Parkin Promotes Airway Inflammatory Response to Interferon Gamma.

PURPOSE: Increased type 2 interferon (i.e., IFN-γ) signaling has been shown to be involved in airway inflammation in a subset of asthma patients who often show high levels of airway neutrophilic inflammation and poor response to corticosteroid treatment. How IFN-γ mediates airway inflammation in a mitochondrial dysfunction setting (e.g., Parkin up-regulation) remains poorly understood. The goal of this study was to determine the role of Parkin, an E3 ubiquitin ligase, in IFN-γ-mediated airway inflammation and the regulation of Parkin by IFN-γ. METHODS: A mouse model of IFN-γ treatment in wild-type and Parkin knockout mice, and cultured human primary airway epithelial cells with or without Parkin gene deficiency were used. RESULTS: Parkin was found to be necessary for the production of neutrophil chemokines (i.e., LIX and IL-8) and airway neutrophilic inflammation following IFN-γ treatment. Mechanistically, Parkin was induced by IFN-γ treatment both in vivo and in vitro, which was associated with less expression of a Parkin transcriptional repressor Thap11. Overexpression of Thap11 inhibited Parkin expression in IFN-γ-stimulated airway epithelial cells. CONCLUSIONS: Our data suggest a novel mechanism by which IFN-γ induces airway neutrophilic inflammation through the Thap11/Parkin axis. Inhibition of Parkin expression or activity may provide a new therapeutic target for the treatment of excessive neutrophilic inflammation in an IFN-γ-high environment.

Single cell RNA-sequencing of human precision-cut lung slices: A novel approach to study the effect of vaping and viral infection on lung health.

Vaping is an increasing health threat in the US and worldwide. The damaging impact of vaping on the human distal lung has been highlighted by the recent epidemic of electronic cigarette or vaping use-associated lung injury (EVALI). The pathogenesis of EVALI remains incompletely understood, due to a paucity of models that recapitulate the structural and functional complexity of the human distal lung and the still poorly defined culprit exposures to vaping products and respiratory viral infections. Our aim was to establish the feasibility of using single cell RNA-sequencing (scRNA-seq) technology in human precision-cut lung slices (PCLS) as a more physiologically relevant model to better understand how vaping regulates the antiviral and pro-inflammatory response to influenza A virus infection. Normal healthy donor PCLS were treated with vaping extract and influenza A viruses for scRNA-seq analysis. Vaping extract augmented host antiviral and pro-inflammatory responses in structural cells such as lung epithelial cells and fibroblasts, as well as in immune cells such as macrophages and monocytes. Our findings suggest that human distal lung slice model is useful to study the heterogeneous responses of immune and structural cells under EVALI conditions, such as vaping and respiratory viral infection.

High-fat diet and palmitic acid amplify airway type 2 inflammation.

Introduction: Metabolic dysfunction such as elevated levels of saturated fatty acids (SFA) may play a role in obese asthma, but its contribution to airway inflammation remains unclear. We sought to determine the role of high-fat diet (HFD) and palmitic acid (PA), a major form of SFA, in regulating type 2 inflammation. Methods: Airway samples from asthma patients with or without obesity, mouse models and human airway epithelial cell culture were utilized to test if SFA amplify type 2 inflammation. Results: Asthma patients with obesity had higher levels of airway PA than asthma patients without obesity. HFD increased the levels of PA in mice, and subsequently enhanced IL-13-induced airway eosinophilic inflammation. PA treatment amplified airway eosinophilic inflammation in mice that were previously exposed to IL-13 or house dust mite. IL-13 alone or in combination with PA increased dipeptidyl peptidase 4 (DPP4) release (soluble DPP4) and/or activity in mouse airways and human airway epithelial cells. Inhibition of DPP4 activity by linagliptin in mice pre-exposed to IL-13 or both IL-13 and PA increased airway eosinophilic and neutrophilic inflammation. Discussion: Our results demonstrated the exaggerating effect of obesity or PA on airway type 2 inflammation. Up-regulation of soluble DPP4 by IL-13 and/or PA may serve as a mechanism to prevent excessive type 2 inflammation. Soluble DPP4 may have the therapeutic potential in asthma patients with obesity who have an endotype with mixed airway eosinophilic and neutrophilic inflammation.

