An efficient method for gene silencing in human primary plasmacytoid dendritic cells: silencing of the TLR7/IRF-7 pathway as a proof of concept

Smith, Nikaïa; Vidalain, Pierre‐Olivier; Nisole, Sébastien; Herbeuval, Jean-Philippe

doi:10.1038/srep29891

Cited by 26 publications

(20 citation statements)

References 47 publications

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“…Small interference RNA experiments. Selected siRNA molecules (On-TARGET plus SMARTpool, Dharmacon) were transfected using the DOTAP liposomal transfection reagent (Roche, Switzerland), as described in a previous study 67 . Briefly, human pDCs were isolated from healthy donors by negative selection using the plasmacytoid Dendritic Cell Isolation Kit II (Miltenyi Biotec, Germany), with a purity of >95%, as determined by flow cytometry ( Supplementary Fig.…”

Section: Methodsmentioning

confidence: 99%

Immune-profiling of ZIKV-infected patients identifies a distinct function of plasmacytoid dendritic cells for immune cross-regulation

et al. 2020

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Zika virus (ZIKV) is a mosquito-borne pathogen with increasing public health significance. To characterize immune responses to ZIKV, here we examine transcriptional signatures of CD4 T, CD8 T, B, and NK cells, monocytes, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs) from three individuals with ZIKV infection. While gene expression patterns from most cell subsets display signs of impaired antiviral immune activity, pDCs from infected host have distinct transcriptional response associated with activation of innate immune recognition and type I interferon signaling pathways, but downregulation of key host factors known to support ZIKV replication steps; meanwhile, pDCs exhibit a unique expression pattern of gene modules that are correlated with alternative cell populations, suggesting collaborative interactions between pDCs and other immune cells, particularly B cells. Together, these results point towards a discrete but integrative function of pDCs in the human immune responses to ZIKV infection.

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Section: Methodsmentioning

confidence: 99%

Immune-profiling of ZIKV-infected patients identifies a distinct function of plasmacytoid dendritic cells for immune cross-regulation

et al. 2020

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“…The experiments were performed using protocol as previously described by Smith et al . 63 . Briefly, H4R and CXCR4 Small interfering RNA (siRNA) (ON-TARGET SmartPool, Dharmarcon) were diluted in PBS and added to an equal volume of DOTAP (Roche Applied Sciences).…”

Section: Methodsmentioning

confidence: 99%

Natural amines inhibit activation of human plasmacytoid dendritic cells through CXCR4 engagement

et al. 2017

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Plasmacytoid dendritic cells (pDC) are specialized in secretion of type I interferon in response to pathogens. Here we show that natural monoamines and synthetic amines inhibit pDC activation by RNA viruses. Furthermore, a synthetic analogue of histamine reduces type I interferon production in a mouse model of influenza infection. We identify CXC chemokine receptor 4 (CXCR4) as a receptor used by amines to inhibit pDC. Our study establishes a functional link between natural amines and the innate immune system and identifies CXCR4 as a potential ‘on-off' switch of pDC activity with therapeutic potential.

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“…Freshly isolated pDCs were treated with small interfering RNA (siRNA) as previously described by Smith et al (43). Briefly, freshly isolated pDCs were seeded at 1 3 10 5 pDCs per 100 ml in a 96-well flat-bottom plate.…”

Section: Tim-3 Sirnamentioning

confidence: 99%

Tim-3 is a Marker of Plasmacytoid Dendritic Cell Dysfunction during HIV Infection and Is Associated with the Recruitment of IRF7 and p85 into Lysosomes and with the Submembrane Displacement of TLR9

Schwartz

Clayton

Mujib

et al. 2017

The Journal of Immunology

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In chronic diseases, such as HIV infection, plasmacytoid dendritic cells (pDCs) are rendered dysfunctional, as measured by their decreased capacity to produce IFN-α. In this study, we identified elevated levels of T cell Ig and mucin-domain containing molecule-3 (Tim-3)-expressing pDCs in the blood of HIV-infected donors. The frequency of Tim-3-expressing pDCs correlated inversely with CD4 T cell counts and positively with HIV viral loads. A lower frequency of pDCs expressing Tim-3 produced IFN-α or TNF-α in response to the TLR7 agonists imiquimod and Sendai virus and to the TLR9 agonist CpG. Thus, Tim-3 may serve as a biomarker of pDC dysfunction in HIV infection. The source and function of Tim-3 was investigated on enriched pDC populations from donors not infected with HIV. Tim-3 induction was achieved in response to viral and artificial stimuli, as well as exogenous IFN-α, and was PI3K dependent. Potent pDC-activating stimuli, such as CpG, imiquimod, and Sendai virus, induced the most Tim-3 expression and subsequent dysfunction. Small interfering RNA knockdown of Tim-3 increased IFN-α secretion in response to activation. Intracellular Tim-3, as measured by confocal microscopy, was dispersed throughout the cytoplasm prior to activation. Postactivation, Tim-3 accumulated at the plasma membrane and associated with disrupted TLR9 at the submembrane. Tim-3-expressing pDCs had reduced IRF7 levels. Furthermore, intracellular Tim-3 colocalized with p85 and IRF7 within LAMP1 lysosomes, suggestive of a role in degradation. We conclude that Tim-3 is a biomarker of dysfunctional pDCs and may negatively regulate IFN-α, possibly through interference with TLR signaling and recruitment of IRF7 and p85 into lysosomes, enhancing their degradation.

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An efficient method for gene silencing in human primary plasmacytoid dendritic cells: silencing of the TLR7/IRF-7 pathway as a proof of concept

Cited by 26 publications

References 47 publications

Immune-profiling of ZIKV-infected patients identifies a distinct function of plasmacytoid dendritic cells for immune cross-regulation

Immune-profiling of ZIKV-infected patients identifies a distinct function of plasmacytoid dendritic cells for immune cross-regulation

Natural amines inhibit activation of human plasmacytoid dendritic cells through CXCR4 engagement

Tim-3 is a Marker of Plasmacytoid Dendritic Cell Dysfunction during HIV Infection and Is Associated with the Recruitment of IRF7 and p85 into Lysosomes and with the Submembrane Displacement of TLR9

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