Our research during this period involved studying ileitis using the TnfΔΑRE model through single-cell RNA sequencing (scRNA-seq) (Objective 1). We identified genes in fibroblast subsets that are activated early, before visible inflammation, suggesting they play a crucial role in TNF-dependent chronic inflammation. Using spatial transcriptomics, we mapped distinct fibroblast populations in chronic intestinal inflammation, revealing common immune functions and unique activation pathways influenced by their surroundings. We also discovered new communication pathways between fibroblasts and immune cells, which we aim to further explore in Objective 3.
Furthermore, we investigated the origins of fibroblasts during chronic inflammation and fibrosis using the CARLIN mouse model crossed with TnfΔΑRE mice (Objective 2). By optimizing doxycycline administration, we traced the differentiation of stromal cells using scRNA-seq. Our findings from lineage tracing in TnfΔΑRE mice suggest that a specific fibroblast subpopulation contributes to intestinal inflammation, particularly in deeper layers, characteristic of both mouse and human ileitis.
To understand the transition of fibroblasts from inflammation to fibrosis, we prioritized IL-6 family cytokines identified from integrated analyses of mouse models and human samples. These cytokines, notably IL-6, OSM, and LIF, were upregulated by a distinct type of activated macrophage in the submucosal layer of TnfΔΑRE mice. In experiments with isolated fibroblasts, we explored their role in activating fibrosis-related pathways, using advanced gene-editing techniques for efficient gene silencing.
Additionally, we analyzed datasets from chronic inflammation models and human fibroblasts to identify key transcription factors involved in fibrosis. We developed screening assays to validate these findings ex vivo and refine our understanding of fibrosis mechanisms. Overall, our research aims to uncover novel insights into the mechanisms driving chronic intestinal inflammation and fibrosis, paving the way for new therapeutic strategies
