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. 2024 Apr 10;15(4):e0029824.
doi: 10.1128/mbio.00298-24. Epub 2024 Mar 5.

A human milk oligosaccharide prevents intestinal inflammation in adulthood via modulating gut microbial metabolism

Kasey M Schalich  1 , Matthew A Buendia  1 , Harpreet Kaur  1 , Yash A Choksi  2 , M Kay Washington  3 , Gabriela S Codreanu  4   5 , Stacy D Sherrod  4   5 , John A McLean  4   5 , Richard M Peek Jr  2 , Sari A Acra  1 , Steven D Townsend  4 , Fang Yan  1   2   6
Affiliations

A human milk oligosaccharide prevents intestinal inflammation in adulthood via modulating gut microbial metabolism

Kasey M Schalich et al. mBio. .

Abstract

Observational evidence suggests that human milk oligosaccharides (HMOs) promote the growth of commensal bacteria in early life and adulthood. However, the mechanisms by which HMOs benefit health through modulation of gut microbial homeostasis remain largely unknown. 2'-fucosyllactose (2'-FL) is the most abundant oligosaccharide in human milk and contributes to the essential health benefits associated with human milk consumption. Here, we investigated how 2'-FL prevents colitis in adulthood through its effects on the gut microbial community. We found that the gut microbiota from adult mice that consumed 2'-FL exhibited an increase in abundance of several health-associated genera, including Bifidobacterium and Lactobacillus. The 2'-FL-modulated gut microbial community exerted preventive effects on colitis in adult mice. By using Bifidobacterium infantis as a 2'-FL-consuming bacterial model, exploratory metabolomics revealed novel 2'-FL-enriched secretory metabolites by Bifidobacterium infantis, including pantothenol. Importantly, pantothenate significantly protected the intestinal barrier against oxidative stress and mitigated colitis in adult mice. Furthermore, microbial metabolic pathway analysis identified 26 dysregulated metabolic pathways in fecal microbiota from patients with ulcerative colitis, which were significantly regulated by 2'-FL treatment in adult mice, indicating that 2'-FL has the potential to rectify dysregulated microbial metabolism in colitis. These findings support the contribution of the 2'-FL-shaped gut microbial community and bacterial metabolite production to the protection of intestinal integrity and prevention of intestinal inflammation in adulthood.IMPORTANCEAt present, neither basic research nor clinical studies have revealed the exact biological functions or mechanisms of action of individual oligosaccharides during development or in adulthood. Thus, it remains largely unknown whether human milk oligosaccharides could serve as effective therapeutics for gastrointestinal-related diseases. Results from the present study uncover 2'-FL-driven alterations in bacterial metabolism and identify novel B. infantis-secreted metabolites following the consumption of 2'-FL, including pantothenol. This work further demonstrates a previously unrecognized role of pantothenate in significantly protecting the intestinal barrier against oxidative stress and mitigating colitis in adult mice. Remarkably, 2'-FL-enhanced bacterial metabolic pathways are found to be dysregulated in the fecal microbiota of ulcerative colitis patients. These novel metabolic pathways underlying the bioactivities of 2'-FL may lay a foundation for applying individual oligosaccharides for prophylactic intervention for diseases associated with impaired intestinal homeostasis.

