Browsing by Author "Arrieta, Marie-Claire"
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Item Open Access A critical assessment of the “sterile womb” and “in utero colonization” hypotheses: implications for research on the pioneer infant microbiome(2017-04-28) Perez-Muñoz, Maria E; Arrieta, Marie-Claire; Ramer-Tait, Amanda E; Walter, JensAbstract After more than a century of active research, the notion that the human fetal environment is sterile and that the neonate’s microbiome is acquired during and after birth was an accepted dogma. However, recent studies using molecular techniques suggest bacterial communities in the placenta, amniotic fluid, and meconium from healthy pregnancies. These findings have led many scientists to challenge the “sterile womb paradigm” and propose that microbiome acquisition instead begins in utero, an idea that would fundamentally change our understanding of gut microbiota acquisition and its role in human development. In this review, we provide a critical assessment of the evidence supporting these two opposing hypotheses, specifically as it relates to (i) anatomical, immunological, and physiological characteristics of the placenta and fetus; (ii) the research methods currently used to study microbial populations in the intrauterine environment; (iii) the fecal microbiome during the first days of life; and (iv) the generation of axenic animals and humans. Based on this analysis, we argue that the evidence in support of the “in utero colonization hypothesis” is extremely weak as it is founded almost entirely on studies that (i) used molecular approaches with an insufficient detection limit to study “low-biomass” microbial populations, (ii) lacked appropriate controls for contamination, and (iii) failed to provide evidence of bacterial viability. Most importantly, the ability to reliably derive axenic animals via cesarean sections strongly supports sterility of the fetal environment in mammals. We conclude that current scientific evidence does not support the existence of microbiomes within the healthy fetal milieu, which has implications for the development of clinical practices that prevent microbiome perturbations after birth and the establishment of future research priorities.Item Open Access A Primary Epithelial-Immune Cell Co-Culture Model to Investigate the Interactions between the Intestinal Epithelium and Innate Immune Cells in Response to Clostridioides difficile toxins(2024-01-04) Smith, Lauren; MacNaughton, Wallace; Hirota, Simon; Geuking, Markus; Arrieta, Marie-ClaireClostridioides difficile is a leading cause of healthcare-associated diarrhea in the United States, with symptoms ranging from mild diarrhea to toxic megacolon. Upon disruption of the colonic epithelium by C. difficile toxins A and B (TcdAB), epithelial cells are thought to signal to underlying immune cells including group-3 innate lymphoid cells (ILC3s) and macrophages to mount an effective immune response. ILC3s and macrophages both modulate pathogen defense (including C. difficile), tissue repair, and inflammation. ILC3s do so largely through interleukin- 22 (IL-22) secretion, and macrophages do so through phagocytosis and the secretion of a wide array of cytokines and chemokines. However, pathways by which the intestinal epithelium responds to C. difficile TcdAB to affect underlying immune cells, such as ILC3s and macrophages, remain poorly understood. We therefore hypothesized that the response to C. difficile toxins throughout the colonic epithelium and underlying tissue is modulated by interactions between the intestinal epithelium, ILC3s and IL-22, and macrophages. To test this hypothesis and address the limitations of cancer cell lines and single lineage models, we developed two novel primary epithelial cell-innate immune cell co-culture models to investigate epithelial-immune interactions in response to C. difficile toxins in vitro. Co-culture of monolayers with either MNK-3 (ILC3-like) cells or bone marrow derived macrophages did not influence monolayer permeability in response to TcdAB. However, in the process of characterizing these models, we established that MNK-3 cells and macrophages remained viable in co-culture and released relevant mediators with respective stimulation. While MNK-3 cells were not activated in co-culture, macrophages were activated by both monolayer media and epithelial presence in co-culture. With these novel experiments, we have provided groundwork for the further development of multi-lineage models.Item Open Access Cluster-specific associations between the gut microbiota and behavioral outcomes in preschool-aged children(2024-03-21) van de Wouw, Marcel; Wang, Yanan; Workentine, Matthew L.; Vaghef-Mehrabani, Elnaz; Barth, Delaney; Mercer, Emily M.; Dewey, Deborah; Arrieta, Marie-Claire; Reimer, Raylene A.; Tomfohr-Madsen, Lianne; Giesbrecht, Gerald F.Abstract Background The gut microbiota is recognized as a regulator of brain development and behavioral outcomes during childhood. Nonetheless, associations between the gut microbiota and behavior are often inconsistent among studies in humans, perhaps because many host-microbe relationships vary widely between