A cell-free TLR5high MSC membrane nanoparticle therapy for Crohn's disease: Targeted immunomodulation via the flagellin/TLR5 axis

Inflammatory bowel disease (IBD), including Crohn's disease (CD; Th1/Th17-driven) and ulcerative colitis (Th2-skewed), lacks therapies correcting T-cell imbalance. Current cytokine-focused treatments remain ineffective, while mesenchymal stromal cells (MSCs) therapies are hindered by inherent heterogeneity and challenges related to cell viability maintenance, batch-to-batch consistency, and standardization. This study aimed to (Kaplan, 2015 (1)) identify MSCs subtypes targeting CD pathology, Sebastian and Siegmund (2024) (2) create MSCs-mimicking nanoparticles, and (Li et al., 2016 (3)) propose a "deconstructed cell therapy" framework. Using colon datasets and CD blood samples, Th1/Th17-macrophage dysregulation was mapped. Transcriptomic screening of eight MSCs sources identified dental pulp-derived TLR5^high-MSCs as superior Th1/Th17 inhibitors compared to umbilical cord TLR5^low-MSCs. In colitis models, TLR5^high-MSCs intercepted gut flagellin (Fla), blocking macrophage TLR5/NF-κB to restore T-cell balance. Decellularized MSCs membranes were engineered into nanovesicles (TLR5^high-CMNP), which showed 3.7-fold higher Fla. affinity than antibodies and suppressed Th1/Th17 activity in vitro. In murine colitis, TLR5^high-CMNP achieved comparable efficacy to MSCs (e.g., 68.9 % reduction in MPO scores), while avoiding challenges associated with live-cell administration - such as embolism occurrence (0 % vs. 24 % in MSCs), need for viability maintenance, and potential variability in TLR5 expression. Bioinformatic analysis confirmed TLR5 as pivotal for MSCs specificity, enabling tailored nanoparticle design. This study highlights TLR5^high-CMNP as a safer, cell-free MSCs alternative and introduces a paradigm prioritizing precise immune checkpoint targeting (e.g., Fla./TLR5) over broad cytokine suppression, resolving IBD therapeutic ambiguity through scalable biomimetic nanomaterials.