4. Discussion
During the recent years, more and more emphasis has been put on
investigation of the link between inflammatory processes arising during
pregnancy and neonate development, pregnancy complications as well as
long-term outcomes affecting further development of the
newborn.35-39 Several studies have shown deregulation
of ABC and SLC transporters40-43 in inflammatory
conditions of placental tissue, however, there is no information about
the effect of inflammation on the expression of OCTN1 and OCTN2
transporters in human placenta so far. Therefore, we aimed to
characterize the gene expression pattern for both OCTN transporters in
response to inflammatory models comprising trophoblast cell lines andex vivo cultured human placental villous explant models.
Both transporters have been associated with inflammatory processes in
other non-placental tissues.14 In particular, the
presence of polymorphic variants of OCTN1/2 were observed in
rheumatoid arthritis44 and intestinal
inflammation-based diseases such as inflammatory bowel
disease45, Crohn’s disease46-48 or
ulcerative colitis.49 Moreover, deregulation ofOCTN2 expression and activity by the state of inflammation was
described in the intestine, even though with rather contradictory
results.50,51 In this study, we show that the gene
expression of both OCTN transporters can be affected when an
inflammatory environment is present in placenta. OCTN1 expression
significantly increased when human placental villous explants were
exposed to LPS. Increase was observed also upon the exposition to TNF-α
but not reaching statistical significance. Similar pattern was observed
in HTR-8/SVneo cell line following the exposition to TNF-α or also IFN-γ
for 72 h. These data indicate possible induction of OCTN1 gene
expression under conditions of bacterial inflammation followed by
release of pro-inflammatory cytokines. Our data are in line with the
study showing upregulation of OCTN1 in response to TNF-α in human
fibroblast-like synoviocyte cell line MH7A used as a model of rheumatoid
arthritis, the chronic inflammatory disease.52 On the
other hand, Li et al.53 describe that LPS-induced
inflammation could inhibit expression and activity of OCTN1/2 in
alveolar epithelial cells (A549). Similarly, study Ling et
al.54 showed that the expression of both OCTN
transporters was significantly decreased in LPS-induced inflammation in
the lactating rat mammary gland at different lactation stages, which
indicates distinct regulatory pathways for OCTN1 expression and role in
various tissues. Even though mediating transport of several endogenous
as well as exogenous compounds OCNT1 seems to show high transport
efficacy for ergothioneine, ensuring thereby protection of cells and
tissues from oxidative stress and/or inflammatory
damage.12,55,56 A study of Shimizu et
al.56 demonstrate that up-regulation of OCTN1 and
consecutive uptake of ergothioneine serve as a feedback mechanism for
preventing further inflammatory damage of the cells. TNF-α and IFN-γ, as
one of the most important inflammatory cytokines play an important role
in maintaining pregnancy.57,58 Couple of studies have
shown a cytotoxic effect of higher TNF-α and IFN-γ levels in human
trophoblast.59,60 Elevated TNF-α concentration in
maternal serum have been reported in early pregnancy associated with
preeclampsia61 or first trimester spontaneous
abortion.62 Moreover, increased TNF-α levels have been
associated with a number of adverse effects reported early in pregnancy,
such as gestational hypertension63 and gestational
diabetes mellitus.64 Placenta itself can be a source
of cytokines including TNF-α, with levels increasing from early
pregnancy to elevated levels shortly before
delivery.65 During the first trimester, significant
endogenous expression of TNF-α has been detected also in extravillous
trophoblasts.66 Based on these data, we hypothesize
that upon external inflammatory stimulation with TNF-α, IFN-γ and
possibly also LPS, upregulation of OCTN1 may serve as a
protective tool for the placental and fetal tissue and thereby aiming
for maintaining optimal fetal development even in the potentially
harmful inflammatory environment. Such mechanism might be even more
essential during the earlier phases of pregnancy, which is in line with
upregulation of OCTN1 being clearly seen especially in the
HTR-8/SVneo cells, the first trimester trophoblast-derived cell line,
comprising, however two populations, trophoblast and stromal
cells.67,68
In the case of OCTN2 transporter, up-regulation of the gene expression
was observed following IFN-γ treatment of HTR-8/SVneo cells, while
decreased OCTN2 expression was seen with TNF-α and LPS in human
placental explants and with TNF-α in BeWo cells. BeWo cells, represent
one of the commonly used placental models, maintaining many
characteristics of human cytotrophoblasts thereby being used to study
the placental uptake of various nutrients e.g.
glucose,69 amino acids70,71 and
iron72 and evaluate the expression and role of several
transporters.16,73 It should be, however, noted that
due to its cancerous origin, the model exhibit pro-tumorous changes that
are inconsistent with healthy tissue and drive the interpretation of
obtained data challenging and their value rather limited. Our results
are in agreement with the study of Console et al.74showing increased expression of OCTN2 in HEK293 cells and
cell-derived exosomes upon exposition to IFN-γ. Moreover, essential role
of INF-γ in the defense against microbial infections through the
increased expression of OCTN2 was reported by another
study.75 The study Console et al.74further suggests that OCTN2 may be increased in association with
restoration of intestinal homeostasis under inflammatory stress.
Increased OCTN2 expression was further seen in Caco-2 cells due to the
treatment with IFN-γ and TNF-α.50 However, another
study shows a decrease in the expression of OCTN2 after exposition to
mixture of proinflammatory cytokines TNF-α, IL-1β and IFN-γ in the human
normal colon FHC cell line. This decrease further lead to reduced
L-carnitine content and decreased proliferation of the FHC
cells.51 The regulation reported by these authors was
shown to involve PPARγ/RXRα pathways.
No upregulation of OCTN1/2 was, however, observed following stimulation
with HMGB1 or in the presence of elevated IL-6 levels, which might
reflect more specific patterns in regulation of OCTN1 during
inflammation in placental cells. In human placenta, HMGB1 is a nuclear
factor that is released from damaged cells acting extracellularly as an
endogenous danger signal mainly through the TLR2 and TLR4
receptors.76 Subsequent release of pro-inflammatory
cytokines further promote the inflammation. Unlike TLR4, TLR2 was
detected mainly in first trimester cytotrophoblast, but not in term
syncytium. Moreover, in inflammatory state such as preeclampsia, the
expression TLR4 significantly increases, when compared to normal
pregnancy.77,78 Thereby we hypothesize, that the
ability of our placental/trophoblast models including healthy term
placenta explants to respond to exogenously applied HMGB1 and reveal any
changes in OCTN1/2 expression might be caused by low expression of
relevant HMGB1-responsive receptors and pathways.
To sum up, our results clearly demonstrated that inflammatory
environment could lead to affected expression of placentalOCTN1/2 genes. We speculate that the upregulation of OCTN1can serve as a protective mechanism to placental tissue allowing for
optimal fetal development. Moreover, we suggest that the exposure of
placental cells to certain inflammatory markers and cytokines may affect
fetal carnitine homeostasis due to OCTN2 deregulation. Following IFN-γ
exposition, significant increase in OCTN2 levels should provide placenta
with enhanced supply of L-carnitine, leading to stimulated of energy
production through the fatty acid oxidation, while LPS and TNF-α show
the opposite tendency through the decrease in OCTN2expression. This study is the first to show the deregulation of
placental OCTN1/2 transporters upon inflammatory environment. Further
studies will be needed to reveal the impact of these gene-deregulations
on the placenta transporter protein expression and the real consequences
for placental tissue transport function and fetal development.