Manuel Luis Wolfson

Progesterone is essential for protecting against LPS-induced pregnancy loss. LIF as a potential mediator of the anti-inflammatory effect of progesterone.

Lipopolysaccharide (LPS) administration to mice on day 7 of gestation led to 100% embryonic resorption after 24 h. In this model, nitric oxide is fundamental for the resorption process. Progesterone may be responsible, at least in part, for a Th2 switch in the feto-maternal interface, inducing active immune tolerance against fetal antigens. Th2 cells promote the development of T cells, producing leukemia inhibitory factor (LIF), which seems to be important due to its immunomodulatory action during early pregnancy. Our aim was to evaluate the involvement of progesterone in the mechanism of LPS-induced embryonic resorption, and whether LIF can mediate hormonal action. Using in vivo and in vitro models, we provide evidence that circulating progesterone is an important component of the process by which infection causes embryonic resorption in mice. Also, LIF seems to be a mediator of the progesterone effect under inflammatory conditions. We found that serum progesterone fell to very low levels after 24 h of LPS exposure. Moreover, progesterone supplementation prevented embryonic resorption and LPS-induced increase of uterine nitric oxide levels in vivo. Results show that LPS diminished the expression of the nuclear progesterone receptor in the uterus after 6 and 12 h of treatment. We investigated the expression of LIF in uterine tissue from pregnant mice and found that progesterone up-regulates LIF mRNA expression in vitro. We observed that LIF was able to modulate the levels of nitric oxide induced by LPS in vitro, suggesting that it could be a potential mediator of the inflammatory action of progesterone. Our observations support the view that progesterone plays a critical role in a successful pregnancy as an anti-inflammatory agent, and that it could have possible therapeutic applications in the prevention of early reproductive failure associated with inflammatory disorders.

Inflammatory Agents Involved in Septic Miscarriage

Even though the understanding of the cause of early pregnancy loss due to chromosomal abnormalities has improved, there is a dearth of knowledge of the causes of loss in euploid conceptuses. Maternal infections are a cause of abort in humans, but the mechanisms are not clear, so we have developed a murine model to study the mechanism of septic abortion by inducing embryonic resorption (ER) with lipopolysaccharide (LPS). We demonstrated that augmented production of nitric oxide (NO) and prostaglandins (PG) is involved in ER, and that inhibitors of their synthesis could prevent ER. Also, we observed an increase in the oxidative damage, evidenced by nitration of tyrosine proteins, due to the peroxynitrite anion. Since an association between chronic marijuana smoking and early miscarriage has been shown in women, we studied the participation of anandamide (AEA), the principal endocannabinoid, on the mechanism of action of LPS. We showed that LPS-induced NO synthesis and tissue damage were mediated by AEA, and that this endotoxin inhibited AEA degradation and increased its synthesis. These results suggest that several inflammatory molecules participate in the mechanism of early pregnancy loss and that their modulation could be useful tools to prevent it.

Lipopolysaccharide-induced murine embryonic resorption involves changes in endocannabinoid profiling and alters progesterone secretion and inflammatory response by a CB1-mediated fashion

Genital tract infections are a common complication of human pregnancy that can result in miscarriage. We have previously shown that a lipopolysaccharide (LPS) induces embryonic resorption in a murine model of inflammatory miscarriage. This is accompanied by a dramatic decrease in systemic progesterone levels associated with a robust pro-inflammatory response that results in embryo resorption. Here, we tested the hypothesis that the endogenous cannabinoid system (eCS), through cannabinoid receptor 1 (CB1), plays a role in regulating progesterone levels and, therefore, the pro-inflammatory response. We show that LPS treatment in pregnant mice causes significant changes in the eCS ligands, which are reversed by progesterone treatment. We further show the CB1-KO mice maintain higher plasma progesterone levels after LPS treatment, which is associated with a feebler uterine inflammatory response and a significant drop in embryo resorption. These data suggest that manipulation of CB1 receptors and/or ligands is a potential therapeutic avenue to decrease infection-induced miscarriage.

Progesterone reverts LPS-reduced FAAH activity in murine peripheral blood mononuclear cells by a receptor-mediated fashion.

Increased anandamide concentrations are associated with pregnancy failure. Anandamide levels are regulated by the fatty acid amide hydrolase (FAAH). The aim of the study was to investigate the role of progesterone (P) on FAAH modulation in murine peripheral blood mononuclear cells (PBMC) under septic conditions. We observed that in vivo administration of LPS to non-pregnant (NP) mice decreased FAAH activity of PBMC while in pregnant mice no changes in FAAH activity were observed. NP animals administered with P had a similar response to LPS as the pregnant animals. Also, NP mice injected with P antagonist and P showed that the effect of P on LPS-reduced FAAH activity was impaired. Furthermore, LPS produced a decrease in the ratio of PR-B/PR-A in NP animals. Our results showed that, in our model the endotoxin decreased PBMCs FAAH activity and this condition was reverted by P in a receptor-mediated fashion.

Growth-associated protein-43 is degraded via the ubiquitin-proteasome system.

