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. 2011 Nov;7(11):e1002374.
doi: 10.1371/journal.ppat.1002374. Epub 2011 Nov 10.

CD11b⁺, Ly6G⁺ cells produce type I interferon and exhibit tissue protective properties following peripheral virus infection

Matthew A Fischer  1 , Michael L Davies, Irene E Reider, Erica L Heipertz, Melanie R Epler, Janet J Sei, Molly A Ingersoll, Nico Van Rooijen, Gwendalyn J Randolph, Christopher C Norbury
Affiliations

CD11b⁺, Ly6G⁺ cells produce type I interferon and exhibit tissue protective properties following peripheral virus infection

Matthew A Fischer et al. PLoS Pathog. 2011 Nov.

Abstract

The goal of the innate immune system is containment of a pathogen at the site of infection prior to the initiation of an effective adaptive immune response. However, effector mechanisms must be kept in check to combat the pathogen while simultaneously limiting undesirable destruction of tissue resulting from these actions. Here we demonstrate that innate immune effector cells contain a peripheral poxvirus infection, preventing systemic spread of the virus. These innate immune effector cells are comprised primarily of CD11b⁺Ly6C⁺Ly6G⁻ monocytes that accumulate initially at the site of infection, and are then supplemented and eventually replaced by CD11b⁺Ly6C⁺Ly6G⁺ cells. The phenotype of the CD11b⁺Ly6C⁺Ly6G⁺ cells resembles neutrophils, but the infiltration of neutrophils typically occurs prior to, rather than following, accumulation of monocytes. Indeed, it appears that the CD11b⁺Ly6C⁺Ly6G⁺ cells that infiltrated the site of VACV infection in the ear are phenotypically distinct from the classical description of both neutrophils and monocyte/macrophages. We found that CD11b⁺Ly6C⁺Ly6G⁺ cells produce Type I interferons and large quantities of reactive oxygen species. We also observed that depletion of Ly6G⁺ cells results in a dramatic increase in tissue damage at the site of infection. Tissue damage is also increased in the absence of reactive oxygen species, although reactive oxygen species are typically thought to be damaging to tissue rather than protective. These data indicate the existence of a specialized population of CD11b⁺Ly6C⁺Ly6G⁺ cells that infiltrates a site of virus infection late and protects the infected tissue from immune-mediated damage via production of reactive oxygen species. Regulation of the action of this population of cells may provide an intervention to prevent innate immune-mediated tissue destruction.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Phagocytic cells control the systemic spread of virus from the ear pinnae and tissue damage at the site of infection.
Wild-type (A–D, G, H) or MAFIA (E, F, G, H) mice were infected i.d. in the ear pinnae with VACV. Ear pinnae or ovaries were harvested at various days post-infection. Ear (A, C, E) or ovary (D, F) lysates were used in a plaque assay to determine viral replication kinetics in vivo. B) Immune cells were isolated from infected ear pinnae and identified as either T cells (open circles; TCRβ+, CD90+) or phagocytes (filled squares; CD11b+, CD90-, NK1.1-, CD19-) using flow cytometry. Infected ear pinnae (C, E) and ovaries (D, F) were harvested from clodronate liposome-(C, D; filled circles) or vehicle-(C, D; open squares) treated wild-type mice and AP20187-(E, F; filled circles) or vehicle-(E, F; open squares) treated MAFIA mice. G, H) The tissue damage in wild-type mice treated with clodronate liposomes (filled squares) or vehicle (filled circles), or MAFIA mice treated with AP20187 (open squares) was documented on indicated days post infection, by measuring lesion size (G) or loss of tissue (H). Data is representative of at least three independent experiments and data shown represent mean cell numbers or virus titers +/- SEM from four ears at each time point and mean lesion size or tissue damage +/- SEM from 10 ears mice at each time point are depicted in (G, H).
Figure 2
Figure 2. Ly6C+Ly6G- and Ly6C+Ly6G+ subpopulations of cells at the site of VACV infection.
