Simon A. Hirota’s research while affiliated with University of Calgary and other places

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Publications (19)


A84 THE USE OF PHYTASE TO ENHANCE DIETARY IRON AND ZINC ABSORPTION - A SCOPING REVIEW
  • Article

February 2025

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3 Reads

Journal of the Canadian Association of Gastroenterology

S Hirota

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N Adamidi

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T Chondrou

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[...]

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Background Phytic acid is abundant in plant-based diets and acts as a micronutrient inhibitor for humans and non-ruminant animals. Phytases are enzymes that break down phytic acid, releasing micronutrients and enhancing their bioavailability, particularly iron and zinc. Deficiencies in iron and zinc are common in patients with the inflammatory bowel diseases and in those in developing countries consuming phytic acid-rich diets. Aims This literature review aimed to summarize findings from human intervention studies on the interactions between phytic acid, phytase and micronutrient bioavailability. Methods An extensive PubMed search (01/01/1990 to 08/02/2024) was conducted using MeSH terms (phytic acid, phytase, IP6, “inositol hexaphosphate,” micronutrient, magnesium, calcium, iron, zinc). Eligible studies included human intervention trials investigating the bioavailability of micronutrients following a) phytase supplementation, b) consumption of phytic acid-rich foods, or c) consumption of dephytinised foods. In-vitro, animal, cross-sectional, and non-English studies were excluded. Results 3055 articles were identified. After title and full-text review 40 articles were eligible. Another 2 were identified after cross-checking reference lists from included papers, resulting in 42 included articles. Most studies exploring the efficacy of exogenous phytase (9 of 11, 82%) or the efficacy of food dephytinisation (11 of 14, 79%) presented augmented iron and zinc bioavailability. In agreement to this, most phytic acid-rich food feeding studies (13 of 17, 77%) showed compromised iron and zinc bioavailability. Conclusions Strong evidence supports decreased iron and zinc bioavailability in phytic acid-rich diets and the improvement potential with phytase interventions. Further studies are needed in larger populations. Funding Agencies None