Parkin promotes airway inflammatory response to interferon gamma.

Background: Increased type 2 interferon (i.e., IFN-γ) signaling has been shown to be involved in airway inflammation in a subset of asthma patients who often show high levels of airway neutrophilic inflammation and poor response to corticosteroid treatment. How IFN-γ mediates airway inflammation in a mitochondrial dysfunction setting (e.g., Parkin up-regulation) remains poorly understood. The goal of this study was to determine the role of Parkin, an E3 ubiquitin ligase, in IFN-γ-mediated airway inflammation and the regulation of Parkin by IFN-γ. Results: Using a mouse model of IFN-γ treatment in wild-type and Parkin knockout mice, and cultured human primary airway epithelial cells with or without Parkin gene deficiency, we found that Parkin was necessary for the production of neutrophil chemokines (i.e., KC and IL-8) and airway neutrophilic inflammation. Mechanistically, Parkin was induced by IFN-γ treatment both in vivo and in vitro, which was associated with less expression of a Parkin transcriptional repressor Thap11. Overexpression of Thap11 inhibited Parkin expression in IFN-γ-stimulated airway epithelial cells. Conclusions: Our data suggests a novel mechanism by which IFN-γ induces airway neutrophilic inflammation through the Thap11/Parkin axis. Inhibition of Parkin expression or activity may provide a new therapeutic target for the treatment of excessive neutrophilic inflammation in an IFN-γ high environment.

Bacterial DNA amplifies neutrophilic inflammation in IL-17-exposed airways.

Background: Neutrophilic asthma (NA) is associated with increased airway interleukin (IL)-17 and abnormal bacterial community such as dominance of nontypeable Haemophilus influenzae (NTHi), particularly during asthma exacerbations. Bacteria release various products including DNA, but whether they cooperate with IL-17 in exaggerating neutrophilic inflammation is unclear. We sought to investigate the role of bacteria-derived DNA in airway neutrophilic inflammation related to IL-17-high asthma and underlying mechanisms (e.g. Toll-like receptor 9 (TLR9)/IL-36γ signalling axis). Methods: Bacterial DNA, IL-8 and IL-36γ were measured in bronchoalveolar lavage fluid (BALF) of people with asthma and healthy subjects. The role of co-exposure to IL-17 and bacterial DNA or live bacteria in neutrophilic inflammation, and the contribution of the TLR9/IL-36γ signalling axis, were determined in cultured primary human airway epithelial cells and alveolar macrophages, and mouse models. Results: Bacterial DNA levels were increased in asthma BALF, which positively correlated with IL-8 and neutrophil levels. Moreover, IL-36γ increased in BALF of NA patients. Bacterial DNA or NTHi infection under an IL-17-high setting amplified IL-8 production and mouse lung neutrophilic inflammation. DNase I treatment in IL-17-exposed and NTHi-infected mouse lungs reduced neutrophilic inflammation. Mechanistically, bacterial DNA-mediated amplification of neutrophilic inflammation is in part dependent on the TLR9/IL-36γ signalling axis. Conclusions: Bacterial DNA amplifies airway neutrophilic inflammation in an IL-17-high setting partly through the TLR9 and IL-36γ signalling axis. Our novel findings may offer several potential therapeutic targets including TLR9 antagonists, IL-36γ neutralising antibodies and DNase I to reduce asthma severity associated with exaggerated airway neutrophilic inflammation.

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.

Airway epithelial immunoproteasome subunit LMP7 protects against rhinovirus infection.

Immunoproteasomes (IP) serve as an important modulator of immune responses to pathogens and other pathological factors. LMP7/ß5i, one of the IP subunits, plays a critical role in autoimmune diseases by downregulating inflammation. Rhinovirus (RV) infection is a major risk factor in the exacerbations of respiratory inflammatory diseases, but whether LMP7 regulates RV-mediated inflammation in the lung particularly in the airway epithelium, the first line of defense against RV infection, remains unclear. In this study, we determined whether airway epithelial LMP7 promotes the resolution of RV-mediated lung inflammation. Inducible airway epithelial-specific LMP7-deficient (conditional knockout, CKO) mice were generated to reveal the in vivo anti-inflammatory and antiviral functions of LMP7. By using LMP7-deficient primary human airway epithelial cells generated by CRISPR-Cas9, we confirmed that airway epithelial LMP7 decreased pro-inflammatory cytokines and viral load during RV infection. Additionally, airway epithelial LMP7 enhanced the expression of a negative immune regulator A20/TNFAIP3 during viral infection that may contribute to the anti-inflammatory function of LMP7. We also discovered that induction of LMP7 by a low dose of polyinosinic:polycytidylic acid (PI:C) reduced RV-mediated inflammation in our CKO mice infected with RV. Our findings suggest that airway epithelial LMP7 has anti-inflammatory and antiviral functions that is critical to the resolution of RV-mediated lung inflammation. Induction of airway epithelial LMP7 may open a novel avenue for therapeutic intervention against RV infection.

