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. 2010 Dec 16;5(12):e15567.
doi: 10.1371/journal.pone.0015567.

A fluorescence reporter model defines "Tip-DCs" as the cellular source of interferon β in murine listeriosis

Philipp Dresing  1 , Stephanie Borkens, Magdalena Kocur, Sonja Kropp, Stefanie Scheu
Affiliations

A fluorescence reporter model defines "Tip-DCs" as the cellular source of interferon β in murine listeriosis

Philipp Dresing et al. PLoS One. .

Abstract

Production of type I interferons, consisting mainly of multiple IFNα subtypes and IFNβ, represents an essential part of the innate immune defense against invading pathogens. While in most situations, namely viral infections, this class of cytokines is indispensable for host survival they mediate a detrimental effect during infection with L. monocytogenes by rendering macrophages insensitive towards IFNγ signalling which leads to a lethal bacterial pathology in mice. Due to a lack of suitable analytic tools the precise identity of the cell population responsible for type I IFN production remains ill-defined and so far these cells have been described to be macrophages. As in general IFNβ is the first type I interferon to be produced, we took advantage of an IFNβ fluorescence reporter-knockin mouse model in which YFP is expressed from a bicistronic mRNA linked by an IRES to the endogenous ifnb mRNA to assess the IFNβ production on a single cell level in situ. Our results showed highest frequencies and absolute numbers of IFNβ+ cells in the spleen 24 h after infection with L. monocytogenes where they were located predominately in the white pulp within the foci of infection. Detailed FACS surface marker analyses, intracellular cytokine stainings and T cell proliferation assays revealed that the IFNβ+ cells were a phenotypically and functionally further specialized subpopulation of TNF and iNOS producing DCs (Tip-DCs) which are known to be essential for the early containment of L. monocytogenes infection. We proved that the IFNβ+ cells exhibited the hallmark characteristics of Tip-DCs as they produced iNOS and TNF and possessed T cell priming abilities. These results point to a yet unappreciated ambiguous role for a multi-effector, IFNβ producing subpopulation of Tip-DCs in controlling the balance between containment of L. monocytogenes infection and effects detrimental to the host driven by IFNβ.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. L. monocytogenes challenge induces highest numbers of IFNβ/YFP+ cells 24 hpi. in the the spleen.
IFNβmob/mob or wt mice were infected with sublethal doses of L. monocytogenes as determined by LD50 survival experiments for the stated timepoints and route of infection (data not shown). The bars show the percentages (A) and absolute numbers (B) of YFP+ cells as measured by flow cytometry in the respective organs. The data shown is from at least two independent experiments with two mice per timepoint.
Figure 2
Figure 2. Peak frequencies of IFNβ expressing cells after L. monocytogenes infection in spleen, liver and mLNs.
The peaks of IFNβ production were determined by timecourse experiments and infection dose titrations for spleen, mLNs and liver, respectively. IFNβ/YFP+ cells from the spleen and the liver are shown 24 h after i.v. infection with 106 or i.p. infection with 107 CFU of L. monocytogenes. IFNβ producing cells in the mLNs are presented 48 h after i.v. infection with 105 or i.p. infection with 106 CFU of L. monocytogenes. The cell populations were electronically gated on CD19− CD3ε− live cells. YFP gating was adjusted to equally treated wt references (not shown). The data shown is representative for at least two independent experiments with two mice per timepoint.
Figure 3
Figure 3. IFNβ/YFP expressing cells are located within the foci of infection in the splenic white pulp.
(A) Shown are spleen sections from IFNβmob/mob mice 24 h after i.v. injection of 106 CFU of L. monocytogenes. YFP+ cells were detected using a cross reacting polyclonal α-GFP antibody. Signals were amplified with tyramide-FITC for YFP and B220 (shown in green) and tyramide-BIO and Streptavidin-Cy3 for α-L. monocytogenes, Gr-1, CD11b, B220 (shown in red). Nuclei in grey stained with DAPI. (B) Presented are high power confocal micrographs of spleen sections from IFNβmob/mob mice stained as described in (A). Nuclei were stained with DAPI shown in blue. The scale equals 10 µm. The micrographs are representative of at least two independent experiments.
Figure 4
Figure 4. Simultaneous visualization of IFNβ/YFP production and the cellular state of infection after L. monocytogenes challenge.
(A) Spleen sections from IFNβmob/mob mice 24 h after i.v. injection of 106 CFU of L. monocytogenes were stained as described before. Arrows or arrowheads point at IFNβ/YFP expressing cells that are infected or not infected, respectively. (B)–(C) Confocal micrographs of spleen sections from IFNβmob/mob mice were prepared as described before. Nuclei were stained with DAPI shown in blue. (C) Shown is an orthogonal section of a z-stack series with the main x/y plane presented as a maximum projection of the z-stack. (D) BMDMs and BMDCs from IFNβmob/mob and wt mice were generated as described before and were infected with the stated MOIs of GFP expressing L. monocytogenes, stimulated with 50 µg/ml poly (I:C) or mock treated for 14 h, respectively,. Extracellular bacteria were killed 1 h p.i. by addition of gentamicin. Cellular IFNβ/YFP production and bacterial load was analyzed by flow cytometry. YFP+ (blue line) and YFP− (red line) cells were gated and overlaid in histograms showing their state of infection as determined by GFP. YFP gating was adjusted to the respective wt control (not shown). The experiments shown were repeated twice with similar results.
Figure 5
Figure 5. IFNβ/YFP producing cells reveal key features of Tip-DCs after L. monocytogenes infection.
(A) IFNβmob/mob mice were i.v. infected with 106 CFU of L. monocytogenes for 24 h. The spleens were removed and YFP+ cells shown as red dots were analysed in comparison to YFP− cells shown in grey by backgating of flow cytometric data. (B) Gating strategy for FACS sorting of YFP+ cells from the spleen against YFP− CD11bhi, YFP− bona fide TIP-DCs and YFP− cDCs. (C) 104 cells of the given cell populations were sorted into PBS and plated on blood agar plates. After incubation at 37°C for 14 h the bacterial colonies were counted. (D) Sorted cell populations were stained for intracellular Mac-3 and analyzed by flow cytometry. The distributions of Mac-3 expression within the sorted populations are shown as histograms. (E) iNOS and TNF expression from sorted splenic cell populations was determined after intracellular staining via flow cytometry. The gating for iNOS+ and TNF+ cells was adjusted to the particular staining of cDCs. Ig matched isotype controls were used on sorted total splenocytes. The data shown is representative of at least two independent experiments with spleens from at least two mice pooled for FACS sorting.
Figure 6
Figure 6. IFNβ+ cells show T cell priming capacities.
YFP+, Tip-DCs, cDCs and macrophages were sorted out of spleens from IFNβmob/mob mice (C57BL/6) 24 h after infection with 106 CFU of L. monocytogenes as shown before. After irradiation with 3000 rad these cells were used as stimulators in an allogenic MLR. Proliferation of CD4+ CD62L+ naive responder T cells from allogenic BALB/c and isogenic C57BL/6 responder mice in response to the sorted cell subsets was measured by the incorporation of 3H-Thymidine. The data is representative for two independent experiments with cells originating from at least three mice per group.
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