Journal: Journal of Virology

Article Title: Early Innate Recognition of Herpes Simplex Virus in Human Primary Macrophages Is Mediated via the MDA5/MAVS-Dependent and MDA5/MAVS/RNA Polymerase III-Independent Pathways ▿

doi: 10.1128/JVI.01106-10

Figure Lengend Snippet: HSV and PAMPs induce IFN, ISGs, and proinflammatory cytokines in human primary macrophages. (A to C and G) Macrophages were infected with HSV-1 (17+, 5 × 105 PFU/ml, MOI of 1) or transfected with poly(I:C) [t-poly(I:C); 2 μg/ml] or poly(dA:dT) [t-poly(dA:dT); 2 μg/ml]. (A to C and G) After 6 h, after which RNA was harvested and mRNA accumulation of IFN-β, IFN-λ1, CXCL10, and TNF-α was assayed by real-time PCR. (D, E, and H) In similar experiments, cell supernatants were harvested after 20 h and assayed for the presence of TNF-α, CXCL10, and CCL3 by ELISA. (E, F, I, and K) Macrophages were infected with HSV or stimulated with IFN-α (100 IU/ml) or the agonist for TLR2 (Pam3CSK4, 100 ng/ml), TLR3 [poly(I:C), 30 μg/ml], TLR7/8 (R848, 1 μg/ml), or TLR9 (ODN2216, 2 μg/ml). After 20 h of stimulation, supernatants were collected and protein levels assayed by ELISA. (J and L) Cells were infected with HSV-1 (17+ or KOS, 5 × 105 PFU/ml, MOI of 1) or HSV-2 (MOI of 1). Supernatants were harvested at 18 h and assayed by ELISA for CXCL10 and CCL3. The real-time PCR data are depicted as relative units (RU), which is a fold change in gene expression normalized to that of the endogenous reference gene 18S rRNA and is relative to the no template control (NTC) calibrator. The results depicted are means ± standard deviations (SD) within the shown experiment. Experiments were done five times (A and B) or two times (C to L), with similar findings.

Article Snippet: Anti-human TLR2 neutralizing antibodies (ImmunoKontact and Imgenex) were added at a final concentration of 8.5 μg/ml 30 min prior to virus stimulation.

Techniques: Infection, Transfection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Gene Expression, Control

Journal: Journal of Virology

Article Title: Early Innate Recognition of Herpes Simplex Virus in Human Primary Macrophages Is Mediated via the MDA5/MAVS-Dependent and MDA5/MAVS/RNA Polymerase III-Independent Pathways ▿

doi: 10.1128/JVI.01106-10

Figure Lengend Snippet: HSV-1 infection activates cytokine expression independently of TLR2. Cells were infected with HSV-1 (17+, MOI of 1) or stimulated with TLR2 ligand Pam3CSK4 (100 ng/ml) in the presence or absence of TLR2 neutralizing antibodies (8.5 μg/ml). After 6 h, RNA was harvested, and after 18 h, supernatants were collected. Accumulation of TNF-α and IFN-β mRNA was assessed by real-time PCR (B and C), and the amounts of secreted TNF-α, CXCL10, and CCL3 were measured by ELISA (A, D, and E). RU, relative units. The results depicted are means ± SD from one of two experiments showing similar results.

Article Snippet: Anti-human TLR2 neutralizing antibodies (ImmunoKontact and Imgenex) were added at a final concentration of 8.5 μg/ml 30 min prior to virus stimulation.

Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay