Abstract
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.
Highlights
Plasmacytoid dendritic cells are rare innate immune cells that comprise about 0.5% of peripheral blood mononuclear cells (PBMCs). They migrate into tissues and are known as “natural” producers of interferon alpha (IFNα). pDCs constitutively express toll-like receptor (TLR)-7 and TLR-9, as well as interferon regulatory factor (IRF)-7, enabling them to detect viral nucleic acids and to quickly secrete type I IFNs (IFNα and IFNβ), which bind neighboring cells and induce hundreds of IFN stimulated genes (ISGs), initiating antiviral defenses
Some reported that circulating pDCs are functionally intact [6, 7], but the majority reported impairment after stimulation with various TLR ligands [1,2,3,4,5], which was attributed to toxic effects of tumor necrosis factor α (TNFα) [5] and direct inhibitory effects of hepatitis C virus (HCV) proteins [8] [9]
We explored an alternative explanation: the possibility that intrahepatic pDCs remain functional during chronic HCV infection but generate an IFNA mRNA signal that is too low to be detected in extracts of whole liver tissue
Summary
Plasmacytoid dendritic cells (pDCs) are rare innate immune cells that comprise about 0.5% of peripheral blood mononuclear cells (PBMCs) They migrate into tissues and are known as “natural” producers of interferon alpha (IFNα). Some reported that circulating pDCs are functionally intact [6, 7], but the majority reported impairment after stimulation with various TLR ligands [1,2,3,4,5], which was attributed to toxic effects of tumor necrosis factor α (TNFα) [5] and direct inhibitory effects of HCV proteins [8] [9]. In contrast to these inhibitory effects, HCV RNA stimulates pDCs by activating TLR-7 and RIG-I [10,11,12,13]
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