Functional characterization of the gut microbiota and associated immune response in patients with “Potential Celiac Disease” (PDC)

• Grant: FC 007/2016
• Title: Functional characterization of the gut microbiota and associated immune response in patients with Potential Celiac Disease
• Duration: Triennial Project
• Principal Investigator: Federica Ricci, Department of Biomedical, Experimental and Clinical Sciences “Mario Serio” University of Florence, Florence, Italy
• Tutor (Head Lab): Prof. Antonino Salvatore Calabrò, Department of Biomedical, Experimental and Clinical Sciences
  “Mario Serio”, University of Florence, Florence, Italy

Publications originated from the Project:

• Niccolai E, Baldi S, Ricci F, Russo E, Nannini G, Menicatti M, Poli G, Taddei A, Bartolucci G, Calabrò AS, Stingo FC, Amedei A. Evaluation and comparison of short chain fatty acids composition in gut diseases. World J Gastroenterol. 2019 Sep 28; 25(36):5543-5558. doi: 10.3748/wjg.v25.i36.5543. PMID: 31576099. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767983
• Baldi S, Menicatti M, Nannini G, Niccolai E, Russo E, Ricci F, Pallecchi M, Romano F, Pedone M, Poli G, Renzi D, Taddei A, Calabrò AS, Stingo FC, Bartolucci G, Amedei A. Free Fatty Acids Signature in Human Intestinal Disorders: Significant Association between Butyric Acid and Celiac Disease. Nutrients. 2021 Feb 26; 13(3):742. doi: 10.3390/nu13030742. PMID:33652681. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996737
• Federica R, Edda R, Daniela R, Simone B, Giulia N, Gabriele L, Marta M, Marco P, Gianluca B, Elena N, Matteo C, Serena S, Matteo R, Amedeo A, Salvatore CA. Characterization of the “gut microbiota-immunity axis” and microbial lipid metabolites in atrophic and potential celiac disease. Front Microbiol. 2022 Sep 30;13:886008. doi: 10.3389/fmicb.2022.886008. PMID: 36246269; PMCID: PMC9561818. https://pubmed.ncbi.nlm.nih.gov/36246269/

THE STUDY

Project rationale and aims

Classic celiac disease (CD) is a clinical form of celiac characterized by a specific serology, by genetic predisposition and histologic changes in the intestinal mucosa (Figure 1). There are individuals that, despite the presence of specific antibodies and HLA genetic susceptibility, have an architecturally normal intestinal mucosa (Figure 1). These individuals are defined patients with Potential Celiac Disease (PDC). The aim of the study is to investigate on adaptive immune response and the composition of the gut microbiota in CD/PCD patients. We expected to obtain an accurate identification of bacterial groups having discriminatory properties in the CD spectrum balance and contemporary to asses a correlated immunological specific profile, which may already be indicative and “predictive” of the future development of villous atrophy in patients with PCD.

Figure 1: Represental image of classic celiac disease and potential celiac disease

Research plan and results obtained

1) We evaluated T cells in duodenal biopsies of CD and PCD patients. The phenotypic characterization of T cells revealed a lower percentage of CD4+ T cells in PCD patients compared to CD patients and the presence of potential γδ lymphocytes only CD patients.
The functional characterization of T cell showed high percentages of lymphocytes producing IFN-γ both CD and PCD patients.
Moreover, the analysis showed in PCD patients a number increased of IL-17+ T cells (Th17) and a high percentage of T cell co‐producing IL17, IL4 and IFN‐γ (Th0/Th17).
In addition to, PCD patients compared to CD patients showed lower percentages of IL4+ T cells (Th2) and T cells co-producing IFN-gamma, IL17 (Th1/Th17).
Furthermore, compared to CD patients, PCD patients reported a higher percentage of immunosuppressive Tregs.
2) We compared the microbiota composition of CD and PCD duodenal biopsy. We observed an abundance of Firmicutes, Bacteroidetes, and Actinobacteria in both CD and PCD patients and high percentages of Proteobacteria and Fusobacteria in adult CD and PCD patients. Intriguingly, no statistically different microbial taxa or bacterial grouping were displayed between the two groups.

Experimental design and methodologies

1) Phenotypical and functional evaluation of adaptive immune response.
Duodenal mucosa samples were dissociated to isolate tissue infiltrating lymphocytes (TILs). TILs were magnetically isolated with anti-human CD3+ microbeads and cloned under limiting dilution. Single T-cell clones (Tcc) were seeded, in microwells (0.3 cells/well) containing RPMI 1640 supplemented with 10% FBS HyClone in the presence of 2×106 irradiated peripheral blood mononuclear cells, phytohemagglutinin and recombinant human interleukin-2. At weekly intervals, 2 × 106 irradiated PBMCs and IL-2 were added to each micro-culture to maintain the expansion of growing clones. The T clones were evaluated for their surface markers and functional properties. Surface markers analysis was done by means of FACS analysis using fluorochrome-conjugated anti CD4, anti CD8. To assess the cytokines’ production Tcc were resuspended at a concentration of 106 cells/ml medium and cultured for 48 h
in the presence of phytohemagglutinin. Cell-free supernatants were collected and assayed for IFN-γ, IL-4, IL-17, and IL-10 with test ELISA to identify the different T cells subsets.
2) Characterization of microbiota composition.
Total DNA was extracted from duodenal mucosa samples of CD and PCD patients using the DNeasy PowerLyzer PowerSoil Kit. The quality and quantity of extracted DNA was assessed using the Qubit Fluorometer and then genomic DNA was frozen at -20°C. Extracted DNA samples were sent to IGA Technology Services where amplicons of the V3–V4 region of the bacterial 16s rRNA were obtained and sequenced in paired-end (2×300 bp) on the MiSeq Illumina platform, according to the Illumina 16S Metagenomic Sequencing Library Preparation protocol. The sequences obtained were statistically analyzed.

Potential pitfalls and caveats

The results of our project will be submitted to a scientific journal. We will work to characterize fecal microbiota of CD/PCD patients and short-chain fatty acids produced by intestinal bacteria in order to obtain a more complete intestinal analysis.

Conclusions and Discussion

Immunological analysis showed differences in the intestinal mucosa of patients. In PCD patients, we observed a lower percentage of CD4+ T cells, no potential γδ lymphocytes, higher percentage of Th17, Th0/Th17 and Treg. Moreover, the intestinal mucosa of PCD patients showed lower percentages of Th2 and Th1/Th17.
CD4+ T cells play a key role in inflammatory response trigged by gluten and γδ lymphocytes have an important role in the pathogenesis of CD. So, we hypothesized that the lower percentage of CD4+ T cells and the absence of γδ lymphocytes in PCD patients might be a sign of a weaker immune response to gluten.
IL-17 is a pro-inflammatory cytokine but a number of studies underlined the involvement in preventing gut mucosa inflammation and the Th2 subpopulation induces the release of IL-4, triggering the activation of B lymphocytes which produce anti tTG2 autoantibodies; on the other hand, the Th1/Th17 subpopulation determines the damage of the enterocytes leading to the atrophy of the intestinal villi. We
hypothesize that increasing of Th17 and Th0/Th17 and decreasing of Th2 and Th1/Th17 might counteract inflammation and contribute to prevent the mucosal damage. Moreover, the higher percentage of immunosuppressive Tregs, acting as a brake of inflammation, in PCD condition could contribute to absence of damages in patients who have all the elements to develop CD.
No statistically different microbial taxa or bacterial grouping were displayed between CD/PCD patients, suggesting the existing of the same microbial architecture in both conditions.