IRF5 and IRF8 modulate the CAL‐1 human plasmacytoid dendritic cell line response following TLR9 ligation

Abstract

Synthetic oligonucleotides (ODNs) containing CpG motifs stimulate human plasmacytoid dendritic cells (pDCs) to produce type-1 interferons (IFNs) and proinflammatory cytokines. Previous studies demonstrated that interferon regulatory factors (IRFs) play a central role in mediating CpG-induced pDC activation. This work explores the inverse effects of IRF5 and IRF8 (also known as IFN consensus sequence-binding protein) onCpG-dependent gene expression in the human CAL-1 pDC cell line. This cell line shares many of the phenotypic and functional properties of freshly isolated human pDCs. Results from RNA interference and microarray studies indicate that IRF5 upregulates TLR9-driven gene expression whereas IRF8 downregulates the same genes. Several findings support the conclusion that IRF8 inhibits TLR9-dependent gene expression by directly blocking the activity of IRF5. First, the inhibitory activity of IRF8 is only observed when IRF5 is present. Second, proximity ligation analysis shows that IRF8 and IRF5 colocalize within the cytoplasm of resting human pDCs and cotranslocate to the nucleus after CpG stimulation. Taken together, these findings suggest that IRF5 and IRF8, two transcription factors with opposing functions, control TLR9 signaling in human pDCs. Keywords:CpG oligonucleotide r Dendritic cell r IRF8, IRF5 r TLR9 Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionPlasmacytoid dendritic cells (pDCs) function at the interface between the innate and adaptive immune systems and play a critical role in the host's response to infectious pathogens [1,2]. Considerable effort has been invested in clarifying the molecular mechanisms through which pDCs perform these vital functions. In humans, pDCs constitutively express TLR 9 enabling them to sense unmethylated CpG motifs expressed by microbial pathogens [3][4][5].Correspondence: Dr. Dennis M. Klinman e-mail: klinmand@mail.nih.govSynthetic oligodeoxynucleotides expressing such CpG motifs mimic the ability of bacterial DNA to stimulate pDCs [6]. In particular, "K" class phosphorothioate oligonucleotide expressing immunostimulatory CpG motif (CpG ODN) have been extensively studied in clinical trials and generate a response characterized by the production of type-1 IFNs, proinflammatory cytokines, and chemokines [7,8]. The release of type-1 IFNs is mediated in a cell type and species-specific manner by "IFN regulating factors" (IRFs) [9]. Recent studies examined which members of the human IRF family regulated the IFN response of CpG-stimulated human pDCs. IRF5 was found to play a vital role in the upregulation of type-1 IFNs and proinflammatory cytokines, represented by respectively [10]. Unexpectedly, IRF8 was found to inhibit Published 2015. This article is a U.S. Government work and is in the public domain in the USA www.eji-journal.eu 648Folkert Steinhagen et al. Eur. J. Immunol. 2016. 46: 647-655 the stimulatory activity of IRF5, a result inconsistent with reports showing that IRF8 boost...

Regulatory role of SphK1 in TLR7/9‐dependent type I interferon response and autoimmunity

“…42 Induction of IFN, TNF-α, or IL-6 could not be detected in CAL-1 cells stimulated with the TLR3 ligand poly (I:C), even after prolonged incubation (data not shown). This is likely due to expression of TLR7/9 by CAL-1 cells as reported previously, 43 but not TLR3. As previous F I G U R E 3 SphK1 regulates nuclear import of IRF3, IRF7, and NF-κB and downstream MAPK signaling.…”

B Cell-Intrinsic Role for IRF5 in TLR9/BCR-Induced Human B Cell Activation, Proliferation, and Plasmablast Differentiation

“…Lysates were loaded into a Covaris sonication microtube and sonicated in a Covaris S2 immersion sonicator with a duty cycle of 5%, intensity 2, at 200 burst for 15 cycles at 1 min each. Lysates were then precleared with 75 µL of a 50% agarose protein a/g bead slurry for 1 h. IRF5 immunoprecipitation (IP) was performed overnight at 4°C on precleared lyates using 4 µg of validated IRF5 antibody (Abcam#: ab124792) ( 34 ). A 40% protein a/g bead slurry was added to each IP for 3 h to allow conjugation of IRF5 antibody to beads.…”

SOCS1 and SOCS3 Target IRF7 Degradation To Suppress TLR7-Mediated Type I IFN Production of Human Plasmacytoid Dendritic Cells

“…The human pDC cell line CAL-1 (kindly provided by Drs. T. Maeda and S. Kamihira, Department of Island Medicine, Nagasaki University, Japan) was cultured in complete RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% FCS (29). Sixteen hours prior to stimulation, 1 3 10 6 CAL-1 cells/well were starved with 1 ml serumreduced medium (1% FCS) in a 48-well plate and then stimulated with 5 mg R837 or 3.3 3 10 6 genome heat-inactivated IAV for the indicated time.…”

“…Immunofluorescence staining was performed as described previously (29,30). Briefly, primary pDCs remained unstimulated or stimulated with R837 or heatinactivated IAV for 3 h. Cells were then fixed in 3% paraformaldehyde and permeabilized with 0.1% saponin.…”