β2-adrenergic signals downregulate the innate immune response and reduce host resistance to viral infection - PubMed (original) (raw)

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Graphical abstract

Figure 1.

Figure 1.

β2-AR signaling regulates viral clearance and resistance to MCMV infection. (A) Survival rate of WT mice infected with MCMV at LD50. Mice were treated (filled brown circles) or not (empty black circles) with Clenbuterol in drinking water during 7 d before infection and throughout the experiment (pool of two independent experiments; Mantel-Cox test, *, P < 0.05). (B) Survival rate of Adrb2−/− mice (filled blue circles) and control Adrb2+/+ littermates (empty black circles) after infection with MCMV at LD50 (pool of three independent experiments; Mantel-Cox test, *, P < 0.05). (C) Viral titers in the spleens of Adrb2−/− mice (filled blue bars) and control Adrb2+/+ littermates (empty bars) at 4.5 d pi. Mice were grouped according to high (left) and low (right) viral loads (pool of two independent experiments; each point represents one mouse; unpaired t test, *, P < 0.05). (D) H&E staining of spleen sections after MCMV infection at LD50. Histopathological lesions were scored from grade 0 to 4, and a color code was attributed to each score. Scale bars = 50 µm. (E) Histopathological analysis of the spleens of infected Adrb2−/− and control Adrb2+/+ littermates 4.5 d pi with MCMV at LD50. Scoring is based on the grading and color code shown in D. The mice were divided into two groups according to their viral loads as shown in B. The frequency of mice with a given pathological score is shown for each group of mice (pool of two independent experiments; n = 12–14 per group).

Figure S1.

Figure S1.

Viral clearance and tissue damage in the liver of Adrb2−/− mice after MCMV infection. (A) Viral titer in the liver of Adrb2−/− mice (filled blue bars) and control Adrb2+/+ littermates (empty bars) at 44 h, 3.5 d, and 4.5 d pi (pool of two independent experiments per time point; each point represents one mouse). (B) Viral titer in the spleen of Adrb2−/− mice (filled blue bars) and control Adrb2+/+ littermates (empty bars) at 44 h and 3.5 d (pool of two independent experiments per time point; each point represents one mouse). (C) H&E staining of liver sections after MCMV infection at LD50. Shown are examples for the grading used to determine tissue damage and the color code. Scale bars = 100 µm. (D) Histological inflammatory scores of livers from Adrb2−/− mice and control Adrb2+/+ littermates 4.5 d pi with MCMV at LD50. Scoring was based on the grading shown in C. (E) ALT activity in the serum of Adrb2−/− mice (filled blue circles) and control Adrb2+/+ littermates (empty circles) at 4.5 d pi (one experiment). (F) Frequency of NK, B, T, and NK T cells, eosinophils, neutrophils, monocytes, macrophages, pDCs, and classical DCs (cDCs) in the spleen of Adrb2−/− mice (filled blue bars) and control littermates (empty bars) at steady state (pool of two independent experiments; each point represents one mouse). (G) Frequency of NK, B, and T cells among total CD45.2+ cells in the liver of Adrb2−/− mice (filled blue bars) and control littermates (empty bars) at steady state (pool of two independent experiments; each point represents one mouse). uni, uninfected.

Figure S2.

Figure S2.

β2-AR deficiency does not alter the trafficking of major immune cell subsets upon MCMV infection. (A–C) Immune cell subsets were analyzed in the (A) blood, (B) spleen, and (C) liver of Adrb2−/− mice (filled blue bars) and control littermates (empty bars) at 44 h pi (pool of three independent experiments; each point represents one mouse). (A) Total number of CD45.2+ cells, B cells (CD45.2+, CD19+), monocytes (CD45.2+, TCRβ−, NKp46−, CD19−, GR-1+), neutrophils (CD45.2+, GR-1high), NK cells (CD45.2+, TCRβ−, CD19−, NKp46+, NK1.1+) and T cells (CD45.2+, CD19− TCRβ+). (B) Total number of CD45.2+ cells, NK cells (CD45.2+, TCRβ−, CD19−, Ly6G−, NKp46+, NK1.1+), B cells (CD45.2+, TCRβ−, NKp46−, Ly6G−, CD19+), T cells (CD45.2+, TCRβ+, CD19−, NKp46−, Ly6G−), monocytes (CD45.2+, TCRβ−, NKp46−, Ly6G−, CD19−, CD11c−, CD11bhigh, Ly6Chigh), neutrophils (CD45.2+, TCRβ−, NKp46−, CD19−, Ly6G+), classical DCs (cDCs; CD45.2+, TCRβ−, NKp46−, CD19−, Ly6G−, CD11blow, CD11c+, MHC-II+), and pDCs (CD45.2+, TCRβ−, NKp46−, CD19−, Ly6G−, CD11b−, CD11c+, Ly6C+). (C) Total number of CD45.2+ cells, ILC1s (CD49a+, CD49b−), NK cells (CD49a−, CD49b+), B cells, T cells, monocytes, neutrophils, cDCs, and pDCs. (D and E) Frequency of Ki67+ cells among total B, T, and NK cells and ILC1s in the spleen (D) and liver (E) of Adrb2−/− mice (filled blue bars) and control littermates (empty bars) at 44 h pi (pool of two to three independent experiments; each point represents one mouse). uni, uninfected.