Early-life fungal colonization and diet-induced obesity model
A Gnotobiotic colonization and diet-induced obesity model. Germ-free dams were gavaged twice with the Oligo-MM12 consortium alone (B; gray) or in combination with C. albicans (B + C; orange), R. mucilaginosa (B + R; pink) or M. restricta (B + M; purple; Males—SD: nB = 23, nB+C = 8, nB+R = 10, nB+M = 12; Females—SD: nB = 18, nB+C = 8, nB+R = 8, nB+M = 12; Males—HFHS: nB = 22, nB+C = 10, nB+R = 8, nB+M = 12; Females—HFHS: nB = 20, nB+C = 12, nB+R = 7, nB+M = 10). Twice in the first week of life, offspring (F1) were exposed to the fungal species corresponding to their colonization group. F1 were weaned onto standard diet (SD) or high-fat-high-sucrose diet (HFHS) until 12 weeks of age. Graphics from BioRender.com. https://BioRender.com/y96j811. B Fungal colony count in fecal samples at 6–7 weeks of age. One mouse sampled per cage (SD: nB+C = 6, nB+R = 8; HFHS: nB+C = 6, nB+R = 7). Two-way ANOVA with Sidak’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. C, D Fecal ITS DNA at 12 weeks in males and females, respectively (Male—SD: nB = 13, nB+C = 7, nB+R = 7, nB+M = 8; Male—HFHS: nB = 16, nB+C = 8, nB+R = 8, nB+M = 8; Female—SD: nB = 14, nB+C = 8, nB+R = 8, nB+M = 8; Female—HFHS: nB = 14, nB+C = 8, nB+R = 6, nB+M = 8). Data represented as mean ± SEM; n indicates number of mice sampled.
Early-life fungal colonization modulates bacterial community composition
A–C Bacterial beta-diversity at 3 weeks based on Bray–Curtis dissimilarity index assessed by permutational ANOVA between bacteria only and B + C (nB = 16, nB+C = 16), B + R (nB = 16, nB+R = 16) and B + M (nB = 13, nB+M = 16), respectively. D Bacterial relative abundance at 3 weeks between bacteria only and B + C (nB = 16, nB+C = 16), B + R (nB = 16, nB+R = 16) and B + M (nB = 13, nB+M = 16), respectively. E–G Bacterial beta-diversity at 12 weeks between bacteria only and B + C (SD: nB = 16, nB+C = 16; HFHS: nB = 16, nB+C = 16), B + R (SD: nB = 16, nB+R = 16; HFHS: nB = 16, nB+R = 14) and B + M (SD: nB = 14, nB+M = 16; HFHS: nB = 16, nB+M = 17), respectively. Fungi and diet effect based on Bray–Curtis Dissimilarity Index assessed by permutational ANOVA. H, I Bacterial relative abundance at 12 weeks between SD-fed or HFHS-fed bacteria only and B + C (SD: nB = 16, nB+C = 16; HFHS: nB = 16, nB+C = 16), B + R (SD: nB = 16, nB+R = 16; HFHS: nB = 16, nB+R = 14) and B + M (SD: nB = 14, nB+M = 16; HFHS: nB = 16, nB+M = 17), respectively. A–C, E–G Ellipses represent 95% CI. Dots indicate SD and triangles indicate HFHS. D, H, I Data represented as mean relative abundance. Normality assessed by Shapiro–Wilk test and differences calculated by unpaired T-test (two-sided) or Mann–Whitney test (two-sided) accordingly. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a source data file; n indicates number of mice sampled. SD standard diet, HFHS high-fat-high-sucrose diet.
Fungal colonization modulates the fecal metabolome and microbiome metabolic function
A–C Fecal metabolome in 12-week fecal samples between B and B + C (SD: nB = 16, nB+C = 16; HFHS: nB = 16, nB+C = 16), B + R (SD: nB = 16, nB+R = 16; HFHS: nB = 16, nB+R = 14) and B + M (SD: nB = 14, nB+M = 16; HFHS: nB = 16, nB+M = 17), respectively. Fungi and diet effect based on Euclidean distance and assessed by permutational ANOVA. Ellipses represent 95% CI. Dots indicate SD and triangles indicate HFHS. D Fold change analysis of fecal metabolome between B and B + C mice fed SD diet (nB = 16, nB+C = 16). E Normalized concentrations of metabolites with significant fold change in (D) between B and B + C mice fed SD diet (nB = 16, nB+C = 16). F Fold change analysis of fecal metabolome between B and B + C mice fed HFHS diet (nB = 16, nB+C = 16). G Normalized concentrations of metabolites with significant fold change in (F) between B and B + C mice fed HFHS diet (nB = 16, nB+C = 16). H Fold change analysis of fecal metabolome between B and B + R mice fed SD diet (nB = 16, nB+R = 16). I Normalized concentrations of metabolites with significant fold change in (H) between B and B + R mice fed SD diet (nB = 16, nB+C = 16). D, F, H Each dot represents an individual metabolite. Highlighted metabolites identified by non-parametric t-tests (two-sided) have p.adjust (false discover rate adjustment) <0.05 denoted by the dashed horizontal line and fold change ≥2 or ≤0.5 denoted by the dashed vertical lines. E, G, I Concentrations underwent median normalization, square root transformation and Pareto scaling. Each dot represents an individual mouse. A–C, E, G, I B = gray; B + C = orange; B + R = pink; B + M = purple. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a source data file; n indicates number of mice sampled. SD standard diet, HFHS high-fat-high-sucrose diet.
Gut fungi differentially modulate obesity development
A, B Body weight from 3–12 weeks with trapezoidal area under the curve (AUC) in males and females, respectively (Male—SD: nB = 23, nB+C = 8, nB+R = 10, nB+M = 12; Male—HFHS: nB = 22, nB+C = 9, nB+R = 8, nB+M = 12; Female—SD: nB = 18, nB+C = 8, nB+R = 8, nB+M = 12; Female—HFHS: nB = 20, nB+C = 12, nB+R = 7, nB+M = 10). Significant differences indicated by asterisk for SD and dots for HFHS. C Fat mass percentage independent of sex (SD: nB = 41, nB+C = 16, nB+R = 18, nB+M = 20; HFHS: nB = 42, nB+C = 21, nB+R = 15, nB+M = 21). D, E WAT wet weight in males and females, respectively (Male—SD: nB = 23, nB+C = 8, nB+R = 10, nB+M = 12; Male—HFHS: nB = 22, nB+C = 9, nB+R = 8, nB+M = 12; Female—SD: nB = 17, nB+C = 8, nB+R = 8, nB+M = 12; Female—HFHS: nB = 20, nB+C = 10, nB+R = 7, nB+M = 10). F Representative WAT H&E-stained sections capture at 20X for each colonization group on SD or HFHS. G Proportion of adipocytes within size ranges 5–40 um, 40–70 um, >70 um (SD: nB = 26, nB+C = 11, nB+R = 13, nB+M = 13; HFHS: nB = 25, nB+C = 10, nB+R = 7, nB+M = 11). Data represents mean ± SEM. Significant differences defined by Two-way ANOVA with Dunnett’s multiple comparisons test relative to B only group within either diet. C–E, G Outliers removed with ROUT (Q = 1%). A–E B = gray; B + C = orange; B + R = pink; B + M = purple. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a source data file; n indicates number of mice sampled. SD standard diet, HFHS high-fat-high-sucrose diet, gWAT gonadal white adipose tissue.
Gut fungi modulate oral glucose tolerance
A, B Oral glucose tolerance test (OGTT) with trapezoidal total glucose area under the curve (AUC) in males and females, respectively (Male—SD: nB = 23, nB+C = 8, nB+R = 10, nB+M = 12; Male—HFHS: nB = 22, nB+C = 9, nB+R = 8, nB+M = 12; Female—SD: nB = 17, nB+C = 8, nB+R = 8, nB+M = 12; Female—HFHS: nB = 20, nB+C = 12, nB+R = 7, nB+M = 10). Data represents mean ± SEM. Significant differences defined by Two-way ANOVA with Dunnett’s multiple comparisons test relative to B only group within either diet. B = gray; B + C = orange; B + R = pink; B + M = purple. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Significant differences indicated by asterisk for SD and dots for HFHS. Source data are provided as a source data file; n indicates number of mice sampled. SD standard diet, HFHS high-fat-high-sucrose diet.

