# A37 POSTBIOTIC BACTERIAL MEMBRANE VESICLES FROM B. SUBTILIS DELIVER NOD2 LIGANDS AND PROMOTE IN VITRO WOUND RE-EPITHLIALIZATION THROUGH RIPK2 SIGNALING

## TL;DR

This study shows that bacterial membrane vesicles from B. subtilis can deliver NOD2 ligands and promote wound healing in intestinal cells through RIPK2 signaling.

## Contribution

The first demonstration that B. subtilis-derived membrane vesicles deliver NOD2 ligands and enhance epithelial regeneration via RIPK2 signaling.

## Key findings

- B. subtilis membrane vesicles induce IL8 and CXCL1 expression in a NOD2-dependent manner.
- MVs promote in vitro wound re-epithelialization, which is blocked by RIPK2 inhibition.
- Wound healing is enhanced by synergistic PAMPs and requires RIPK2 signaling.

## Abstract

Bacterial membrane vesicles (MVs) are bilipid nanoparticles secreted as a conserved method of intercellular communication. MVs from Gram-positive bacteria carry diverse bacterial products and represent a unique opportunity in the growing field of postbiotics. This native machinery for mediating microbe-host interactions makes MVs a promising tool for intestinal drug delivery in the context of Crohn’s disease (CD). CD is a chronic inflammatory bowel disease characterized by relapsing inflammation of the digestive tract. CD is associated with (1) loss of function mutations in the host gene encoding the nuclear-binding oligomerization domain 2 (NOD2) pattern recognition receptor and (2) a gut microbiome with reduced capabilities to generate NOD2 stimulating muropeptides from peptidoglycan. Nod2-/- mice have decreased barrier integrity and impaired epithelial restitution upon challenge. However, there are currently no therapeutic strategies targeting NOD2 for CD management.

The aims of this study were to (1) leverage MVs as a biological system for NOD2 ligand delivery to intestinal epithelial cells and (2) determine the roll of probiotic MVs and bacterial ligand synergy in wound re-epithelialization.

MVs were purified from B. subtilis through filtration and ultracentrifugation. MVs were characterized through nanoparticle tracking analysis using NanoSight300. WT and NOD2-/- HCT116 cells, a human colorectal carcinoma cell line, were utilized for IL-8 secretion quantification by ELISA, gene expression by qPCR, and in vitro scratch wound assay by Incucyte quantification.

Here, we demonstrate for the first time that MVs from B. subtilis deliver NOD2 ligands in vitro. Treatment with isolated MVs induces IL8 secretion and CXCL1 expression in a NOD2-dependent manner. Furthermore, MVs promote re-epithelialization after in vitro scratch wounding. This effect was completely blunted by the addition of an inhibitor for RIPK2, a downstream transducer required for NOD2 signaling. Through bacterial pathogen-associated molecular patterns (PAMPs) screening, we found that wound re-epithelialization is promoted by PAMP synergy but dependent on RIPK2 signaling.

This work suggests that delivery of muropeptides by probiotic-derived MVs is a promising strategy to promote epithelial regeneration during injury through targeting NOD2. Future directions will work towards validating these findings in human ileal primary cells and murine models of intestinal injury.

CIHRMedicine by Design