NFκB-dependent increase of kynurenine pathway activity in human placenta: inhibition by sulfasalazine.

Catabolism of tryptophan via the kynurenine pathway is up-regulated in the human placenta by infection, resulting in the release of pro-inflammatory and neuroactive metabolites into the fetal circulation. In this study we determined if activation of NFκB is involved in the inflammation-induced increase of kynurenine pathway activity in the human placenta. Placentae obtained after elective caesarian section at 37-40 weeks gestation (n=8), and explants (35-40 mg) prepared from terminal villi were incubated under standard conditions in the presence of 10 µg/ml LPS for 24 or 48 h; duplicates of each explant were incubated either with or without 5mM sulfasalazine added to the medium. Expression of mRNAs for key kynurenine-forming enzymes, indoleamine 2,3-dioxygrenase (IDO) and tryptophan 2,3-doalxygenase (TDO) and the inflammatory cytokines TNFα and IL6 was studied by RT-PCR. Kynurenine output by explants was measured in samples in the incubation medium by absorbance at 363nm after separation from other metabolites using an HPLC technique. Expression of IDO, TDO, TNFα and IL6 mRNAs was increased with LPS treatment, a response mitigated by the presence of sulfasalazine (P<0.01, P<0.01, P=0.03 &P=0.04). Kynurenine output into the culture medium increased with LPS treatment but this was also prevented by sulfasalazine at 24h (mean ± SEM; 412.1 ± 40 vs. 147.7 ± 48.9 nM/mg, P=0.01) and 48 h (636 ± 39.1 vs. 135.5 ± 29.8 nM/mg, P=0.001, respectively). Sulfasalazine inhibited the LPS induction of both the kynurenine pathway and pro-inflammatory cytokines in the placenta, implicating NFκB in the LPS effect. Direct measurement of NFκB activity showed that sulfasalazine decreased NFκB activation under both control and LPS-treated conditions. These observations show that kynurenine pathway activity in the human placenta is increased by a NFκB dependent pathway, and suggests a new therapeutic strategy for the management of pregnancies with in utero infection.

Localisation of indoleamine 2,3-dioxygenase and kynurenine hydroxylase in the human placenta and decidua: implications for role of the kynurenine pathway in pregnancy.

Indoleamine 2,3-dioxygenase (IDO) has been implicated in contributing to immunotolerance in early pregnancy, but the presence in the term placenta of mRNAs for enzymes that produce other biologically active kynurenine end-products suggests other functions for kynurenine pathway metabolites. The aim of this study was to investigate the localisation of two key enzymes - IDO and kynurenine hydroxylase (KYN-OHase) - in first trimester decidua and in the human placenta across pregnancy. Using immunocytochemistry, it was shown that there was strong expression of IDO and KYN-OHase in stromal and glandular epithelial cells of first trimester decidua. In first and second trimester placenta, IDO and KYN-OHase were localised to the syncytiotrophoblast, stroma and macrophages. IDO and KYN-OHase mRNAs were also identified, and the enzymes appear to be functional because kynurenine and 3-hydroxy-anthranilic acid (respective products of the activity of these enzyme) were released into the medium when first trimester placental explants were maintained in culture for 48h. In term placenta, both IDO and KYN-OHase immunoreactivities were confined mainly to vascular endothelial cells of villous blood vessels, and to macrophages within the fetal villus, whereas syncytial staining was very weak or absent. The shift of expression of these enzymes away from the syncytiotrophoblast to fetal endothelial cells in terminal villi suggests that the function of the enzymes may change from a role in immunosuppression at the maternal-fetal interface in early pregnancy, to one associated with regulation of fetoplacental blood flow or placental metabolism in late gestation.