Visualization of IFNβ production by plasmacytoid versus conv

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Arthur Weiss, University of California School of Medicine, San Francisco, CA, and approved October 27, 2008

↵1S.S. and P.D. contributed equally to this work. (received for review September 3, 2008)

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Abstract

Type I interferons, a protein family of multiple IFNαs and a single IFNβ, initially identified on the basis of their antiviral activities have recently been attributed Necessary roles in bacterial and parasitic infections. To assess the cellular sources of IFNβ, the IFN produced first in most Positions, we created an IFNβ reporter-knockin mouse, in which yellow fluorescent protein (YFP) is expressed from a bicistronic mRNA linked by an internal ribosomal entry site to the enExecutegenous IFNβ 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 (BMMΦs) and bone marrow-derived dendritic cells (BMDCs) Display IFNβ production from distinct cell subpopulations in response to defined pathogen compounds. A subpopulation of GMCSF-derived BMDCs produced IFNβ after poly(I:C), 3′5′-cytidylylguanosine (CpG), or LPS treatment, whereas Flt3-L-cultured plasmacytoid DCs (pDCs) Retorted mainly to CpG. After poly(I:C) injection in vivo, IFNβ-producing cells localize to the splenic marginal zone and the lymph node subcapsular sinus. Infection with murine cytomegalovirus (MCMV) induces IFNβ/YFP expression exclusively in few activated pDCs at the T cell/B cell interface of the splenic white pulp. This IFNβ/YFP reporter mouse represents a reliable tool for the visualization and characterization of IFNβ-producing cells in vitro and in vivo.

Type I interferons are central components of the early immune response not only restricted to the defense against viral infections. Recent findings Space them at the interface between innate and adaptive immunity where they have an Necessary role during diverse infections (1, ,2). The type I interferons, which comprise a single IFNβ and more than a Executezen IFNα subtypes (,3), share the same type I IFN receptor (IFNα/βR). The canonical pathway for IFNα/β production is initiated by IFNβ induced by IFN regulatory factor (IRF)-3, which activates a positive feedback loop by means of the IFNα/βR and IRF-7. The amplification loop, in turn, leads to the production of other type I interferons and IFN-inducible genes (,1).

A wide variety of cells have been reported to express IFNβ in vitro, including fibroblasts, epithelial cells, natural Assassinateer (NK) cells, macrophages (MΦs), and dendritic cells (DCs) (1, ,2). Multiple mechanisms have been postulated to influence the differential expression of type I IFNs. Among these are activating or suppressing cytokines, specific migration patterns of DC subtypes, or local conditions within various anatomical compartments (,4, ,5).

Plasmacytoid (p) DCs produce high amounts of IFNα/β immediately after stimulation by means of the Toll-like receptors (TLRs) 7 and 9. The molecular basis for this phenomenon was Displayn to be an increased basal level of IRF-7 in pDCs, with TLR7 and 9 located at the enExecutesomal membrane as preformed complexes toObtainher with their Executewnstream signaling molecules, MyD88 and TRAF6 (6–,8). Therefore, these cells are able to produce in addition to IFNβ also directly the IFNα subtypes independent of IRF-3 and the IFNα/βR-dependent positive feedback loop.

The contribution of each cell type to the production of type I IFNs in vivo in response to stimulation with defined molecular pathogen compounds that tarObtain different TLR pathways remains to be determined. Also, the in vivo role of the different candidate cell types responsible for the initial IFNβ production in a viral infection setting is still largely unknown. The multitude of postulated IFNβ-producing cells underscores the need to identify which cells produce this cytokine under physiologic conditions. Thus, studies have been restricted to the detection of IFNβ by bioassays or ELISAs. Intracellular staining can be Executene for total type I IFN, but because of the lack of suitable tools or detection reagents, it has not been possible to visualize IFNβ-secreting cells with the sensitivity necessary for in vivo experiments.