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Early-life gut mycobiome core species modulate metabolic health in mice
  • Article
  • Full-text available

February 2025

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11 Reads

The gut microbiome causally contributes to obesity; however, the role of fungi remains understudied. We previously identified three core species of the infant gut mycobiome (Rhodotorula mucilaginosa, Malassezia restricta and Candida albicans) that correlated with body mass index, however their causal contributions to obesity development are unknown. Here we show the effects of early-life colonization by these fungal species on metabolic health in gnotobiotic mice fed standard (SD) or high-fat-high-sucrose (HFHS) diets. Each species resulted in bacterial microbiome compositional and functional differences. R. mucilaginosa and M. restricta increased adiposity in mice fed SD, while only R. mucilaginosa exacerbated metabolic disease. In contrast, C. albicans resulted in leanness and resistance to diet-induced obesity. Intestinal nutrient transporter expression was unaffected by the presence of fungi in jejunal enteroids, yet the immune landscape in white adipose tissue was distinctly impacted by each fungal species, suggesting that these phenotypes may be a result of fungal immune regulation. This work revealed that three common fungal colonizers have distinct causal influences on obesity and metabolic inflammation and justifies the consideration of fungi in microbiome research on host metabolism.

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PRISMA 2020 flow diagram for new systematic reviews, which included searches of databases and registers only; * all records were identified through PubMed database.
Intervention studies exploring the impact of phytase on micronutrient absorption.
Intervention studies exploring the impact of phytic acid on micronutrient absorption.
Intervention studies exploring the impact of food dephytinization on micronutrient absorption.
Dietary Phytic Acid, Dephytinization, and Phytase Supplementation Alter Trace Element Bioavailability—A Narrative Review of Human Interventions

Background: Phytic acid is abundant in plant-based diets and acts as a micronutrient inhibitor for humans and non-ruminant animals. Phytases are enzymes that break down phytic acid, releasing micronutrients and enhancing their bioavailability, particularly iron and zinc. Deficiencies in iron and zinc are significant public health problems, especially among populations with disease-associated malnutrition or those in developing countries consuming phytic acid-rich diets. This narrative review aimed to summarize findings from human intervention studies on the interactions between phytic acid, phytase, and micronutrient bioavailability. Methods: An extensive PubMed search (1 January 1990 to 8 February 2024) was conducted using MeSH terms (phytic acid, phytase, IP6, “inositol hexaphosphate,” micronutrient, magnesium, calcium, iron, zinc). Eligible studies included human intervention trials investigating the bioavailability of micronutrients following (a) phytase supplementation, (b) consumption of phytic acid-rich foods, or (c) consumption of dephytinized foods. In vitro, animal, cross-sectional, and non-English studies were excluded. Results: 3055 articles were identified. After the title and full-text review, 40 articles were eligible. Another 2 were identified after cross-checking reference lists from included papers, resulting in 42 included articles. Most studies exploring the efficacy of exogenous phytase (9 of 11, 82%) or the efficacy of food dephytinization (11 of 14, 79%) demonstrated augmented iron and zinc bioavailability. Most phytic acid-rich food-feeding studies (13 of 17, 77%) showed compromised iron and zinc bioavailability. Conclusions: Strong evidence supports decreased iron and zinc bioavailability in phytic acid-rich diets and significant improvements with phytase interventions. Studies of longer periods and within larger populations are needed.


Dietary Phytic Acid, Dephytinisation and Phytase Supplementation Alter Micronutrient Bioavailability – a Literature Review of Human Interventions

November 2024

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3 Reads

Background: Phytic acid is abundant in plant-based diets and acts as a micronutrient inhibitor for humans and non-ruminant animals. Phytases are enzymes that break down phytic acid, releasing micronutrients and enhancing their bioavailability, particularly iron and zinc. Deficiencies in iron and zinc are significant public health problems, especially among populations with disease-associated malnutrition or those in developing countries consuming phytic acid-rich diets. This literature review aimed to summarize findings from human intervention studies on the interactions between phytic acid, phytase and micronutrient bioavailability. Methods: An extensive PubMed search (01/01/1990 to 08/02/2024) was conducted using MeSH terms (phytic acid, phytase, IP6, "inositol hexaphosphate," micronutrient, magnesium, calcium, iron, zinc). Eligible studies included human intervention trials investigating the bioavailability of micronutrients following a) phytase supplementation, b) consumption of phytic acid-rich foods, or c) consumption of dephytinised foods. In vitro, animal, cross-sectional, and non-English studies were excluded. Results: 3055 articles were identified. After title and full-text review, 40 articles were eligible. Another 2 were identified after cross-checking reference lists from included papers, resulting in 42 included articles. Most studies exploring the efficacy of exogenous phytase (9 of 11, 82%) or the efficacy of food dephytinisation (11 of 14, 79%) demonstrated augmented iron and zinc bioavailability. In agreement with this, most phytic acid-rich food feeding studies (13 of 17, 77%) showed compromised iron and zinc bioavailability. Conclusions: Strong evidence supports decreased iron and zinc bioavailability in phytic acid-rich diets and significant improvements with phytase interventions. Studies exploring these effects for longer periods and within larger populations are needed.


Inflammasome activation links enteric Salmonella Typhimurium infection to a rapid, cytokine-dependent increase in intestinal mucin release