individuals. This study aims to stratify children based on their gut microbiota composition (i.e., clusters) and to identify novel gut microbiome cluster-specific associations between the stool metabolomic pathways and child behavioral outcomes. Methods Stool samples were collected from a community sample of 248 typically developing children (3–5 years). The gut microbiota was analyzed using 16S sequencing while LC-MS/MS was used for untargeted metabolomics. Parent-reported behavioral outcomes (i.e., Adaptive Skills, Internalizing, Externalizing, Behavioral Symptoms, Developmental Social Disorders) were assessed using the Behavior Assessment System for Children (BASC-2). Children were grouped based on their gut microbiota composition using the Dirichlet multinomial method, after which differences in the metabolome and behavioral outcomes were investigated. Results Four different gut microbiota clusters were identified, where the cluster enriched in both Bacteroides and Bifidobacterium (Ba2) had the most distinct stool metabolome. The cluster characterized by high Bifidobacterium abundance (Bif), as well as cluster Ba2, were associated with lower Adaptive Skill scores and its subcomponent Social Skills. Cluster Ba2 also had significantly lower stool histidine to urocanate turnover, which in turn was associated with lower Social Skill scores in a cluster-dependent manner. Finally, cluster Ba2 had increased levels of compounds involved in Galactose metabolism (i.e., stachyose, raffinose, alpha-D-glucose), where alpha-D-glucose was associated with the Adaptive Skill subcomponent Daily Living scores (i.e., ability to perform basic everyday tasks) in a cluster-dependent manner. Conclusions These data show novel associations between the gut microbiota, its metabolites, and behavioral outcomes in typically developing preschool-aged children. Our results support the concept that cluster-based groupings could be used to develop more personalized interventions to support child behavioral outcomes. Video AbstractItem Open Access The Cystic Fibrosis Microbiome and its Association with Incident Infections with Mycobacteroides (Mycobacterium) abscessus(2021-05-13) Bharadwaj, Lalit; Parkins, Michael; Storey, Douglas; Arrieta, Marie-Claire; Surette, MichaelInfection with M. abscessus complex (MABC) is increasingly detected within CF populations. MABC infection has been associated with exaggerated lung function decline and poses significant treatment complexities. We performed a retrospective case-control study of twenty-one patients with MABC infection matching each to two randomly identified age (+/-2 yrs) and gender-matched uninfected controls. Total genomic DNA from sputum was extracted, amplified and Illumina MiSeq paired-end sequencing of the hypervariable V3 region of the 16S rRNA gene was performed. Demographics and dynamic variables of disease were recorded and compared between groups. 174 sputum samples (median 8, IQR (6-12)) from MABC cases and 42 control samples were assessed. The sputum microbiota from patients who would develop MABC infection in the subsequent two years differed from controls (p=0.038, R2 = 2.5%, PERMANOVA). In particular, sputum from MABC cases – prior to its identification – had higher alpha-diversity; Shannon diversity (p=0.023), Observed species (p=0.042) and lower P. aeruginosa relative and absolute abundance (p=0.035). We observed significant changes in community structure over time during potent antibacterial therapies, returning to baseline upon their discontinuation. These data suggest that sputum microbiome analysis and P. aeruginosa bioburden should be evaluated in multi-center studies as potential biomarkers to predict MABC infection and treatment response.Item Open Access Dietary Manipulation at Pre-Conception and During Development Influence Metabolism and Gut Microbiota in Rats(2021-02-01) Chleilat, Fatima; Reimer, Raylene; Shearer, Jane; Arrieta, Marie-ClaireBackground. Obesity is a complex disease, modulated by a plethora of factors, including gut microbiota, early post-natal nutrition, parental diet and epigenetics. Objective. This dissertation examines how dietary manipulation during the early post-natal period and during pre-conception alters metabolic, microbial and epigenetic outcomes in rats. Specifically, the objectives were to determine: 1) the impact of human milk oligosaccharide (HMO) supplementation on health status in weanling rats; 2) the impact of a paternal high protein (HP) diet on offspring metabolic health; 3) whether supplementation of a methyl donor cocktail (HF/S+M) before conception attenuates the deleterious metabolic and epigenetic effects of a paternal high fat/ sucrose diet intergenerationally; 4) the impact of paternal prebiotic consumption on microbial and metabolic outcomes in fathers and offspring. Methods. 