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. 2013 Oct 15;191(8):4223-34.
doi: 10.4049/jimmunol.1300910. Epub 2013 Sep 18.

Identifying the Initiating Events of anti-Listeria Responses Using Mice With Conditional Loss of IFN-γ Receptor Subunit 1 (IFNGR1)

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Free PMC article

Identifying the Initiating Events of anti-Listeria Responses Using Mice With Conditional Loss of IFN-γ Receptor Subunit 1 (IFNGR1)

Sang Hun Lee et al. J Immunol. .
Free PMC article

Abstract

Although IFN-γ is required for resolution of Listeria monocytogenes infection, the identities of the IFN-γ-responsive cells that initiate the process remain unclear. We addressed this question using novel mice with conditional loss of IFN-γR (IFNGR1). Itgax-cre(+)Ifngr1(f/f) mice with selective IFN-γ unresponsiveness in CD8α(+) dendritic cells displayed increased susceptibility to infection. This phenotype was due to the inability of IFN-γ-unresponsive CD8α(+) dendritic cells to produce the initial burst of IL-12 induced by IFN-γ from TNF-α-activated NK/NKT cells. The defect in early IL-12 production resulted in increased IL-4 production that established a myeloid cell environment favoring Listeria growth. Neutralization of IL-4 restored Listeria resistance in Itgax-cre(+)Ifngr1(f/f) mice. We also found that Itgax-cre(+)Ifngr1(f/f) mice survived infection with low-dose Listeria as the result of a second wave of IL-12 produced by Ly6C(hi) monocytes. Thus, an IFN-γ-driven cascade involving CD8α(+) dendritic cells and NK/NKT cells induces the rapid production of IL-12 that initiates the anti-Listeria response.

