In of pro-inflammatory pathways. Earlier it was

In our study we used tri-sodium citrate as capping agent of GNPs. Difference
in capping agents could have an impact in toxicity of gold nanoparticles. When
20 nm GNP was coated with citrate and compared with 11-mercaptoundecanoic acid
(MUA) coating, no induction of cytotoxicity was observed in HepG2 cells (46).
However, genotoxicity was observed in cells exposed to citrate coated GNP, but
not in cells exposed to MUA coated GNPs (46). In
our study, citrate coated 5, 15, and 30 nm GNPs did not show cytotoxicity and genotoxicity (data not shown). Patra et
al demonstrated that citrate coated GNPs could induce cytotoxicity in A549
cells even at a dose of 30 nM in contrast to our data (47). This conflicting data could be interpreted to the size of the GNPs
being used as well as the source of capping agent. The morphology change in
A549 cells after GNP treatment was also distinct from our study, which could be
a secondary factor for the difference in cytotoxicity  (47). Positive and negative-surface charged modification in GNPs could
also impact cytotoxicity.

Cancer-related inflammation has been suggested to be
one of the newer hallmarks of cancer (48). Molecular mechanisms linking inflammation to cancer have begun to
unravel. TLRs have been known to be one of the major innate immune players
responsible for the activation of a myriad of pro-inflammatory pathways. Earlier
it was believed that TLRs are expressed only by the immune cells. However, it
is now known that a wide-range of TLRs is also present on the surface of tumor (49). Recently,
TLR 2, 4 and 9 are known to be expressed in lung cancer cell lines. Moreover, several studies have demonstrated that
unrestrained TLR activation may promote chronic inflammation culminating in
tumor progression (50, 51).
Thus, it is indicated that TLRs are one of the vital connecting links between
chronic inflammation and cancer. Since, uncontrolled TLR signaling has been correlated
with cancer progression, use of TLR antagonists could be a promising strategy
for preventing tumor progression.  Amongst
all the TLRs, several studies have reported that TLR4 and TLR9 upregulation is
responsible for carcinogenesis (52, 53). Since
the different sized gold nanoparticles that were used in our study did not
display cytotoxicity in A549 cells, we hypothesized that such particles could
be used as immunomodulators to downmodulate cancer-related inflammation by
antagonizing TLR signaling. When A549 cells were stimulated with either LPS or
CpG-ODN, we observed significant increase in the expression of pro-inflammatory
cytokines including TNF-?, IL-1?, and IL-6 compared to untreated cells. The levels of
these cytokines have been shown to be enhanced during lung cancer (54-56).
Additionally, other pro-inflammatory genes that are upregulated in lung cancer
include iNOS, NF-kB, and p38 (57-59).
NF-kB is potently induced by TNF-?, thereby implying the increase in
NF-kB expression, while both TNF-? and IL-1? activate p38 signaling pathway (60, 61).
Similar to the expression of pro-inflammatory cytokines, expression of iNOS,
NF-kB, and p38 were enhanced when A549 cells were activated with either LPS or
CpG-ODN compared to control cells (Figure 5A, B). When A549 cells were treated
with GNPs only, the expression of the pro-inflammatory genes came down to the
level expressed by untreated control A549 cells. However, before
stimulation with LPS, pre-treatment of A549 cells with different sized GNPs led
to significant downregulation in the expression of pro-inflammatory genes when
compared to A549 cells treated with CpG-ODNs only.
Interestingly, the expression of IL-6 did not alter significantly when the
cells were pre-treated with GNPs and stimulated with LPS compared to LPS only
stimulated cells (Figure 5A). However, the expression of the remaining
pro-inflammatory genes were significantly attenuated when pre-treated with GNPs
and activated with LPS (Figure 5A, C). This suggests that GNPs are specifically
attenuate TLR4 signaling by downregulating IL-1? and TNF-?-mediated NF-?B and p38 activation in lung cancer cells. Lung cancer progression is also associated
with expression of both pro-inflammatory cytokines including IL-8, IL-12, IFN-? and anti-inflammatory cytokines including IL-4 and IL-10 (62).
Whether GNP pre-treated A549 cells are able to alter the expression of these
cytokines after stimulating with either LPS or CpG-ODNs need to be investigated
further.  To the best of our knowledge, this
is the first study that has used 5, 15, and 30 nm citrate-capped GNPs and have
defined their anti-inflammatory properties when lung adenocarcinoma A549 cell
lines have been used and stimulated with TLR agonists, LPS and CpG-ODNs.
Further studies need to be designed to assess if different surface modification
of GNPs have similar effect in A549 cells activated with LPS and CpG-ODNs.
Furthermore, it will also be important to examine whether GNP-bioconjugates
impart similar anti-inflammatory effects.