October 2024

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79 Reads

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2 Citations

The host restricts Salmonella enterica serovar Typhimurium infection of the gut via inflammasome-dependent sloughing of infected epithelial cells. Here we determined that concurrent caspase 1/11-dependent release of the goblet cell-derived mucin, Muc2, into the intestinal lumen also controls Salmonella burdens in infected mice. The increased release of mucins from goblet cells in the cecum and nearby proximal colon, and the subsequent thickening of the protective mucus barrier layer in the distal colon, were all dependent on the cytokines interleukin (IL)-18 and IL-22, as deficiencies in either cytokine resulted in reduced mucin secretion. Supplementation of IL-18 into IL-22 deficient mice restored mucin secretion, indicating that IL-22 acted upstream of IL-18 secretion during infection. In contrast, IL-18 and IL-22 independent signaling through Nlrp6 underlies only a modest, infection-induced increase in mucin secretion from goblet cells in the distal colon. These findings reveal that inflammasome signaling orchestrates multiple levels of protection centered on the intestinal epithelium, including pyroptosis and expulsion of infected enterocytes, as well as the release of mucins by goblet cells in the cecum and along the length of the colon. Our studies underscore the pivotal, multi-faceted role of inflammasome signaling in promoting host defense at the intestinal mucosal surface.


Challenges in IBD Research 2024: Preclinical Human IBD Mechanisms

May 2024

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176 Reads

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6 Citations

Inflammatory Bowel Diseases

Preclinical human inflammatory bowel disease (IBD) mechanisms is one of 5 focus areas of the Challenges in IBD Research 2024 document, which also includes environmental triggers, novel technologies, precision medicine, and pragmatic clinical research. Herein, we provide a comprehensive overview of current gaps in inflammatory bowel diseases research that relate to preclinical research and deliver actionable approaches to address them with a focus on how these gaps can lead to advancements in IBD interception, remission, and restoration. The document is the result of multidisciplinary input from scientists, clinicians, patients, and funders and represents a valuable resource for patient-centric research prioritization. This preclinical human IBD mechanisms section identifies major research gaps whose investigation will elucidate pathways and mechanisms that can be targeted to address unmet medical needs in IBD. Research gaps were identified in the following areas: genetics, risk alleles, and epigenetics; the microbiome; cell states and interactions; barrier function; IBD complications (specifically fibrosis and stricturing); and extraintestinal manifestations. To address these gaps, we share specific opportunities for investigation for basic and translational scientists and identify priority actions.


Evaluating the Dynamics of Cyp3a11, MRP2 and MDR1 Modulation by IL-22

May 2024

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4 Reads

Physiology

Background: IL-22 is a cytokine produced by a number of immune cell populations, including type 3 innate lymphoid cells. We have shown previously that IL-22 suppresses the expression Cyp3a11 in the mouse intestine, a gene that encodes a drug metabolizing. In addition, IL-22 reduces the expression of MRP2 and MDR1, the gene that encodes as Multidrug resistance protein 2 or 1 involve in the drug detoxification. Modulation of these proteins can influence the oral bioavailability of drugs. Aims: The aim of this study was to define the existence of reversible modulation of IL-22 on Cyp3a11, MRP2 and MDR1 expression and protein level or activity in the intestinal epithelium. Methods: In vitro enteroids of the mouse small intestine were cultured in 3D in the presence/absence of IL-22 for 5 days. Cyp3a11, MRP2 and MDR1 transcript expression was evaluated by qPCR followed by protein analysis to define their modulation and activity at varying timepoints after IL-22 treatment cessation. Results: IL-22 reduced the expression and activity of Cyp3a11 which normalized soon after stopping IL-22 treatment. This action of IL-22 are made without any modulation of protein level of Cyp3a11. In the same way, IL-22 reduces the expression and the protein level of MRP2 and MDR1, an effect normalizes after treatment cessation. Conclusions: IL-22 suppresses the expression and the activity of Cyp3a11 without any modulation of the protein level unlike at the level of MRP2 and MDR1 which are reduced. These data suggest that regulation of gene expression, protein or enzyme activity by IL-22 may have variable effects: Cyp3a11 is ATP-independent, whereas MRP2 and MDR1 are ATP-dependent. This direct or indirect enzymatic modulation of these proteins must be taken into account when assessing the role of the drug in the biology of the intestinal mucosa. Dr. Lloyd Sutherland Chair in IBD/GI Research/Weston Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.


