Novel Bacteroides Vulgatus strain protects against gluten-induced break of human celiac gut epithelial homeostasis: a pre-clinical proof-of-concept study

Abstract

Background and aims: We have identified a decreased abundance of microbial species known to have a potential anti-inflammatory, protective effect in subjects that developed Celiac Disease (CeD) compared to those who did not. We aim to confirm the potential protective role of one of these species, namely Bacteroides vulgatus, and to mechanistically establish the effect of bacterial bioproducts on gluten-dependent changes on human gut epithelial functions.

Methods: We identified, isolated, cultivated, and sequenced a unique novel strain (20220303-A2) of B. vulgatus found only in control subjects. Using a human gut organoid system developed from pre-celiac patients, we monitored epithelial phenotype and innate immune cytokines at baseline, after exposure to gliadin, or gliadin plus B. vulgatus cell free supernatant (CFS).

Results: Following gliadin exposure, we observed increases in epithelial cell death, epithelial monolayer permeability, and secretion of pro-inflammatory cytokines. These effects were mitigated upon exposure to B. vulgatus 20220303-A2 CFS, which had matched phenotype gene product mutations. These protective effects were mediated by epigenetic reprogramming of the organoids treated with B. vulgatus CFS.

Conclusions: We identified a unique strain of B. vulgatus that may exert a beneficial role by protecting CeD epithelium against a gluten-induced break of epithelial tolerance through miRNA reprogramming.

Impact: Gut dysbiosis precedes the onset of celiac disease in genetically at-risk infants. This dysbiosis is characterized by the loss of protective bacterial strains in those children who will go on to develop celiac disease. The paper reports the mechanism by which one of these protective strains, B. vulgatus, ameliorates the gluten-induced break of gut epithelial homeostasis by epigenetically re-programming the target intestinal epithelium involving pathways controlling permeability, immune response, and cell turnover.

Fig. 1. Fold change (FC) of signature genes expressed in organoids of potential CeD (PCeD) compared to healthy controls (HC, N = 2), acute (ACD, N = 2) and remission (GFD, N = 2)).

Markers of acute celiac activation related to goblet cells signature, chemokines and barrier [SDS1] were found already altered in both PCeDs. Triplicate for each organoid. One-way ANOVA. (P values: *<0.05, **<0.01, ***<0.001), ****<0.0001.

Fig. 2. The protective effect of B. Vulgatus-A2 CFS against PTG-induced cell death and increased epithelial paracellular permeability is dose-dependent.

Dose-dependent effect of B. vulgatus-A2 CFS on cell viability (a) and on epithelial paracellular permeability (b) of PCeD organoids (N = 2) exposed to PTG. N of experiments/organoid = 3. One-way ANOVA test (*) P < 0.05, (***)P < 0.001, (****) P < 0.0001. (**) P < 0.01, (***) P < 0.001.

Fig. 3. Cytokine release analysis (N = 2 per treatment) with average bars in pg/ml from two bio replicates, each with technical duplicates.

Error bars are representative of the standard deviation of the averages.

Fig. 4. Effect of B. vulgatus-A2 CFS on miR-152-3p expression in both PCeD and HC organoids.

High-throughput miRNAs analysis was performed by Real-Time PCR using pre-custom plates for the inflammatory response and the autoimmunity focus in PCeD vs. HC organoids at basal condition (a) and after B. vulgatus-A2 CFS treatment (b), and in PCeD organoids treated with B. vulgatus-A2 CFS vs. untreated ones (c) (n = 3 organoids derived monolayers/group). Histograms represent the mean ± SEM. *P < 0.05, ****P < 0.0001.

Fig. 5. Effect of B. vulgatus-A2 CFS on miRNAs expression in both PCeD and HC organoids.

Modulation of miRNAs expression in B. vulgatus-A2 CFS-treated PCeD organoids (white bars) vs. untreated PCeD organoids (black bars) (a). Differential expression of miRNAs in HC (black bars) and PCeD organoids (white bars) after the treatment with B. vulgatus-A2 CFS (b). For both the analyses, a high-throughput miRNAs analysis was performed by Real-Time PCR using pre-custom plates for the inflammatory response and the autoimmunity focus (n = 3 organoids derived monolayers/group). Histograms represent the mean ± SEM. ****P < 0.0001.