1) Weanling male and female rats consumed 2’Fucosyllactose and 3’Sialylactose HMO-fortified diet for 8 weeks. Fathers consumed 2) a diet high in protein, 3) a high fat/ sucrose diet fortified with a methyl donor cocktail of betaine, choline, folic acid and vitamin B12, or 4) a high prebiotic fiber diet. All three paternal studies underwent their dietary intervention for 9 weeks. Offspring consumed a control diet for 13 weeks. Anthropometric, glucocentric and gut microbiota outcomes were measured in all four studies. Results. The primary findings include: HMO supplementation improved intestinal permeability, gut barrier function, and gut microbial composition in females while reducing weight gain and inflammatory cytokines in males; 2) Paternal HP diet reduced adiposity and altered epigenetic markers intergenerationally. Offspring had improved insulin sensitivity; 3) Paternal HF/S+M intake improved paternal reproductive outcomes and intergenerational gut microbial, epigenetic and metabolic outcomes; 4) Paternal prebiotic intake improved paternal gut microbiota with lesser effects in offspring. Conclusion. Our results provide evidence of early post-natal HMO supplementation and paternal HP, HF/S+M and prebiotic intake, as important modulators of gut microbial, epigenetic and metabolic outcomes.Item Open Access Divergent maturational patterns of the infant bacterial and fungal gut microbiome in the first year of life are associated with inter-kingdom community dynamics and infant nutrition(2024-02-07) Mercer, Emily M.; Ramay, Hena R.; Moossavi, Shirin; Laforest-Lapointe, Isabelle; Reyna, Myrtha E.; Becker, Allan B.; Simons, Elinor; Mandhane, Piush J.; Turvey, Stuart E.; Moraes, Theo J.; Sears, Malcolm R.; Subbarao, Padmaja; Azad, Meghan B.; Arrieta, Marie-ClaireAbstract Background The gut microbiome undergoes primary ecological succession over the course of early life before achieving ecosystem stability around 3 years of age. These maturational patterns have been well-characterized for bacteria, but limited descriptions exist for other microbiota members, such as fungi. Further, our current understanding of the prevalence of different patterns of bacterial and fungal microbiome maturation and how inter-kingdom dynamics influence early-life microbiome establishment is limited. Results We examined individual shifts in bacterial and fungal alpha diversity from 3 to 12 months of age in 100 infants from the CHILD Cohort Study. We identified divergent patterns of gut bacterial or fungal microbiome maturation in over 40% of infants, which were characterized by differences in community composition, inter-kingdom dynamics, and microbe-derived metabolites in urine, suggestive of alterations in the timing of ecosystem transitions. Known microbiome-modifying factors, such as formula feeding and delivery by C-section, were associated with atypical bacterial, but not fungal, microbiome maturation patterns. Instead, fungal microbiome maturation was influenced by prenatal exposure to artificially sweetened beverages and the bacterial microbiome, emphasizing the importance of inter-kingdom dynamics in early-life colonization patterns. Conclusions These findings highlight the ecological and environmental factors underlying atypical patterns of microbiome maturation in infants, and the need to incorporate multi-kingdom and individual-level perspectives in microbiome research to improve our understandings of gut microbiome maturation patterns in early life and how they relate to host health. Video AbstractItem Open Access Divergent maturational patterns of the infant bacterial and fungal gut microbiome in the first year of life are associated with inter-kingdom community dynamics and infant nutrition(2024-02-07) Mercer, Emily M.; Ramay, Hena R.; Moossavi, Shirin; Laforest-Lapointe, Isabelle; Reyna, Myrtha E.; Becker, Allan B.; Simons, Elinor; Mandhane, Piush J.; Turvey, Stuart E.; Moraes, Theo J.; Sears, Malcolm R.; Subbarao, Padmaja; Azad, Meghan B.; Arrieta, Marie-ClaireAbstract Background The gut microbiome undergoes primary ecological succession over the course of early life before achieving ecosystem stability around 3 years of age. These maturational patterns have been well-characterized for bacteria, but limited descriptions exist for other microbiota members, such as fungi. Further, our current understanding of the prevalence of different patterns of bacterial and fungal microbiome maturation and how inter-kingdom dynamics influence early-life microbiome establishment is limited. Results We examined individual shifts in bacterial and fungal alpha diversity from 3 to 12 months of age in 100 infants from the CHILD Cohort Study. We identified divergent patterns of gut bacterial or fungal microbiome maturation in over 40% of infants, which were characterized by differences in community composition, inter-kingdom dynamics, and microbe-derived metabolites in urine, suggestive of alterations in the timing of ecosystem transitions. Known microbiome-modifying factors, such as formula feeding and delivery by C-section, were associated with atypical bacterial, but not fungal, microbiome maturation patterns. Instead, fungal microbiome maturation was influenced by prenatal exposure to artificially sweetened beverages and the bacterial microbiome, emphasizing the importance of inter-kingdom dynamics in early-life colonization patterns. Conclusions These findings highlight the ecological and environmental factors underlying atypical patterns of microbiome maturation