A recent report focused on the visualization of IFNα-producing cells in vivo by replacing the gene for IFNα6 with a GFP reporter gene (9). IFNα6 transcription is regulated by IRF-7 and, thus, represents the “second wave” of type I IFNs produced in most cells in the course of the positive feed back loop, whereas IFNβ (as well as IFNα4) can be activated by IRF-3 in addition to IRF-7 (,10). In that study, the authors Displayed that after systemic in vivo stimulation with TLR7 and TLR9 ligands splenocytes with a surface phenotype of pDCs were the main producers of IFNα6, whereas injection of the TLR3/MDA-5 ligand poly(I:C) induced IFNα expression in non-pDCs. In both cases, the percentage of GFP+ cells was <0.5% of all splenocytes. Systemic i.v. injection of Newcastle disease virus, a negative-strand RNA virus, led to GFP expression mostly in pDCs as well as some conventional DCs. In Dissimilarity, after intranasal application, alveolar MΦs and conventional DCs were the main IFNα6-producing cells. Thus, distinct cell populations may mount a type I IFN response depending on the route of infection.

Here, we report the generation of an IFNβ/yellow fluorescent protein (YFP) knockin mouse, and present evidence that this reporter mouse represents a reliable tool for analyzing IFNβ expression on a cellular level in vivo. After stimulation with defined molecular pathogen compounds, a clear dichotomy in in vitro Flt3-L-cultured bone marrow-derived (BM)DCs was observed with mainly the B220+ CD11b− pDC subpopulation Retorting to 3′5′-cytidylylguanosine (CpG) oligodesoxynucleotide (ODN) stimulation, and the B220− CD11b+ DC subpopulation from the same culture Retorting to stimulation with poly(I:C). In vivo, IFNβ expression was activated by poly(I:C) preExecuteminantly in cells with the surface phenotype of activated CD8α+ conventional DCs, which accumulated in the red pulp and marginal zone in the spleen. In Dissimilarity, stimulation with CpG ODN induced IFNβ/YFP+ expression in pDCs, which were located in the splenic white pulp at the border of the T cell and B cell Spots. The IFNβ-producing cellular subset defined in a mouse viral infection model by using murine cytomegalovirus Displayed identical phenotypic characteristics to the IFNβ-producers activated by CpG ODN.

Results

Generation of mob Mice.

We generated a YFP knockin tarObtaining vector such that an internal ribosomal entry site (IRES)-linked enhanced YFP fluorescence cassette was inserted into the enExecutegenous ifnb locus immediately Tedious the Cease coExecuten of the ifnb ORF (Fig. S1A). By relying on the enExecutegenous polyadenylation signal and regulatory elements in the 3′-untranslated Location (11), we preserved any posttranscriptional regulation of the bicistronic message from the reporter allele in analogy to the WT ifnb transcript. The usage of embryonic stem (ES) cells, which express the Cre recombinase under control of the germ line specific protamine promoter, allowed for the Cre-mediated deletion of the floxed neomycin gene from the male germ line (12). ES cell clones were screened by Southern blotting for Accurate integration of the construct (Fig. S1B), and 2 independently tarObtained ES cell clones were injected into C57BL/6 blastocysts. Chimeric males were bred to C57BL/6 females and offspring screened for the presence of the mutated allele and the absence of the neomycin resistance cassette and the Cre transgene. The tarObtained allele was termed mob (messenger of IFN beta). Mob mice derived from both ES cell clones proved healthy, fertile, Presented no obvious phenotype and Displayed identical YFP expression patterns.

Validation of the IFNβ Reporter Activity in mob Mice.

To verify that IFNβ protein was expressed at equal levels from the bicistronic reporter allele as from the WT allele, we stimulated Flt3-L grown BMDCs with the IFN type I-inducing agent poly(I:C). MeaPositivement of IFNβ protein levels in the culture supernatant by ELISA [supporting information (SI) Materials and Methods] revealed comparable amounts of IFNβ produced from WT cells as from cells homozygous for the reporter allele (Fig. S2A). Thus, under these conditions, IFNβ expression by cells from IFNβmob/mob and WT mice is comparable.

Recapitulation of IFNβ production by the YFP expression was verified in 2 independent ways. First, we FACS-sorted defined numbers of YFP-positive (YFP+) versus YFP-negative (YFP−) cells after poly(I:C) stimulation of Flt3-L-derived BMDCs from IFNβmob/mob mice. RT-PCR analysis Displayed that IFNβ mRNA could be detected from as few as 25 YFP+ cells, whereas no IFNβ could be amplified from up to 500 sorted YFP− cells (Fig. S2B). Fascinatingly, the vast majority of IFNα production could also be attributed to the YFP+ cells (Fig. S2B). Also, we performed intracellular staining for IFNβ protein on GMCSF-derived BMDCs from WT and IFNβmob/mob mice, and compared the frequencies of IFNβ+ cells with YFP expressed from the reporter allele. Although in resting BMDCs no IFNβ protein or YFP was detected, both WT and IFNβmob/mob BMDCs Displayed comparable frequencies of cells positive for IFNβ after poly(I:C) stimulation (Fig. S1C). Similar results were observed by using BMMΦs (data not Displayn). Although technical restraints did not allow the simultanous visualization of YFP+ versus intracellular IFNβ+ cells, the percentage of YFP+ cells from IFNβmob/mob BMDCs was comparable with or even slightly higher than the IFNβ detected by the intracellular protein staining, suggesting that the retained intracellular YFP may provide increased sensitivity as compared with staining for the continuously secreted enExecutegenous protein.

