Toll-like receptor 9 in plasmacytoid dendritic cells fails to detect parvoviruses

Abstract

Toll-like receptor 9 (TLR9) recognizes genomes of double-stranded DNA (dsDNA) viruses in the endosome to stimulate plasmacytoid dendritic cells (pDCs). However, how and if viruses with single-stranded DNA (ssDNA) genomes are detected by pDCs remain unclear. Here we have shown that despite the ability of purified genomic DNA to stimulate TLR9 and despite the ability to enter TLR9 endosomes, ssDNA viruses of the Parvoviridae family failed to elicit an interferon (IFN) response in pDCs.

Fig 1

Murine bone marrow cells and pDCs do not respond to murine parvovirus. (A) Total bone marrow cells from mice of indicated backgrounds were transfected with CpG 2216 (5 μM) or infected with MVMp (20,000 genomes/cell). Supernatants were collected after 24 h, and IFN-α levels were measured by ELISA. (B) Flt3L pDCs were transfected with CpG 2216 (5 μM) or infected with MVMp (20,000 genomes/cell). Cells were collected after 8 h for analysis of IFN-α and IL-6 and IL-12p40 cytokine mRNA levels by RT-qPCR, and supernatants were collected after 24 h for analysis of IFN-α, IL-6, and IL-12p40 protein levels by ELISA. (C) Flt3L pDCs from C57BL/6 mice were transfected with CpG 2216 (5 μM) or infected with VSV (multiplicity of infection [MOI] = 5), MVMp (20,000 genomes/cell), AAV-2 WT (10,000 genomes/cell), or AAV-2 GFP (10,000 genomes/cell). After 24 h, cell supernatant was collected, and IFN-α and IL-6 protein levels were measured by ELISA.

Fig 2

MVMp enters bone marrow cells and Flt3L pDCs. (A) Total bone marrow cells were infected with MVMp (20,000 genomes/cell) for 3 h, and then cells were treated with neuraminadase (NA) for 1 h to remove bound virions from the cell surface. Cells were stained with anticapsid antibody and 4′6-diamidino-2-phenylindole (DAPI) after being spun onto spot slides. Representative images are shown. (B) Flt3L pDCs were infected with MVMp (20,000 genomes/cell) for 4 h and then incubated with or without neuraminidase for 2 h. After extensive washing, cells were lysed and subjected to qPCR to quantify the number of MVMp genomes that had entered the cells. (C) Flt3L pDCs were infected with serial dilutions of MVMp ranging from 20,000 genomes/cell to 2,500 genomes/cell for 24 h. Cells were then stimulated with CpG 2216 (1 μM), and after 24 h, supernatants were collected and IFN-α and IL-6 protein levels were quantified by ELISA.

Fig 3

MVMp genomic DNA stimulates pDCs via TLR9 and MyD88. Flt3L pDCs from C57BL/6, TLR9-deficient, or MyD88-deficient mice were transfected with CpG2216 (5 μM) or MVMp genomic DNA (10,000 genome equivalents/cell) or infected with VSV (MOI = 5) or with MVMp (10,000 genomes/cell). Cell supernatants were collected after 24 h, and IFN-α (top) or IL-6 (bottom) protein levels were measured by ELISA.

Fig 4

MVMp localization within TLR9 LRO. RAW264.7 cells expressing TLR9-GFP were infected with MVMp (20,000 genomes/cell) for 5.5 h. Cells were fixed and stained with anti-LAMP2 and anti-MVMp capsid antibodies and then subjected to confocal microscopy. Representative images are shown.