TLR7-mediated Type I and Type III interferon production and TLR7 movement within primary human plasmacytoid dendritic cells is stimulated by Influenza virus (67.22)

Abstract

Abstract Human plasmacytoid dendritic cells (pDC) recognize single stranded RNA and double stranded DNA viruses through endosomal TLR7 and -9, respectively, resulting in production of type I and III interferons (IFN). pDC produce both IFN-α and IFN-λ upon Influenza (Flu) virus stimulation; this induction of IFN is dependent on signaling through TLR7 since the TLR7 inhibitor, IRS661, inhibited Flu-induced IFN production in pDC, but not HSV-induced IFN, which signals through TLR9. Cathepsins (Cat), cysteine endolysosomal proteases, are essential for antigen processing in flu-stimulated murine DCs. We found that the broad Cat inhibitor, Z-FM-FMK, inhibited flu and HIV-induced IFN-α production by pDC, both which require processing to make their RNA accessible to TLR7, but had no effect on the small molecule TLR7 ligand Imiquimod-induced IFN production. Flu is known to enter cells via the endocytic pathway, with viral membrane fusion with the endolysosomal membrane required for the release of its viral RNA into the cytoplasm and its subsequent replication. Using ImageStream imaging flow cytometry, we observed that TLR7 is constitutively present in the EEA1 (early)- and LAMP1 (late)-positive endosomes in pDC, and, upon stimulation with flu, TLR7 co-localization with LAMP1-positive endosomes increases. Taken together, these results indicate that recognition of Flu in pDC requires virus endocytosis, Cat processing, and results in TLR7 movement from early to late endosomes and IFN production.