Cutting Edge: Oseltamivir Decreases T Cell GM1 Expression and Inhibits Clearance of Respiratory Syncytial Virus: Potential Role of Endogenous Sialidase in Antiviral Immunity

Abstract

The sialoglycosphingolipid GM1 is important for lipid rafts and immune cell signaling. T cell activation in vitro increases GM1 expression and increases endogenous sialidase activity. GM1 expression has been hypothesized to be regulated by endogenous sialidase. We tested this hypothesis in vivo using a mouse model of respiratory syncytial virus (RSV) infection. RSV infection increased endogenous sialidase activity in lung mononuclear cells. RSV infection increased lung CD8+ T cell surface GM1 expression. Activated CD8+ T cells in the lungs of RSV-infected mice were GM1high. Treatment of RSV-infected mice with the sialidase/neuraminidase inhibitor oseltamivir decreased T cell surface GM1 levels. Oseltamivir treatment decreased RSV-induced weight loss and inhibited RSV clearance. Our data indicate a novel role for an endogenous sialidase in regulating T cell GM1 expression and antiviral immunity. Also, oseltamivir, an important anti-influenza drug, inhibits the clearance of a respiratory virus that lacks a neuraminidase gene, RSV.

Differential Regulation of GM1 and Asialo-GM1 Expression by T Cells and Natural Killer (NK) Cells in Respiratory Syncytial Virus Infection

“…Rabbit polyclonal anti-ASGM1 Ab (Wako Chemicals USA, Richmond, VA) and isotype control Ab (rabbit gamma globulin; Jackson Immunoresearch, West Grove, PA) were labeled with PE-conjugated anti-rabbit IgG Fab fragments according to the manufacturer's protocol, and 1 g of Ab was used per 2 ϫ 10 5 cells (Zenon Rabbit IgG Labeling Kit; Molecular Probes, Eugene, OR). We used 0.1 g of FITC-labeled cholera toxin B-subunit (CtxB-FITC, SigmaAldrich, St. Louis, MO) per 2 ϫ 10 5 cells to stain GM1 as previously described (56). RSV-specific CD8 ϩ T cells were stained with RSV M2 protein-specific PE-labeled H-2K d tetramers (RSV-tetramer), or influenza NP-specific PE-labeled H-2K d tetramers (flu-tetramer; Beckman Coulter, Fullerton, CA) as a negative control as previously described (56).…”

“…We used 0.1 g of FITC-labeled cholera toxin B-subunit (CtxB-FITC, SigmaAldrich, St. Louis, MO) per 2 ϫ 10 5 cells to stain GM1 as previously described (56). RSV-specific CD8 ϩ T cells were stained with RSV M2 protein-specific PE-labeled H-2K d tetramers (RSV-tetramer), or influenza NP-specific PE-labeled H-2K d tetramers (flu-tetramer; Beckman Coulter, Fullerton, CA) as a negative control as previously described (56). Samples were analyzed using a LSR II flow cytometer (BD Biosciences, San Jose, CA), and 2-5 ϫ 10 4 live lymphocytes per lung were analyzed based on forward and sidescatter properties.…”

“…Cells were counted then treated and stained for cell surface molecules (CD3, CD4, CD8, and CD49b) and intracellular IFN-␥ as previously described (56). For intracellular cytokine staining, cells were incubated in RPMI/10% heat-inactivated FBS/1 M ionomycin (Sigma, St. Louis, MO), 10 ng/mL PMA (Sigma),7 L/10 mL Golgi Stop (BD Pharmingen, San Jose, CA) for 6 h at 37°C and 5% CO 2 .…”

“…We previously showed that activated CD8 ϩ T cells in the lungs of RSV-infected mice have increased cell surface GM1 expression (56). We hypothesized that GM1 and ASGM1 are coordinately expressed by NK and T cells.…”

“…GM1 is incorporated into the RSV viral envelope (55). At the level of the host immune response, RSV-specific and IFN-␥-expressing CD8 ϩ T cells have increased GM1 expression in vivo, and decreased Tcell GM1 expression in the context of RSV infection was associated with reduced viral clearance and reduced disease severity (56). Defining the role of GSLs in RSV infection and pathogenesis may lead to targeted therapies aimed at reducing viral replication and/or ensuing immunopathology.…”