Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus

Abstract

We have previously shown that mice inoculated intranasally with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus [AcNPV]) are protected from a lethal challenge by influenza virus. However, the precise mechanism of induction of this protective immune response by the AcNPV treatment remained unclear. Here we show that AcNPV activates immune cells via the Toll-like receptor 9 (TLR9)/MyD88-dependent signaling pathway. The production of inflammatory cytokines was severely reduced in peritoneal macrophages (PECs) and splenic CD11c(+) dendritic cells (DCs) derived from mice deficient in MyD88 or TLR9 after cultivation with AcNPV. In contrast, a significant amount of alpha interferon (IFN-alpha) was still detectable in the PECs and DCs of these mice after stimulation with AcNPV, suggesting that a TLR9/MyD88-independent signaling pathway might also participate in the production of IFN-alpha by AcNPV. Since previous work showed that TLR9 ligands include bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides, we also examined the effect of baculoviral DNA on the induction of innate immunity. Transfection of the murine macrophage cell line RAW264.7 with baculoviral DNA resulted in the production of the inflammatory cytokine, while the removal of envelope glycoproteins from viral particles, UV irradiation of the virus, and pretreatment with purified baculovirus envelope proteins or endosomal maturation inhibitors diminished the induction of the immune response by AcNPV. Together, these results indicate that the internalization of viral DNA via membrane fusion mediated by the viral envelope glycoprotein, as well as endosomal maturation, which releases the viral genome into TLR9-expressing cellular compartments, is necessary for the induction of the innate immune response by AcNPV.

FIG. 1.

Immune system activation of macrophages by heat-denatured or gp64-deficient AcNPV. (A) Purified particles of the mutant virus, AcNPVΔ64, lack gp64, as assayed by immunoblotting. (B) The production of TNF-α in RAW264.7 cells (10⁶ cells/well) inoculated with AcNPV (5 μg/ml) (bar 2) or AcNPVΔ64 (5 μg/ml) (bar 3) was determined 24 h after inoculation by a sandwich ELISA. 1 is an uninfected control. Data are shown as means ± SD. (C) AcNPV and PGN were incubated at 70°C for 30 min. Treated and untreated samples were inoculated into RAW264.7 cells (10⁶ cells/well) and incubated for 24 h. The production of TNF-α was determined by a sandwich ELISA. Data are shown as means ± SD.

FIG. 2.

Immune system activation by AcNPV in macrophages is not mediated by gp64. (A) Wild-type gp64 and a deletion mutant lacking the transmembrane region of the gp64 envelope protein (gp64ΔTM) were expressed in Sf-9 cells. Whole-cell lysates and culture supernatants were subjected to SDS-PAGE under reducing conditions and visualized by immunoblotting with an antihexahistidine monoclonal antibody. Lane 1, cells transfected with pIB/V5-His; lanes 2 and 3, cells transfected with pIBgp64ΔTM/V5-His and pIBgp64/V5-His, respectively. The heavy chains of the antibody are indicated by asterisks. (B) Purified AcNPV virions (lane 2) and gp64ΔTM (lane 3) were analyzed by SDS-PAGE and Coomassie blue staining. Lane 1, molecular mass markers. (C) Activation of mouse macrophage RAW264.7 cells (10⁶ cells/well) treated with the indicated amounts of AcNPV or gp64ΔTM. The production of TNF-α and IL-6 in culture supernatants after 24 h of incubation was determined by sandwich ELISAs. PGN was used as a positive control. Data are shown as means ± SD. (D) Production of IFN-α in RAW264.7 cells (10⁶ cells/well) inoculated with AcNPV (5 μg/ml) or gp64ΔTM (5 μg/ml), as determined by a sandwich ELISA after 24 h of incubation. Data are shown as means ± SD. (E) Production of TNF-α in RAW264.7 cells (10⁶ cells/well) inoculated with AcNPV (20 μg/ml) or PGN (2.5 μg/ml), with or without a pretreatment with the indicated amounts of gp64ΔTM for 2 h at 37°C. After 24 h of incubation, the production of TNF-α in culture supernatants was determined by a sandwich ELISA. Data are shown as means ± SD.

FIG. 3.

AcNPV activates PECs and DCs in a MyD88/TLR9-dependent manner. (A) PECs (2 × 10⁵ cells/well) from wild-type (C57BL/6) or MyD88-, TLR2-, TLR4-, or TLR9-deficient mice were stimulated with the indicated amounts of AcNPV or loxoribine. The production of IL-12 p40 in culture supernatants was measured by a sandwich ELISA. Data are shown as means ± SD. (B) Northern blot analysis of murine macrophage cells stimulated with AcNPV. PECs (6 × 10⁶ cells/well) from wild-type or MyD88- or TLR9-deficient mice were stimulated with AcNPV (10 μg/ml) for the indicated times. Total RNAs were extracted and subjected to Northern blot analysis. (C) Splenic CD11c⁺ DCs were prepared from wild-type or MyD88-, TLR4-, or TLR9-deficient mice and enriched by magnetic cell sorting. Splenic DCs (10⁵ cells/well) were stimulated with the indicated amounts of AcNPV or loxoribine for 24 h. The production of IL-12 p40 in supernatants was measured by a sandwich ELISA. Data are shown as means ± SD.