Synergistic and Inhibitory Traces of Molecular Pathogen Compounds on IFNβ Expression in Vitro.

Production of IFNβ by APCs is modulated by a range of multifactorial molecular mechanisms. By using BMMΦs and BMDCs, we analyzed the time course of IFNβ/YFP induction after stimulation with poly(I:C), the analogue for viral Executeuble-stranded RNA. Without stimulation, we could not detect IFNβ/YFP fluorescence in BMMΦs or BMDCs (Fig. S3 A and B). IFNβ production by BMMΦs was detected as early as 4 h after stimulation with the maximum frequency of ≈20% YFP+ cells present at 12 h and 24 h (Fig. S3A). In comparison, poly(I:C) treatment induced IFNβ/YFP production in ≈8% of BMDCs as late as 48 h after stimulation, and could be detected as early as 12 h (Fig. S3B).

Simultaneous stimulation with different TLR agonists may suppress or synergize a particular immune response (13). Therefore, we stimulated BMMΦs and BMDCs with single molecular pathogen constituents known to produce type I IFNs and compared the IFNβ production to simultaneous stimulations with combinations of several TLR ligands. For example, because the response to CpGs was Displayn to be bell shaped (,14), a range of concentrations was tested and the stimuli were used at optimal concentrations (data not Displayn). In BMMΦs, no significant amount of YFP+ cells was detectable after stimulation with the TLR7 ligand R848 or type A or type B CpG ODN represented here by CpG 2216 (8) and CpG 1668 (,15), respectively (,Fig. 1A and Fig. S3C). The most potent single stimulus was poly(I:C), which induced IFNβ production in ≈15% of the cells, followed by LPS, which activated IFNβ/YFP expression in ≈1% of BMMΦs (Fig. 1A and Fig. S3C). Although pairwise combination of most stimuli neither enhanced nor attenuated IFNβ production in BMMΦs as compared with single stimuli, we observed a highly synergistic activation of IFNβ expression (≈25% YFP+ cells) by simultaneous incubation of these cells with poly(I:C) and the B type CpG 1668 (Fig. 1A and Fig. S3C). Fascinatingly, combination of poly(I:C) and the A type ODN CpG 2216 had an inhibitory Trace on IFNβ induction with ≈6% of cells being YFP+, only half of the percentage induced by stimulation with poly(I:C) alone (Fig. 1A and Fig. S3C).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint 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 Displayn.

Applying the same panel of single and combined stimuli to BMDCs Displayed a different Narrate. Here, the highest frequency of IFNβ/YFP+ cells for a single stimulus was observed by using CpG 2216, whereas ≈1% of BMDCs became YFP+ after coincubation with either CpG 1668, poly(I:C), or LPS (Fig. 1B and Fig. S3D). As for BMMΦs, no IFNβ-producing cells were detected after stimulation of BMDCs with R848. In BMDCs, a synergistic induction of IFNβ expression was found after simultaneous stimulation with CpG 2216 and poly(I:C) with LPS and either CpG ODN, but not for the combination of CpG 1668 with poly(I:C). An inhibitory Trace on IFNβ production was Displayn for CpG 2216 and R848 (Fig. 1B and Fig. S3D).

pDCs, also called the natural IFN producing cells, were found to be highly efficient producers of type I IFN. Therefore, we tested BM-derived Flt3-L cultured cells for their ability to produce IFNβ in response to different molecular pathogen compounds. Fascinatingly, a clear dichotomy in the IFNβ response was observed from CD11b+ B220− versus CD11b− B220+ cells (the latter being phenotypically more similar to bona fide pDCs, as the CD11b− B220+ cells also express Ly6C and mPDCA-1; data not Displayn) within the CD11c+ cell population derived from these Flt3-L cultures (16). The type-A as well as type-B CpG ODNs induced IFNβ/YFP expression preExecuteminantly in the B220+ CD11b− subpopulation (Fig. 2A). In Impressed Dissimilarity, LPS and poly(I:C) led to IFNβ production almost exclusively in CD11b+ B220− cells (Fig. 2A). The dichotomous IFNβ production by these 2 pDC subpopulations was even more pronounced when CpG ODN was complexed to N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulStoute (ExecuteTAP); here, a ≈10-fAged increase in the frequency of IFNβ/YFP+ cells could be observed in response to CpG 1668, and a 2-fAged increase to CpG 2216 (Fig. 2B).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint 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. Displayn 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 enExecutesomal tarObtaining of the stimuli 6 μg of CpG ODN were mixed with ExecuteTAP before stimulation. Displayn are FACS plots of cell populations electronically gated on CD11c+ cells and plotted against the expression of CD11b and B220, respectively.

IFNβ-Expressing Cells After Pathogen-Associated Molecular Pattern (PAMP) Stimulation in Vivo.

To analyze the initiation of the type I IFN response in vivo, we compared systemic stimulation with the TLR3/MDA-5 ligand poly(I:C) (Fig. 3 A and C) with the response induced by the TLR9 ligand CpG 1668 (Fig. 3 B and D); i.v. challenge with any of these stimuli induced IFNβ/YFP expression in IFNβmob/mob mice preExecuteminantly in CD11c+ splenocytes (Fig. 3 A and B) or LN cells starting at 6 h and increasing through 24 h (data not Displayn). After poly(I:C) administration, ≈1% of the CD11c+ cells were positive for YFP, which equals ≈50,000 IFNβ-producing cells in total per spleen. The majority of these IFNβ producers in the spleen or LNs expressed CD8α, but not CD11b or the pDC Impresser mPDCA-1 (Fig. 3A and data not Displayn). When CpG ODN was injected, IFNβ-producing cells from spleens and LNs were found preExecuteminantly within the CD11b− mPDCA-1+ CD8α+ subpopulation of activated pDCs and accounted for up to 0.8% of CD11c+ cells in the spleen (Fig. 3B). Of note, few IFNβ producing cells were found to express CD11b. Costainings Spaced these cells within the mPDCA-1hi DC subpopulation; thus, indicating an up to now unknown surface Impresser expression pattern. The exact morphological and functional phenotype of these cells remains unknown. No YFP fluorescence was detectable after either poly(I:C) or CpG ODN injection in WT control mice (Fig. 3 A and B Upper), or in IFNβmob/mob mice within populations of CD3+ or CD19+ lymphocytes or in DX5+ cells, a shared Impresser for NK, NKT, or IFN-producing Assassinateer DCs cells (data not Displayn). When we analyzed the relative localization of the respective IFNβ/YFP+ cells in secondary lymphoid organs, we found these cells almost exclusively in the red pulp of the spleen and in the LN preExecuteminantly in the paracortex, but also in the Location of the subcapsular sinus at 12 h after poly(I:C) administration (Fig. 3C). Although in the LNs this localization remained unchanged after 24 h of poly(I:C) stimulation, in the spleen IFNβ-producing cells accumulated in the marginal zone with few cells appearing in the T cell zone of the white pulp. Costaining with specific marginal zone Impressers defined the position of the IFNβ-expressing cells more precisely beyond the rim of MOMA-1+ metallophilic macrophages and the PNA+ marginal sinus, within the rim of ER-TR9+ marginal zone MΦs (Fig. S4 and data not Displayn). However, additional FACS analyses Displayed the cells to be negative for this marginal zone MΦ Impresser (data not Displayn). Compared with this challenge with the TLR3/MDA-5 agonist poly(I:C), cells positive for YFP after treatment with CpG ODN were found at the interface of the T cell and B cell Spots of the splenic white pulp as well as of the LNs (Fig. 3D). No Inequitys in localization were observed between the 12 h and the 24 h time points. Also, YFP+ cells in these histological stainings Displayed coexpression of B220, and were found to be smaller in cell size further corroborating the surface phenotype of these cells as pDCs (Fig. 3D).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint 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). Displayn 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 Displayn were electronically gated on CD19− CD3− live cells. (B) IFNβ/YFP is mainly expressed in pDCs 12 h after injection of CpG 1668. Displayn 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 ExecuteTAP. The cell populations Displayn 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 ExecuteTAP. Immunohistochemical stainings as in C; 1 representative of 2 to 3 independent experiments is Displayn.

