ISRE-Reporter Mouse Reveals High Basal and Induced Type I IFN Responses in Inflammatory Monocytes

Abstract

Type I and type III interferons (IFNs) are critical for controlling viral infections. However, the precise dynamics of the IFN response have been difficult to define in vivo. Signaling through type I IFN receptors leads to interferon-stimulated response element (ISRE)-dependent gene expression and an antiviral state. As an alternative to tracking IFN, we used an ISRE-dependent reporter mouse to define the cell types, localization, and kinetics of IFN responding cells during influenza virus infection. We find that measurable IFN responses are largely limited to hematopoietic cells, which show a high sensitivity to IFN. Inflammatory monocytes display high basal IFN responses, which are enhanced upon infection and correlate with infection of these cells. We find that inflammatory monocyte development is independent of IFN signaling; however, IFN is critical for chemokine production and recruitment following infection. The data reveal a role for inflammatory monocytes in both basal IFN responses and responses to infection.

Keywords: influenza; innate immunity; interferon; monocytes.

Figure 1.. The Mx1^gfp Allele Functions As a Reporter for Type I and Type III IFN Responses

(A and B) Mx1^gfp, Mx1^gfp/Ifnar1^−/−, and Mx1^gfp/Stat2^−/− mice were i.v. injected with 100 μg of PIC, and GFP expression in the spleen was measured at 24 hr. (C and D) Mx1^gfp, Mx1^gfp/Ifnar1^−/−, and Mx1^gfp/ Stat2^−/− mice were i.n. injected with 50 μg of PIC, and GFP expression was measured at 24 hr. Arrow indicates IFNAR1-independent reporter expression in epithelial cells. (A) and (C) show mean ± SD for n = 3 animals. (B) and (D) show representative FACs plots. See also Figure S1.

Figure 2.. Basal IFN Responses Are Present at High Levels in Ly6C^hi Monocytes

(A and B) Bone marrow and spleen cells from untreated B6 or Mx1^gfp mice were stained for T cells (CD3⁺), B cells (CD19⁺), DCs (CD11c⁺), NK cells (NK1.1⁺), neutrophils (Ly6C⁺Ly6G⁺CD11b⁺), or monocytes (Ly6C⁺Ly6G⁻CD11b⁺), and GFP expression was examined. (C and D) Bone marrow or spleen was harvested from the indicated untreated mice, and GFP expression on Ly6C^hiLy6G⁻CD11b⁺ monocytes was analyzed. (E) qPCR on sorted spleen Ly6C^hiLy6G⁻CD11b⁺ and Ly6C^loLy6G⁻CD11b⁺ monocytes from untreated Mx1^gfp and Mx1^gfp/Stat2^−/− mice. (B) shows mean ± SD for n = 4 animals. (D) shows mean ± SD for n = 3 animals. (E) shows mean ± SD for n = 3 animals. **indicates significance of p< 0.01 using unpaired t test. (A) and (C) show representative FACs plots. See also Figure S2.

Figure 3.. Exposure to PIC Induces a Systemic IFN Response

(A–C) Mx1^gfp mice were i.v. injected with 100 μg of PIC, and GFP expression in the spleen was measured at days 0–5 post-treatment (A and B) or 2, 4, or 6 hr post-treatment (C). (D and E) Mx1^gfp mice were i.v. injected with 100 μg of PIC, and GFP expression was measured in T cells (CD3⁺), B cells (CD19⁺), DCs (CD11c⁺), NK cells (NK1.1⁺), neutrophils (Ly6C⁺Ly6G⁺CD11b⁺), and monocytes (Ly6C⁺Ly6G⁻CD11b⁺) in the spleen at 24 hr. Inset histogram shows basal and induced GFP expression in Ly6C^hi monocytes. (F and G) Mx1^gfp mice were i.v. or i.n. injected with 50 μg of PIC, and GFP expression in organs was measured at 24 hr. (B), (C), (E), and (G) show mean ± SD for n = 2–3 animals. (A), (D), and (F) show representative FACs plots. See also Figure S3.

