Introduction: Despite strict adherence to a GFD, nearly one-fifth of patients with celiac disease (CeD) remain non-responsive with persistent mucosal injury. The mechanisms driving this poor mucosal regenerative capacity are unknown. We studied intestinal stem cell (ISC) niche dynamics and inflammatory markers in Gluten-Free-Diet (GFD) responsive versus non-responsive CeD to uncover mechanisms underlying persistent epithelial injury.
Methods: We prospectively evaluated duodenal biopsies from 60 patients (n-30 responsive & 30-non-responsive) with CeD evaluated at baseline and after ≥12 months on a gluten-free diet. We assessed stem cell niche dynamics including epithelial integrity, cellular turnover (Ki-67, γ-H2AX), stem cell dynamics (Lgr5, BMI1), Wnt signalling (β-catenin), Paneth cell function (β-defensin) using immunohistochemistry and for inflammatory cytokines (IL-15, IL-21) using immunofluorescence.
Results: Following ≥12 months of GFD, markers of epithelial integrity improved significantly: the % of patients exhibiting severe villin loss dropped from 28.3% to 4.8% (p<0.001), indicating significant restoration of the epithelial brush border, while MUC2 showed near-restoration (p = 0.056). Regarding cellular turnover and DNA damage, villous Ki-67 declined from a median of 10% to 5.5% (p=0.012), and villous γ-H2AX was significantly reduced (p=0.0156). In terms of stem cell dynamics and Paneth cell function, Lgr5 expression more than doubled from 6.00 to 12.00 (p<0.0001), β-defensin increased from 28% to 49% (p<0.0001), and membranous β-catenin remained stable (p=0.18). When comparing responders versus non-responders, responders achieved superior recovery with higher MUC2 (95% vs. 80%,p=0.032), lower villous Ki-67 (3%vs.10%,p=0.075), and near-complete suppression of IL-15 (0.25%vs.4.02%,p=0.009) and IL-21 (0% vs. 9.13%,p<0.001). In contrast, non-responders showed persistent villin loss and elevated crypt Ki-67 (74.3%vs.67.8%,p=0.124), while BMI1, Lgr5, and β-defensin showed no significant differences between groups.
Conclusion: GFD restores epithelial integrity, reduces DNA damage, and expands Lgr5⁺ stem cells in CeD. However, non-responders show persistent IL-15/IL-21-driven inflammation and failed stem cell activation, highlighting the need for targeted therapies.