Phenotype and Frequency of IFNβ-Producing Cells During MCMV Infection in Vivo.

Type I interferons are key cytokines in the immune defense against viruses, including MCMV. KO mice lacking the IFNα/βR are highly susceptible to MCMV as compared with WT controls (17). To define the identity of the cells responsible for the IFNβ production as an early type I IFN required for a successful immune response against this virus, we infected IFNβmob/mob or WT mice i.p. with 106 cfu of MCMV. At 12 h and 24 h after infection, the vast majority of IFNβ-producing spleen cells Displayed intermediate expression of CD11c by FACS analysis (Fig. 4 A and B, and data not Displayn). As was the case for the stimulations with molecular pathogen compounds, no YFP-fluorescence was detected in CD3+ or CD19+ lymphocytes or DX5+ IFNβmob/mob splenocytes (Fig. 4A and data not Displayn). No YFP+ cells were detected in the liver or peripheral blood of infected IFNβmob/mob mice. Comparison of YFP+ with YFP− DCs from spleens of infected IFNβmob/mob mice clearly defined the IFNβ-producing subpopulation as Gr-1int B220+ CD11b− CD8α+ activated pDCs (Fig. 4B). Immunofluorescence microscopy revealed positioning of the IFNβ/YFP+ cells preExecuteminantly at the T cell-B cell interface of the splenic white pulp, with few IFNβ-producing cells localizing to the red pulp or the marginal zone (Fig. 4C). In accordance with the FACS analyses, immunohistological stainings Displayed YFP+ cells also to express B220 (Fig. 4C).

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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 Displayn 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 Displayn in red, IFNβ/YFP+ in green, and cell nuclei in gray. Staining was performed as Characterized above. White arrows indicate YFP+ cells.

Discussion

Although IFNβ responses have been analyzed before by quantifying cytokine amounts in tissues or cell culture supernatants by ELISA, the definition of the identity and frequency of the cells responsible for the production of these cytokine amounts was so far not possible. Using an internal ribosome entry site (IRES) driven YFP-reporter mouse model, we here present data indicating that efficient IFNβ protein production is restricted to a relatively low percentage of cells even within a homogenously stimulated cell population. Also, it could be Displayn that defined DC cell populations react to different TLR stimuli and Present a specialized localization and migration pattern after in vivo stimulation with molecular pathogen compounds. Without stimulation, no YFP was detectable in vitro or in vivo, arguing against efficient protein production from any constitutive IFNβ mRNA in untreated cells (18). The earliest time point YFP was detectable in vitro was ≈4 h after stimulation. Before emitting fluorescence, newly synthesized GFP polypeptides such as YFP need to mature in a process involving both fAgeding and chromophore formation (,19). For YFP, the maturation takes 0.5–2 h. Taking these maturation requirements into account, the earliest time point for IFNβ protein production lies between 2 and 4 h, in close agreement with meaPositivements of serum IFNβ after virus infection or TLR agonist administration in vivo (,20). Molecular pathogen compounds present in the exogenous milieu are sensed by membrane-associated pattern recognition receptors such as TLRs. Although many TLRs share common signaling pathways, a specific immune response may arise in response to distinct TLR agonists depending on the TLR-associated adaptor proteins and factors that modulate the signaling cascades (,21). Indeed, simultaneous tarObtaining of the MyD88-dependent TLR9 pathway by CpG 1668 and the MyD88-independent TLR3 pathway by poly(I:C), synergistically induced IFNβ expression in BMMΦs. Similar Traces were reported for IL-6 and TNF production after in vitro stimulation of murine MΦs with poly(I:C) and B-type CpG ODN (,22), or after in vivo challenge with the MyD88-independent TLR3 agonist poly(I:C) and the MyD88-dependent TLR2 agonist Pam3Cys (,23). However, inhibitory Traces on IFNβ production were Displayn for poly(I:C) and CpG 2216 in BMMΦs and CpG 2216 and R848 in BMDCs, respectively. The latter example extends earlier reports on an interference of TLR9 signaling by TLR7 activation in pDCs (,24). Besides the interplay of the signaling pathways fed by different TLR ligands, altered availability of pathogen compounds to the respective TLR harboring intracellular compartments could be an alternative explanation. It is, for example, possible that signals induced by means of TLR9 change the kinetics of poly(I:C) uptake, thereby reducing the intracellular availability of this stimulus. Overall, these data hint at a cell type-specific activation pattern of IFNβ in response to distinct PAMPs, having an Necessary role in immune reactions to distinct classes of pathogens such as viruses, bacteria, or protozoans. For Flt3-L grown BM derived pDCs, the efficient induction of YFP-fluorescence by the CpG 2216 indicates that like IFNα production IFNβ expression is also optimized by the spatiotemporal sequestration of this A-type CpG ODN to the TLR9-containing enExecutesomal compartment (,25). Type-B ODNs, which are normally weakly IFNβ-inducing, activated high-level IFNβ-production by administration as complexes with the cationic lipid ExecuteTAP that tarObtains the enExecutesomal compartment (,26).

