Characterization of the "gut microbiota-immunity axis" and microbial lipid metabolites in atrophic and potential celiac disease

Abstract

Introduction: Potential celiac disease (pCD) is characterized by genetic predisposition, positive anti-endomysial and anti-tissue transglutaminase antibodies, but a normal or almost normal jejunal mucosa (e.g., minor histological abnormalities without villous atrophy). To gain further insights into basic mechanisms involved in the development of intestinal villous atrophy, we evaluated and compared the microbial, lipid, and immunological signatures of pCD and atrophic CD (aCD).

Materials and methods: This study included 17 aCD patients, 10 pCD patients, and 12 healthy controls (HC). Serum samples from all participants were collected to analyze free fatty acids (FFAs). Duodenal mucosa samples of aCD and pCD patients were taken to evaluate histology, tissue microbiota composition, and mucosal immune response.

Results: We found no significant differences in the mucosa-associated microbiota composition of pCD and aCD patients. On the other hand, in pCD patients, the overall abundance of serum FFAs showed relevant and significant differences in comparison with aCD patients and HC. In detail, compared to HC, pCD patients displayed higher levels of propionic, butyric, valeric, 2-ethylhexanoic, tetradecanoic, hexadecanoic, and octadecanoic acids. Instead, aCD patients showed increased levels of propionic, isohexanoic, and 2-ethylhexanoic acids, and a lower abundance of isovaleric and 2-methylbutyricacids when compared to HC. In addition, compared to aCD patients, pCD patients showed a higher abundance of isobutyric and octadecanoic acid. Finally, the immunological analysis of duodenal biopsy revealed a lower percentage of CD4⁺ T lymphocytes in pCD infiltrate compared to that observed in aCD patients. The functional characterization of T cells documented a pro-inflammatory immune response in both aCD and pCD patients, but the pCD patients showed a higher percentage of Th0/Th17 and a lower percentage of Th1/Th17.

Conclusion: The results of the present study show, for the first time, that the duodenal microbiota of patients with pCD does not differ substantially from that of aCD; however, serum FFAs and local T cells displayed a distinctive profile between pCD, aCD, and HC. In conclusion, our result may help to shed new light on the "gut microbiota-immunity axis," lipid metabolites, and duodenal immune response in overt CD and pCD patients, opening new paradigms in understanding the pathogenesis behind CD progression.

Keywords: T cells; celiac disease; cytokines; fatty acids; immune response; microbiota; potential celiac disease.

FIGURE 1

Taxonomic composition of aCD and pCD patients’ microbiota Coordinate plot showing the relative abundance of the five most abundant phyla in each aCD and pCD duodenal biopsy.

FIGURE 2

Boxplots reporting alpha diversity indices (respectively, Richness, Shannon index, and Chao1 index) in aCD and pCD samples. Alpha diversity indexes are composite indexes reflecting abundance and consistency. Boxes represent the interquartile range (IQR) between the first and third quartiles (25th and 75th percentiles, respectively), and the horizontal line inside the box defines the median. Whiskers represent the lowest and highest values within 1.5 times the IQR from the first and third quartiles, respectively. P-values less than 0.05 were considered statistically significant.

FIGURE 3

Multivariate representations of the entire sample set. (A) Complete hierarchical clustering based on Euclidean distance of all identified OTUs, (B) complete hierarchical clustering based on Euclidean distance of the top five most informative OTUs, (C) principal coordinate analysis (PCoA) using Bray-Curtis dissimilarity as a distance metric.

FIGURE 4

Boxplots representing the overall abundance of FFAs in aCD patients, pCD patients, and healthy controls (μmol/L). P-values of the intergroup comparisons were assessed with Kruskal–Wallis test. P-values less than 0.05 were considered statistically significant. The asterisks * represent p-values, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Percentage of CD4 + and CD8 + T-cell clones, respectively, obtained from the duodenal mucosa of patients with atrophic CD (A) and potential CD (B).

FIGURE 6

(A) The cytokine phenotype percentage of CD4⁺ T-cell clones obtained from the duodenal mucosa of patients with atrophic CD (orange) and potential CD (blue). P-values of the intergroup comparisons were assessed with Fisher’s exact test. P-values less than 0.05 were considered statistically significant. The asterisks * represent p-values, *p < 0.05. (B) The cytokine phenotype percentage distribution of CD8⁺ T-cell clones obtained from the duodenal mucosa of patients with atrophic CD (orange) and potential CD (blue).