A56 EVALUATING THE DYNAMICS OF CYP3A11 AND REG3G MODULATION BY IL-22

February 2024

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6 Reads

Journal of the Canadian Association of Gastroenterology

Background IL-22 is a cytokine produced by a number of immune cell populations, including type 3 innate lymphoid cells. We have shown previously that IL-22 suppresses the expression Cyp3a11 in the mouse intestine, a gene that encodes a drug metabolizing enzyme that accounts for roughly 70% of total drug metabolism. In addition, IL-22 induces the expression of Reg3g, the gene that encodes an antimicrobial C-type lectin that has bactericidal activity against Gram+ bacteria in the small intestine. Modulation of these proteins can influence both the oral bioavailability of drugs and microbial composition. Aims The aim of this study was to define the existence of reversible modulation of IL-22 on Cyp3a11 and Reg3g expression in the intestinal epithelium. Methods In vitro enteroids from each part of the mouse small intestine were cultured in 3D in the presence or absence of IL-22 for 5 days. Cyp3a11 and Reg3g transcript expression was evaluated by qPCR followed by protein analysis to define their modulation at varying timepoints after IL-22 treatment cessation. Results IL-22 reduced Cyp3a11 expression, an effect that normalized the day after treatment cessation. However, 4 days after treatment cessation there was rebound hyperexpression of Cyp3a11 in duodenal enteroids, which normalized the following day. IL-22 reduced the expression and activity of Cyp3a11 which normalized soon after stopping IL-22 treatment. In contrast, IL-22 induced the expression of Reg3g, an effect that lasted up to 5 days after treatment cessation. The effect of IL-22 on Cyp3a11 expression and activity was rapidly reversed in contrast to the effect on Reg3g which was more prolonged. Conclusions IL-22 suppresses the expression of Cyp3a11 and induces the expression of Reg3g. The reversible nature of these effects is different between both, with the increased expression of Reg3g 5 days after IL-22 treatment cessation. These data suggest that the regulation of gene expression, protein or enzyme activity by IL-22 may exhibit varying temporal effects and should be considered when assessing its role in intestinal mucosal biology. Funding Agencies This work was funded by the Dr. Lloyd Sutherland Chair in IBD/GI Research and the Weston Foundation.


A62 EXAMINING THE ACTIVATION OF XENOBIOTIC RECEPTORS PXR AND AHR IN COLONOIDS USING MICROBIAL METABOLITES AND CHEMICAL LIGANDS

February 2024

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3 Reads

Journal of the Canadian Association of Gastroenterology

Background The Aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) are vital xenobiotic receptors activated by foreign substances, playing pivotal roles in regulating chemical metabolism. Traditionally, these receptors have been closely linked to their roles in mediating responses to toxic compounds. However, recent findings have revealed their newfound significance in maintaining gut homeostasis and regulating inflammatory processes. Aims We investigated the dual roles of AhR and PXR activation. We started with dosage response experiments to determine optimal treatment conditions, such as treatment concentration and exposure time. Subsequently, we compared transcriptomic responses induced by chemical ligands with those from microbial metabolites, aiming to understand how AhR and PXR activation can yield both adverse and beneficial effects. Methods Using mouse 3D colonoids and 2D monolayer cultures, PXR and AhR were activated using the microbial metabolites indole-3-propionic acid (IPA) and indole-3-pyruvic acid (IPyA), respectively. Concentrations were determined through dosage response experiments. Changes to PXR and AhR target gene expression were measured using qPCR. We compared the gene induction by IPA and IPyA to responses driven by the chemical ligands pregnenolone 16α-carbonitrile (PCN) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively. Results In 3D cell culture, PCN induced PXR target genes Cyp2c55 and Abcb1a, while IPA had no significant effect. Cyp3a11 was absent. Both IPyA and TCDD induced AhR genes, Cyp1a1 and Cyp1b1, with varying induction, and Cyp1a2 showed no response. In 2D cell culture, PCN significantly increased Cyp2c55 gene expression, but neither PCN nor IPA significantly induced Abcb1a. In 3D culture, TCDD significantly induced Cyp1a1 and Cyp1b1, while IPyA only induced Cyp1a1, revealing differences in AhR gene induction between 2D and 3D cell culture models. Conclusions Our research reveals contrasting gene induction patterns following AhR and PXR activation through microbial metabolites and chemical ligands. These varied responses highlight the multifaceted roles these receptors play within the gut environment. This study enhances our understanding of xenobiotic receptors' contributions to maintaining gut homeostasis and regulating inflammatory processes, shedding light on the interplay between ligands and receptors. Methods First, we isolate the crypts of mice to create 3D organoids, were we have access to the basolateral side. To capture the true biological system, we convert the 3D culture to 2D. This enables us to explore both the apical and basolateral sides, retaining the crucial polarity of the cells. Funding Agencies CIHR


