| 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.
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