Lactobacillus reuteri DSM 17938 feeding of healthy newborn mice regulates immune responses while modulating gut microbiota and boosting beneficial metabolites

Abstract

Early administration of Lactobacillus reuteri DSM 17938 (LR) prevents necrotizing enterocolitis and inhibits regulatory T-cell (Treg)-deficiency-associated autoimmunity in mice. In humans, LR reduces crying time in breastfed infants with colic, modifies severity in infants with acute diarrheal illnesses, and improves pain in children with functional bowel disorders. In healthy breastfed newborns with evolving microbial colonization, it is unclear if early administration of LR can modulate gut microbiota and their metabolites in such a way as to promote homeostasis. We gavaged LR (10⁷ colony-forming units/day, daily) to C57BL/6J mice at age of day 8 for 2 wk. Both male and female mice were investigated in these experiments. We found that feeding LR did not affect clinical phenotype or inflammatory biomarkers in plasma and stool, but LR increased the proportion of Foxp3⁺ regulatory T cells (Tregs) in the intestine. LR also increased bacterial diversity and the relative abundance of p_Firmicutes, f_Lachnospiraceae, f_Ruminococcaceae, and genera Clostridium and Candidatus arthromitus, while decreasing the relative abundance of p_Bacteriodetes, f_Bacteroidaceae, f_Verrucomicrobiaceae, and genera Bacteroides, Ruminococcus, Akkermansia, and Sutterella. Finally, LR exerted a major impact on the plasma metabolome, upregulating amino acid metabolites formed via the urea, tricarboxylic acid, and methionine cycles and increasing tryptophan metabolism. In conclusion, early oral administration of LR to healthy breastfed mice led to microbial and metabolic changes which could be beneficial to general health.NEW & NOTEWORTHY Oral administration of Lactobacillus reuteri DSM 17938 (LR) to healthy breastfed mice promotes intestinal immune tolerance and is linked to proliferation of beneficial gut microbiota. LR upregulates plasma metabolites that are involved in the urea cycle, the TCA cycle, methionine methylation, and the polyamine pathway. Herein, we show that LR given to newborn mice specifically increases levels of tryptophan metabolites and the purine nucleoside adenosine that are known to enhance tolerance to inflammatory stimuli.

Keywords: Bacteroidetes; Firmicutes; biomarker; healthy; intestine; lactobacillus reuteri; metabolites; microbiota; newborn; probiotic.

Graphical abstract

Fig. 1.

Effect of Lactobacillus reuteri (LR) on inflammatory biomarkers in healthy breastfed mice. A: normal histology representatives of the liver, lung, small intestine, and colon of wild-type control (WTC) vs. wild-type LR fed (WTL). B: levels of fecal calprotectin assessed by ELISA. C: levels of fecal chemokine CXCL1/KC assessed by ELISA; w, week. D: plasma levels of IFNγ and IL-4 assessed by ELISA. E: percentage of CD44⁺ T cells of nonregulatory T-cells (Tregs) analyzed by flow cytometry. MLN, mesenteric lymph nodes. F: percentage of Foxp3⁺ T cells (Tregs) in CD4⁺ T-cell populations analyzed by flow cytometry. Both male and female mice were investigated in these experiments. A total of 16 mice (n = 8 in WTC and n = 8 in WTL) were employed for the analysis to compare the groups between WTL and WTC. In addition, for the different ages of WT mice, n = 6 in 2 wk, n = 6 in 3 wk, n = 6 in 10 wk, and n = 7 in 28 wk of age (B and C). P < 0.05 indicates significance, determined by two-tailed, one-way ANOVA corrected for multiple comparisons with Tukey posttests for B and C; nonpaired Student’s t test for 2 group comparisons.

Fig. 2.

Effect of Lactobacillus reuteri (LR) on α diversity and β diversity of gut microbiota. A: Shannon α diversity: wild-type LR fed (WTL) vs. wild-type control (WTC), P = 0.0011. B: evenness of α diversity: WTL vs. WTC, P = 0.0008. C: 2-dimensional principle coordinates analysis (PCoA) plot of clusters of WTC vs. WTL: P1 vs. P2 (left), P1 vs. P3 (middle), and P2 vs. P3 (right). WTC: black dots; WTL: red dots. WTL vs. WTC: P = 0.001. PERMANOVA, permutational multivariate ANOVA.