Growth‐associated protein‐43 (GAP‐43) is a phosphoprotein whose expression in neurons is related to the initial establishment and remodeling of neural connections. GAP‐43 gene expression is known to be regulated at both the transcriptional and the postranscriptional levels. However, very little is known about the cellular mechanism involved in the degradation of this protein. Ubiquitin (Ub) is well known for its role in targeting cytoplasmic proteins for degradation by the 26S proteasome. The ubiquitin‐proteasome system (UPS) consists of a conserved cascade of three enzymatic components that attach Ub covalently to various substrates and control the degradation of protein involved in several important cellular processes. In this study, we investigated the degradation of GAP‐43 in transfected NIH 3T3 cells and neuronal cultures. We found that the proteasome inhibitors, lactacystin and MG132 increased the cellular GAP‐43 level, leading to the accumulation of polyubiquitinated forms of this protein in transfected cells and that the Ub‐proteasome pathway is also involved in the turnover of this protein in neurons. We conclude based on our findings that GAP‐43 is a substrate of the UPS.

Lipopolysaccharide-induced murine embryonic resorption involves nitric oxide mediated inhibition of the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

The initial inactivation of prostaglandins (PGs) is mediated by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). PGs are potent mediators of several biological processes, including inflammation and reproduction. In uterus, PGs play a key role in infection-induced pregnancy loss, in which concentration of this mediator increased. This process is accompanied with the induction of nitric oxide synthase expression and a marked increase in uterine levels of nitric oxide. There is no information concerning nitric oxide contribution to potential changes in PG catabolism, but experimental evidence suggests that nitric oxide modulates PG pathways. The specific objectives of the study were to evaluate the protein expression of HPGD (15-PGDH) and to characterize the nitric oxide-dependent regulation of this enzyme in a model of lipopolysaccharide (LPS)-induced embryonic resorption. Results show that LPS decreased HPGD protein expression and augmented PGE synthase activity; therefore, PGE₂ levels increased in uterus in this inflammatory condition. Just as LPS, the treatment with a nitric oxide donor diminished HPGD protein expression in uterine tissue. In contrast, the inhibition of nitric oxide synthesis both in control and in LPS-treated mice increased 15-PGDH levels. Also, we have found that this enzyme and PGE₂ levels are not modulated by peroxynitrite, an oxidant agent derived from nitric oxide. This study suggests that LPS and nitric oxide promote a decrease in the ability of the uterus for PG catabolism during bacterially triggered pregnancy loss in mice.

Opposite Effects of Methanandamide on Lipopolysaccharide-Induced Prostaglandin E2 and F2α Synthesis in Uterine Explants from Pregnant Mice

Prostaglandins (PG) are effective abortifacients and are important mediators of lipopolisaccharide (LPS)-induced embryonic resorption (ER). Besides, anandamide (AEA) has been described as one of the major endocannabinoids present in the uterus suggesting that it might play a role in reproduction. It has been reported that high levels of AEA are associated with pregnancy failure and that LPS increases AEA production. Also, it has been observed that AEA modulates PG production in different tissues. In this sense, we studied whether LPS-induced PG production is modulated by AEA and we also assessed the effect of this endocannabinoid on PG metabolism in an in vitro model. Uterine explants from BALB/c implantation sites were cultured in the presence of LPS plus cannabinoid receptor (CB) specific antagonists and PG production was assessed. Then, we studied the effect of exogenous AEA on different steps of PG metabolic pathway. We showed that AEA is involved in LPS-induced PG biosynthesis. Also, we observed that AEA exerts opposite effects on PGE2 and PGF2α biosynthesis, by inhibiting PGE2 production and increasing PGF2α levels. We suggest that AEA could be involved in the mechanisms implicated in LPS-induced ER. A better understanding of how AEA could be affecting ER could help developing specific interventions to prevent this pathology.

Progesterone modulates the LPS-induced nitric oxide production by a progesterone-receptor independent mechanism.

Genital tract infections caused by Gram-negative bacteria induce miscarriage and are one of the most common complications of human pregnancy. LPS administration to 7-day pregnant mice induces embryo resorption after 24h, with nitric oxide playing a fundamental role in this process. We have previously shown that progesterone exerts protective effects on the embryo by modulating the inflammatory reaction triggered by LPS. Here we sought to investigate whether the in vivo administration of progesterone modulated the LPS-induced nitric oxide production from peripheral blood mononuclear cells from pregnant and non-pregnant mice. We found that progesterone downregulated LPS-induced nitric oxide production by a progesterone receptor-independent mechanism. Moreover, our results suggest a possible participation of glucocorticoid receptors in at least some of the anti-inflammatory effects of progesterone.

Endocannabinoid system and pregnancy

The endocannabinoid system (eCS), is a complex system, comprising the main endogenous ligands anandamide and 2-arachidonoyl glycerol, the cannabinoid receptors CB1 and CB2 and the biosynthetic and degrading enzymes. Cumulative evidence shows that the eCS plays an important role in reproduction, from egg fertilization to parturition. Therefore, alterations in this system, either by recreation/therapeutic use of cannabis or deregulation of the endogenous cannabinoids, might lead to adverse pregnancy outcomes, including retardation in embryo development, poor blastocyst implantation, inhibition of decidualization, miscarriage and compromised placentation. Nevertheless, the molecular mechanisms by which the eCS participates in different stages of pregnancy remain poorly understood. In this review, we will examine the evidence from animal and human studies to support the role of the eCS in implantation, early-to-late pregnancy and placentation as well as the difficulties of targeting this system for treatment of female infertility.