Placental mTOR signaling and fetal growth restriction in placental malaria

Abstract

© 2017 Dr. Kris Genelyn Bernardino DimasuayBackground: Low birthweight is a major global health issue contributing to about 70% of all neonatal deaths. Malaria in pregnancy is a leading cause of low birthweight responsible for ~900,000 low birthweight deliveries and ~200,000 infant deaths annually. Placental Plasmodium falciparum malaria can trigger intervillositis, a local inflammatory response more strongly associated with low birthweight than placental malaria infection alone. In malaria-endemic regions, almost 50% of all low birthweight cases are attributed to fetal growth restriction. The underlying mechanisms of fetal growth restriction in placental malaria are still unknown but a study implicated impaired placental amino acid transport in placenta malaria-associated intervillositis. Fetal growth is highly dependent on placental amino acid transport. Placental mechanistic target of rapamycin (mTOR) signaling is a nutrient sensing pathway that regulates the expression and activity of amino acid transporters in the syncytiotrophoblast, the nutrient transporting epithelium of the placenta. Decreased placental mTOR signaling has been associated with reduced activity of placental amino acid transporters that lead to decreased fetal growth in animal models and human cases. Here, the present study investigated the potential role of placental mTOR signaling in the pathogenesis of fetal growth restriction in placental malariaassociated intervillositis by putting emphasis on reduced amino acid uptake. Also, the present study investigated the possible contribution of placental autophagy, a process negatively regulated by mTOR, to alter amino acid uptake in placental malariaassociated intervillositis. Methods: Placental villous tissue biopsies sampled at delivery from Malawian women were grouped into uninfected placentas (n = 17) and placental malaria without (n = 7) and with (n = 14) intervillositis. Western blotting was done to quantify levels of expression of mTOR downstream targets (rps6, 4EBP-1 and Akt) and autophagic markers (LC3-II:LC3-I ratio, Rab7, ATG4B and p62). Immunofluorescence staining coupled with image analysis were used to quantify the density of autophagosomes (LC3B puncta), lysosomes (LAMP1 puncta), and their colocalization in the syncytiotrophoblast. In addition, an in vitro model of placental malaria-associated intervillositis was developed, using trophoblast cells and monocyte-malaria infected red blood cell co-cultures, to establish mTOR signaling as a mechanistic link between placental malaria-associated intervillositis and reduced amino acid uptake. Results: The first study provided evidence that 1) placental mTOR activity is lower in cases of placental malaria with intervillositis; and 2) decreased placental mTOR activity is associated with reduced placental amino acid uptake and lower birthweight. Using the trophoblast cell in vitro model, it was demonstrated that 1) placental mTOR signaling is a possible mechanistic link between placental malaria-associated intervillositis and decreased amino acid uptake; and 2) constitutive mTOR activation partially restores amino acid uptake. These results suggest that placental mTOR signaling inhibition mediates the decrease in amino acid uptake in placental malariaassociated intervillositis. The second study provided evidence that 1) decreased placental mTOR activity (such as we found in placental malaria with intervillositis) is associated with increased autophagosome formation; 2) there were more LC3B and LAMP1 puncta in the syncytiotrophoblast from women with placental malaria with intervillositis, although there was no biologically relevant increase in colocalization of these different puncta; and 3) expression of Rab7, a protein that mediates autophagosome and lysosome fusion, was lower in placental malaria with intervillositis. These results seem to indicate a block in the late stage of autophagy. Conclusion: This thesis provides strong support for the hypothesis that inhibition of placental mTOR signaling mechanistically links inflammation in placental malaria and reduced placental amino acid uptake and this may contribute to the pathogenesis of fetal growth restriction in placental malaria. Furthermore, this thesis demonstrated the detrimental impact of placental malaria-associated intervillositis on placental autophagy. Dysregulated placental autophagy could negatively affect amino acid uptake and could worsen placental inflammation, and thereby may also contribute to the pathogenesis of fetal growth restriction. There is an urgent need to identify mechanisms linking placental malaria to LBW. The work presented in this thesis will facilitate the development of interventions aimed at minimizing the effect of malaria on birthweight that could be used to complement existing malaria control measures. Interventions involving possible mTOR agonists may enhance placental nutrient uptake, which could improve fetal growth and pregnancy outcomes in women with malaria