A Commensal Bifidobacterium longum Strain Prevents Gluten-Related Immunopathology in Mice through Expression of a Serine Protease Inhibitor

Abstract

Microbiota-modulating strategies, including probiotic administration, have been tested for the treatment of chronic gastrointestinal diseases despite limited information regarding their mechanisms of action. We previously demonstrated that patients with active celiac disease have decreased duodenal expression of elafin, a human serine protease inhibitor, and supplementation of elafin by a recombinant Lactococcus lactis strain prevents gliadin-induced immunopathology in the NOD/DQ8 mouse model of gluten sensitivity. The commensal probiotic strain Bifidobacterium longum NCC2705 produces a serine protease inhibitor (Srp) that exhibits immune-modulating properties. Here, we demonstrate that B. longum NCC2705, but not a srp knockout mutant, attenuates gliadin-induced immunopathology and impacts intestinal microbial composition in NOD/DQ8 mice. Our results highlight the beneficial effects of a serine protease inhibitor produced by commensal B. longum strains.IMPORTANCE Probiotic therapies have been widely used to treat gastrointestinal disorders with variable success and poor mechanistic insight. Delivery of specific anti-inflammatory molecules has been limited to the use of genetically modified organisms, which has raised some public and regulatory concerns. By examining a specific microbial product naturally expressed by a commensal bacterial strain, we provide insight into a mechanistic basis for the use of B. longum NCC2705 to help treat gluten-related disorders.

Keywords: celiac; commensal; gluten; microbiota; probiotic; serpin.

FIG 1

B. longum srp⁺ and L. lactis-elafin are equally effective in preventing gliadin immunopathology in mice. (A) NOD/DQ8 mice were sensitized with cholera toxin and pepsin-trypsin-digested gliadin 1× per week for 3 weeks. Nonsensitized mice (controls) received cholera toxin alone. Subsequently, mice were treated daily with B. longum srp⁺, L. lactis-elafin, or PBS–20% glycerol and simultaneously challenged with gliadin 3× per week for 2 weeks. Control mice received no bacterial treatment. (B) CD3⁺ intraepithelial lymphocytes in small-intestinal villus tips were quantified and expressed as the number of IELs per 100 enterocytes. Mice treated with srp-expressing B. longum srp⁺ had significantly lower numbers of IELs than gliadin-sensitized mice receiving no bacterial treatment. Further, B. longum srp⁺ treatment resulted in numbers of IELs similar to those seen with mice treated with L. lactis expressing elafin. ***, P < 0.001; ****, P < 0.0001. (C) Representative images were captured at ×40 magnification. Data are shown as means ± standard errors of the means (SEM). Statistical significance determinations were performed by ANOVA followed by Bonferroni post hoc analysis. Control, nonsensitized, no treatment; Ll-E, gliadin plus L. lactis expressing elafin; Bl srp⁺, gliadin plus wild-type (WT) B. longum NCC2705; Vehicle, gliadin, no treatment (n = 3 to 6/group).

FIG 2

B. longum constitutively expressing srp inhibits human neutrophil elastase activity in vitro. (A) srp mRNA levels were quantified from various B. longum strains. Expression of srp was higher in B. longum srp(Con) than in B. longum srp⁺ (****, P < 0.0001). No srp mRNA was detected in B. longum Δsrp (n = 4). (B) Inhibitory capacities of various B. longum strains were tested in vitro, as measured by fluorescence produced via cleavage of FITC-elastin substrate at 1.5, 3.125, 6.25, 12.5, 25, and 50 mU/ml, expressed as relative fluorescence units (RFU) (n = 3/group). Compared to B. longum Δsrp, B. longum srp⁺ inhibited cleavage of elastin by human neutrophil elastase (HNE) in vitro at 1.5 mU/ml (P < 0.01), resulting in lower RFU. In the same assay, B. longum srp(Con) further inhibited HNE across all concentrations of HNE compared to B. longum Δsrp, resulting in lower RFU. As well, B. longum Δsrp did not inhibit HNE, as the levels of cleavage of FITC-elastin determined by RFU produced were not significantly different between B. longum Δsrp and buffer alone at any concentration of HNE added. Buffer, HNE alone; srp⁺, B. longum srp⁺; Δsrp, B. longum Δsrp; srp(Con), B. longum srp(Con). ND, not detectable. Data are shown as means ± SEM. Statistical significance determinations were performed using the Kruskal-Wallis test. *, P < 0.05 (versus B. longum Δsrp); **, P < 0.01 (versus B. longum Δsrp).