Figure S3.

Figure S3.

Inflammatory cytokine response in MCMV-infected Adrb2−/− and Adrb2LysMCre mice. (A) Kinetics of CXCL1, TNF-α, IL-6, IL-10, MIP-1α (CCL3), MCP-1 (CCL2), IL-12p70, and IFN-γ production in the serum of _Adrb2−/−_mice (filled blue circles) and control littermates (empty circles) after infection with MCMV at LD50 (pool of two to three experiments per time point). (B) Concentrations of IL-12p70, MCP-1 (CCL2), and MIP-1α (CCL3) in the serum of Adrb2LysMCre mice (filled green circles) and control littermates (empty circles) 44 h pi (pool of three independent experiments; each point represents one mouse). (C) Total number of IFN-γ–producing NK T cells in the liver (left) and spleen (right) of Adrb2−/− mice (filled blue bars) and control littermates (empty bars) at 44 h pi (pool of three independent experiments; each point represents one mouse). (D) Frequency of GzB expressing NK cells in the spleen and liver and frequency of GzB-expressing ILC1s in the liver of Adrb2−/− mice (filled blue bars) and control littermates (empty bars) at 44 h pi (pool of three independent experiments; each point represents one mouse). (E) Total number of Ly49H+ NK cells in the spleen (left) and liver (right) of Adrb2−/− mice (filled blue bars) and control littermates (empty bars) at 44 h pi (pool of three independent experiments; each point represents one mouse). uni, uninfected.

Figure 2.

Figure 2.

β2-AR signals downregulate the cytokine and chemokine responses to MCMV. (A) Concentrations of CXCL1, TNF-α, IL-6, IL-10, MIP-1a (CCL3), MCP1 (CCL2), IL-12p70, and IFN-γ in the serum of Adrb2−/− (filled blue circles) and control Adrb2+/+ littermates (empty circles) at 44 h pi (pool of three independent experiments; each point represents one mouse; unpaired t test, *, P < 0.05). (B) Concentrations of CXCL1, TNF-α, IL-6, and IL-10 in the serum of Adrb2LysMCre mice (filled green circles) and controls (empty circles) at 44 h pi (pool of three independent experiments; each point represents one mouse). (C) Survival of Adrb2LysMCre mice (filled green circles) and control littermates (empty circles) after infection with MCMV at LD50 (pool of three independent experiments). uni, uninfected mice; n.s., not significant.

Figure 3.

Figure 3.

β2-AR signals regulate IFN-γ production by liver NK cells. (A) Splenocytes from Adrb2+/+ and Adrb2−/− littermates were stimulated in vitro with anti-NK1.1 mAb-coated plates in the presence (filled black bars) of noradrenaline (10 µM) or diluent (control, empty bars). The frequency of CD107a (left) and IFN-γ–producing NK cells (right) 4 h after stimulation is shown (each point represents one mouse; means ± SEM are shown; unpaired t test, *, P < 0.05). (B) Gating strategy used to identify NK cells (CD45+, CD3−, CD8−, CD19−, Ly6G−, NKp46+, NK1.1+, CD49a−, CD49b+) and ILC1s (CD45+, CD3−, CD8−, CD19−, Ly6G−, NKp46+, NK1.1+, CD49a+, CD49b−) in the liver. (C and D) Frequency of IFN-γ–producing NK cells and ILC1s in the liver (C) and spleen (D) of Adrb2−/− mice (filled blue bars) and control Adrb2+/+ littermates (empty bars) at 44 h pi (pool of three independent experiments; each point represents one mouse; unpaired t test, *, P < 0.05). (E) Frequency of IFN-γ–producing Ly49H+ (left) and Ly49H− (right) NK cells in the liver of Adrb2−/− mice (filled blue bars) and control Adrb2+/+ littermates (empty bars) at 44 h pi (pool of three independent experiments; each point represents one mouse; unpaired t test, *, P < 0.05; **, P < 0.005). (F) Concentration of IFN-γ in the serum of Adrb2Ncr1iCre mice (filled purple circles) and control littermates (empty circles) at 44 h pi (pool of three independent experiments; each point represents one mouse). (G) Frequency of IFN-γ–producing NK cells in the liver of Adrb2Ncr1iCre mice (filled purple bars) and control littermates (empty bars) at 44 h pi (pool of three independent experiments; each point represents one mouse). (H) Survival rate of Adrb2Ncr1iCre mice (filled purple circles) and control littermates (empty circles) after infection with MCMV at LD50 (pool of four independent experiments). uni, uninfected.

Figure 4.

Figure 4.

Differential regulation of the inflammatory cytokine response by β2-AR signaling in hematopoietic and nonhematopoietic cells. (A) Concentration of CXCL1, TNF-α, IL-6, IL-10, IL-12p70, and IFN-γ in the serum of (WT→Adrb2−/−) BM-chimeras (filled red circles) and (WT→Adrb2+/+) BM-chimeras (empty red circles) at 44 h pi (pool of three independent experiments; each point represents one mouse; unpaired t test, *, P < 0.05; **, P < 0.005). (B) Concentration of CXCL1, TNF-α, IL-6, IL-10, IL-12p70, and IFN-γ in the serum of (_Adrb2−/−_→WT) BM-chimeras (filled orange circles) and (_Adrb+/+_→WT) BM-chimeras (empty orange circles) at 44 h pi (pool of three independent experiments; each point represents one mouse; unpaired t test, *, P < 0.05; **, P < 0.005). Data obtained for uninfected mice (uni) are shown.

Figure 5.

Figure 5.

β2-AR signaling in nonhematopoietic cells regulates resistance to MCMV infection independently of catecholaminergic innervation. (A) Survival rate of (_Adrb2−/−_→WT; filled orange circles), (Adrb2+/+→WT; empty orange circles), and (Adrb2−/−_→_Adrb2−/−; filled black circles) BM chimeras after MCMV infection at LD50 (pool of one to two experiments). (B) Survival rate of (WT→_Adrb2−/−; filled red circles), (WT→_Adrb2+/+; empty red circles), and (Adrb2−/−_→_Adrb2−/−; filled black circles) BM chimeras after MCMV infection at LD50 (pool of one to two experiments; Mantel-Cox test, *, P < 0.05). (C) Survival rate of Adrb2−/− mice (filled circles) and control littermates (empty circles) with (blue circles) or without (black circles) NK cell depletion with anti-NK1.1 mAb treatment 2 d before MCMV infection (pool of three independent experiments; Mantel-Cox test, *, P < 0.05; **, P < 0.005). (D) Experimental design for the experiments presented in E, F, and G. (E) Immunofluorescence analysis of liver sections from mice treated with 6-OHDA or PBS as control. Endothelial cells from blood vessels were stained with anti-CD31 antibody (left, white staining; right, green staining in overlaid images). TH+ nerves were stained with anti-TH antibodies (middle, white staining; right, red staining in overlaid images; scale bars = 50 µm). (F) Serum IFN-γ concentrations of 6-OHDA–treated mice (filled circles) and control mice (empty circles) with NK1.1 cell depletion (purple circles) or without NK1.1 cell depletion (black circles) after infection with MCMV at LD50 (pool of two independent experiments; each point represents one mouse; Mann-Whitney U test, *, P < 0.05; **, P < 0.05). (G) Survival rate of 6-OHDA treated mice (filled circles) and control mice (empty circles) with NK1.1 cell depletion (purple circles) or without NK1.1 cell depletion (black circles) after infection with MCMV at LD50 (pool of two independent experiments; Mantel-Cox test, *, P < 0.05). (H) Model: β2-AR signaling induced by catecholamines produced systemically in the blood circulation act on nonhematopoietic cells to modulate proinflammatory signals. This modulation downregulates the NK cell IFN-γ response, which is necessary for efficient viral clearance. This pathway reduces the control of viral replication, increases the severity of spleen lesions, and dampens host resistance to infection. uni, uninfected.