Toll-Like Receptor 3 Is a Mediator of the Innate Immune Response to Cytomegalovirus in Immature Dendritic Cells.

Dendritic cells (DCs), contribute to the initiation of immune responses to viral infection. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and initiate antimicrobial immune responses. TLR3 in DCs recognizes viral double-stranded RNA and triggers downstream signals to activate the NF?B and the interferon ß promoter. Double-stranded RNA may also be produced by double-stranded DNA viruses, such as HCMV, through bidirectional transcription from the genome during infection.

Here we investigated whether TLR3 mediates the interaction between monocyte-derived immature DCs (iDCs) and HCMV after either active viral replication or viral penetration. We observed that HCMV strains differ in their interactions with iDCs. Strains that show no tropism for DCs, such as AD169, only penetrate iDCs, whereas the DC-tropic strains, e.g. TB40-E, actively replicate in iDCs. This difference provides an opportunity to study different forms of virus-DC interaction. Genome-wide expression array analysis showed that although 23 genes encoding cytokines, chemokines, and transcription factors are upregulated in iDCs after incubation with either strain, subsets of genes are induced specifically by DC-tropic or DC-nontropic strains. Only interaction with the DC-tropic HCMV strain TB40E, which replicates and produces mature virions, led to up-regulation of the TLR3 gene as well as genes downstream of TLR3 in the TLR3-signaling pathway, including class I interferon genes, NF?B, TRAF family member-associated NFKB activator (TANK), TANK-binding kinase 1 (TBK1), CXCL10, and CXCL11. The DC-nontropic HCMV strain AD169, which penetrates iDCs without replicating, did not upregulate genes of the TLR3 pathways. For selected genes, array data were confirmed by quantitative real-time PCR assay and ELISA to detect the gene products.

To further confirm that the DC-tropic HCMV strain TB40E interacts with iDCs via TLR3, we transfected DCs with TLR3-specific siRNA prior to infection. TLR3 gene expression was potently silenced, while levels of the hALAS housekeeping gene mRNA remained normal. After these transfected DCs were infected with TB40E, HCMV-induced TLR3 gene expression was still markedly downregulated (−219 x), as were the downstream genes of the TLR3-signaling pathway (IFNa, −2.8 x; IFNß, −12.8 x; NF?B, −7.7 x; CCL5, −14.4 x; CXCL10, −16.5 x; CXCL11, −10.9 x). In contrast, TLR3 siRNA alone did not significantly modulate the expression of NF?B, CCL5, CXCL10, and class I interferons. Our results are consistent with those of McWirther et al., who reported that mice with a deficiency of TBK1 which is downstream of TLR3 show marked defects in IFNa and IFNß gene expression after viral infection or after engagement of TLR3 by double-stranded RNA.

Thus, a key mediator of HCMV-DC interaction, which activates both a MyD88-dependent pathway that leads to early NF?B activation and a MyD88-independent pathway that leads to a class I interferon response (IFNa and IFNß) via interferon regulatory factor 3 (IRF3). This activation of the TLR3 signalling pathway was not observed when the DC-nontropic HCMV strain AD169 penetrated DCs without replicating. The identification of pathways that enhance innate antiviral immune responses may provide new avenues of therapeutic intervention for viral infections.