Human enteric defensin 5 protects intestinal barrier integrity via cell state-dependent P2Y11-FAK-Rac1 signaling

Human enteric α-defensin 5 (HD5) is an antimicrobial peptide and a key effector of intestinal mucosal innate immunity. Paradoxically, HD5 has also been shown to enhance Shigella infection by inducing filopodia-like extensions via activation of the P2Y11 receptor. However, the broader physiological significance of the HD5-P2Y11 interaction remains poorly understood. Here, using primary human colonic epithelial cells, human colonic organoids, and a gut-on-a-chip model, we uncover a previously unappreciated role of HD5 in establishing and preserving the human intestinal barrier by promoting epithelial cell adhesion under stress conditions. Mechanistically, HD5 activates a previously unrecognized P2Y11-FAK signaling axis, leading to phosphorylation of FAK and paxillin and promoting focal adhesion formation, with Rac1 acting downstream to support epithelial adhesion and barrier integrity. Strikingly, this FAK-centered signaling operates independently of PKA, revealing a cell state-dependent bifurcation in HD5-P2Y11 signaling: in well-adhered epithelial cells, HD5 engages PKA to induce exploratory protrusions, whereas in suspended or injured epithelial cells, HD5 preferentially engages FAK to restore adhesion and reinforce barrier integrity. Together, these results identify HD5 as a human-specific epithelial signaling molecule that links innate immune defense to epithelial adhesion dynamics and contributes to mucosal barrier homeostasis.