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.