Keywords: 2′-fucosyllactose; Bifidobacterium longum subsp. infantis; human milk oligosaccharide; inflammatory bowel diseases; intestinal barrier; pantothenate; the gut microbiota.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
2′-FL modifies the gut microbial composition in adult mice. Adult mice received 2′-FL (1 mg/mL) in drinking water for 7 (7D, n = 5) or 28 (28D, n = 7) days. Mice receiving regular drinking water only were used as the control (Con., n = 5). The fecal bacteria were analyzed by WGS sequencing. (A) Relative abundance (R.A.%) taxa plot depicting the relative abundance of the top 15 families of 2′-FL consumption. (B) The weighted unifrac PCoA coordinates for the three groups. (C) 2′-FL-induced alterations in the R.A.% of the Bifidobacteriaceae, Lactobacillaceae, Eggerthellaceae, Oscillospiraceae, Desulfovibrionaceae, and Rikenellaceae families compared to the control group. (D) Heatmap illustrating the relative abundance Z score for all 42 identified genera across the control, 7D 2′-FL, and 28D 2′-FL groups. (E) Species that were significantly (P < 0.05) upregulated (Lactobacillus, Bifidobacterium, and Enterorhabdus) or downregulated (Anaerotruncus) in the 28D 2′-FL group compared to the control group within the four significantly altered genera were identified. For panel C, different letters between groups (a, b, or c) indicate that the two groups are significantly different; any common letters between groups indicate that they are not significantly different.
Fig 2
Fig 2
2′-FL-modulated gut microbial community plays a role in ameliorating colitis in adult mice. (A) Study design. Donors include mice with (2′-FL donor) or without (control donor) 2′-FL treatment. Feces were collected for three consecutive days from donor groups and pooled within groups for FMT to recipient mice after recipient mice received broad-spectrum antibiotic treatment and a subsequent 48-hour washout period. FMT was administered for three consecutive days, after which mice received no treatment for three consecutive days before treatment with DSS or water as a control. (B) Representative hematoxylin and eosin staining of colonic sections. (C) Colon injury and inflammation scores. (D) Quantification of TNF mRNA expression by RT-PCR. The average mRNA expression levels in the antibiotics + water group were set as 100%, and the mRNA expression level of each mouse was compared with the average. (E) Colonic sections were immunostained with an anti-ZO-1 antibody and a FITC-labeled secondary antibody (green). Nuclei were stained with DAPI (blue). Membrane (white arrows) and intracellular (yellow arrows) ZO-1 distributions are shown.
Fig 3
Fig 3
Bifidobacterium infantis growth and metabolism of 2′-fucosyllactose in vitro. (A and B) B. infantis strains ATCC 15702 (A) and ATCC 15697 (B) were grown in RPMI 1640 medium (no glucose) with and without supplementation with 2′-FL at indicated concentrations and OD600 was recorded. (C) Kinetics of ATCC 15702 growth in the presence of H2O2 with and without supplementation with 2′-FL at indicated concentrations. (D) B. infantis strain ATCC 15702 was cultured in reinforced clostridial medium with and without 2′-FL at 10 mg/mL for 7 hours (mid-logarithmic growth phase). Metabolites in the medium only (medium control, MC), medium with 2′-FL (MF), supernatant from B. infantis culture in MC (C), and supernatant from B. infantis in MF (F) were identified by metabolomics. Metabolite names in red are enriched (secreted), while those in blue are depleted (consumed). Levels 1, 2, and 3 metabolites (green) were identified with high confidence, while levels 4 and 5 were not and thus were not considered for further analysis. (E) Fold change for significant (P < 0.05) F vs C supernatant [secreted (red) and consumed (blue)] levels 1, 2, and 3 metabolites. (F) Normalized peak intensity values for the three secreted metabolites identified in panel E between 2′-FL and control supernatants, depicting actual abundance. (G) Abundance (CPM, counts per million) of the pantothenate biosynthetic process pathway for Bifidobacterium pseudolongum identified in mice feces. One data point represents one mouse (N.D., not detected). *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control within the same time point. For panels A–C, asterisks are color coded to represent the condition that is significant, compared to the control (black). At least three independent bacterial culture experiments were performed.
Fig 4
Fig 4
