Critical Role of Plasmacytoid Dendritic Cells in Regulating Gene Expression and Innate Immune Responses to Human Rhinovirus-16

Abstract

Though human rhinoviruses (HRVs) are usually innocuous viruses, they can trigger serious consequences in certain individuals, especially in the setting of impaired interferon (IFN) synthesis. Plasmacytoid dendritic cells (pDCs) are key IFN producing cells, though we know little about the role of pDC in HRV-induced immune responses. Herein, we used gene expression microarrays to examine HRV-activated peripheral blood mononuclear cells (PBMCs) from healthy people, in combination with pDC depletion, to assess whether observed gene expression patterns were pDC dependent. As expected, pDC depletion led to a major reduction in IFN-α release. This was associated with profound differences in gene expression between intact PBMC and pDC-depleted PBMC, and major changes in upstream regulators: 70–80% of the HRV activated genes appeared to be pDC dependent. Real-time PCR confirmed key changes in gene expression, in which the following selected genes were shown to be highly pDC dependent: the transcription factor IRF7, both IL-27 chains (IL-27p28 and EBI3), the alpha chain of the IL-15 receptor (IL-15RA) and the IFN-related gene IFI27. HRV-induced IL-6, IFN-γ, and IL-27 protein synthesis were also highly pDC dependent. Supplementing pDC-depleted cultures with recombinant IL-15, IFN-γ, IL-27, or IL-6 was able to restore the IFN-α response, thereby compensating for the absence of pDC. Though pDC comprise only a minority population of migratory leukocytes, our findings highlight the profound extent to which these cells contribute to the immune response to HRV.

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“…Antigen-presenting dendritic cells are essential for detecting invading pathogens and activating other immune cells; therefore, they play a role in both innate and adaptive immune response. Previous work from our research group confirmed pDC as the primary and vital IFNα producer cell type (Xi et al 2015;Xi et al 2017), and as expected, we saw that the pDC marker CLEC4C was more highly expressed in the IFN-high group at baseline than in the IFN-low group, both in whole blood and PBMC (Whole blood, Table 4:1; PBMC, Figure 4:5). CLEC4C remained as a significant differentially expressed gene in the transcriptome analysis, indicating that pDC numbers at baseline are a key factor predicting the magnitude of the IFNα response to viral stimulation.…”

“…LILRA4 was previously identified as being stably expressed in unstimulated pDC compared to other immune cell types (Cho et al 2008). CLEC4C was recognised as a pDC specific cell marker in 2001 (Dzionek et al 2001), and we previously showed that it is markedly reduced in pDC depleted cell populations (Xi et al 2017), therefore we used those two markers to detect pDC population ( Figure 2:1:A). We compared whole blood to PBMC, and as expected the pDC frequency was higher in PBMC than in whole blood ( Figure 2:1:B).…”

“…Although pDC form only a fraction of immune cells, they are crucial in antiviral immunity as shown with pDC depletion experiments (Xi et al 2017). Our laboratory has previously shown that pDCs comprise more than 90% of all IFNα producing cells, with monocytes 10% and other dendritic cells less than 1% (Xi et al 2015).…”

“…CLEC4C and LILRA4 are cell surface proteins that are expressed exclusively on pDC (Cao & Bover 2010;Xi et al 2017). CLEC4C is a C-type lectin domain family 4 member C (also known as CD303 and BDCA-2) known to regulate antigen presentation and type I interferon production (Dzionek et al 2001).…”

“…In our results, CLEC4C performed consistently across the two samples types and methods but LILRA4 did not, especially when using RT-PCR on whole blood samples. The available evidence suggests that LILRA4 gene expression may be more influenced by pDC activation status than CLEC4C gene expression(Cho et al 2008;Xi et al 2017) thereby making LILRA4 gene expression unstable as a marker for estimating pDC numbers. Therefore technical factors and/or biological variability (e.g.…”