Inhibition of placental mTOR signaling provides a link between placental malaria and reduced birthweight

BACKGROUND: Placental Plasmodium falciparum malaria can trigger intervillositis, a local inflammatory response more strongly associated with low birthweight than placental malaria infection alone. Fetal growth (and therefore birthweight) is dependent on placental amino acid transport, which is impaired in placental malaria-associated intervillositis. Here, we tested the hypothesis that mechanistic target of rapamycin (mTOR) signaling, a pathway known to regulate amino acid transport, is inhibited in placental malaria-associated intervillositis, contributing to lower birthweight. METHODS: We determined the link between intervillositis, mTOR signaling activity, and amino acid uptake in tissue biopsies from both uninfected placentas and malaria-infected placentas with and without intervillositis, and in an in vitro model using primary human trophoblast (PHT) cells. RESULTS: We demonstrated that (1) placental mTOR activity is lower in cases of placental malaria with intervillositis, (2) placental mTOR activity is negatively correlated with the degree of inflammation, and (3) inhibition of placental mTOR activity is associated with reduced placental amino acid uptake and lower birthweight. In PHT cells, we showed that (1) inhibition of mTOR signaling is a mechanistic link between placental malaria-associated intervillositis and decreased amino acid uptake and (2) constitutive mTOR activation partially restores amino acid uptake. CONCLUSIONS: Our data support the concept that inhibition of placental mTOR signaling constitutes a mechanistic link between placental malaria-associated intervillositis and decreased amino acid uptake, which may contribute to lower birthweight. Restoring placental mTOR signaling in placental malaria may increase birthweight and improve neonatal survival, representing a new potential therapeutic approach

Role of Myeloid Cell-Specific TLR9 in Mitochondrial DNA-Induced Lung Inflammation in Mice

Mitochondrial dysfunction is common in various pathological conditions including obesity. Release of mitochondrial DNA (mtDNA) during mitochondrial dysfunction has been shown to play a role in driving the pro-inflammatory response in leukocytes including macrophages. However, the mechanisms by which mtDNA induces leukocyte inflammatory responses in vivo are still unclear. Moreover, how mtDNA is released in an obese setting has not been well understood. By using a mouse model of TLR9 deficiency in myeloid cells (e.g., macrophages), we found that TLR9 signaling in myeloid cells was critical to mtDNA-mediated pro-inflammatory responses such as neutrophil influx and chemokine production. mtDNA release by lung macrophages was enhanced by exposure to palmitic acid (PA), a major saturated fatty acid related to obesity. Moreover, TLR9 contributed to PA-mediated mtDNA release and inflammatory responses. Pathway analysis of RNA-sequencing data in TLR9-sufficient lung macrophages revealed the up-regulation of axon guidance molecule genes and down-regulation of metabolic pathway genes by PA. However, in TLR9-deficient lung macrophages, PA down-regulated axon guidance molecule genes, but up-regulated metabolic pathway genes. Our results suggest that mtDNA utilizes TLR9 signaling in leukocytes to promote lung inflammatory responses in hosts with increased PA. Moreover, TLR9 signaling is involved in the regulation of axon guidance and metabolic pathways in lung macrophages exposed to PA

Placental mTOR signaling and fetal growth restriction in placental malaria

© 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

Role of Myeloid Cell-Specific TLR9 in Mitochondrial DNA-Induced Lung Inflammation in Mice

