Toll‐like Receptor 9 constitutively traffics from the ER to the lysosome prior to stimulation with CpG DNA

Abstract

The innate recognition of unmethylated CpG dinucleotide motifs present in microbial DNA is mediated through Toll‐like receptor 9 (TLR9). This immunostimulatory effect has been harnessed for therapeutic purposes in vaccines as adjuvant and in cancer therapy. However, recognition of self DNA complexes by TLR9 leads to autoimmune pathogenesis. Control of TLR9 localization contributes to discrimination between self and microbial DNA. The objective of our research is to elucidate the molecular mechanism that regulates TLR9 localization and trafficking. To determine if TLR9 traffics constitutively through the secretory pathway, we developed two in‐vitro assays. A TLR9 chimeric construct, with a furin cleavage site, demonstrates that TLR9 constitutively traffics to the Golgi. Lectin blotting of TLR9 glycan modifications indicates that TLR9 traffics through the Golgi to reach the trans‐Golgi network. Cell fractionation experiments further demonstrate that TLR9 constitutively traffics to lysosomes. Constitutively trafficked TLR9 may initiate signaling in response to CpG DNA since the Golgi disrupting agent Brefeldin A inhibits late, but not early TLR9 signaling events. Together, our data support a model where TLR9 constitutively traffics through Golgi to reach lysosome where signaling is initiated in response to CpG DNA. Supported by NIH grant K22CA113705 awarded to C.A. Leifer.