| Abstract |
Toll-like Receptor 9 (TLR9) is an endosomal receptor found highly
expressed in B cells and plasmacytoid dendritic cells (pDCs). It is
activated by unmethylated CpG DNA and synthetic CpG
oligodeoxyribonucleotides (ODNs) to produce pro-inflammatory cytokines
(TNFalpha, IL-12) and type I interferons (IFNs). It senses viral and
bacterial pathogens and activates adaptive immune responses. Dysregulation
of TLR9 signaling can lead to progression and regression of various
cancers and autoimmune diseases. Thus, modulation of TLR9 activity is an
attractive target for immunotherapy. Due to scarcity of pDCs in the blood
and a lack of a suitable model for studying of TLR9, knowledge about this
receptor is lacking. The goal of this project was to characterize two
experimental model cell lines (THP-1 TLR9 mCherry and CAL-1 TLR9 mCherry)
for in vitro studies of TLR9, use them to study TLR9 expression,
trafficking and signaling in pDC-like cells and investigate the role of
Rab11a and Rab39a in these processes. Both cell lines express doxycycline-
inducible TLR9 mCherry. The results showed that IL-4, GM-CSF-
differentiated THP-1 TLR9 mCherry cells resemble immature dendritic cells
in their ability to induce IFNbeta1 and TNFalpha in response to CpG ODNs.
Undifferentiated CAL-1 TLR9 mCherry cells potently induced IFNbeta1 in
response to CpG ODNs, validating their pDC-like properties. In IL-4,
GM-CSF-differentiated THP-1 TLR9 mCherry cells TLR9 was primarily found in
LAMP1-positive vesicles (late endosomes/lysosomes) before and after
stimulation, indicating that TLR9 may reside and interact with its ligands
in late endosomes. In CAL-1 TLR9 mCherry cells, the majority of TLR9
colocalized with late endosomes/lysosomes before and after stimulation
with CpG ODN 2216, a CpG ligand thought to signal from early endosomes.
Stimulation of TLR9 with CpG ODN 2006, a CpG ligand thought to signal from
late endosomes, in CAL-1 TLR9 mCherry cells led to a decrease in TLR9-
LAMP1 colocalization, indicating that TLR9 may be signaling and
interacting with this ligand from other endosomal compartments. Rab11a
regulates TLR4-induced IRF3 activation and IFNbeta production, but its
role in TLR9 signaling is unknown. Rab39a has been identified as a
potential regulator of TLR9 signaling, but its exact role is still unclear.
The results showed that both Rab11a and Rab39a may potentially play a role
in TLR9-induced IFNbeta1 signaling in IL-4, GM-CSF-differentiated THP-1
TLR9 mCherry cells. However, further experiments are required to validate
these findings. In conclusion, the ability to study TLR9 expression,
subcellular localization and response upon ligand stimulation, makes both
THP-1 TLR9 mCherry and CAL-1 TLR9 mCherry cells promising experimental
models for studying TLR9 signaling and trafficking in vitro.
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