MAFIA (A–F), CX3CR1+/GFP (G) or wild-type (H) mice were infected in the ear pinnae with VACV and the ears harvested at various days post infection (A, B) or on day 5 post infection (C–H). A) The phagocyte population (CD11b+, CD90-, NK1.1-, CD19-) in the ear was analyzed (using a gating strategy outlined in Fig. S3) for expression of GFP (driven by the CD115 promoter) and CD11b. B) The GFP+CD11b+ population from A) was analyzed for expression of Ly6C and Ly6G. C–G) The resulting Ly6C+Ly6G- (open histograms, top row) and Ly6C+Ly6G+ (open histograms, bottom row) populations were analyzed for the expression of various intracellular (C) or cell surface (D–F) markers using antibodies or GFP as a surrogate for CX3CR1 expression (G) and compared to CD11b- cells (filled histogram). H) Cells from ear pinnae 5 days post-infection were magnetically sorted based on Ly6G expression, fixed onto slides using cytofugation, and stained. Data shown are representative of at least three independent experiments in which three mice were examined at each time point.
Figure 3
Figure 3. Both Ly6C+Ly6G- and Ly6C+Ly6G+ subpopulations extravasate into the site of infection.
A) Mice were treated i.v. with either non-depleting anti-CD11b (filled circles) or isotype control (open squares) and infected i.d. in the ear pinnae with VACV. Ear pinnae were harvested at various times post-infection and the phagocyte population was then analyzed for the Ly6C+Ly6G- (top) and Ly6C+Ly6G+ (bottom) subpopulations. B) Mice either untreated (left) or treated once (as opposed to the repeated treatment in Fig. 1) with clodronate liposomes (right) were injected with fluorospheres and then infected i.d. with WR VACV. After 5 days of infection, ear pinnae were harvested and Ly6C+Ly6G- (top) and Ly6C+Ly6G+ (bottom) phagocytes were analyzed for fluorosphere uptake using flow cytometry. Data are representative of at least three independent experiments and mean phagocyte numbers +/- SEM from four ears at each time point are depicted in A).* represents p<0.05 using a Student's T-test.
Figure 4
Figure 4. Ly6C+Ly6G- and Ly6C+Ly6G+ cells have distinct functional profiles.
Mice were infected i.d. in the ear pinnae with VACV. Ear pinnae were harvested at 5 days post-infection. The Ly6C+Ly6G- (top, open histograms) and Ly6C+Ly6G+ (bottom, open histograms) CD11b+ monocyte subpopulations were analyzed for the expression of various cytokines/effector molecules using antibodies (A-D) or a reactive dye triggered by exposure to ROS (E) and compared to CD11b- cells (filled histograms). For TNF-α staining, cells were first incubated with CpG DNA. Data are representative of at least three independent experiments in which the phenotype of at least two mice were examined independently.
Figure 5
Figure 5. Depletion of Ly6G+ cells leads to increased tissue damage at the site of VACV infection.
Mice were treated with vehicle (A-left, B, D, E - open squares), isotype control antibody (F-open squares) or anti-Ly6G antibody (A-right, B, D, E, F - filled circles) and infected i.d. in the ear pinnae with VACV. A) The presence of Ly6C+Ly6G- and Ly6C+Ly6G+ monocytes populations in the ear pinnae was measured 5 days post-infection. B) Replicating virus titers were measured at various times post infection. C-F) The tissue damage in mice treated with anti-Ly6G antibody for the entire course of infection (C, D, E) or after day 10 post infection (F, arrowed) was documented visually on indicated days post infection (C), by measuring lesion size (D) or loss of tissue (E, F). Data are representative of at least three independent experiments. Mean virus titers +/- SEM from four ears at each time point are depicted in (B). Mean lesion size or tissue damage +/- SEM from five mice at each time point are depicted in (D, E). * represents p<0.05 using a Student's T-test.
Figure 6
Figure 6. Loss of NADPH-oxidase activity leads to increased tissue damage at the site of VACV infection.
A–D) Wild-type (open squares) or GP91-/- (filled circles) mice were infected i.d. in the ear pinnae with VACV. A) Replicating virus titers in the ear pinnae were measured at various times post infection. The tissue damage in GP91-/- mice was documented visually on day 9 (B), or by measuring lesion size (C) or loss of tissue (D) at various times post infection. E, F) GP91-/- mice were treated with anti-Thy1 (closed circles) or isotype control (open squares) and lesion size (E) and tissue damage (F) quantified at the days shown. Mean virus titers +/- SEM from four ears mice at each time point are depicted in (A). Mean lesion size or tissue damage +/- SEM from ten ears at each time point are depicted in (C, D, E F). * represents p<0.05 using a Student's T-test.
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