A38 INVESTIGATION OF POST-TRANSLATIONAL MODIFICATIONS IN SERUM OF CROHN’S DISEASE PATIENTS USING A PROTEOMICS APPROACH

March 2023

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15 Reads

Journal of the Canadian Association of Gastroenterology

Background Canada has one of the highest prevalences of Crohn’s disease (CD) worldwide. More specifically, fibrostenotic CD is a phenotype with prolonged chronic inflammation and fibrotic strictures often resistant to anti-inflammatory therapies and characterized by luminal narrowing that ultimately requires surgery. Proteins play an essential role in disease pathogenesis, and post-translational modifications (PTMs) can alter their properties. PTMs have been frequently implicated in human diseases. However, they have yet to be explored in the context of CD, which could lead to new avenues for a better understanding of disease mechanisms and the discovery of biomarkers. Purpose Identify post-translational modifications in serum proteins of CD patients. Method Serum samples from patients with strictures or inflammatory phenotype (without strictures) (n=4 per group), as diagnosed by intestinal ultrasound, were analyzed using a shotgun-proteomics approach. Protein identification and PTM prediction were performed with FragPipe. Identified mass shifts determined by an open search in FragPipe were mapped to possible PTMs and confirmed via unimod.org. Statistical significance analysis was performed with MSstatsPTM. Result(s) The prediction analysis identified 363 potential modification sites, including artifacts and chemical derivatives. The addition of all potential PTMs in the analysis would lead to false positives; therefore, it was selected five of the most abundant mass shifts mapped to true PTMs: cysteine oxidation, serine methylation, and three modifications of the protein n-termini (formaldehyde adduct, carbamylation, and formylation). Standard proteomics analysis identified 3635 unique peptides and 317 unique proteins. The addition of the predicted PTMs increased the number of peptides by 9.8%, reaching 3994 unique sequences. Interestingly, a very subtle increase was observed on the protein level, where only two additional proteins were identified. Of the PTMs identified, methylation of a serine residue on the variable chain of immunoglobulin (IGLV1-47) was statistically enriched in inflammatory samples (5.74 fold change, adj. p-value = 0.041). The variable chain participates in the antigen recognition process, and modification of its amino acids could impact antibody specificity. Additionally, structuring patients showed two modifications on thrombin: oxidation of cysteine and methylation of serine. Thrombin was previously shown to be elevated in CD patients compared to healthy controls. As both modifications were not present in inflammatory patients, they constitute potential biomarkers for specific diagnosis of the structuring disease. Conclusion(s) The observed gain in peptide identification demonstrates the diversification promoted by PTMs and exhibits their importance in proteomics studies. Even though the identified modifications require further validation, they can shed light on new players of CD pathogenesis and suggest novel biomarkers for disease diagnosis. Disclosure of Interest None Declared


Citations (8)


... This increase in goblet cell populations was accompanied by a consistent upregulation of Muc2 expression, as shown by immunohistochemical staining (Fig. 3G) and increased Muc2 protein levels in colonic tissue detected by ELISA (Fig. 3H). Muc2 staining reflects the functional status of goblet cells and the integrity of the mucus layer, with increased expression indicating enhanced goblet cell activity and improved intestinal barrier function [40]. Collectively, our findings underscore the protective effect of WKB on intestinal barrier function in DSS-induced colitis. ...