FIG. 4.

IFN production by AcNPV is mediated by a MyD88/TLR9-independent process. (A) PECs (2 × 10⁵ cells/well) and splenic CD11c⁺ DCs (1 × 10⁵ cells/well) were prepared from wild-type or MyD88- or TLR9-deficient mice and stimulated with the indicated amounts of AcNPV or loxoribine for 24 h. The production of IFN-α in culture supernatants was measured by a sandwich ELISA. Data are shown as means ± SD. (B) Northern blot analysis of murine macrophage cells stimulated with AcNPV. PECs (6 × 10⁶ cells/well) from wild-type or MyD88- or TLR9-deficient mice were stimulated with AcNPV (10 μg/ml) for the indicated times. Total RNAs were then extracted and subjected to Northern blot analysis.

FIG. 5.

Activation of mouse macrophage cell line by AcNPV DNA. (A) Methylation status of genomic DNA. Genomic DNAs obtained from AcNPV, Sf-9 cells, E. coli, and 293T cells were digested with the methylation-sensitive restriction enzyme HpaII. Undigested (−) and digested (+) samples were analyzed by agarose gel electrophoresis. (B) RAW264.7 cells (10⁶ cells/well) were treated with AcNPV DNA (5 μg/ml) or PGN (2.5 μg/ml) in the absence (−) or presence (+) of liposomes for 24 h, and the production of TNF-α in culture supernatants was determined by a sandwich ELISA. Data are shown as means ± SD. (C) Activation of RAW264.7 cells (10⁶ cells/well) inoculated with untreated or UV-inactivated AcNPV (5 μg/ml) in the presence or absence of liposomes was assessed by the production of TNF-α in culture supernatants. Data are shown as means ± SD.

FIG. 6.

AcNPV DNA induces NF-κB activation through human TLR9. (A) 293T cells were transfected with an empty or human TLR9 expression vector together with a pELAM luciferase reporter plasmid. Twenty-four hours after transfection, the cells were stimulated with digested or undigested AcNPV DNA (10 μg/ml). hCpG (10 μg/ml) was used as a positive control. The luciferase activity was determined at 24 h posttransfection and expressed as the level of induction compared with that detected in cells transfected with the human TLR9 expression vector alone. Data are shown as means ± SD. (B) Immunofluorescence micrographs of 293T cells transfected with an N-terminal Flag-tagged human TLR9 expression vector and stained with an anti-Flag (M2) monoclonal antibody. The intracellular (left) and cell surface (right) expression of TLR9 is shown. Nuclei were stained with propidium iodide (PI). Samples were observed by confocal microscopy. (C) The surface and intracellular expression of human TLR9 in 293T cells transfected with an N-terminal Flag-tagged human TLR9 expression vector (+) or an empty vector (−) and stained with an anti-Flag monoclonal antibody was examined by fluorescence-activated cell sorting.

FIG. 7.

AcNPV requires endosomal maturation to induce immune system activation in macrophages. (A) RAW264.7 cells (10⁶ cells/well) were stimulated with AcNPV (5 μg/ml), mCpG (200 ng/ml), LPS (10 ng/ml), or PGN (2.5 μg/ml) at the indicated concentrations of chloroquine. After 24 h of incubation, the production of TNF-α in culture supernatants was determined by a sandwich ELISA. Chloroquine was added to the cells 2 h before stimulation. Data are shown as means ± SD. (B) RAW264.7 cells (10⁶ cells/well) were treated with AcNPV (5 μg/ml) or LPS (10 ng/ml) and with the indicated concentrations of endosomal maturation inhibitors. After 24 h of incubation, the production of TNF-α in culture supernatants was determined by a sandwich ELISA. The inhibitors were added to the cells 2 h before stimulation. Data are shown as means ± SD.

FIG. 8.

AcNPV penetrates macrophages through the phagocytic pathway. (A) 293T and RAW264.7 cells (10⁶ cells/well) were inoculated with a recombinant baculovirus possessing the luciferase gene under the control of the CAG promoter, AcCAGluc (49) (10 and 20 μg/ml). Cells were harvested 24 h after infection, and relative luciferase activities were determined. (B) 293T and RAW264.7 cells (10⁶ cells/well) were inoculated with AcCAGluc (40 μg/ml), washed extensively after 1 h of adsorption, and harvested after 4 or 6 h of incubation. The presence of the p39 capsid protein in cells inoculated with AcNPV was determined by immunoblotting with an anti-p39 monoclonal antibody.