Figure 6.

S1P₁ receptor agonism actively suppresses recruitment of innate immune cells through down-regulation of chemokine expression on lung endothelial cells. (A) Relative mRNA expression of chemokines from purified lung endothelial cell populations at 36-hours post influenza virus infection. (B) Protein expression of CCL2 and CXCL10 measured by ELISA on FACS purified vascular and lymphatic lung endothelial cells 48 hours post influenza virus infection. (C) LysM-GFP mice were infected with 1 × 10⁴ PFU of influenza virus. Two days later bone marrow cells were harvested from infected LysM-GFP mice and transferred into C57BL/6J mice that were either left uninfected or infected with 1 × 10⁴ PFU of influenza virus 2-hours prior. Mice receiving bone marrow cells from LysM-GFP positive mice were treated with either vehicle or CYM-5442 (2mg/kg 1, 13, 25, and 37 hours post-infection) and 48-hours post-infection total lung cells were harvested from lung homogenates and the total numbers of GFP expressing granulocytes (CD11b+, Ly6G+) and macrophages/monocytes (CD11b+F480+) were quantified. (D) Shows the total number of LysM-GFP positive granulocytes and macrophages/monocytes per lung of uninfected or infected mice treated with either vehicle or CYM-5442. Exogenous intratracheal administration of chemokine restores recruitment of innate immune cells however, does not resurrect cytokine/chemokine production after CYM-5442 treatment. (E) Mice were infected with 1 × 10⁴ PFU WSN influenza virus and either Vehicle (water), or CYM5442 (2mg/kg 1,13, 25 and 37 hours-post-infection) were administered to mice i.t.. Following administration of CYM-5442, recombinant mouse CCL2 was administered (50μg) i.t. directly into the lungs. The bar graph shows the total numbers of macrophages/monocytes, neutrophils and NK cells in the lung 48 hours post-influenza virus infection. (F) Pro-inflammatory cytokines and chemokines were measured in BALF 48 hours post-infection by ELISA in mice treated with vehicle, CYM-5442 or CYM-5442 + rmCCL2 as in E. Data represent average SEM from 5 mice/group. *, p < 0.05; **, p < 0.005; ***, p < 0.0005. Results are representative of two independent experiments.