Down regulation of macrophage IFNGR1 exacerbates systemic <i>L</i>. <i>monocytogenes</i> infection

<div><p>Interferons (IFNs) target macrophages to regulate inflammation and resistance to microbial infections. The type II IFN (IFNγ) acts on a cell surface receptor (IFNGR) to promote gene expression that enhance macrophage inflammatory and anti-microbial activity. Type I IFNs can dampen macrophage responsiveness to IFNγ and are associated with increased susceptibility to numerous bacterial infections. The precise mechanisms responsible for these effects remain unclear. Type I IFNs silence macrophage <i>ifngr1</i> transcription and thus reduce cell surface expression of IFNGR1. To test how these events might impact macrophage activation and host resistance during bacterial infection, we developed transgenic mice that express a functional FLAG-tagged IFNGR1 (fGR1) driven by a macrophage-specific promoter. Macrophages from fGR1 mice expressed physiologic levels of cell surface IFNGR1 at steady state and responded equivalently to WT C57Bl/6 macrophages when treated with IFNγ alone. However, fGR1 macrophages retained cell surface IFNGR1 and showed enhanced responsiveness to IFNγ in the presence of type I IFNs. When fGR1 mice were infected with the bacterium <i>Listeria monocytogenes</i> their resistance was significantly increased, despite normal type I and II IFN production. Enhanced resistance was dependent on IFNγ and associated with increased macrophage activation and antimicrobial function. These results argue that down regulation of myeloid cell IFNGR1 is an important mechanism by which type I IFNs suppress inflammatory and anti-bacterial functions of macrophages.</p></div

fGR1 macrophages respond to type I IFNs and retain high cell surface IFNGR1 expression.

<p>Naïve peritoneal macrophages were treated with 100 U/mL of IFNβ A) for 6 hrs to determine surface IFNGR1 expression; B) 5, 30, 60 min to evaluate STAT1 pY701 by western blot. pSTAT1 bands were normalized to β-actin loading control, Arbitrary Units (AU); C) for 24 hrs and MHC I expression determined by flow cytometry. D) Macrophages were pre-treated with 100 U/mL of IFNβ for 6 hrs before stimulation with 100 U/mL of IFNγ for 24 hrs and MHC II up regulation determined. All MFIs were normalized to their respective unstimulated controls and pooled from multiple experiments (n = 3 independent experiments, 1–3 mice per experiment, *Two-Tailed t-test).</p

IFNγ is required for increased resistance of fGR1 mice to <i>L</i>. <i>monocytogenes</i> infection.

<p>C57Bl/6 and fGR1 mice were treated with PBS or 500 μg of α-IFNγ depletion antibody 24 hrs before infection with a low dose (5 x 10³ CFUs) of <i>L</i>. <i>monocytogenes</i>. A) Bacterial burdens were determined in both the liver and spleen 72 hpi. (Data pooled from 3 independent experiments, 3–5 mice per group, *Mann-Whitney Test). The splenic inflammatory monocytes were analyzed by flow cytometry for the surface expression of B) IFNGR1 and C) pro-inflammatory activation markers MHC II, CD80, and CD64. (MFIs are pooled from 2-independent experiments, 3–5 mice per group, *Two-Tailed T-test).</p

Heightened pro-inflammatory phenotype of fGR1 inflammatory monocytes during <i>L</i>. <i>monocytogenes</i> infection.

<p>Splenocytes from WT (blue) and fGR1 (red) mice 96 hpi <i>L</i>. <i>monocytogenes</i> infection were analyzed for A) FLAG expression on inflammatory monocytes (CD11b^hi, Ly6C^hi, Ly6G^lo, gated <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006388#ppat.1006388.s004" target="_blank">S3A Fig</a>) and neutrophils (CD11b^hi, Ly6C^hi, Ly6G^hi). B) IFNGR1 and C) “M1”-like pro-inflammatory activation markers MHC II, CD80, and CD64 on splenic inflammatory monocytes. (Representative data of one independent experiment, n = 5–10 mice per group. Experiment repeated at least three times. *Two-Tailed t-test).</p

fGR1 expression does not alter basal IFNγ responsiveness.

<p>Peritoneal macrophages from naïve fGR1 and WT mice were A) stimulated for 30 min with increasing concentrations of IFNγ, B) 100 U/mL of IFNγ for 5, 30, 60 min and pY701 STAT1 determined by western blots. pSTAT1 blots were normalized to β-actin, Arbitrary Units (AU). C) Naïve peritoneal macrophages (CD90.2 -, CD11b^hi, F480^hi) from IFNAR KO (green), C57Bl/6 (blue), and fGR1 (red) were stimulated with 100 U/mL of IFNγ for 24 hrs and MHC II up regulation evaluated by flow cytometry. MFIs were normalized to their respective unstimulated controls and pooled from multiple experiments (n = 3 independent experiments, 1–3 mice per experiment).</p

fGR1 mice are more resistant to <i>L</i>. <i>monocytogenes</i> infection.

<p>WT C57Bl/6 (blue) and fGR1 (red) mice were infected intravenously with 1.0 x 10⁴ CFUs of <i>L</i>. <i>monocytogenes</i>. Bacterial burdens were determined in both the A) liver and B) spleen at 24, 48, 72 and 96 hpi. (All time points are pooled from at least 3 independent experiments, 3–5 mice per group per experiment, *Two-Tailed t-test).</p

fGR1 expression increases macrophage cell intrinsic ability to kill bacteria.

<p>A). Bacterial burdens from C57Bl/6 (blue) and fGR1 (red) mice 60 hpi. Flow cytometry was used to determine the B) frequency of monocytes infected by GFP-expressing <i>L</i>. <i>monocytogenes (Lm)</i> 60 hpi, and C) MFI of GFP-<i>Lm</i> within the inflammatory monocyte population (Data pooled from 3 independent experiments, 3–5 mice per group). D) Inflammatory monocytes were FACS sorted and lysed to determine <i>Lm</i> bacterial burdens 60 hpi. (Representative CFUs from 2 independent experiments, 3 mice per groups per experiment). pHrodo <i>S</i>. <i>aureus</i> Bioparticles were incubated for 30 min with enriched myeloid cells isolated from 60 hpi spleen and the E) MFI and F) frequency of pHrodo positive inflammatory monocytes was determined by flow cytometry (Representative data from 2 independent experiments, 3 mice per group per experiment). G) Spleens were depleted of CD90.2+, IgM+, NK1.1+, Ly6G+ cells and CD11b+ cells were analyzed by fluorescent microscopy. Arrows indicate colocalization of <i>Lm</i> and LAMP-1. Unbiased quantification of the H) relative distribution of the number of <i>Lm</i> per CD11b+ cell and I) percentage of <i>Lm</i> colocalized with LAMP-1 per CD11b+ cell. (Representative data from 2 independent experiments, 3 mice per group, *Two-Tailed T-test).</p