Human Bronchial Epithelial Cell Culture Models for Cigarette Smoke and Vaping Studies.

Despite the continuing public health efforts to stop or reduce smoking, cigarette smoke use remains popular in the youth and adult population. A recent surge in the use of electronic cigarette and vaping products has created another major health challenge in public health. There is an urgent need to use physiologically relevant models to study the health effect of smoking or vaping in human subjects. Airway diseases such as bronchitis (Landman et al., CMAJ 191:E1321-E1331, 2019; Goniewicz, et al. Harm Reduct J 17:91, 2020; Xie et al., JAMA Netw Open 3:e2020816, 2020) have been described in people who smoke, vape, or both. Here, we will describe methods to collect, expand, and culture human airway epithelial cells from endobronchial brushings and expose these cells cultured at the air-liquid interface to cigarette smoke or electronic cigarette vapor.

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.

Tollip interaction with STAT3: a novel mechanism to regulate human airway epithelial responses to type 2 cytokines.

BACKGROUND: Toll-interacting protein (Tollip) is one of the key negative regulators in host innate immunity. Genetic variation of Tollip has been associated with less Tollip expression and poor lung function in asthmatic patients, but little is known about the role of Tollip in human airway type 2 inflammatory response, a prominent feature in allergic asthma. OBJECTIVE: Our goal was to determine the role and underlying mechanisms of Tollip in human airway epithelial responses such as eotaxin to type 2 cytokine IL-13. METHODS: Tollip deficient primary human airway epithelial cells from 4 healthy donors were generated by the gene knockdown approach and stimulated with IL-13 to measure activation of transcription factor STAT3, and eotaxin-3, an eosinophilic chemokine. RESULTS: Following IL-13 treatment, Tollip deficient cells had significantly higher levels of STAT3 activation and eotaxin-3 than the scrambled control counterpart, which was reduced by a STAT3 inhibitor. Interaction between Tollip and STAT3 proteins was identified by co-immunoprecipitation. CONCLUSION: Our results, for the first time, suggest that Tollip inhibits excessive eotaxin-3 induction by IL-13, in part through the interaction and inhibition of STAT3. These findings lend evidence to the potential of a STAT3 inhibitor as a therapeutic target, especially for type 2 inflammation-high asthmatics with Tollip deficiency.

Parkin, an E3 ubiquitin ligase, enhances airway mitochondrial DNA release and inflammation.

INTRODUCTION: Parkin (Park2), an E3 ubiquitin ligase, is critical to maintain mitochondrial function by regulating mitochondrial biogenesis and degradation (mitophagy), but recent evidence suggests the involvement of Parkin in promoting inflammation. In the present study, we determined if Parkin regulates airway mitochondrial DNA (mtDNA) release and inflammatory responses to type 2 cytokine interleukin (IL)-13 and allergens. METHODS: We measured Parkin mRNA expression in brushed bronchial epithelial cells and mtDNA release in the paired bronchoalveolar lavage fluid (BALF) from normal subjects and asthmatics. Parkin-deficient primary human tracheobronchial epithelial (HTBE) cells generated using the CRISPR-Cas9 system were stimulated with IL-13. To determine the in vivo function of Parkin, Parkin knockout (PKO) and wild-type (WT) mice were treated with IL-13 or allergen (house dust mite, HDM) in the presence or absence of mtDNA isolated from normal mouse lungs. RESULTS: Parkin mRNA expression in asthmatic airway epithelium was upregulated, which positively correlated with the levels of released mtDNA in BALF. IL-13-stimulated HTBE cells increased Parkin expression. Moreover, IL-13 induced mtDNA release in Parkin-sufficient, but not in Parkin-deficient HTBE cells. PKO (vs WT) mice attenuated airway mtDNA release and inflammation following IL-13 or HDM treatments. mtDNA amplified airway inflammation in mice treated with IL-13 or HDM. Notably, Parkin also mediated mtDNA-induced exacerbation of airway inflammation. CONCLUSION: Our research findings suggest that Parkin promotes mtDNA release and inflammation in airways, thus improving our understanding of the complex role of Parkin and mitochondrial dysfunction in asthma pathogenesis.

