A15 CX3CR1+ MYELOID CELL-INTRINSIC NOD2 SIGNALING LIMITS CROHN’S DISEASE-ASSOCIATED INTESTINAL FIBROSIS

TL;DR

This study shows that NOD2 signaling in CX3CR1+ myeloid cells helps prevent intestinal fibrosis in Crohn’s disease by maintaining a healing-friendly environment in the gut.

Contribution

The study identifies a novel role for NOD2 signaling in CX3CR1+ myeloid cells in preventing intestinal fibrosis through regulation of stromal-immune interactions.

Findings

NOD2 deficiency in CX3CR1+ myeloid cells leads to reduced CD206+ macrophages and disrupted tissue repair in fibrotic intestines.

FAET cells transdifferentiate into fibroblasts instead of endothelial cells in the absence of NOD2 signaling.

Restoring NOD2 signaling in CX3CR1+ myeloid cells supports wound healing and prevents fibrosis in a mouse model of Crohn’s disease.

Abstract

Crohn’s disease (CD) is a chronic inflammatory bowel disease that often progresses to fibrostenotic disease, requiring surgical intervention. Intestinal fibrosis (IF) is a debilitating outcome of chronic inflammation, excessive collagen deposition, abnormal cellular functions and defective tissue remodeling. CD stems from a complex interplay of host genetics, immune dysregulation, microbial imbalance, and environmental cues. Notably, loss-of-function variants in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) – a cytosolic innate-immune receptor and the strongest genetic risk factor for CD – increase susceptibility to IF. Yet, the cell-intrinsic mechanisms by which NOD2 deficiency promotes IF remain unclear.

(1) To identify the cellular and molecular mediators that predispose the host with the CD-associated risk gene, NOD2 to IF. (2) To investigate how NOD2 dysfunction mediates inter- and intra-cellular rewiring during IF?

We employed a single-cell RNA-sequencing (scRNA-seq) approach to explore the cellular landscape in our CD-mouse model, where littermate wild-type (WT) and Nod2-/- mice were challenged with a chronic inflammatory insult regime, using dextran sulfate sodium (cDSS) for 3 cycles. Complementary approaches such as quantitative real-time PCR (qRT-PCR), immunohistochemistry (IHC), RNA-in situ hybridization (RNA-ISH), flow cytometric analyses and conditional-knockout mice are used to validate cell populations and molecular signatures linked with IF.

Leveraging single-cell transcriptomics analysis, we identified the emergence of a unique cluster of Pi16+ progenitor cells, termed FAET (Fibroblast and Endothelial Transition) cells in the fibrotic gut. Our in silico cell-fate mapping analysis revealed FAET cells transdifferentiate into inflammation-associated fibroblasts (IAFs) instead of endothelial cells (EndoCs), causing loss of crypt microvasculature in Nod2-/- mice. Cellular interactome analysis and genetic studies revealed that CX3CR1+ myeloid cell-intrinsic NOD2 signaling maintains CX3CR1+CD206+ gut resident macrophages, supporting an anatomical niche conducive to FAET-to-EndoCs transition over IAFs, thereby aiding wound healing. Consistently, ileal biopsies from CD patients with NOD2 variants exhibit reduced CD206+ macrophages and disrupted pro-restitutive stromal-immune interaction networks.

CX3CR1+ myeloid cell-intrinsic NOD2 signaling defines a pro-restitutive stromal-immune niche that limits intestinal fibrosis associated with CD. Our study offers new cellular and molecular insights into devising complementary and transformative therapeutic strategies to prevent IF in CD patients.

CIHRThe American Association of Immunologists (AAI) Intersect Fellowship Program for Computational Scientists and Immunologists