Mitochondrial dysfunction is common in various pathological conditions including obesity. Release of mitochondrial DNA (mtDNA) during mitochondrial dysfunction has been shown to play a role in driving the pro-inflammatory response in leukocytes including macrophages. However, the mechanisms by which mtDNA induces leukocyte inflammatory responses in vivo are still unclear. Moreover, how mtDNA is released in an obese setting has not been well understood. By using a mouse model of TLR9 deficiency in myeloid cells (e.g., macrophages), we found that TLR9 signaling in myeloid cells was critical to mtDNA-mediated pro-inflammatory responses such as neutrophil influx and chemokine production. mtDNA release by lung macrophages was enhanced by exposure to palmitic acid (PA), a major saturated fatty acid related to obesity. Moreover, TLR9 contributed to PA-mediated mtDNA release and inflammatory responses. Pathway analysis of RNA-sequencing data in TLR9-sufficient lung macrophages revealed the up-regulation of axon guidance molecule genes and down-regulation of metabolic pathway genes by PA. However, in TLR9-deficient lung macrophages, PA down-regulated axon guidance molecule genes, but up-regulated metabolic pathway genes. Our results suggest that mtDNA utilizes TLR9 signaling in leukocytes to promote lung inflammatory responses in hosts with increased PA. Moreover, TLR9 signaling is involved in the regulation of axon guidance and metabolic pathways in lung macrophages exposed to PA

Identified by 18S Ribosomal RNA Gene Sequences

Trichomonads are obligate anaerobes generally found in the digestive and genitourinary tract of domestic animals. In this study, four trichomonad isolates were obtained from carabao, dog, and pig hosts using rectal swab. Genomic DNA was extracted using Chelex method and the 18S rRNA gene was successfully amplified through novel sets of primers and undergone DNA sequencing. Aligned isolate sequences together with retrieved 18S rRNA gene sequences of known trichomonads were utilized to generate phylogenetic trees using maximum likelihood and neighbor-joining analyses. Two isolates from carabao were identified as Simplicimonas similis while each isolate from dog and pig was identified as Pentatrichomonas hominis and Trichomitus batrachorum, respectively. This is the first report of S. similis in carabao and the identification of T. batrachorum in pig using 18S rRNA gene sequence analysis. The generated phylogenetic tree yielded three distinct groups mostly with relatively moderate to high bootstrap support and in agreement with the most recent classification. Pathogenic potential of the trichomonads in these hosts still needs further investigation

New Hosts of Simplicimonas similis

Trichomonads are obligate anaerobes generally found in the digestive and genitourinary tract of domestic animals. In this study, four trichomonad isolates were obtained from carabao, dog, and pig hosts using rectal swab. Genomic DNA was extracted using Chelex method and the 18S rRNA gene was successfully amplified through novel sets of primers and undergone DNA sequencing. Aligned isolate sequences together with retrieved 18S rRNA gene sequences of known trichomonads were utilized to generate phylogenetic trees using maximum likelihood and neighbor-joining analyses. Two isolates from carabao were identified as Simplicimonas similis while each isolate from dog and pig was identified as Pentatrichomonas hominis and Trichomitus batrachorum, respectively. This is the first report of S. similis in carabao and the identification of T. batrachorum in pig using 18S rRNA gene sequence analysis. The generated phylogenetic tree yielded three distinct groups mostly with relatively moderate to high bootstrap support and in agreement with the most recent classification. Pathogenic potential of the trichomonads in these hosts still needs further investigation

Microbiological Quality of Fresh Produce from Open Air Markets and Supermarkets in the Philippines

This study is the first in the Philippines to conduct a comprehensive assessment of the prevalence of bacterial pathogens and somatic phages in retailed fresh produce used in salad preparation, namely, bell pepper, cabbage, carrot, lettuce, and tomato, using culture and molecular methods. Out of 300 samples from open air and supermarkets, 16.7% tested positive for thermotolerant Escherichia coli, 24.7% for Salmonella spp., and 47% for somatic phages. Results show that counts range from 0.30 to 4.03 log10 CFU/g for E. coli, 0.66 to ≥2.34 log10 MPN/g for Salmonella spp., and 1.30 to ≥3.00 log10 PFU/g for somatic phages. Statistical analyses show that there was no significant difference in the microbial counts between open air and supermarkets (α=0.05). TaqMan and AccuPower Plus DualStar real-time polymerase chain reaction (RT-PCR) was used to confirm the presence of these organisms. The relatively high prevalence of microorganisms observed in produce surveyed signifies reduction in shelf-life and a potential hazard to food safety. This information may benefit farmers, consumers, merchants, and policy makers for foodborne disease detection and prevention

