Individual liver plasmacytoid dendritic cells are capable of producing IFNα and multiple additional cytokines during chronic HCV infection

Plasmacytoid dendritic cells (pDCs) are “natural” interferon α (IFNα)-producing cells. Despite their importance to antiviral defense, autoimmunity, and ischemic liver graft injury, because DC subsets are rare and heterogeneous, basic questions about liver pDC function and capacity to make cytokines remain unanswered. Previous investigations failed to consistently detect IFNα mRNA in HCV-infected livers, suggesting that pDCs may be incapable of producing IFNα. We used a combination of molecular, biochemical, cytometric, and high-dimensional techniques to analyze DC frequencies/functions in liver and peripheral blood mononuclear cells (PBMCs) of hepatitis C virus (HCV)-infected patients, to examine correlations between DC function and gene expression of matched whole liver tissue and liver mononuclear cells (LMCs), and to determine if pDCs can produce multiple cytokines. T cells often produce multiple cytokines/chemokines but until recently technical limitations have precluded tests of polyfunctionality in individual pDCs. Mass cytometry (CyTOF) revealed that liver pDCs are the only LMC that produces detectable amounts of IFNα in response TLR-7/8 stimulation. Liver pDCs secreted large quantities of IFNα (~2 million molecules of IFNα/cell/hour) and produced more IFNα than PBMCs after stimulation, p = 0.0001. LMCs secreted >14-fold more IFNα than IFNλ in 4 hours. Liver pDC frequency positively correlated with whole liver expression of “IFNα-response” pathway (R2 = 0.58, p = 0.007) and “monocyte surface” signature (R2 = 0.54, p = 0.01). Mass cytometry revealed that IFNα-producing pDCs were highly polyfunctional; >90% also made 2–4 additional cytokines/chemokines of our test set of 10. Liver BDCA1 DCs, but not BDCA3 DCs, were similarly polyfunctional. pDCs from a healthy liver were also polyfunctional. Our data show that liver pDCs retain the ability to make abundant IFNα during chronic HCV infection and produce many other immune modulators. Polyfunctional liver pDCs are likely to be key drivers of inflammation and immune activation during chronic HCV infection.浆细胞样树突状细胞（plasmacytoid dendritic cells, pDCs）是一类"天然"的α干扰素（interferon α, IFNα）产生细胞。尽管其在抗病毒防御、自身免疫以及缺血性肝移植损伤中发挥关键作用，但由于树突状细胞（dendritic cells, DCs）亚群具有稀有性与异质性，有关肝脏pDC的功能及其细胞因子产生能力的基础科学问题仍未得到解答。既往研究未能在丙型肝炎病毒（hepatitis C virus, HCV）感染的肝脏组织中持续检测到IFNα mRNA，这提示pDC可能无法产生IFNα。本研究结合分子生物学、生物化学、流式细胞术以及高维分析技术，对HCV感染患者的肝脏及外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）中的DC频率与功能展开分析，探究DC功能与匹配的全肝组织、肝脏单个核细胞（liver mononuclear cells, LMCs）基因表达之间的相关性，并明确pDC是否能够产生多种细胞因子。T细胞通常可分泌多种细胞因子/趋化因子，但直至近期，技术局限仍阻碍了对单个pDC多功能性的检测。质谱流式细胞术（mass cytometry, CyTOF）结果显示，肝脏pDC是唯一一类可在经Toll样受体7/8（Toll-like receptor 7/8, TLR-7/8）刺激后产生可检测水平IFNα的LMC。肝脏pDC可分泌大量IFNα（约200万个IFNα分子/细胞/小时），且刺激后其IFNα分泌量显著高于PBMCs（p=0.0001）。4小时内，LMC分泌的IFNα量较干扰素λ（interferon λ, IFNλ）高14倍以上。肝脏pDC的频率与全肝"IFNα应答"通路的基因表达（R²=0.58, p=0.007）以及"单核细胞表面"特征基因的表达（R²=0.54, p=0.01）均呈正相关。质谱流式细胞术分析显示，产IFNα的pDC具有高度的多功能性；在本研究检测的10种细胞因子/趋化因子中，超过90%的pDC还可同时产生2~4种其他细胞因子/趋化因子。肝脏BDCA1阳性树突状细胞（BDCA1 DCs）可呈现类似的多功能性，而BDCA3阳性树突状细胞（BDCA3 DCs）则无此特性。健康肝脏来源的pDC也表现出多功能特性。本研究数据表明，在慢性HCV感染过程中，肝脏pDC仍具备产生大量IFNα以及多种其他免疫调节因子的能力。具有多功能性的肝脏pDC可能是慢性HCV感染期间炎症反应与免疫激活的关键驱动因素。