Introduction
Non-celiac gluten/wheat sensitivity (NCG/WS) is a clinical condition in which gluten/wheat ingestion evokes, while withdrawal ameliorates or reverts gastrointestinal (GI) and extra-GI symptoms. The pathogenetic mechanisms of NCG/WS are poorly understood and no diagnostic biomarker is so far available. NCG/WS diagnosis is established by excluding celiac disease (CD) and wheat allergy (WA), evaluating response to a gluten-free diet (GFD) and the effects of gluten reintroduction. Initial evidence suggests a central role played by the gut microbiota in NCG/WS.
Aims & Methods
This study aimed to identify: i) microbiota species valid as biomarkers for rapid and non-invasive NCG/WS diagnosis; ii) the effects of GFD on microbiota in NCG/WS. Subjects with a clinical suspect of either NCG/WS or CD and healthy volunteers (Ctrl) were enrolled while on a free diet. A thorough work-up (including clinical, endoscopic, allergologic and serological evaluations) was applied to diagnose NCG/WS or CD while excluding WA. Instructions for GFD were provided to all participants. Stool samples for gut microbiome were obtained from NCG/WS and CD before (t0) and after 3, 7, 30 and 200 days of GFD (t1, t2, t3, t4). Stool samples from Ctrl were obtained at t0, t1, t2, and t3. The presence and severity of GI (GI Symptom Rating scale) and extra-GI symptoms of NCG/WS were recorded at each time point (t0-t4). NCG/WS diagnosis was confirmed in participants with a 30% improvement in symptom scores in t3 vs. t0. Metagenome from stools was shotgun-sequenced. Data were processed and microbiome profiled with MetaPhlAn v.4.1 (Jun.23). Alpha-diversity (Shannon index, SI), beta-diversity and relative abundances were computed. Principal coordinates analysis was performed (Bray-Curtis dissimilarity test) and groups were compared (PERMANOVA test). Differences in SI at t0 among cohorts were assessed (Mann-Whitney U-test). Species present at any time point in at least 10 subjects were assessed. Candidate biomarkers were validated in newly diagnosed NCG/WS and CD cohorts. Species varying relative abundance during the GFD were identified (linear mixed model and Friedman's ANOVA). P-values were combined with the harmonic mean and corrected for multiple testing (Benjamini-Hochberg method, q-value).
Results
Twenty-eight NCG/WS (F, 37±16 yrs), 11 CD (9 F, 32±15 yrs) and 37 Ctrl (25 F, 33±14 yrs) were enrolled. Alpha-diversity at t0 was comparable (p>0.19) in NCG/WS (SI=4.0), CD (SI=3.7) and Ctrl (SI=4.1), whereas beta-diversity was different among groups (p=0.026). A total of 507 species were identified. The candidate biomarker Clostridiaceae bacterium (SGB47672) showed lower abundance in NCG/WS vs. Ctrl (q<0.2), data confirmed in validation cohorts (172 NCG/WS, 243 CD). Alpha-diversity was preserved (p>0.05) in NCG/WS and CD up to t4 and in Ctrl (t3) in GFD, whereas concerning changes in relative abundance (q<0.2; p<0.05) we identified a reduction of Bifidobacterium longum (SGB17248).
Conclusion
We showed that NCG/WS exhibits a peculiar metagenomic signature with C. bacterium (SGB47672) being a potential biomarker for NCG/WS diagnosis. Our study suggests that the use of probiotic supplements can help to restore B. longum (SGB17248) deficiency during GFD. Beyond integrating GFD treatment, probiotics containing Bifidobacteria can positively contribute by exerting anti-inflammatory and protective effects on the intestinal epithelial barrier in NCG/WS.
References
Volta U, De Giorgio R, Caio G, Uhde M, Manfredini R, Alaedini A. Nonceliac Wheat Sensitivity: An Immune-Mediated Condition with Systemic Manifestations. Gastroenterol Clin North Am. 2019 Mar;48(1):165-182
Beghini F, McIver LJ, Blanco-Míguez A, Dubois L, Asnicar F, Maharjan S, Mailyan A, Manghi P, Scholz M, Thomas AM, Valles-Colomer M, Weingart G, Zhang Y, Zolfo M, Huttenhower C, Franzosa EA, Segata N. Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery 3. Elife. 2021 May 4;10:e65088.
Yao S, Zhao Z, Wang W, Liu X. Bifidobacterium Longum: Protection against Inflammatory Bowel Disease. J Immunol Res. 2021 Jul 23;2021:8030297.
