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Cellosaurus publication CLPUB00540

Publication number CLPUB00540
Authors Wang C.
Title Characterization of a model cell line to study Toll-like receptor 9 signaling and trafficking.
Citation Thesis MSc (2019), Norwegian University of Science and Technology, Norway
Web pages https://hdl.handle.net/11250/2613188
Abstract Toll-like receptors (TLRs) are a family of innate immune receptors that function to mount an immune response upon recognition of molecules associated with infection or injury. Toll-like receptor 9 (TLR9) is expressed predominantly in endosomes of plasmacytoid dendritic cells (pDCs), where it is responsible for recognition of unmethylated CpG DNA derived from virus or bacteria. Activated TLR9 initiates immune responses which are important in antiviral immunity. Upon activation, TLR9 locates to early endosomes and initiates the production of interferons (IFNs) through transcription factor IRF7. Further sorting of TLR9 to late endosomes stimulates the production of proinflammatory cytokines like TNFalpha and IL-12B through transcription factor NF-kappaB. In pDCs, TLR9 can trigger potent anti-tumor immunity, however, the receptor has also been found involved in driving tumor progression. Since pDCs are rare in the human blood, this project sought to establish and characterize a model cell system that resembles human pDCs. A THP-1 cell line with inducible expression of TLR9 mCherry was characterized and optimized for studying TLR9 signaling and trafficking. Different differentiation protocols were applied to the THP-1 TLR9 mCherry cells before cells were induced to express TLR9 and stimulated with CpG. PMA differentiation of these cells failed to produce a potent IFNbeta1 response in response to CpG. In contrast, GM-CSF and IL-4-differentiated and undifferentiated THP-1 TLR9 mCherry cells mimicked pDC responses and induced marked levels of IFNbeta1, as well as TNFalpha, in response to CpG. Previous findings in HEK293 cells indicated a role for the GTPase Rab39a in TLR9 signaling. Undifferentiated THP-1 TLR9 mCherry cells were used as a model cell line for studying how Rab39a silencing might affect TLR9 signaling. siRNA experiments targeting Rab39a revealed an increased tendency of IFNbeta1 and TNFalpha mRNA levels in response to CpG in undifferentiated THP-1 TLR9 mCherry cells. Combined, this project provides a novel model system to study TLR9 signaling and trafficking and suggests that Rab39a might be involved in regulation of signaling from TLR9.
Cell lines CVCL_XI47; THP-1 TLR9 mCherry