Indole-3-lactic acid associated with Bifidobacterium-dominated microbiota significantly decreases inflammation in intestinal epithelial cells

Abstract

Background: Bifidobacterium longum subsp. infantis (B. infantis) is a commensal bacterium that colonizes the gastrointestinal tract of breast-fed infants. B. infantis can efficiently utilize the abundant supply of oligosaccharides found in human milk (HMO) to help establish residence. We hypothesized that metabolites from B. infantis grown on HMO produce a beneficial effect on the host.

Results: In a previous study, we demonstrated that B. infantis routinely dominated the fecal microbiota of a breast fed Bangladeshi infant cohort (1). Characterization of the fecal metabolome of binned samples representing high and low B. infantis populations from this cohort revealed higher amounts of the tryptophan metabolite indole-3-lactic acid (ILA) in feces with high levels of B. infantis. Further in vitro analysis confirmed that B. infantis produced significantly greater quantities of the ILA when grown on HMO versus lactose, suggesting a growth substrate relationship to ILA production. The direct effects of ILA were assessed in a macrophage cell line and intestinal epithelial cell lines. ILA (1-10 mM) significantly attenuated lipopolysaccharide (LPS)-induced activation of NF-kB in macrophages. ILA significantly attenuated TNF-α- and LPS-induced increase in the pro-inflammatory cytokine IL-8 in intestinal epithelial cells. ILA increased mRNA expression of the aryl hydrogen receptor (AhR)-target gene CYP1A1 and nuclear factor erythroid 2-related factor 2 (Nrf2)-targeted genes glutathione reductase 2 (GPX2), superoxide dismutase 2 (SOD2), and NAD(P) H dehydrogenase (NQO1). Pretreatment with either the AhR antagonist or Nrf-2 antagonist inhibited the response of ILA on downstream effectors.

Conclusions: These findings suggest that ILA, a predominant metabolite from B. infantis grown on HMO and elevated in infant stool high in B. infantis, and protects gut epithelial cells in culture via activation of the AhR and Nrf2 pathway.

Keywords: Aryl-hydrocarbon receptor; Indole-3-lactic acid; Milk oligosaccharides; Nuclear factor erythroid 2–related factor 2.

Fig. 1

Increased ILA production in fecal samples from infants with high levels of bifidobacteria. Determination of microbiota in infant fecal samples using 16S, Bif-TRFLP, and BLIR assays. a Fecal samples showed low and high distributions of Bifidobacterium among breast fed infants (n = 18) previously characterized (1). In fecal samples with a high level of Bifidobacterium, the distribution of species and subspecies is shown in different shades of green (note B. longum subsp. infantis dominating all of the high Bifidobacterium feces). b PCoA plots of weighted and c unweighted β diversity was significantly different between high and low Bifidobacterium groups. d Metabolite analyses of infant fecal samples by LC-MS/MS (72). e High Bifidobacterium samples showed highly significant differences in ILA production (p < 0.0001) compared to low Bifidobacterium samples. Student t-test was used to determine significance with corresponding P values considered statistically significant in *p < 0.05, **p < 0.01, *** p = 0.0001

Fig. 2

ILA inhibits LPS-induced inflammation in macrophage and intestinal epithelial cells. a SEAP activity in RAW blue macrophage cells exposed to media alone, LPS alone, or with different concentrations of ILA for 1 h prior to addition of LPS. b IL-8 from IECs exposed to media alone, LPS alone for 18 h or with different concentrations of ILA for 1 h prior to LP. Data is expressed as mean ± SEM, n = 3–7 and Student t-test was used to determine significance with corresponding P values considered statistically significant; *** p < 0.0001 compared to no treatment control or ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.0001 compared to LPS alone

Fig. 3

ILA inhibits TNF-α induced inflammation in intestinal epithelial cells. mRNA expression in IEC exposed to media alone, TNF-α or TNF-α in the presence of different concentrations of ILA for 1 h. a IL-8, b beta-defensin2, c SERT and d TPH1 mRNA expression. Data is expressed as mean ± SEM, n = 6 for each condition and Student t-test was used to determine significance with corresponding P values considered statistically significant (*p < 0.05, **p < 0.01, *** p < 0.0001)

Fig. 4

Cell density or presence of ILA affects AhR localization. a AhR cytosolic protein expression in IEC plated at high density (100% confluency) versus low confluency (50% confluency). b AhR nuclear protein expression in high and low density IECs (p = ns). c AhR cytosolic protein expression in high density IEC treated with ILA. d AhR nuclear protein expression in high density IEC treated with ILA. Protein was pooled from 6 individual plates of cells. Data is expressed as mean ± SEM; n = 2 and student t-test was used to determine significance with corresponding P values considered statistically significant in *p < 0.05, **p < 0.01, *** p < 0.0001

Fig. 5

AhR or Nrf2 inhibition prevents ILA-induced response in intestinal epithelial cells. mRNA expression in IEC treated with ILA alone or with AhR or Nrf2 antagonist. a CYP1A1 b GPX2 c NQO1 d SOD2 e IL-8 mRNA. Data is expressed as mean ± SEM with n = 6–8 of relative expression values fitted with a 3-paramenter dose response curve vs the log-transformed ILA concentration (Prism, v. 8.0, GraphPad Software, San Diego, CA). Statistical significance of the effect of each treatment on the means of relative expression of each target gene at the highest ILA concentration (10 mM) was determined using a one-way ANOVA analysis with a TukeyHSD posthoc comparison (R, version 3.5.2 (1)). For IL-8, the statistical differences between the means in the absence of ILA was as also determined by this method. Different letters represent significantly different means. A summary of statistics can be found in Supplemental Table 3

Fig. 6

Proposed model of ILA activation. ILA activates AhR which increases expression of AhR regulated gene CYP1A1 as well as increasing activity of Nrf2. Activated Nrf2 leads to increased expression of cytoprotective genes to reduce oxidative cellular stress brought about by inflammatory agents such as LPS or TNF-α