citations

                    IRF-5 and NF-kB p50 co-regulate IFN-ß and IL-6 expression in TLR9-stimulated human plasmacytoid dendritic cells.
                

Authors:

Steinhagen F, McFarland AP, Rodriguez LG, Tewary P, Jarret A, Savan R, Klinman DM.

In:

Source: Eur J Immunol
Publication Date: (2014)
Issue: 7: 43(7)

Research Area:

Immunotherapy / Hematology

Basic Research

Cells used in publication:

Cal-1: Species: human; Tissue Origin: blood

Platform:

Nucleofector® I/II/2b

4D-Nucleofector® 96-well Systems

Experiment

siRNA, ODN with nucleofected with DN-100 ans SF solution in the 96Well Shuttle System The authors aimed to identify the key signaling pathways responsible for the up-regulation of IFN-ß and IL-6 in CpG stimulated human plasmacytoid dendritic cells (pDCs). For stimulation specific synthetic oligonucleotides were used expressing CpG motifs (so called “K” ODNs which are clinically relevant) which mimic the bacterial DNA trigger for toll-like receptor 9 ( TLR9). After they have shown that the human plasmacytoid DC cell line CAL-1 is mimicking the response of human pDCs to “K” ODN stimulation, CAL-1 cells were transfected using the Nucleofector™ 96-well Shuttle™ System with siRNAs against various interferon regulatory factor (IRFs) to evaluate their contribution to the regulation of IFN-ß or IL-6 expression in CpG-stimulated pDCs. They were able to achieve knockdown efficiencies of 70–85% on mRNA level leading to an effective depletion of target proteins (shown by Western Blot) without any siRNA-mediated off-target effects. The study showed that IRF-5 and NF-?B p50 are key co-regulators of IFN-ß and IL-6 expression following TLR9-mediated activation of human pDCs.

Abstract

Synthetic oligonucleotides (ODN) expressing CpG motifs mimic the ability of bacterial DNA to trigger the innate immune system via TLR9. Plasmacytoid dendritic cells (pDCs) make a critical contribution to the ensuing immune response. This work examines the induction of antiviral (IFN-ß) and pro-inflammatory (IL-6) cytokines by CpG-stimulated human pDCs and the human CAL-1 pDC cell line. Results show that interferon regulatory factor-5 (IRF-5) and NF-?B p50 are key co-regulators of IFN-ß and IL-6 expression following TLR9-mediated activation of human pDCs. The nuclear accumulation of IRF-1 was also observed, but this was a late event that was dependant on type 1 IFN and unrelated to the initiation of gene expression. IRF-8 was identified as a novel negative regulator of gene activation in CpG-stimulated pDCs. As variants of IRF-5 and IRF-8 were recently found to correlate with susceptibility to certain autoimmune diseases, these findings are relevant to our understanding of the pharmacologic effects of "K" ODN and the role of TLR9 ligation under physiologic, pathologic, and therapeutic conditions.

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