Pantothenate reduces colitis and preserves intestinal epithelial barrier in mice. Adult mice received either water (control) or pantothenate (Panto., 1 mM) in drinking water for 14 days prior to induction of colitis by DSS or TNBS. (A and B) Representative hematoxylin and eosin (H&E) staining of colon sections and colon injury and inflammation scores for mice with DSS treatment. (C and D) Representative H&E staining of colon sections and inflammation scores for mice with TNBS treatment. (E) Quantification of plasma FITC-Dextran in mice in the DSS experimental model. (F) Colonic sections from the DSS models were immunostained with an anti-ZO-1 antibody and a FITC-labeled secondary antibody (green).
Fig 5
Fig 5
Pantothenate preserves the intestinal epithelial barrier and inhibits oxidative stress in intestinal epithelial cells. (A) Caco2 cells were pretreated with 20 or 40 µg/mL of D-calcium pantothenate for 2 hours before treatment with 50 µM of H2O2 for indicated times. Transepithelial electrical resistance was recorded. (B) YAMC cells were treated with H2O2 (20 µM) with and without co-treatment with pantothenate at 0.1 mM for 4 hours. Cells were fixed for immunostaining using an anti-ZO-1 antibody and a FITC-labeled secondary antibody (green). Nuclei were stained with DAPI (blue). White arrows represent the disruption of the tight junction. (C) YAMC cells were treated with pantothenate at 0.025 and 0.1 mM for 6 hours. Cellular lysates were collected for the GPX activity measured as nanomoles of NADPH oxidized/minute. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to 50 µM H2O2 within the same indicated time point. For panel A, asterisks are color coded to represent the condition that is significant compared to H2O2 (blue). At least three independent bacterial culture experiments were performed.
Fig 6
Fig 6
Unique and significantly enriched gut microbial metabolic pathways by 2′-FL consumption in adult mice. Adult mice received 2′-FL treatment as described in Fig. 1. (A) Microbial metabolic pathways identified by the HUMAnN3 program were compared between the control, 2′-FL 7D, and 2′-FL 28D groups. A total of 34 pathways were identified as uniquely detectable after 2′-FL consumption, but not in any control mice, including four pathways only after 7 days 2′-FL, 9 pathways after 7 or 28 days 2′-FL, and 21 pathways after 28 days 2′-FL. (B) Metabolic pathway abundance [bar blots, in reads per kilobase (RPK)] normalized by mapped reads per individual animal and the names of their respective pathway byproducts and products are illustrated for these 34 pathways. Pathways are grouped according to one of the three experimental group(s) they were identified in (from panel A) and are further organized into super pathway functions. (C) Of the 233 common pathways between all three study groups, 99 were significantly different in abundance (P < 0.05) in the 2′-FL 28D group (98 pathways) or the 2′-FL 7D and 28D groups (one pathway) compared to the control group. The 99 significantly enriched pathways are displayed in a hierarchically clustered heatmap to illustrate the changes in enrichment. (D) Functional categorization of the 99 significantly different pathways into the top 10 biological functions illustrated in a circus plot.
Fig 7
Fig 7
Microbial metabolic pathways that are altered in patients with UC are regulated by 2′-FL consumption in adult mice. (A) Table of characteristics for UC patients. Human stool samples were analyzed by WGS and the HUMAnN3 program. (B) Volcano plot comparing the significance of the coefficient of correlation (effect size) of the 372 microbial metabolic pathways between non-IBD and UC patients, including 33 pathways that were significantly positively correlated/enriched (red, correlation coefficient > 2 and FDR < 0.05) with ulcerative colitis, and 31 pathways that were significantly negatively correlated/depleted (blue, correlation coefficient < −2 and FDR < 0.05) with ulcerative colitis. (C) Comparison of significantly altered microbial metabolic pathways in UC patients (FDR < 0.05) and after 2′-FL consumption in adult mice (FDR < 0.05) identified 26 common differentially abundant pathways. (D) Plotting the coefficient of correlation for the 26 common ulcerative colitis and 2′FL-regulated pathways identified nine pathways depleted in UC but enriched by 2′FL (red), three pathways depleted by UC and only detected after 2′-FL consumption (green), four pathways enriched by UC and only detected after 2′-FL consumption (blue), and 10 pathways enriched by UC and 2′-FL (black). (E) Direct comparison of the coefficient of correlation (effect size) for the nine pathways enriched after 2′-FL 28D consumption in mice that are also depleted in UC patient fecal microbes. 2′-FL was compared to the control, and UC patients were compared to non-IBD patients. (F) Circos plot categorizing common super pathway functions for the four groups of pathways identified in panel D.
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