FIG 3

B. longum srp mediates the protective effect observed in mice. NOD/DQ8 mice were sensitized with cholera toxin and pepsin-trypsin-digested gliadin 1× per week for 3 weeks. Nonsensitized mice (controls) received cholera toxin alone. Subsequently, sensitized mice were treated daily with B. longum srp⁺ (srp⁺), B. longum Δsrp (Δsrp), or B. longum srp(Con) and simultaneously challenged with gliadin 3× per week for 2 weeks. Control mice received PBS–20% glycerol. (A) CD3⁺ intraepithelial lymphocytes in small-intestinal villus tips were quantified and expressed as IELs per 100 enterocytes. Mice treated with srp-expressing B. longum srp⁺ or B. longum srp(Con) had significantly lower numbers of IELs than B. longum Δsrp-treated mice. Representative images were captured at ×40 magnification (n = 10 to 11/group). (B) Small-intestinal sections were subjected to H&E staining, and villus (V) and crypt (C) lengths were measured via light microscopy, expressed as V:C ratios. B. longum srp(Con) treatment in gliadin-sensitized mice resulted in significantly higher V:C ratios than B. longum Δsrp treatment. Representative images were captured at ×10 magnification (n = 10 to 11/group). (C) Paracellular permeability was restored in sensitized NOD/DQ8 mice treated with B. longum strains expressing srp, i.e., B. longum srp⁺ and B. longum srp(Con). Proximal small-intestinal sections were mounted on Ussing chambers to measure ex vivo paracellular permeability, expressed as ⁵¹Cr-EDTA flux (n = 7 to 8/group). (“Hot sample” refers to the collection of mucosal buffer after addition of ⁵¹Cr-EDTA [100%]. All serosal samples collected afterwards are compared to the hot sample.) Data are shown as means ± SEM. Control, nonsensitized, no treatment; srp⁺, B. longum srp⁺; Δsrp, B. longum Δsrp; srp(Con), B. longum srp(Con). Statistical significance determinations were performed by ANOVA followed by Bonferroni post hoc analysis. ***, P < 0.001; *, P < 0.05.

FIG 4

Treatment with gliadin and srp-expressing B. longum shifts fecal microbiota profiles. The figure presents principal-coordinate analysis plots of 16S data in NOD/DQ8 mice. (A) Gliadin induces a shift in β-diversity as calculated using the Unifrac unweighted distance method (P < 0.001). Microbial compositions were different between mice receiving B. longum Δsrp and those receiving B. longum srp⁺ (P < 0.05); those receiving B. longum Δsrp and those receiving B. longum srp(Con) (P < 0.001); and those receiving B. longum srp⁺ and those receiving B. longum srp(Con) (P < 0.001) (n = 5 to 6/group). (B) Gliadin also shifted β-diversity as assessed using Bray-Curtis dissimilarity parameters (P < 0.005). Microbial compositions are significantly different between B. longum Δsrp and B. longum srp⁺ (P < 0.05); between B. longum Δsrp and B. longum srp(Con) (P < 0.005); and between B. longum srp⁺ and B. longum srp(Con) (P < 0.005). Each circle represents an individual fecal sample (n = 5 to 6/group). Control, nonsensitized, no treatment; srp⁺, B. longum srp⁺; Δsrp, B. longum Δsrp; srp(Con), B. longum srp(Con). Statistical analyses were performed via PERMANOVA in QIIME. Plots were constructed in R.

FIG 5

Fecal genera affected by B. longum expressing srp. (A) Results of examinations of genus-level composition of fecal microbiota after B. longum treatment in NOD/DQ8 mice are depicted as an average percentage corresponding to each group in stacked column charts. (B) Genera significantly differing in relative abundances between groups. Data are shown as box and whisker plots (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Control, nonsensitized, no treatment; srp⁺, B. longum srp⁺; Δsrp, B. longum Δsrp; srp(Con), B. longum srp(Con) (n = 5 to 6/group). Statistical analyses were performed via Kruskal-Wallis testing followed by FDR (q < 0.05).

FIG 6

B. longum NCC2705 is detected in the gastrointestinal tract of treated mice. (A) The relative abundances of Bifidobacteria genus members as determined by 16S rRNA sequencing were similar for all groups of NOD/DQ8 mice (n = 5 to 6/group). SI, small intestine. (B) Strain-specific primers detected B. longum Δsrp, B. longum srp⁺, and B. longum srp(Con) in small-intestinal DNA extracted from all bacterially treated mice. Control, nonsensitized, no treatment; srp⁺, B. longum srp⁺; Δsrp, B. longum Δsrp; srp(Con), B. longum srp(Con) (n = 5/group). Statistical analyses were performed via Kruskal-Wallis testing followed by FDR determinations (q < 0.05).