Fig. 3.

Effect of Lactobacillus reuteri (LR) on the relative abundance of bacterial community in healthy breastfed mice. A: relative abundance of Firmicutes and Bacteroidetes at phylum level. B: heatmap of bacteria for the top 20 genus from random forest importance to compare with wild-type control (WTC) vs. wild-type LR fed (WTL). C: relative abundance of Lactobacilli at genus level.

Fig. 4.

Calculated linear discriminant analysis (LDA) scores to evaluate the up- or downregulation of bacteria by oral feeding Lactobacillus reuteri (LR) to healthy breastfed mice. The right bars are green indicating upregulation by LR; the left bars are red indicating downregulation by LR. Bacterial classification in parenthesis: p, phylum level; c, class level; o, order level; f, family level; and g, genus level.

Fig. 5.

Effect of Lactobacillus reuteri (LR) on plasma metabolomics profile in healthy breastfed mice. A: counts of biochemical (metabolites) up- or downregulated by LR oral feeding. Red indicates upregulation and the green indicates downregulation. B: heatmap of plasma metabolites in each individual mouse classified as groups of wild-type control (WTC) (black) and wild-type LR fed (WTL) (red). C: 2-dimensional principle components analysis (PCA) plot of metabolome clusters of WTC (black dots) and WTL (red dots). Each dot represents 1 sample. PC1 vs. PC2 (top), PC1 vs. PC3 (middle), and PC2 vs. PC3 (bottom); P = 0.001. PERMANOVA, permutational multivariate ANOVA.

Fig. 6.

Heatmap of altered metabolites of amino acid pathway compared the groups of wild-type control (WTC) vs. wild-type Lactobacillus reuteri fed (WTL).

Fig. 7.

The upregulated metabolites in amino acid pathways by Lactobacillus reuteri (LR) in healthy breastfed mice. A: metabolites that are significantly upregulated by LR in urea cycle. B: metabolites that are significantly upregulated by LR in tricarboxylic acid (TCA) cycle. C: metabolites that are significantly upregulated in methionine metabolism cycle. DMTPA, 2,3-dihydroxy-5-methylthio-4-pentenoate; MTA, 5-methylthio-adenosine; SAH, S-adenosyl-homocysteine. Data represent the average fold changes (>1, dotted lines) of wild-type LR fed (WTL) compared with wild-type control (WTC) with P < 0.05 between groups analyzed by Metabolon; n = 8 mice per group.

Fig. 8.

Upregulated tryptophan metabolites in amino acid pathways by Lactobacillus reuteri (LR) in healthy breastfed mice. A: metabolites that are significantly upregulated by LR in urea cycle. Data represent the average fold changes (>1, dotted line) of wild-type LR fed (WTL) compared with wild-type control (WTC) with P < 0.05 between groups analyzed by Metabolon; n = 8 mice per group. B: tryptophan metabolism. Upregulated metabolites by LR are indicated by arrows.

Fig. 9.

Plasma adenosine and inosine detected by metabolomics analysis. The comparisons of 3 groups are as indicated. MRS, deMan-Rogosa-Sharpe. #P < 0.05, wild-type by Lactobacillus reuteri (LR) fed (WTL) vs. wild-type control (WTC). *P < 0.05, **P < 0.01, WTL vs. WT no treatment; n = 8 mice per group.

Fig. 10.

Summarized connections among the amino acid pathway affected by Lactobacillus reuteri (LR)in healthy breastfed mice. Arrows indicate amino acid metabolites upregulated by LR in plasma. Three major circles affected by LR demonstrated as urea cycle, tricarboxylic acid (TCA) cycle, and methionine methylation cycle. Changed metabolites also related to polyamine pathway. SAM, S-adenosylmethionine; MTA, 5-methylthio-adenosine; SAH, S-adenosyl-homocysteine.