Interleukin 1 Receptor-Like 1 (IL1RL1) Promotes Airway Bacterial and Viral Infection and Inflammation.

Interleukin 1 receptor-like 1 (IL1RL1), also known as suppression of tumorigenicity 2 (ST2), is the receptor for interleukin 33 (IL-33) and has been increasingly studied in type 2 inflammation. An increase in airway IL-33/ST2 signaling in asthma has been associated with eosinophilic inflammation, but little is known about the role of ST2 in neutrophilic inflammation. Airway Mycoplasma pneumoniae and human rhinovirus (HRV) infections are linked to neutrophilic inflammation during acute exacerbations of asthma. However, whether ST2 contributes to M. pneumoniae- and HRV-mediated airway inflammation is poorly understood. The current study sought to determine the functions of ST2 during airway M. pneumoniae or HRV infection. In cultured normal human primary airway epithelial cells, ST2 overexpression (OE) increased the production of neutrophilic chemoattractant IL-8 in the absence or presence of M. pneumoniae or HRV1B infection. ST2 OE also enhanced HRV1B-induced IP-10, a chemokine involved in asthma exacerbations. In the M. pneumoniae-infected mouse model, ST2 deficiency, in contrast to sufficiency, significantly reduced the levels of neutrophils following acute (≤24 h) infection, while in the HRV1B-infected mouse model, ST2 deficiency significantly reduced the levels of proinflammatory cytokines KC, IP-10, and IL-33 in bronchoalveolar lavage (BAL) fluid. Overall, ST2 overexpression in human epithelial cells and ST2 sufficiency in mice increased the M. pneumoniae and HRV loads in cell supernatants and BAL fluid. After pathogen infection, ST2-deficient mice showed a higher level of the host defense protein lactotransferrin in BAL fluid. Our data suggest that ST2 promotes proinflammatory responses (e.g., neutrophils) to airway bacterial and viral infection and that blocking ST2 signaling may broadly attenuate airway infection and inflammation.

Immunoproteasomes as a novel antiviral mechanism in rhinovirus-infected airways.

Rhinovirus (RV) infection is involved in acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). RV primarily infects upper and lower airway epithelium. Immunoproteasomes (IP) are proteolytic machineries with multiple functions including the regulation of MHC class I antigen processing during viral infection. However, the role of IP in RV infection has not been explored. We sought to investigate the expression and function of IP during airway RV infection. Primary human tracheobronchial epithelial (HTBE) cells were cultured at air-liquid interface (ALI) and treated with RV16, RV1B, or interferon (IFN)-λ in the absence or presence of an IP inhibitor (ONX-0914). IP gene (i.e. LMP2) deficient mouse tracheal epithelial cells (mTECs) were cultured for the mechanistic studies. LMP2-deficient mouse model was used to define the in vivo role of IP in RV infection. IP subunits LMP2 and LMP7, antiviral genes MX1 and OAS1 and viral load were measured. Both RV16 and RV1B significantly increased the expression of LMP2 and LMP7 mRNA and proteins, and IFN-λ mRNA in HTBE cells. ONX-0914 down-regulated MX1 and OAS1, and increased RV16 load in HTBE cells. LMP2-deficient mTECs showed a significant increase in RV1B load compared with the wild-type (WT) cells. LMP2-deficient (compared with WT) mice increased viral load and neutrophils in bronchoalveolar lavage (BAL) fluid after 24 h of RV1B infection. Mechanistically, IFN-λ induction by RV infection contributed to LMP2 and LMP7 up-regulation in HTBE cells. Our data suggest that IP are induced during airway RV infection, which in turn may serve as an antiviral and anti-inflammatory mechanism.