in infants, and the need to incorporate multi-kingdom and individual-level perspectives in microbiome research to improve our understandings of gut microbiome maturation patterns in early life and how they relate to host health. Video AbstractItem Embargo Effects of a Paternal Diet High in Animal Protein versus Plant Protein on Offspring Metabolic and Microbial Outcomes in a Rodent Model(2023-11-17) Patterson, Riley; Reimer, Raylene; Shearer, Jane; Arrieta, Marie-Claire; Thompson, JenniferObesity and type 2 diabetes are influenced by genetic and environmental factors, including diet. In addition to the well-known influence of maternal nutrition on offspring health, paternal diet has also been shown recently to program lifelong disease risk in their offspring. For instance, a paternal diet high in casein protein demonstrated numerous protective effects for their offspring including reduced body fat percentage, enhanced insulin sensitivity and higher satiety hormone levels compared to paternal high fat or control diets. Based on mounting evidence of this paternal influence, as well as dietary guidance to consume more plant products, the objective of this thesis was to examine the effects of animal versus plant protein on paternal and offspring health. Five-week-old male Sprague Dawley rats (n=36) were randomized into three dietary intervention groups for 8-11 weeks: 1) control AIN-93; 2) high animal casein protein (AP); and 3) high plant pea protein (PP). They were then mated. Offspring were challenged with a high fat/sucrose diet (HFD) from 10-16 weeks of age. Metabolic and microbial outcomes assessed in the fathers and offspring included body composition (DXA), glucose tolerance and insulin sensitivity (OGTT and ITT, respectively), gut microbial composition (16S rRNA sequencing), and gene expression (RT-PCR). The PP diet directly altered paternal hepatic microRNA expression and gut microbial profiles fostering improved metabolic functioning including enhanced insulin sensitivity and improved lipid metabolism and intestinal integrity depicted in decreased hepatic triglyceride and serum LPS levels, respectively. In contrast, adult offspring from fathers fed the AP diet exhibited increased adiposity, altered lipid metabolism, and dysregulated satiety hormones that were accompanied by altered miRNA expression and modified gut microbiota following 6-week exposure to a HFD. Overall, a paternal diet high in pea protein had numerous beneficial effects on the fathers’ metabolism and gut microbiota but had minimal effects on their offspring. Whereas a paternal diet high in animal protein during the preconception period had minimal effects on the fathers but programmed an increased predisposition to metabolic dysfunction in their adult offspring when unmasked by HFD exposure.Item Open Access Metaproteomic profiling of fungal gut colonization in gnotobiotic mice(2022-02-22) Pettersen, Veronika K.; Dufour, Antoine; Arrieta, Marie-ClaireAbstract Background Eukaryotic microbes can modulate mammalian host health and disease states, yet the molecular contribution of gut fungi remains nascent. We previously showed that mice exclusively colonised with fungi displayed increased sensitivity to allergic airway inflammation and had fecal metabolite profiles similar to germ-free mice. This marginal effect on the host metabolome suggested that fungi do not primarily use metabolites to modulate the host immune system. Methods To describe functional changes attributed to fungal colonisation, we performed mass spectrometry-based analyses of feces (Label-Free Quantitative; LFQ) and the small intestine (labeling with Tandem Mass Tag; TMT) of gnotobiotic mice colonised with defined consortia of twelve bacterial species, five fungal species, or both. We also evaluated the effect of microbiome perturbances on the metaproteome by analysing feces from mouse pups treated with an antibiotic or antifungal. Results We detected 6675 proteins in the mice feces, of which 3845 had determined LFQ levels. Analysis of variance showed changes in the different gnotobiotic mouse groups; specifically, 46% of 2860 bacterial, 15% of 580 fungal, and 76% of 405 mouse quantified proteins displayed differential levels. The antimicrobial treatments resulted in lasting changes in the bacterial and fungal proteomes, suggesting that the antimicrobials impacted the entire community. Fungal colonisation resulted in changes in host proteins functional in innate immunity as well as metabolism, predicting specific roles of gut fungi on host systems during early developmental stages. Several of the detected fungal proteins (3% of 1492) have been previously reported as part of extracellular vesicles and having immunomodulating properties. Using an isobaric labelling TMT approach for profiling low abundant proteins of the jejunal tissue, we confirmed that the five fungal species differentially impacted the host intestinal proteome compared to the bacterial consortium. The detected changes in mouse jejunal proteins (4% of 1514) were mainly driven by metabolic proteins. Conclusions We used quantitative proteomic profiling of gnotobiotic conditions to show how colonisation