Reference:

WKB ameliorates DSS-induced colitis through inhibiting enteric glial cells activation and altering the intestinal microbiota
Inflammasome activation links enteric Salmonella Typhimurium infection to a rapid, cytokine-dependent increase in intestinal mucin release

... Their etiology and pathogenesis are not well established, but it is speculated to be multifactorial and involves genetic, environmental, immune and microbial factors. The microbiota, which includes a constellation of archaea, bacteria, fungi, viruses, protists and helminths, was associated with IBD susceptibility, clinical aspects, disease activity or remission, and response to treatment [2]. ...

Challenges in IBD Research 2024: Preclinical Human IBD Mechanisms

Inflammatory Bowel Diseases

... Shotgun proteomics were carried out as described previously by [30][31][32] and was performed on the previously described flash-frozen VL and soleus muscle tissue that was stored at -80 °C after sacrifice of the animals. Muscle samples were added to a lysing tube with 500 uL of lysis buffer (2% SDS, 200 mM ammonium bicarbonate, protease inhibitor tablets) and 4-5 small stainless steel homogenizing beads. ...

NR4A1 modulates intestinal smooth muscle cell phenotype and dampens inflammation‐associated intestinal remodeling

... 3,4 However, the results of sex differences were not clear when an animal model of experimental colitis was used. In these models, for example, Painsipp et al. 13 found that the female mice suffer more depression-like behaviors and the males more anxiety, other authors, even though using female and male rodents in their studies, did not make a sex differentiation on behavior, 21,22 and others did not find differences between sexes. 9,16,18 Regarding the motor behaviors, there are scarcely any reports examining whether, in the colitis models, the sex affects these behaviors. ...

Recruitment of α4β7 monocytes and neutrophils to the brain in experimental colitis is associated with elevated cytokines and anxiety-like behavior

Journal of Neuroinflammation

... For instance, Blautia is reduced in gut microbiotas of Crohn's disease and colorectal cancer patients (3,4), and work from our group showed supplementation of this Blautia isolate can reduce colitis severity and colitis-associated sociability deficits in mice (5). However, other studies correlate Blautia abundance with irritable bowel syndrome and ulcerative colitis (6)(7)(8). As species-level distinctions were not made in these analyses, further investigation into this genus is warranted to understand its impact on human health. ...

Colitis-associated microbiota drives changes in behaviour in male mice in the absence of inflammation
  • Citing Article
  • March 2022

Brain Behavior and Immunity

... Breastfeeding is crucial for promoting a healthy gut microbiota in infants. Human milk contains beneficial components such as bioactive compounds and human milk oligosaccharides (HMOs), which selectively foster the growth of beneficial bacteria like Bifidobacteria (222)(223)(224)(225)(226)(227)(228)(229)(230)(231)(232)(233)(234)(235)(236). For CS-born infants, breastfeeding can partially mitigate the microbial disturbances caused by the lack of maternal vaginal microbiota exposure. ...

Xenobiotic receptors and the regulation of intestinal homeostasis - harnessing the chemical output of the intestinal microbiota
  • Citing Article
  • December 2021

AJP Gastrointestinal and Liver Physiology

... The gut microbiota also has a direct impact on host metabolism, as gut microorganisms are capable of breaking down complex food compounds that cannot be digested by human enzymes, such as certain types of fiber and non-absorbable carbohydrates [42]. This breakdown results in the production of key metabolites, such as SCFAs, which are products of bacterial fermentation [43]. SCFAs, including acetate, propionate, and butyrate, are absorbed by intestinal epithelial cells and utilized as a source of energy, thereby contributing to intestinal health and the prevention of inflammation [44,45]. ...

Intestinal microbiota shapes gut physiology and regulates enteric neurons and glia

... To a lesser extent, altered locomotion was observed in the animals with a depleted microbiome, including germ-free (GF) animals or animals treated with an antibiotic cocktail (ABX). However, a consistent outcome regarding the influence of microbial depletion on locomotion has not been observed in the field, possibly due to the differences in animal models, strains, ages, and sexes [13][14][15][16][17][18][19][20][21][22] . Therefore, whether and how the gut microbiota impacts host locomotion is still unclear. ...

Behavioural adaptations after antibiotic treatment in male mice are reversed by activation of the aryl hydrocarbon receptor
  • Citing Article
  • August 2021

Brain Behavior and Immunity