Lammers, K. M.; Vazquez, D. S.; Segura, V.; Hollmann, J.; Huebner, W.; Morre, S. A.; Huser, T.; Sousa, C.; Dodero, V. I.

Wheat gluten is the main trigger of gluten-related disorders such as celiac disease, in which the proteolysis-resistant, 33-mer peptide is a key immunodominant factor. It has been reported that the 33-mer forms oligomers and thin-plate superstructures that accumulate in the Caco-2 intestinal epithelial cell line, although their biological effects remain only partially understood. Here, we screened the effects of these nanostructures on Toll-like receptors in Caco-2 cells to investigate their effect on the early innate immune response. Our findings showed a specific upregulation of TLR5 mRNA in response to 33-mer gliadin superstructures, although no inflammatory response was detected based on CXCL1 and CXCL8 levels. Moreover, immunofluorescence analysis revealed partial co-localization with TLR5. We further found that the 33-mer superstructures attenuate flagellin-induced TLR5/NF{kappa}B signaling in reporter cells, similar to a blocking anti-hTLR5-IgA2 antibody. Molecular docking suggested that the 33-mer peptide occupies the concave region of TLR5 as well as 54% of the TLR5-TLR5 dimerization interface. The interference with the TLR5 dimerization domain could explain the experimental attenuation of flagellin-mediated TLR5 activation. Given that TLR5 contributes not only to inflammation but also to intestinal homeostasis, our findings suggest that the formation of 33-mer nanostructures upon its accumulation may increase the local concentration of 33-mer, interfering with TLR5-mediated mucosal immune responses. These findings provide a rationale for investigating the role of TLR5 and 33-mer aggregates in clinical samples from patients with celiac disease and other gluten-related disorders.