CD200R1 Supports HSV-1 Viral Replication and Licenses Pro-Inflammatory Signaling Functions of TLR2

The CD200R1:CD200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. We generated CD200R1−/− mice and employed them to explore both the role of CD200R1 in regulating macrophage signaling via TLR2 as well as the host response to an in vivo, TLR2-dependent model, herpes simplex virus 1 (HSV-1) infection. CD200R1−/− peritoneal macrophages demonstrated a 70–75% decrease in the generation of IL-6 and CCL5 (Rantes) in response to the TLR2 agonist Pam2CSK4 and to HSV-1. CD200R1−/− macrophages could neither up-regulate the expression of TLR2, nor assemble a functional inflammasome in response to HSV-1. CD200R1−/− mice were protected from HSV-1 infection and exhibited dysfunctional TLR2 signaling. Finally, both CD200R1−/− mice and CD200R1−/− fibroblasts and macrophages showed a markedly reduced ability to support HSV-1 replication. In summary, our data demonstrate an unanticipated and novel requirement for CD200R1 in “licensing” pro-inflammatory functions of TLR2 and in limiting viral replication that are supported by ex vivo and in vivo evidence

CD200R1 Supports HSV-1 Viral Replication and Licenses Pro-Inflammatory Signaling Functions of TLR2

The CD200R1:CD200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. We generated CD200R1−/− mice and employed them to explore both the role of CD200R1 in regulating macrophage signaling via TLR2 as well as the host response to an in vivo, TLR2-dependent model, herpes simplex virus 1 (HSV-1) infection. CD200R1−/− peritoneal macrophages demonstrated a 70–75% decrease in the generation of IL-6 and CCL5 (Rantes) in response to the TLR2 agonist Pam2CSK4 and to HSV-1. CD200R1−/− macrophages could neither up-regulate the expression of TLR2, nor assemble a functional inflammasome in response to HSV-1. CD200R1−/− mice were protected from HSV-1 infection and exhibited dysfunctional TLR2 signaling. Finally, both CD200R1−/− mice and CD200R1−/− fibroblasts and macrophages showed a markedly reduced ability to support HSV-1 replication. In summary, our data demonstrate an unanticipated and novel requirement for CD200R1 in “licensing” pro-inflammatory functions of TLR2 and in limiting viral replication that are supported by ex vivo and in vivo evidence

CD200R1 controls expression of TLR2 in macrophages.

<p>(A, B) The expression of CD200R1 (A) or TLR2 (B) on the surface of WT (circles), TLR2KO (squares), or CD200R1KO (triangles) elicited peritoneal macrophages (MΦ) before (–, closed symbols) or after HSV-1 infection (+, open symbols) was determined by flow cytometry. The lack of expression of CD200R1 or TLR2 on the surface of CD200R1KO or TLR2KO macrophages, respectively, served as internal controls. Data representative of 3 experiments (preparations from individual mice n = 3 WT and KO are shown). An unpaired, two tailed Student’s t-test was used to determine statistical significance of independent experiments.</p

CD200R1 regulates replication of HSV-1 in cells.

<p>(A, B) Flow cytometric analysis of GFP expression in CD200R1^+/+ (WT) and CD200R1^−/− (KO) mouse embryonic fibroblasts (MEFs; A) and elicited peritoneal macrophages (B) at various time points following infection with ICP8-GFP virus (MOI 10∶1). Histograms of each sample were generated. A gate was set on the uninfected control for each condition, which excluded 99% of the uninfected cells. This gate was applied to the paired infected samples to calculate the % GFP+ cells. Data representative of 3 experiments. (C) CD200R1 expression on MEFs (left panel) or elicited peritoneal macrophages (MΦ, right panel) was determined using immunofluorescence staining. Scale bar = 10 µm. Data representative of 3–4 experiments (MΦ from n = 6, WT and KO).</p

CD200R1 licenses pro-inflammatory signaling in peritoneal macrophages.

<p>(A) CD200R1^+/+ (WT) or CD200R1^−/− (KO) elicited peritoneal macrophages were stimulated with either HSV-1 (MOI 10∶1, left column), Pam₂CSK₄ (100 ng/ml, center column), LPS (100 ng/ml, right column), or medium for 24, 48, or 72 h. IL-6 (upper row, pg/ml), CCL5/Rantes (middle row, pg/ml), and CCL2/MCP1 (lower row, pg/ml) levels were measured by ELISA. Data shown are mean and SD of representative experiment (total of 3 experiments, n = 4, WT and KO). An unpaired, two tailed Student’s t-test was used to determine statistical significance of independent experiments, p values; * p<0.05, † p<0.01. (B) Supernatant IL-1β levels from WT and KO elicited peritoneal macrophages (left panels) or bone marrow macrophages (right panels) 16 h after addition of HSV-1 (MOI 10∶1) or after a 3 h stimulation with LPS (100 ng/ml) followed by ATP (1 mM) for 1 h. ELISA results are representative of 3 experiments. (C) WT and KO elicited peritoneal macrophages were cultured for 20 h with HSV-1 (MOI 10∶1). Media (left lanes) and cells (right lanes) were analyzed by SDS-PAGE followed by Western blot for cleavage of pro-IL-1β to IL-1β. Data representative of 3 experiments (n = 4, WT; n = 5, KO).</p