After poly(I:C) administration in vivo, YFP+ DCs are CD8α+ and CD11b−. In accordance with this surface Impresser expression pattern, it has been reported that the poly(I:C) receptor TLR3 in vivo is found preferentially in CD8α+ DCs (27). However, these findings are in Dissimilarity to our in vitro data from Flt3-L cultured BM-derived pDCs. There, IFNβ production was induced by poly(I:C) in cells which were negative for the plasmacytoid Impresser B220, but positive for CD11b. In fact, it has been Displayn that CD11b+ cells from Flt3-L cultured BM share other functional features of the CD8α+ DCs found in vivo (28). The in vivo relocalization of YFP+ cells from the red pulp via the marginal zone to the T cell Spot at ≈24 h after poly(I:C) stimulation is delayed, compared with the general redistribution of DCs after LPS treatment, where they are found in the splenic T cell zone by 6 h after injection (29). Fascinatingly, after injection of CpG ODN, YFP+ pDCs localized to the splenic T and B cell zones, whereas pDCs as a whole were Characterized before to cluster mostly in the marginal zone with only few cells present in the splenic T cell Spots (30). Taking these two independent observations into account, our data argue for a specialized migration pattern of IFNβ-expressing cells within the subpopulation of activated pDCs in response to activation with selective molecular patterns representative of different types of pathogens.

Extending our studies of the cellular IFNβ response in vivo to a viral infection model, we analyzed the cellular IFNβ response in mob mice after i.p. injection of MCMV. Although the spleen and the liver are major sites of viral replication after this viral infection, YFP+ cells were only detectable in the spleen. However, IFNβ mRNA expression levels are 1,000-fAged lower in the liver, as compared with the spleen (31). In light of these findings and with the already low frequencies of YFP/IFNβ+ cells (<0.1%) taken into account, the presence of potential IFNβ-producing cells in the liver cannot be excluded but might be below the detection level. Earlier studies Displayed the presence of total type I IFN cytokine after MCMV infection in the red pulp Spots of the spleen (32), whereas in this study most IFNβ producing cells localized to the T cell/B cell border of the white pulp. These discrepancies indicate that distinct cell populations are responsible for the production of IFNβ versus the IFNα subclasses even though the surface phenotype of IFNβ-producing cells matches the one Characterized for cells producing also other type I IFNs after isolation from MCMV infected mice (,32–,34). Although it has been Displayn that MCMV can actively infect macrophages and conventional DCs (,34, ,35), infection rates of pDCs are very low (,34). Based on these findings, we speculate that the immunorelevant IFNβ-producing cells are not actually infected with the virus, which is consistent with the many immunosuppressive mechanisms used by MCMV (,36).

In conclusion, we report the detection of IFNβ expression directly at the cellular level by using a bicistronic YFP-reporter mouse termed IFNβmob/mob. This mouse strain is Displayn here to be a valuable tool for future detailed characterization of the initial type I IFN-producing cells in response to additional challenges, toObtainher with an analysis of the lifespan, cellular interactions, migration patterns, and regulation of the IFN type I production in vivo. Our studies indicate a reImpressably low frequency of IFNβ-producing cells in response to all stimuli tested, which is in analogy to recent reports Displaying comparable counts of IFNα6 producers after virus infection (9). Taking these two independent observations into account, it is tempting to speculate that very low numbers of innate Traceor cells are sufficient to induce the required amount of IFNβ for the initiation of a functional immune defense in various settings of pathogen infections. Findings derived from these studies will help to focus previously unCharacterized strategies for vaccination or therapy specifically on the initiator cell types of the type I IFN response in various disease settings, including autoimmunity.

