Effects of Limosilactobacillus reuteri strains PTA-126787 and PTA-126788 on intestinal barrier integrity and immune homeostasis in an alcohol-induced leaky gut model

Abstract

Intestinal barrier is a first line of defense that prevents entry of various harmful substances from the lumen into the systemic environment. Impaired barrier function with consequent translocation of harmful substances into systemic circulation ("leaky gut") is a central theme in many gastrointestinal, autoimmune, mental, and metabolic diseases. Probiotics have emerged as a promising strategy to maintain intestinal integrity and address "leaky gut". Using in silico, in vitro and avian in vivo analyses, we previously showed that two novel L. reuteri strains, PTA-126787 (L. reuteri 3630) and PTA-126788 (L. reuteri 3632), isolated from broiler chickens possess favorable safety profiles. Consistent with a recent study, here we show that L. reuteri 3630 and 3632 are phylogenetically similar to human L. reuteri strains. Daily administration of high doses of L. reuteri 3630 and 3632 to Sprague Dawley rats for 28 days was found to be safe with no adverse effects. More importantly, administration of L. reuteri 3630 and 3632 significantly reduced markers associated with alcohol-induced leaky gut, by downregulating inflammatory cytokines and upregulating anti-inflammatory cytokines in an alcohol model of leaky gut in mice. While L. reuteri 3630 cells and supernatant showed no activation, L. reuteri 3632 cells but not supernatant showed activation of AhR, a key transcription factor that regulates gut and immune homeostasis. L. reuteri 3630 is creamish white in morphology typical of Lactobacillus species and L. reuteri 3632 displays a unique orange pigmentation, which was stable even after passaging for 480 generations. We identified a rare polyketide biosynthetic gene cluster in L. reuteri 3632 that likely encodes for the orange-pigmented secondary metabolite. Similar to L. reuteri 3632 cells, the purified orange metabolite activated AhR. All together, these data provide evidence on the phylogenetic relatedness, safety, efficacy, and one of the likely mechanisms of action of L. reuteri 3630 and 3632 for potential probiotic applications to address "leaky gut" and associated pathologies in humans.

Fig. 1

Maximum likelihood tree of L. reuteri strains based on the concatenation of 81 core bacterial genes using UBCG2. The ten lineages are as described in Li et al. based on the population structure analysis using BAPS. Label colors refer to different vertebrate hosts, green for rodent, red for porcine, blue for human, pink for non-human primates, orange for birds, olive for herbivore, and black for L. reuteri strains 3630 (PTA-126787) and 3632 (PTA-126788). The two L. reuteri strains SD2112 and RC-14, which are part of the commercial probiotic products are highlighted with a star.

Fig. 2

Protective effect of L. reuteri 3632 and 3630 (1:1) on leaky gut and proinflammatory cytokine expression in a chronic-binge-on alcohol model of leaky gut in mice. A & B. Effect of L. reuteri administration for 10 days on leaky gut as measured using FITC labelled dextran (A) and iFABP (B). C-J. Effect of L. reuteri administration for 10 days on the expression of inflammatory (C–H) and anti-inflammatory (I,J) cytokines (fold change) in the intestine. Cytokine levels were measured from the intestine using qPCR. The results represent the fold change in expression of the target gene compared to β actin from 8–12 mice. PF, pair fed; AF, alcohol fed; AFBP, alcohol fed mice treated with L. reuteri 3632 and 3630 in 1:1 ratio. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Fig. 3

AhR activity of L. reuteri 3632 and 3630 whole cells, cell-free supernatants and purified PKS metabolite from L. reuteri 3632. AhR activity assay was performed using HepG2-Lucia™ AhR reporter cells (Invivogen) following manufacturer’s instructions. The AhR agonist 6-Formylindolo[3,2-b]carbazole (FICZ, 1μg final concentration) was used as a positive control. The test articles included L. reuteri 3632 and 3630 whole cells and cell-free supernatants and purified PKS metabolite [50μg in phosphate buffered saline (PBS)]. For preparing whole cells, overnight grown L. reuteri 3632 and 3630 were centrifuged at 10,000 rpm for 10 min at 4 °C, the supernatants were filter sterilized with 0.22μM filter, the whole cells were resuspended in 10 ml of PBS. The media control, whole cells and cell-free supernatant samples were further diluted seven-fold in PBS and twenty microliters of the sample was mixed with 180μl of cells containing 1.5 × 10⁵ cells/ml in a flat-bottom 96-well plate and incubated for 36 h in a CO₂ incubator. Following 36 h of incubation, 20μl of the sample was mixed with 50μl of Quanti-Luc™ and luciferase activity was measured immediately. The RLUs for whole cells, cell-free supernatants and purified PKS metabolite were normalized to either MRS media or PBS. The data represents mean ± standard deviation of 3 independent experiments. ***P < 0.001.

Fig. 4

Identification and characterization of the BGC encoding an orange pigmented PKS metabolite from L. reuteri 3632. A. L. reuteri 3630 (creamish white) and 3632 (orange pigmentation) cell pellets. B. L. reuteri 3632 plasmid map showing the location of the PKS locus (shown in blue). C. L. reuteri 3632 PKS locus showing the organization of 15 genes from pksA to pksO. D. Putative structure of the orange pigmented PKS metabolite as identified by NMR and HRMS. E. Proposed biosynthetic pathway to produce PKS metabolite in L. reuteri 3632.