Figures

Figure 1
Figure 1. Generation of conditional IFNγ receptor gene targeted mice (Ifngr1f/f)
(A) Targeting strategy. H1, BamH1; Neo, neomycin resistance gene; TK, herpes simplex virus thymidine kinase gene; open triangle, loxP site. Open gray boxes indicate external southern probe to verify 5′ and 3′ homologous recombination. Small arrowheads indicate pairs of primers used in PCR to confirm either 5′ homologous recombination (f3/r3) or insertion of loxP sites (f1/r1 and f2/r2). (B) Confirmation of germline transmission by examining 3′ end of targeted allele by southern blotting. +, wild allele; f, conditional allele. (C) Verification of correct insertion of both 34 bp-long loxP sites by PCR. (D) Expression of IFNGR1 was assessed by flow cytometry analysis of spleen, peripheral blood leukocytes (PBLs), and thymus from wild type, Ifngr1f/f, and Ifngr1WU−/− mice. Data are representative of three separate experiments. (E) Confirmation of germline transmission of the Ifngr1WU−/− allele by 5′ southern blotting. (F) Measurement of phosphorylated STAT1 in Ifngr1WU−/− mice after in vitro IFN stimulation (10,000 U/ml IFNα5, 10,000 U/ml IFNβ, and 1,000 U/ml IFNγ) for 10 minutes at 37°C.
Figure 2
Figure 2. Vav-icre+Ifngr1f/f mice lack functional IFNGR1 in hematopoietic cells
(A) Splenic IFNGR1 expression in Vav-icre+Ifngr1f/f mice was measured. (B) After in vitro IFNγ stimulation (1,000 U/ml) for 15 minutes at 37°C, phosphorylated STAT1 in splenocytes from Vav-icre+Ifngr1f/f mice was analyzed. All Data are representative of at least two separate experiments. Gating strategies are depicted in Supplemental Fig. 1A.
Figure 3
Figure 3. Itgax-cre+Ifngr1f/f mice lack functional IFNGR1 in CD8α+/CD103+ DCs
(A) Splenic IFNGR1 expression in Itgax-cre+Ifngr1f/f mice was measured. IFNGR1 levels in the indicated cellular subsets in Itgax-cre+Ifngr1f/f mice compared with Ifngr1f/f mice are summarized in the associated bar graph of panel. (B) FACS analysis in liver and peritoneal cavity to confirm the lack of IFNGR1 expression in hepatic and peripheral CD103+ DCs in Itgax-cre+Ifngr1f/f mice. (C) After in vitro IFNγ stimulation (1,000 U/ml) for 15 minutes at 37°C, phosphorylated STAT1 in splenocytes from Itgax-cre+Ifngr1f/f mice was analyzed. pSTAT1 staining of un-stimulated controls was indistinguishable from that in Ifngr1WU−/− mice (Supplemental Fig. 1B). (D) Selective lack of up-regulation of CD40 in splenic CD8α+ DCs in Itgax-cre+Ifngr1f/f mice after in vitro IFNγ stimulation (500 U/ml) for 18 hr at 37°C. Splenic CD11c+ cells were positively enriched by MACS purification prior to IFNγ stimulation. The wildtype, knockout, and isotype controls for panel (A) and (C) are the same as for Fig. 2 panel (A) and (B) because the flow cytometry was performed at the same time. All Data are representative of at least two separate experiments. Gating strategies are depicted in Supplemental Fig. 1A. *, p ≤ 0.05.
Figure 4
Figure 4. Both Vav-icre+Ifngr1f/f and Itgax-cre+Ifngr1f/f mice display increased susceptibility to Listeria infection
(A) Mice were infected with 2.5 × 105 (left panel) or 1 × 105 (right panel) L. monocytogenes i.p. and the survival were monitored over time. (B and C) Listeria CFUs in spleen, liver, and peritoneum infected with 105 L. monocytogenes i.p. (D) Mice were infected with 1 × 104 (left panel), 5 × 103 (middle panel), or 2.5 × 103 (right panel) L. monocytogenes i.v. and the survival were monitored over time. (E) Listeria CFUs in spleen, liver, and peritoneum at 3 days after i.v. infection with 103 or 104 L. monocytogenes. Each symbol in Listeria CFUs represents an individual mouse and lines represent the mean Log10CFU. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
Figure 5
Figure 5. Early production of IL-12 from CD8α+ DCs is significantly decreased in Listeria-infected Itgax-cre+Ifngr1f/f mice
All mice were infected with 106 L. monocytogenes i.v. (A) Representative flow cytometry plots for IL-12p40 expression from CD8α+ DCs and Ly6Chi monocytes. For each quadrant, gating was based on cells from uninfected controls that were analyzed at every time point (For simplicity, only the uninfected control at 9 hr after infection is shown). (B) Summary of percentages of IL-12p40 positive CD8α+ DCs in the spleen during the first 24 hr of infection (n ≥ 4 at each time point). (C) Percentages of IL-12p40 positive CD8α+ DCs in the spleen from Ifngr1f/f mice and Ifngr1WU−/− mice at 9 hr of infection. (D) The expression of indicated genes in sorted CD8α+ DCs from spleens was determined by qRT-PCR after 9 hr of infection. (E) Summary of percentages of IL-12p40 positive Ly6Chi monocytes in the spleen during the first 24 hr of infection (n ≥ 4 at each time point). *, p ≤ 0.05; **, p ≤ 0.01.
Figure 6
Figure 6. Early production of IFNγ from NK and NKT cells is significantly decreased in Listeria-infected Itgax-cre+Ifngr1f/f mice
All mice were infected with 106 L. monocytogenes i.v. (A) Representative plots for IFNγ from NK, NKT and CD8+ T cells during the first 24 hr of infection. Summary of the percentages of IFNγ positive splenic NK, NKT, and CD8+ T cells is shown in (B), (C), and (D), respectively (n ≥ 4 at each time point). *, p ≤ 0.05.
Figure 7
Figure 7. Depletion of NK/NKT cells producing early IFNγ significantly reduces IL-12p40 production from CD8α+ DCs
(A) Representative FACS plots documenting depletion of NK/NKT cells by anti-NK1.1 mAb (top and second row), depletion of MP CD8+ T cells by anti-CXCR3 mAb (third row), and IL-12p40 production from CD8α+ DCs at 9 hr of infection with 106 L. monocytogenes i.v. (bottom row). (B) Summary of percentages of IL-12p40 positive CD8α+ DCs after treatment with depleting mAbs (n ≥ 5). **, p ≤ 0.01.
Figure 8
Figure 8. Blockade of TNFα prior to Listeria infection reduces both IL-12 production from CD8α+ DCs and IFNγ production from NK/NKT cells
Ifngr1f/f mice were pretreated with indicated mAbs (as described in Materials and Methods) prior to infection with 106 L. monocytogenes i.v. Spleens were harvested at 9 hr after infection, and cells analyzed for expression of IL-12p40 and IFNγ by intracellular cytokine staining. Percentages of IFNγ positive NK cells (A), IFNγ positive NKT cells (B), and IL-12p40 positive CD8α+ DCs (C) are plotted as bar graphs (n ≥ 4). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
Figure 9
Figure 9. Neutralization of IL-4 restores Listeria resistance in Itgax-cre+Ifngr1f/f mice
(A) Spleens were harvested at 9 hr after infection with 106 L. monocytogenes i.v. and analyzed for the expression of indicated genes by qRT-PCR. (B) IL-4 expression in NK and NKT cells sorted from spleens was determined by qRT-PCR after 9 hr of infection with 106 L. monocytogenes i.v. (C) Listeria CFUs in spleen and liver of infected mice at 3 days after infection. Mice were pretreated with 11B11 mAb prior to infection with 103 L. monocytogenes i.v. Data are a combination of two separate experiments. (D) The expression of indicated genes was analyzed by qRT-PCR at 12 hr after infection with 106 L. monocytogenes i.v. Every qRT-PCR data is represented relative to the expression of 18S (ΔCt). In order to facilitate visualization, values were transformed as indicated on the y-axis label. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

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