Impaired placental autophagy in placental malaria.

BACKGROUND: Placental malaria is a major cause of low birthweight, principally due to impaired fetal growth. Intervillositis, a local inflammatory response to placental malaria, is central to the pathogenesis of poor fetal growth as it impairs transplacental amino acid transport. Given the link between inflammation and autophagy, we investigated whether placental malaria-associated intervillositis increased placental autophagy as a potential mechanism in impaired fetal growth. METHODS: We examined placental biopsies collected after delivery from uninfected women (n = 17) and from women with Plasmodium falciparum infection with (n = 14) and without (n = 7) intervillositis. Western blotting and immunofluorescence staining coupled with advanced image analysis were used to quantify the expression of autophagic markers (LC3-II, LC3-I, Rab7, ATG4B and p62) and the density of autophagosomes (LC3-positive puncta) and lysosomes (LAMP1-positive puncta). RESULTS: Placental malaria with intervillositis was associated with higher LC3-II:LC3-I ratio, suggesting increased autophagosome formation. We found higher density of autophagosomes and lysosomes in the syncytiotrophoblast of malaria-infected placentas with intervillositis. However, there appear to be no biologically relevant increase in LC3B/LAMP1 colocalization and expression of Rab7, a molecule involved in autophagosome/lysosome fusion, was lower in placental malaria with intervillositis, indicating a block in the later stage of autophagy. ATG4B and p62 expression showed no significant difference across histological groups suggesting normal autophagosome maturation and loading of cargo proteins into autophagosomes. The density of autophagosomes and lysosomes in the syncytiotrophoblast was negatively correlated with placental amino acid uptake. CONCLUSIONS: Placental malaria-associated intervillositis is associated with dysregulated autophagy that may impair transplacental amino acid transport, possibly contributing to poor fetal growth.

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.

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.

Placental Responses to Changes in the Maternal Environment Determine Fetal Growth.

Placental responses to maternal perturbations are complex and remain poorly understood. Altered maternal environment during pregnancy such as hypoxia, stress, obesity, diabetes, toxins, altered nutrition, inflammation, and reduced utero-placental blood flow may influence fetal development, which can predispose to diseases later in life. The placenta being a metabolically active tissue responds to these perturbations by regulating the fetal supply of nutrients and oxygen and secretion of hormones into the maternal and fetal circulation. We have proposed that placental nutrient sensing integrates maternal and fetal nutritional cues with information from intrinsic nutrient sensing signaling pathways to balance fetal demand with the ability of the mother to support pregnancy by regulating maternal physiology, placental growth, and placental nutrient transport. Emerging evidence suggests that the nutrient-sensing signaling pathway mechanistic target of rapamycin (mTOR) plays a central role in this process. Thus, placental nutrient sensing plays a critical role in modulating maternal-fetal resource allocation, thereby affecting fetal growth and the life-long health of the fetus.

Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2.

Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

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 log 10 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.

First report of Trichomonas tenax infections in the Philippines.

Recent studies have reported Trichomonas tenax as a cause of pleuropulmonary infections in humans. In this study, sputum and vaginal swab samples were collected from patients suffering from respiratory ailments in Rodriguez, Rizal and sex workers attending the social hygiene clinics in Angeles City in Pampanga, Mandaluyong City and Pasay City in Metro Manila, Philippines, respectively. DNA was extracted from samples and the 18S rRNA gene was amplified and sequenced. Phylogenetic trees were constructed using neighbor-joining, maximum-likelihood, maximum parsimony, and Bayesian inference analyses. Results showed that the new primer sets successfully amplified T. tenax from 14 sputum samples and Trichomonas vaginalis from 19 vaginal swab samples. Consequently, all isolates clustered with high bootstrap support and posterior probability values to their respective reference strains in the phylogenetic tree. Thus, the genus Trichomonas formed a highly supported clade with T. vaginalis in the first clade and T. tenax in the second clade. These findings conclude that T. vaginalis is solely present in the genito-urinary tract of females and that T. tenax can be found in the respiratory tract of humans. To our knowledge, this is the first report of detection and identification of T. tenax from sputum samples in the Philippines. However, further studies are still needed to determine the pathogenicity of this organism in humans.