Impaired keratinization activity in perianal Crohn's fistulas is associated with poor prognosis

Perianal fistulas remain a major complication in Crohn's disease, profoundly impacting quality of life. Although often therapy refractory, strong heterogeneity exists between patients, both in clinical behavior and therapy response. In this study, we aimed to identify biological factors involved in the clinical behavior of perianal fistulas, allowing better stratification.

Forty-nine patients were included (Crohn's n = 36, cryptoglandular n = 13). Gene expression was analyzed in internal fistula openings and tracts by RNA sequencing. Data were verified using spatial transcriptomics and functionally in human adult organoid cultures. A gene-module score for keratinization activity was calculated and correlated to the Crohn's TOpClass classification as well as to long-term fistula outcomes.

Perianal fistulas were associated with the development of stratified squamous epithelium, expressing specific keratins (KRT5, KRT7, and KRT13). In human organoid models, exposing intestinal mucosa to a pro-inflammatory cytokine mixture partially induced similar changes, supporting rectal mucosal redifferentiation in response to perianal wounding. A gene-module score based on the keratinization-associated markers showed a decreasing gradient from cryptoglandular to mild Crohn-related to severe Crohn-related fistula (0.448 vs. -0.125 vs. -0.448, P = .018). Interestingly, the score was also predictive of long-term outcomes: Patients with low keratinization often showed progressive disease (14/20 patients), whereas this was not seen in the high keratinization group (0/16, P < .001).

We identify an epithelial redifferentiation process characterized by a keratinization-associated gene module as part of the healing process in perianal fistula. The extent of the keratinization not only distinguished Crohn-related from cryptoglandular fistulas but also aligned with disease severity and healing potential.