a full landscape of expression and functionality of the innate immune signaling pathways in the major human liver cells is still missing. In order to comparatively characterize these pathways, we purified primary human hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells (LSEC), and Kupffer cells (KC) from human liver resections. We assessed mRNA and protein expression level of the major innate immune sensors, as well as checkpoint-inhibitor ligands in the purified cells, and found Toll-like receptors (TLR), RIG-I-like receptors, as well as several DNA cytosolic sensors to be expressed in the liver microenvironment. Amongst the cells tested, KC were shown to be most broadly active upon stimulation with PRR ligands emphasizing their predominant role in innate immune sensing the liver microenvironment. By KC immortalization, we generated a cell line that retained higher innate immune functionality as compared to THP1 cells, which are routinely used to study monocyte/macrophages functions. Our findings and the establishment of the KC line will help to understand immune mechanisms behind antiviral effects of TLR agonists or checkpoint inhibitors, which are in current preclinical or clinical development.

不同肝脏细胞类型均具备免疫学特性，其中包括对初始控制病原体感染至关重要的细胞固有先天免疫功能。然而，目前仍缺乏人类主要肝脏细胞中先天免疫信号通路的表达与功能全貌图谱。为对这些通路开展对比表征研究，本研究从人体肝切除标本中分离纯化了原代人肝细胞（primary human hepatocytes）、肝星状细胞（hepatic stellate cells）、肝窦内皮细胞（liver sinusoidal endothelial cells，LSEC）以及库普弗细胞（Kupffer cells，KC）。本研究对纯化后的细胞中主要先天免疫传感器以及免疫检查点抑制剂配体的mRNA与蛋白表达水平进行了检测，结果发现Toll样受体（Toll-like receptors，TLR）、RIG-I样受体（RIG-I-like receptors）以及多种胞质DNA传感器均在肝脏微环境中有所表达。在本次检测的各类细胞中，库普弗细胞（KC）经模式识别受体（pattern recognition receptors，PRR）配体刺激后展现出最为广泛的激活活性，这凸显了其在肝脏微环境先天免疫感知中的主导作用。通过对库普弗细胞进行永生化构建，本研究获得了一株细胞系，相较于常规用于研究单核细胞/巨噬细胞功能的THP1细胞系，该细胞系保留了更高的先天免疫功能活性。本研究的发现以及该库普弗细胞系的构建，将有助于阐明当前处于临床前或临床研发阶段的Toll样受体激动剂或免疫检查点抑制剂的抗病毒免疫作用机制。