Page 229 - Read Online

Figueroa-Hall et al. TLR4-mediated signaling in CHME-5 cells

model an inflammatory state in the CNS and studied further analysis of ubiquitination in CHME-5 cells is
several signaling proteins that are constitutively warranted to elucidate these events.
expressed and activated in the TLR4 pathway.
Analysis of 2 crucial intracellular TLR4 signaling TLR4, a transmembrane glycoprotein, is part of
proteins, IκBα and NF-κB, were chosen to investigate the innate immune response, and is expressed in
neuroinflammatory signaling in CHME-5 cells. LPS- the CNS, primarily in microglia [17,18] . Consequently,
induced NF-κB p65 activation was consistent with in addition to assessing LPS-induced signaling,
other microglial cell lines [28,37-39] . Given the fact that we were interested in the effects of LPS on TLR4
many studies investigating microglial responses used expression. Increased TLR4 gene expression
the same dose of LPS (1 μg/mL), we are confident following LPS treatment was consistent with reports
that CHME-5 cells were adequately treated to showing increases in TLR4 expression in whole
observe inflammatory responses [36,40,41] ; unlike the blood cells and monocytes as early as 2 and 3 h,
use of a lower dose of LPS (1 ng/mL), which failed to respectively [49,50] . The increase observed in TLR4
activate and release nitric oxide in CHME-5 cells [42] . gene expression in CHME-5 cells following LPS
Additionally, other studies using human primary treatment has been reported to occur through binding
microglia and CHME-5 cells have used similar LPS of the master regulator PU.1 to TLR4 promoter
[51]
dose range (0.1-1 μg/mL) and time points (6 h for RT- regions, in response to endotoxin . The expression
PCR) to investigate inflammatory responses [26,36,37,40,41] . of TLR4 as early as 3 h may be attributed to TLR4
being an early or middle phase gene that peaks at 1 h
The ability of p65 to be activated, translocate into the and 3 h [45] . LPS-induced TLR4 protein expression
nucleus, and bind to NF-κB consensus sequences, increased compared to unstimulated cells as seen in
is crucial in mediating inflammatory responses in a both immunoblot analysis and immunocytochemistry.
timely manner. Here, we demonstrated a second, Activation at 270 min may also be due to late-
more functional mean of NF-κB activation. LPS- phase NF-κB activation, which is attributed to TRIF-
[3]
induced NF-κB binding activity was exhibited in 2 dependent signaling . On the other hand, the lack
waves, at 10 and 90 min. This LPS-induced biphasic of TLR4 protein expression at 180 min may be due
activity, has been previously shown in macrophages to negative regulation. There are several negative
and is attributed to several possibilities: (1) platelet regulators that control TLR4 inflammatory signaling
activating factor (PAF), which is up-regulated at different stages in the signaling pathway, such as
during inflammatory stimuli, is involved in NF-κB selective androgen receptor modulator, RP105, and
nuclear translocation, which in turn produces pro- ST2L, which can be induced as early as 10 min, as
inflammatory cytokines, ultimately up-regulating PAF in the case of IRAK-M [52,53] . Further investigation is
again in a feedback loop or (2) the release of p65 warranted to determine mechanisms for regulation of
is not only attributed to IκBα, but IκBβ as well [43,44] . LPS-induced TLR4 signaling in CHME-5 cells.
NF-κB is first released from IκBα and then from
IκBβ, causing a biphasic response [43] . In fact, we We provide novel images of CHME-5 cells,
also observed that the NF-κB target gene, TNFα, showing TLR4 and CD68 immunofluorescence.
displayed similar biphasic-like expression in CHME-5 Epifluorescence imaging revealed that CD68
cells following LPS treatment, which was also seen in and TLR4 are constitutively expressed and are
HeLa cells [45] . robustly up-regulated following LPS stimulation.
Understandably, up-regulation of CD68 is expected
LPS-induced IκBα activation as demonstrated by due to its state of activation in response to LPS.
phosphorylation is an early (within 10 min), transient Moreover, confocal imaging provided novel
event in CHME-5 cells. Phosphorylation was assessed visualization of the expression of TLR4 and CD68 in
in cytoplasmic fractions; thus it is presumable that unstimulated and LPS-stimulated cells. Furthermore,
after 10 min IκBα translocated into the nucleus or we provided a 3D representation of these proteins
underwent proteasomal degradation [46] . Additionally, in CHME-5 cells, in response to LPS. The ability to
even though the data shows a trend towards IκBα reconstruct cells and observe protein expression in
degradation, the analysis revealed that this was a 3D setting provides spatial awareness based on
not the case during early events in CHME-5 cells. fluorescent intensity. Together, these imaging studies
It may be that in CHME-5 cells, evaluation in whole provide new, qualitative information about CD68/
cell lysates is needed to get a detailed assessment TLR4 expression in CHME-5 cells.
of IκBα degradation, as research indicates that
IκBα is also present in the nucleus [46,47] . IκBα signal- In summary, understanding microglial inflammatory
dependent degradation only occurs in response responses is very important given the instrumental
to ubiquitination of lysine residues [47,48] , therefore role of these cells in the innate CNS immune response
Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ October 19, 2017 229