How maternal exposure to aflatoxin B1 impacts the development of progeny intestinal immune system?

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Early-life exposure to aflatoxin B1 and associated effects on gut microbiota: preliminary results under earlyMYCO project

Aflatoxin B1 (AFB1) produces acute or chronic deleterious health effects in humans and animals. Still, long-term effects derived from initial exposure in early life, a critical period for colonization and development of gut microbiota, has not been fully evaluated. Particularly, aflatoxins could impair gut microbiota and immunity settlement, as they have been proven to cross the placental barrier and can be found in breast milk. We investigated the impact of maternal exposure to AFB1 on early-life microbiota in a mouse model. Females were fed jelly pellets containing 400 µg/kg AFB1 diluted in DMSO (treated animals n=6) or DMSO vehicle alone (control group n=6) during pregnancy and lactation. Faeces from the offspring of both treated and control females were collected immediately after weaning and faecal DNA was extracted and purified. Bacterial taxa diversity and relative abundance were assessed by High-Throughput Sequencing performed in an Illumina Miseq® sequencer, targeting the V3 and V4 regions of the 16S rRNA gene. Operational taxonomic units (OTUs) were determined by clustering reads to 16S reference databases. A hundred and twenty-four (N=124) bacterial genera were found in both groups, 5 were only present in AFB1 treated group and 27 exclusively in control groups. A hundred and fifty-one (N=151) bacterial species were common to both groups, 15 species exclusively found in AFB1 litters and 34 species were exclusively found in control litters. To assess abundance and characterize species diversity and evenness, Shannon diversity index was calculated but no significant differences were found between groups. Although present in both groups, Akkermansia muciniphila and Bacteroides acidifaciens were significantly higher in controls. A. muciniphila colonizes the intestinal tract in childhood and regulates mucus thickness, intestinal barrier integrity and is involved in immune modulation. B. acidifaciens participates in the metabolism of lipids and sugars and activates some cytokines and immune cell receptors. Sulfidogenic bacteria recently related to inflammatory bowel disease such as Desulfovibrio piger and Bilophila wadsworthia were exclusivly found in the treated litters. Early-life gut microbiome is paramount to trigger the gut immune defences, but is far less stable than the adult microbiome. Moreover, previous work identified aflatoxins intake as a potential health hazard in Portuguese children. These preliminary results open an extensive field to further investigate the association between mycotoxins and microbiome, as the latest is increasingly recognized as a major player in a wide spectrum of diseases.This work was funded by FCT/MCTES through national funds, to earlyMYCO (PTDC/MED-TOX/28762/2017), and CESAM (UIDP/50017/2020+UIDB/50017/2020).N/

Aflatoxin B1 and early life gut microbiota: preliminary results under earlyMYCO project

Aflatoxin B1 (AFB1) produces acute or chronic deleterious health effects in humans and animals. Still, long-term effects derived from initial exposure in early-life, a critical period for colonization and development of gut microbiota, has not been fully evaluated, . particularly, effects on gut microbiota and immunity system. This study, performed under the earlyMYCO project, investigated the impact of maternal exposure to AFB1 on early-life microbiota in a mouse model. Females were fed jelly pellets containing 400 µg/kg AFB1 diluted in DMSO (treated animals n=6) or DMSO vehicle alone (control group n=6) during pregnancy and lactation. Faeces from the offspring were collected immediately after weaning and faecal DNA was extracted and purified. Bacterial taxa diversity and relative abundance were assessed by High-Throughput Sequencing performed in an Illumina Miseq® sequencer, targeting the V3 and V4 regions of the 16S rRNA gene. Operational taxonomic units (OTUs) were determined by clustering reads to 16S reference databases. A hundred and twenty-four (N=124) bacterial genera were found in both groups, 5 were only present in AFB1 treated group and 27 exclusively in control groups. A hundred and fifty-one (N=151) bacterial species were common to both groups, 15 species exclusively found in AFB1 litters and 34 species were exclusively found in control litters. Although present in both groups, Akkermansia muciniphila and Bacteroides acidifaciens were significantly higher in controls. A. muciniphila colonizes the intestinal tract in childhood and regulates mucus thickness, intestinal barrier integrity and is involved in immune modulation. B. acidifaciens participates in the metabolism of lipids and sugars and activates some cytokines and immune cell receptors. Sulfidogenic bacteria, recently related to inflammatory bowel disease, such as Desulfovibrio piger and Bilophila wadsworthia were exclusivly found in the treated litters. Early-life gut microbiome triggers the gut immune defences, but is far less stable than the adult microbiome. These preliminary results open an extensive field to further investigate the association between mycotoxins and microbiome, as the latest is increasingly recognized as a major player in a wide spectrum of diseases.This work was funded by FCT/MCTES through national funds, to earlyMYCO (PTDC/MED-TOX/28762/2017), and CESAM (UIDP/50017/2020+UIDB/50017/2020).N/