with selected fungal species impacts the host gut proteome. Our results suggest that an increased abundance of certain gut fungal species in early life may affect the developing intracellular attributes of epithelial and immune cells.Item Open Access The role of intestinal fungi on microbiome ecology, host immune development, and susceptibility to airway inflammation(2023-12-04) van Tilburg Bernardes, Erik; Arrieta, Marie-Claire; Proud, David; McCoy, Kathy D.; McDonald, Braedon A.Early-life microbiome alterations can lead to immune dysregulation and increase susceptibility to asthma. Bacterial changes often precede asthma development in humans and have been causally linked to heightened airway inflammation in mice. Prospective infant studies have also identified fungal microbiome (mycobiome) alterations associated with asthma risk. However, it remains unknown if fungi contribute to the pathogenesis of atopy and asthma. My Ph.D. thesis project aimed to determine the causal role of early-life fungal colonization in immune development and susceptibility to allergic airway inflammation. We determined the role of intestinal fungi on microbiome structure, function, and host immune development in gnotobiotic mice colonized with defined communities of 12 bacteria and/or five fungi. Gut fungi exerted significant ecological pressures to the coexisting bacterial microbiome, and vice versa. Early-life fungal colonization also induced robust host systemic immune changes and influenced the immune phenotype of lung’s inflammatory response to ovalbumin allergen. Antibiotic use also impacts the gut microbiome, leading to an increased risk of asthma. However, the impact of antibiotics on the infant mycobiome is unknown. To investigate mycobiome changes associated with antibiotic treatment we conducted an observational, prospective clinical study of 47 infants (under 6 months of age) who received antibiotics. Antibiotic use decreased bacterial and increased fecal fungal DNA and induced the expansion of Malassezia spp. in infants. To evaluate the effect of colonization with Malassezia spp. on immune development and airway inflammation, we assessed early-life immune readouts and susceptibility to a house-dust mite (HDM) model in mice colonized with or without Malassezia restricta. M. restricta colonization increased intestinal immune responses deemed critical in atopy development, and elevated airway inflammation in HDM-challenged mice. Further evaluation in eosinophil-deficient mice revealed that the observed immune response is partially dependent on this cell type. This translational work demonstrates that fungi are integral components of the intestinal microbiome, causally implicated in host immune development and susceptibility to airway inflammation. Fungal overgrowth and expansion of Malassezia spp. are previously overlooked collateral effects of infant antibiotic use, which may offer a potential strategy to prevent or mitigate pediatric asthma and related conditions.Item Open Access The Role of the Autonomic Nervous System in Mediating the Relationship Between the Gut Microbiota and Social Behaviour(2023-10-17) Zinman, Julia; Giesbrecht, Gerald; Arrieta, Marie-Claire; Campbell, Tavis; Kopala Sibley, DanielSocial competence is profoundly important for child development by influencing personality and quality of life. Recent discoveries indicate that bacteria within the gastrointestinal tract (i.e. gut microbiota), play an important role in social behaviour through connections to the brain (i.e. the microbiota-gut-brain axis). One of these connections occurs through the vagus nerve, which is a prominent member of the parasympathetic nervous system (PNS). We aimed to determine whether the gut microbiota at 3-4 years of age was associated with social behaviour deficits, and whether this relationship was mediated by vagal activity. We used data (n=248) from the prospective APrON study. Stool microbiota was assessed for its bacterial composition using 16s sequencing and its metabolome using liquid chromatography–mass spectrometry. Vagal activity was indexed by respiratory sinus arrhythmia (RSA) collected in lab. Social behaviour was assessed using the Behavioural Assessment System for Children (BASC-2). We used raw and adjusted Spearman correlation analyses to investigate the associations between the gut microbiota, baseline RSA and social behaviour. We investigated whether vagal activity mediates the relationship between the gut microbiota and social behaviour using PROCESS. Greater alpha diversity predicted lower Withdrawal scores at 3-4 years old. Associations between stool taxa and social behaviour were not statistically significant after performing a false detection rate (FDR) correction. Baseline RSA was positively associated with Bullying scores in males but not females. There was no significant mediation effect of baseline RSA on the relationship between the gut microbiota and social behaviour. Ultimately, exploring the intricate relationship between gut microbiota and behaviour in children is of utmost significance, as it holds the potential to revolutionize our understanding of child development and well-being.