Introduction

Saccharomyces boulardii CNCM I-745 probiotic properties have been demonstrated for decades. The exact mechanisms behind these beneficial effects are widely documented, notably anti-inflammatory properties and preservation of the intestinal barrier integrity. However most of the current knowledge was obtained from studies focusing on infectious or inflammatory colonic diseases and very little is known regarding the effects of this yeast on small intestine pathologies.
In previous work, we showed that after oral gavage of p31-43 gliadin peptide, a fragment of gluten-derived peptides that neither binds to HLA class II molecules nor induces T cell activation, is responsible for toxic effects and strong inflammatory responses in the proximal small intestine (Gomes Castro et al., 2019, Ruera C et al, 2020). In this mouse model, intragastric administration of p31-43 elicits mucosal damage in small intestinal together with the production of inflammatory mediators, inflammasome activation, and cell death.

Aims & Methods

Aim: To study the effects of S. boulardii pre-treatment on the inflammatory response in the proximal small intestine in a mouse model of sterile inflammation induced by p31-43.
Materials and methods: Seven week-old C57BL/6 mice were housed under specific pathogen-free conditions and allowed access to autoclaved food and water ad libitum. A three-weeks pretreating phase by gavage with S. boulardii CNCM I-745 (3g/kg/day) or vehicle (Phosphate-buffered saline, PBS), was performed. Then, a single dose of 20µg of p31-43 gliadin peptide (LGQQQPFPPQQPY, at > 95% purity) per mouse or PBS, was delivered by intragastric administration. Mice were euthanized 4 or 16 hours after challenge and proximal small intestine was sampled to perform different histological analysis: villus high/crypt depth (V/C) ratio, intraepithelial lymphocytes (IELs) counting, cell death by TUNEL staining. Assessment of the activation of caspase-1, a central mediator of inflammasome pathway, was performed by Western Blot on small intestinal samples. Statistics were performed using Anova Test.

Results

The evaluation of the histological changes at the proximal small intestine showed that administration of S. boulardii was able to prevent the damage induced by p31-43 for both 4h (p< 0.01) and 16h (<0.0003) after p31-43 treatment. This treatment also prevented mucosal IELs recruitment.
The assessment of cell death by TUNEL staining on sections of the small intestine showed that S. boulardii was effective to block the induction of cell death caused by p31-43 (p< 0.005).
Pretreatment with S boulardii before the p31-43 challenge reduced inflammasome activation by maintaining caspase-1 to basal levels (p<0.01).

Conclusion

Three-week phase of preconditioning with S. boulardii before the p31-43 treatment prevented intestinal macroscopic damage, the increase in IELs numbers, cell death and inflammasome activation. These findings show the beneficial effects of S. boulardii CNCM I-745 in proximal small intestine inflammatory conditions and may be of interest in celiac disease patients.

Disclosure

Nothing to declare

Introduction

The synthetic peptide p31–43, derived from α-gliadin, is known to elicit innate immune responses and epithelial stress in the small intestine. In murine models, intragastric administration of p31–43 reproduces some of the key features of celiac disease (CeD), including reduced villus-to-crypt (V/C) ratio, intraepithelial lymphocyte (IEL) infiltration, production of proinflammatory mediators, inflammasome activation, and epithelial cell death. Previously, we reported that Saccharomyces boulardii CNCM I-745 (S.b.) pre-treated mice effectively prevented histological alterations and inflammation in the proximal small intestine triggered by a single intragastric administration of p31-43 peptide. To better replicate the chronic and progressive nature of small intestinal inflammation observed in human pathology, such as in CeD, we developed a more stringent and sustained model through repeated p31–43 exposure.

Aims & Methods

To evaluate whether the histological and inflammatory changes induced by a three-week intragastric administration of the gliadin peptide p31-43, could be alleviated by concomitant treatment with S.b.
Seven-week-old C57BL/6 mice received daily oral gavage of either S.b. (3 g/kg/day) or vehicle for three weeks. Concurrently, animals were challenged five times per week with 40 µg of the p31-43 gliadin peptide (LGQQQPFPPQQPY) via intragastric administration, either alone or in combination with S.b. (p31-43+S.b.). Sixteen hours after the final challenge, mice were euthanized, and the small intestine was collected for histological and molecular analyses. Histological assessments included V/C ratio, IEL, and Goblet cell count. Caspase-1 activation was evaluated by Western blot (WB) to assess inflammasome activation. Expression of genes related to inflammation and tissue repair was assessed by RT-qPCR. Statistical significance was determined using ANOVA.