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There is link between the constitutive expression of specific TLR4
variants and cancer (11). Sustained TLR4 activation is one of the factors that lead to
carcinogenesis (63).
LPS-mediated TLR4 ligation in A549 and H1299 cells leads to p38 MAPK activation
promoting inflammation, and induction of anti-inflammatory cytokine IL-8 and
TGF-?, leading to resistance of apoptosis
suggesting that TLR4 activation could promote tumor immune escape during cancer-related
inflammation (51). Our
data suggest that GNPs downregulate the p38 MAPK signaling pathway thereby
reducing cancer-related inflammation. Whether, these GNPs affect the expression
of anti-inflammatory cytokines and thereby impact the severity of the
cancer-related inflammation is not known in A549 cells. Production of
anti-inflammatory cytokines by LPS in A549 cells could depend on the length of
stimulation. It could be argued that the induction of pro-inflammatory
cytokines precede the expression of anti-inflammatory cytokines in A549 cells
stimulated by LPS. In fact we observed an early peak of pro-inflammatory genes
when A549 cells were activated by LPS for 3 hrs. Even though the
pro-inflammatory genes were induced after 6 hrs of stimulation, GNP
pre-treatment of A549 cells followed by stimulation did not have similar impact
as it had after 3 hrs activation (data not shown). Therefore, early targeting
of pro-inflammatory pathways could prove beneficial to attenuate cancer-related
inflammation. Utilization of CpG-ODN has been explored as an anti-tumor immune
response as adjuvant therapy by targeting TLR9 pathway (64).
However, TLR9 activation has been demonstrated to augment metastasis of human
lung cancer cell lines and in vivo (12, 65).
Therefore, CpG-based therapies could target TLR9 signaling pathway to attenuate
cancer-related inflammation. Our data suggests that GNPs could target the TLR9
signaling pathway to limit cancer-related inflammation (Figure 5B, D).
Therefore GNPs could not only target the surface TLR, but also can have an
impact on the activation of TLR expressed in endosome. When we compared the
activation of both TLR4 and TLR9 pathways when A549 cells were stimulated with
LPS and CpG-ODN, respectively, we observed that the induction of
pro-inflammatory cytokines was higher when LPS was used as stimulant.
When A549 cells were pre-treated with GNPs, the pro-inflammatory cytokines apart
from IL-6 came down to the level expressed by control cells.
This suggests that GNPs could act with higher efficiency when they target TLR4
signaling pathway.

Our results indicate that 5 nm GNPs are most potent in
downregulating the TLR4 signaling pathway.  Enhanced capability of small-sized (4
nm GNPs) in inducing immunomodulation was observed Tsai
et al (2011). They demonstrated that 4 nm GNPs were most potent in
manipulating inflammatory responses in macrophage cell line RAW264.7. Increased
anti-inflammatory potency was attributed to increased surface area to volume
ratio of small-sized GNPs in this study. Similarly, in our study the 5 nm GNPs
were observed to be most potent in downregulating the inflammatory responses
induced by immunostimulants. Thus, this increased immunomodulatory potential of
5 nm GNPs could be attributed to efficient internalization by A549 cells and
large surface are to volume ratio of 5 nm GNPs. In order to unravel the mechanism
underlying the immunomodulatory potential of 5 nm GNPs, the effect of GNPs’
treatment on the expression of TLR4 (significantly down modulated) by A549
cells was investigated by qPCR and flow cytometric analysis. We observed that 5
nm GNPs impaired the expression of TLR4 mRNA as well prevented the production
of TLR4 (Figure 6A, B). This suggests that the mechanism of action of 5 nm GNPs
in downmodulating TLR4 signaling pathway is by attenuating the TLR4 expression
resulting in diminished TLR4 signaling. This observation is supported by a
study involving C57BL/6 mice, wherein GNP-quercetin conjugates prevented kidney
injury by hampering TLR4/NF-?B expression
and associated inflammatory responses (66).
Moreover, naked GNPs were also observed to alleviate LPS-induced inflammation
in eye by downmodulating TLR4 expression and corresponding NF-?B activation indicating downregulation of TLR4 expression is a
mechanism utilized by GNPs to induce immunomodulation in a variety of
inflammatory conditions (67). Our
data suggest that gold nanoparticles downregulate the expression of TLR4
expression thereby immunomodulating the inflammatory responses in A549 cells