Figure 4.. The IFN Response to Influenza Virus Is Confined to the Lung and Draining Lymph Node

(A and B) Mx1^gfp mice were infected with 10² pfu of PR8 (A) or HALo (B), and GFP expression in organs was examined at days 0–10 post-infection. (C) Mx1^gfp mice were infected with 10² pfu of Cal09, and GFP expression in the lungs was examined. (D) Mx1^gfp mice were infected with increasing doses of PR8, and GFP expression was measured in the lung. (E) Mx1^gfp mice were infected with PR8 or the PR8-NS1 RNA-binding mutant virus (R38A/K41A), and GFP expression was measured in the lung. The bar represents the mean for n = 2 animals for all panels. Data from (A) are shown shaded in (B) and (C) for comparison.

Figure 5.. The IFN Response to Influenza Virus Is Largely Restricted to Hematopoietic Cells in the Lung

(A and B) Mx1^gfp mice were stained for hematopoietic cells (CD45+), epithelial cells (EpCAM+CD45−), or endothelial cells (CD31+) in the lung at day 7 post-infection with 10² pfu of PR8 or at day 1 post-treatment with 50 μg of i.n. PIC. (C and D) Mx1^gfp mice were infected with 10⁶ pfu of PR8 or PR8-NS1 R38A/K41A or treated i.n. with the indicated amount of PIC, and GFP expression was measured in hematopoietic and epithelial cells at day 1. (E and F) Macrophages and MEFs were stimulated with universal type I IFN or infected with PR8 or PR8ΔNS1 at different MOIs, and GFP was measured (E) or viral gene expression was determined (F) at 24 hr post-infection. (B) and (D) show the mean ± SD for n = 3 animals. (A), (C), and (E) show representative FACs plots. See also Figure S4.

Figure 6.. GFP^hi Cells during Influenza Virus Infection Are Infected Ly6C^hi Monocytes

(A and B) Mx1^gfp mice were infected with 10² pfu of PR8, and GFP expression was measured in T cells (CD3⁺), B cells (B220⁺), NK cells (NK1.1 ⁺), alveolar macrophages (CD11c⁺SiglecF⁺), CD11b DCs (CD11c^hiMHCII^hiCD11b⁺CD103⁻), CD103 DCs (CD11c^hiMHCII^hiCD103⁺CD11b⁻), neutrophils (Ly6C⁺Ly6G⁺CD11b⁺), and Ly6C^hi monocytes (Ly6C^hiLy6G⁻CD11b⁺) at day 7 post-infection. (C and D) Mx1^gfp mice were infected with 10⁴ pfu of PR8 and stained for surface M2 protein in T cells, B cells, Ly6C^hi monocytes, or epithelial cells at day 3. (B) shows the mean ± SD for n = 4–6 animals. (D) shows the mean ± SD for 3 animals. (A) and (C) show representative FACs plots. See also Figure S5.

Figure 7.. Ly6C^hi Monocyte Recruitment, but Not Development, Is Dependent on IFN Signaling

(A) Mx1^gfp and Mx1^gfp/Stat2^−/− mice were mock infected or infected with 10² pfu of HALo, and Ly6^hl (Ly6C^hiCD11b⁺Ly6G⁻) and Ly6C^lo (Ly6C^loCD11b⁺ Ly6G⁻) monocytes in the lung were measured by flow cytometry at day 2 post-infection. (B) Mice were infected as in (A), and Ly6C^hi monocyte and neutrophil (Ly6C^loLy6G⁺CD11b⁺) influx was measured by flow cytometry at day 2 post-infection. (C) Mice were infected as in (A), and viral titer in the lung was determined by plaque assay at day 3 post-infection. Titers were below the limit of detection at day 2. (D) Gene expression in the lung of animals from (B) was determined by qPCR at day 2 post-infection. (B) and (D) show mean ± SD for n = 3 animals. (C) shows mean ± SD for n = 5 animals. (A) shows a representative FACs plot. **indicates significance of p < 0.01 using unpaired t test.