Early-life exposure to MYCOtoxins and its impact on health – a case study

Considering the potential impact on health and the scarce data available regarding early-life exposure to mycotoxins, earlyMYCO project (early-life exposure to MYCOtoxins and its impact on health) proposed to answer key questions: are pregnant women and infants until six months exposed to mycotoxins? Is this exposure a health threat? Does this early-life exposure influence the intestinal immune system development? Which is the burden derived from the exposure to mycotoxins? The earlyMYCO pilot study enrolled 19 pairs of mother and children, with a loss to follow-up ranging between 11% and 47% for different moments of observation. The mycotoxins’ biomarkers detected were AFB1, OTA, DON and bZEL in urine samples (mother and children), and AFB1, aZEL, FB1, FB2 and FB3 in breast milk samples. Food consumption data revealed that foods consumed more frequently during the week were bread, dairy products, non-alcoholic drinks (tea and coffee), animal products (meat and fish) and pasta. Regarding infants, 22% were fed with infant formula and 78% were exclusively breastfed. Considering the exposure levels, a low risk of mothers’ exposure to the main mycotoxins analyzed is expected, since urine samples did not reveal detectable levels of these compounds; however, infants’ urine samples presented a DON mean value of 14.8 ng/mL (corresponding to 148.0 μg/kg bw/day through reverse dosimetry), which could represent a risk for this population group. Notably, maternal exposure to AFB1 promoted an increase of overall T cell population, while it also resulted in a selective reduction of cytokine-producing innate lymphoid cells group 2 (ILC2) population in intestine of the progeny. These alterations were associated with decreased expression of Reg3b and Reg3g by the intestinal mucosa of progeny. Thus, these results indicate that maternal exposure to mycotoxins impacts the development of offspring intestinal immune system. An in vitro approach using intestinal cell lines Caco-2 and Caco-2/HT29-MTX models exposed to AFB1 during 24h, confirmed the deleterious effects of AFB1 on intestinal membrane integrity and its effect on mucus layer. To assess the impact of AFB1 on early-life microbiota, faeces from litters of AFB1 treated female mice and controls were assessed by metagenomics. Although the overall diversity (Shannon diversity index) of the microbiome wasn’t affected between groups, the microbiome composition varied between AFB1 and control faecal samples (Bray–Curtis dissimilarity index). In particular, some beneficial species were diminished in the litters from AFB1 treated females. Results emphasized the need for assessing the prenatal and lactation exposure to mycotoxins.This work was funded by FCT/MCTES through national funds, to earlyMYCO (PTDC/MED-TOX/28762/2017), and CESAM (UIDP/50017/2020+UIDB/50017/2020).N/

Ferritin regulates organismal energy balance and thermogenesis

Objective: The ferritin heavy/heart chain (FTH) gene encodes the ferroxidase component of the iron (Fe) sequestering ferritin complex, which plays a central role in the regulation of cellular Fe metabolism. Here we tested the hypothesis that ferritin regulates organismal Fe metabolism in a manner that impacts energy balance and thermal homeostasis. Methods: We developed a mouse strain, referred herein as FthR26 fl/fl, expressing a tamoxifen-inducible Cre recombinase under the control of the Rosa26 (R26) promoter and carrying two LoxP (fl) sites: one at the 5′end of the Fth promoter and another the 3' end of the first Fth exon. Tamoxifen administration induces global deletion of Fth in adult FthR26Δ/Δ mice, testing whether FTH is required for maintenance of organismal homeostasis. Results: Under standard nutritional Fe supply, Fth deletion in adult FthR26Δ/Δ mice led to a profound deregulation of organismal Fe metabolism, oxidative stress, inflammation, and multi-organ damage, culminating in death. Unexpectedly, Fth deletion was also associated with a profound atrophy of white and brown adipose tissue as well as with collapse of energy expenditure and thermogenesis. This was attributed mechanistically to mitochondrial dysfunction, as assessed in the liver and in adipose tissue. Conclusion: The FTH component of ferritin acts as a master regulator of organismal Fe homeostasis, coupling nutritional Fe supply to organismal redox homeostasis, energy expenditure and thermoregulation. Keywords: Iron metabolism, Redox homeostasis, Adipose tissue, Energy expenditure, Adipose tissue, Mitochondri

Circulating adipocyte-derived extracellular vesicles are novel markers of metabolic stress

We recently reported that stressed adipocytes release extracellular vesicles (EVs) that act as “find me” signals to promote macrophage migration and activation. In this study we performed a comprehensive characterization of stressed adipocyte-derived EVs, assessing their antigenic composition, lipidomics, and RNA profiles. Perilipin A was identified as one of the adipose-specific proteins and studied as a potential novel biomarker to detect adipocyte-derived EVs in circulation. Circulating EVs were significantly increased in mice with diet-induced obesity (DIO) and in obese humans with Metabolic Syndrome compared to lean controls. This increase was associated with decreased glucose tolerance in the DIO mice and metabolic dysfunction, elevated insulin and homeostatic model assessment of insulin resistance (HOMA-IR), in the obese humans. EVs from both DIO mice and obese humans were enriched in perilipin A, a central gatekeeper of the adipocyte lipid storehouse and a marker of adipocyte differentiation. In obese humans, circulating levels of EVs enriched in perilipin A were dynamic, decreasing 35% (p<0.05) after a 3-month reduced calorie diet intervention. This translational study provides an extensive characterization of adipocyte-derived EVs. The findings identify perilipin A as a novel biomarker of circulating EVs of adipocyte origin and support the development of circulating perilipin A-positive EVs as indicators of adipose tissue health