Tollip Inhibits IL-33 Release and Inflammation in Influenza A Virus-Infected Mouse Airways

Respiratory influenza A virus (IAV) infection continues to pose significant challenges in healthcare of human diseases including asthma. IAV infection in mice was shown to increase IL-33, a key cytokine in driving airway inflammation in asthma, but how IL-33 is regulated during viral infection remains unclear. We previously found that a genetic mutation in Toll-interacting protein (Tollip) was linked to less airway epithelial Tollip expression, increased neutrophil chemokines, and lower lung function in asthma patients. As Tollip is involved in maintaining mitochondrial function, and mitochondrial stress may contribute to extracellular ATP release and IL-33 secretion, we hypothesized that Tollip downregulates IL-33 secretion via inhibiting ATP release during IAV infection. Wild-type and Tollip knockout (KO) mice were infected with IAV and treated with either an ATP converter apyrase or an IL-33 decoy receptor soluble ST2 (sST2). KO mice significantly lost more body weight and had increased extracellular ATP, IL-33 release, and neutrophilic inflammation. Apyrase treatment reduced extracellular ATP levels, IL-33 release, and neutrophilic inflammation in Tollip KO mice. Excessive lung neutrophilic inflammation in IAV-infected Tollip KO mice was reduced by sST2, which was coupled with less IL-33 release. Our data suggest that Tollip inhibits IAV infection, potentially by inhibiting extracellular ATP release and reducing IL-33 activation and lung inflammation. In addition, sST2 may serve as a potential therapeutic approach to mitigate respiratory viral infection in human subjects with Tollip deficiency

Glyburide treatment in gestational diabetes is associated with increased placental glucose transporter 1 expression and higher birth weight

Use of glyburide in gestational diabetes (GDM) has raised concerns about fetal and neonatal side effects, including increased birth weight. Placental nutrient transport is a key determinant of fetal growth, however the effect of glyburide on placental nutrient transporters is largely unknown. We hypothesized that glyburide treatment in GDM pregnancies is associated with increased expression of nutrient transporters in the syncytiotrophoblast plasma membranes. We collected placentas from GDM pregnancies who delivered at term and were treated with either diet modification (n = 15) or glyburide (n = 8). Syncytiotrophoblast microvillous (MVM) and basal (BM) plasma membranes were isolated and expression of glucose (glucose transporter 1; GLUT1), amino acid (sodium-coupled neutral amino acid transporter 2; SNAT2 and L-type amino acid transporter 1; LAT1) and fatty acid (fatty acid translocase; FAT/CD36, fatty acid transporter 2 and 4; FATP2, FATP4) transporters was determined by Western blot. Additionally, we determined GLUT1 expression by confocal microscopy in cultured primary human trophoblasts (PHT) after exposure to glyburide. Birth weight was higher in the glyburide-treated group as compared to diet-treated GDM women (3764 +/- 126 g vs. 3386 +/- 75 g; p < 0.05). GLUT1 expression was increased in both MVM (+50%; p < 0.01) and BM (+75%; p < 0.01). In contrast, MVM FAT/CD36 (-65%; p = 0.01) and FATP2 (-65%; p = 0.02) protein expression was reduced in mothers treated with glyburide. Glyburide increased membrane expression of GLUT1 in a dose-dependent manner in cultured PHT. This data is the first to show that glyburide increases GLUT1 expression in syncytiotrophoblast MVM and BM in GDM pregnancies, and may promote transplacental glucose delivery contributing to fetal overgrowth.NIH DK89989 National Fund for Scientific and Technological Development (FONDECYT)-Chile postdoctoral grant 317014