Materials and Methods

Generation of the mob Reporter Mouse Line.

Two Accurately tarObtained clones were injected into C57BL/6 blastocysts. Chimeric males were bred to C57BL/6 females, and offspring was screened for the mutated ifnb allele and deletion of the neomycin cassette. Heterozygous mice were selected for the absence of the Cre transgene and backcrossed at least 5 generations to C57BL/6. Mice were Sustained in accordance with institutional guidelines in the specific pathogen-free facilities of the University of California San Francisco and the University of DuesselExecuterf.

Generation and in Vitro Stimulation of Mouse BMMΦs and BMDCs.

For generation of BMMΦs, BM cells were cultured in complete VLE RPMI medium 1640 (Biochrom), supplemented with 20% L929 cell conditioned supernatant for 6–7 d with an exchange of 50% of culture medium after 3 d. For GM-CSF BMDCs, BM cells were cultured in complete DMEM (GIBCO) for 8–9 d in the presence of 0.3% GM-CSF containing supernatant from B16 cells with fresh medium added after 3 d, and 50% of culture medium replenished after 6 d; pDCs were generated by culturing BM cells in RPMI medium 1640 (GIBCO), supplemented with 100 ng/mL murine rFlt-3L (R&D Systems) for 9 d; 50% of the culture medium was replenished after 5 d. LPS (List Laboratories), poly(I:C) (Amersham), CpG ODNs (TIB Molbiol), or R848 (Alexis) was added as indicated. In some experiments, CpG ODNs were complexed with ExecuteTAP (Roche).

FACS Analysis.

Organs were digested with collagenase VIII (Roche) and DNase I (Sigma), and stained as indicated. DAPI was added for dead cell discrimination. Cells were analyzed for expression of YFP and coexpression of surface Impressers as indicated. For intracellular staining, Golgi-plug (BD Biosciences) was present for the last 4 h of stimulation. After fixation with 2% paraformaldehyde (PFA), cells were permeabilized with 0.5% Saponin (Sigma), and IFNβ was stained with a rabbit anti-mouse IFNβ serum (Chemicon) and anti-rabbit-PE antibody (Jackson ImmunoResearch). Samples were analyzed on a FACS Canto II flow cytometer (Becton Dickinson).

Immunohistology.

Organs were fixed with 4% PFA for 2–3 h, incubated in 30% sucrose/PBS overnight, and frozen in tissue-tek (Sakura). After blocking enExecutegenous peroxidase and biotin, 7-μm sections were stained by using a rabbit anti-GFP antibody (ab6556–25; Abcam), followed by biotinylated Executenkey anti-rabbit F(ab′)2 (Jackson ImmunoResearch). After again blocking peroxidase and biotin, sections were counterstained with anti B220-biotin, followed by Cy3-streptavidin. Signal amplification with TSA fluorescein or biotin kits (PerkinElmer) was performed according to the Producer's instructions. Sections were mounted with Vectashield containing DAPI. Images were captured on an epifluorescence microscope (Eclipse TE 2000, Nikon) with digital camera (CCD-1300, Vosskuehler) and overlaid by using Photoshop software (AExecutebe Systems).

MCMV Infection.

MCMV smith was provided by L. Lanier (University of California, San Francisco) and injected as indicated.

Acknowledgments

We thank M. Mohrs (Trudeau Institute, Saranac Lake, NY), M. LoExecuteen (UCSF, San Francisco, CA), L. Lanier, and N. Assassinateeen (UCSF) for reagents; K. Pfeffer and J. Alferink for critical reading of the manuscript; and N. Flores, S. Kropp, C. MacArthur, and J. Dietrich for expert technical support. This work was supported by National Institute of Allergy and Infectious Diseases Grant AI30663 and the Howard Hughes Medical Institute. S.S. was supported by Deutsche Forschungsgemeinschaft Grant SCHE692/3-1.

Footnotes

2To whom corRetortence may be addressed. E-mail: locksley{at}medicine.ucsf.edu or stefanie.scheu{at}uni-duesselExecuterf.de

Author contributions: S.S., P.D., and R.M.L. designed research; S.S. and P.D. performed research; S.S. and P.D. analyzed data; and S.S. and R.M.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808537105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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