Results

Histological analysis revealed that S.b. significantly prevented mucosal alterations induced by p31–43, restoring V/C ratio (p = 0.025) and reducing IEL infiltration (p = 0.08). No significant changes were observed in Goblet cell numbers; however, MUC2 mRNA expression was significantly upregulated after three weeks of p31-43 exposure independently of S.b. intervention (p=0.01). Markers related to epithelial cell proliferation and differentiation (ATOH1/HES1) suggested a trend toward enhanced differentiation of secretory epithelial cells in mice treated with S.b., either alone or in combination with p31-43.
RT-qPCR analysis of genes associated with barrier protection, including AREG, REG3g, and RELMB did not reveal significant changes. However, exposure to p31-43 led to upregulation of proinflammatory genes such as MX1, IFNβ, and CXCL10. Notably, S.b. treatment effectively blocked the p31-43-induced upregulation of CXCL10 (p=0.09). WB further demonstrated that S.b. attenuated caspase-1 activation, indicating suppression of p31-43-induced inflammasome activation (p=0.09).

Conclusion

This study demonstrates that treatment with S.b. effectively impedes the mucosal damage and inflammation induced by gliadin peptide p31-43. Although Goblet cell numbers remained unchanged, the upregulation of MUC2 expression suggests an enhanced mucosal defense response. S.b. also suppressed key inflammatory markers, including CXCL10 and caspase-1 activation. Collectively, these findings support the therapeutic potential of Saccharomyces boulardii CNCM I-745 in managing gliadin-induced intestinal damage and highlight its promise for preventing or alleviating intestinal pathology caused by sterile inflammation.

Disclosure

Biocodex (France) funded this study, but did not play a role in the experiment design or the results analysis

Introduction

Anti-Saccharomyces cerevisiae antibodies (ASCA) and anti-neutrophil cytoplasmic antibodies (ANCA) are well recognized as serological markers in inflammatory bowel diseases (IBD). With the growing evidence of a bidirectional association between IBD and celiac disease (CD), there has been an increased interest in identifying shared biomarkers for both conditions.

Aims & Methods

We aimed to assess seroprevalence of ANCA, IgA and IgG ASCA, in newly diagnosed CD patients. CD diagnosis was set according to 2013 ACG guideline and 2019 ESsCD guideline. Serum collected at the time of diagnosis for all CD patients was analyzed for IgA and IgG ASCA, and ANCA concentration, using INOVA Quanta Lite® assays. Positivity cut-off was defined as ≥ 25U for ASCA and > 30U for ANCA.

Results

Altogether 82 newly diagnosed CD patients, 82.9% female, were recruited, all of them having atrophic mucosal injury on duodenal biopsy sampling and one being seronegative CD. While none of the CD patients had positive ANCA, seroreactivity against Saccharomyces cerevisiae was seen in 32.93% of cases for IgG ASCA. A lower prevalence for IgA ASCA was recorded, of 4.88%, due to associated IgA deficiency. Moreover, 7.32% of newly diagnosed CD patients had equivocal result for IgG ASCA.

Conclusion

A high prevalence of ASCA, particularly IgG type, was seen in newly diagnosed, biopsy-proven CD patients. Future research should investigate the clinical significance of the seroreactivity to IBD-specific antibodies in CD patients.

References

1. Pinto-Sanchez MI, Seiler CL, Santesso N, Alaedini A, Semrad C, Lee AR, Bercik P, Lebwohl B, Leffler DA, Kelly CP, Moayyedi P, Green PH, Verdu EF. Association Between Inflammatory Bowel Diseases and Celiac Disease: A Systematic Review and Meta-Analysis. Gastroenterology. 2020 Sep;159(3):884-903.e31. doi: 10.1053/j.gastro.2020.05.016. Epub 2020 May 13. PMID: 32416141.
2. Damoiseaux JG, Bouten B, Linders AM, Austen J, Roozendaal C, Russel MG, Forget PP, Tervaert JW. Diagnostic value of anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic antibodies for inflammatory bowel disease: high prevalence in patients with celiac disease. J Clin Immunol. 2002 Sep;22(5):281-8. doi: 10.1023/a:1019926121972. PMID: 12405161.

Disclosure

None