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. 2008 Dec 23;105(51):20416-21.
doi: 10.1073/pnas.0808537105. Epub 2008 Dec 16.

Visualization of IFNbeta production by plasmacytoid versus conventional dendritic cells under specific stimulation conditions in vivo

Stefanie Scheu  1 , Philipp Dresing, Richard M Locksley
Affiliations

Visualization of IFNbeta production by plasmacytoid versus conventional dendritic cells under specific stimulation conditions in vivo

Stefanie Scheu et al. Proc Natl Acad Sci U S A. .

Abstract

Type I interferons, a protein family of multiple IFNalphas and a single IFNbeta, initially identified on the basis of their antiviral activities have recently been attributed important roles in bacterial and parasitic infections. To assess the cellular sources of IFNbeta, the IFN produced first in most situations, we created an IFNbeta reporter-knockin mouse, in which yellow fluorescent protein (YFP) is expressed from a bicistronic mRNA linked by an internal ribosomal entry site to the endogenous IFNbeta mRNA. This YFP expression allows spatiotemporal tracking of the initiation of the type I IFN response on a single-cell level. In vitro bone marrow-derived macrophages (BMMPhis) and bone marrow-derived dendritic cells (BMDCs) show IFNbeta production from distinct cell subpopulations in response to defined pathogen compounds. A subpopulation of GMCSF-derived BMDCs produced IFNbeta after poly(I:C), 3'5'-cytidylylguanosine (CpG), or LPS treatment, whereas Flt3-L-cultured plasmacytoid DCs (pDCs) responded mainly to CpG. After poly(I:C) injection in vivo, IFNbeta-producing cells localize to the splenic marginal zone and the lymph node subcapsular sinus. Infection with murine cytomegalovirus (MCMV) induces IFNbeta/YFP expression exclusively in few activated pDCs at the T cell/B cell interface of the splenic white pulp. This IFNbeta/YFP reporter mouse represents a reliable tool for the visualization and characterization of IFNbeta-producing cells in vitro and in vivo.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
In vitro IFNβ/YFP expression induced by molecular pathogen compounds in BMMΦs and BMDCs. BMMΦs (A) or GMCSF-derived BMDCs (B) from WT or IFNβmob/mob mice were stimulated for 24 h with 50 μg/mL poly(I:C), 6 μg/mL CpG 1668, 100 ng/mL LPS, 10 μg/mL R848 alone or in the stated combinations given simultaneously, or were left untreated. Bars represent the percentages of YFP+ cells within CD11b+ cells from BMMΦs or from CD11c+ cells from BMDCs, respectively; 1 representative of 3 independent experiments is shown.
Fig. 2.
Fig. 2.
In vitro IFNβ/YFP expression from Flt3-L-derived BM-derived DCs reveals a dichotomy in the IFNβ response after molecular pathogen compound stimulation. (A) BM cells of WT or IFNβmob/mob mice were grown with 100 ng/mL Flt3-L for 10 d, replated and stimulated for 24 h with 100 ng/mL LPS, 6 μg/mL CpG 1668 or CpG 2216, 50 μg/mL poly(I:C), or left untreated. Shown are FACS plots of cell populations electronically gated on CD11c+ cells and plotted against the expression of CD11b and B220, respectively. (B) Flt3-L-derived BMDCs were generated as in A. For endosomal targeting of the stimuli 6 μg of CpG ODN were mixed with DOTAP before stimulation. Shown are FACS plots of cell populations electronically gated on CD11c+ cells and plotted against the expression of CD11b and B220, respectively.
Fig. 3.
Fig. 3.
In vivo IFNβ/YFP expression induced by poly(I:C) or CpG ODN in spleen and LNs. (A) The majority of IFNβ/YFP+ cells are lymphoid DCs 12 h after injection of poly(I:C). Shown are FACS stains of spleens from mice of the indicated genotype 12 h after i.v. injection of 200 μg of poly(I:C). The cell populations shown were electronically gated on CD19− CD3− live cells. (B) IFNβ/YFP is mainly expressed in pDCs 12 h after injection of CpG 1668. Shown are FACS stains of spleens from mice of the indicated genotype 12 h after i.v. injection of 1.5 nmol of CpG 1668 complexed to DOTAP. The cell populations shown were electronically gated on CD19− CD3− cells. (C) Localization of YFP+ cells in the spleen or LNs 12 h and 24 h after i.v. injection of 200 μg of poly(I:C). YFP+ cells were detected by using a cross-reacting polyclonal anti-GFP serum. Signals were amplified with tyramide-FITC for YFP and tyramide-bio and streptavidin-Cy3 for B220. Nuclei in gray stained with DAPI. (D) Localization of YFP+ cells in the spleen or LNs 12 h and 24 h after i.v. injection of 1.5 nmol of CpG 1668 complexed to DOTAP. Immunohistochemical stainings as in C; 1 representative of 2 to 3 independent experiments is shown.
Fig. 4.
Fig. 4.
Plasmacytoid DCs are the main IFNβ-producing cells after Murine CMV infection. (A) FACS analysis of spleens from IFNβmob/mob or WT mice 12 h after infection with 106 CFU MCMV i.p. identifies the majority of YFP+ cells as CD11cint. The cell populations shown were electronically gated on CD19− CD3− cells. (B) Differential expression of pDC surface antigens on YFP+ versus YFP− cells after MCMV infection. FACS histograms from spleens of MCMV infected IFNβmob/mob mice were gated for YFP+ or YFP− cells and overlayed. Pregating as in A. (C) Localization of IFNβ/YFP+ cells at the T cell/B cell interface of the splenic white pulp of MCMV infected IFNβmob/mob mice. B220+ cells are shown in red, IFNβ/YFP+ in green, and cell nuclei in gray. Staining was performed as described above. White arrows indicate YFP+ cells.
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References

    1. Decker T, Muller M, Stockinger S. The Yin and Yang of type I interferon activity in bacterial infection. Nat Rev Immunol. 2005;5:675–687. - PubMed
    1. Bogdan C, Mattner J, Schleicher U. The role of type I interferons in non-viral infections. Immunol Rev. 2004;202:33–48. - PubMed
    1. van Pesch V, Lanaya H, Renauld JC, Michiels T. Characterization of the murine alpha interferon gene family. J Virol. 2004;78:8219–8228. - PMC - PubMed
    1. Yoneyama H, Matsuno K, Matsushimaa K. Migration of dendritic cells. Int J Hematol. 2005;81:204–207. - PubMed
    1. Liu YJ. IPC: Professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu Rev Immunol. 2005;23:275–306. - PubMed

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