Periodic Reporting for period 1 - BecomingCausal (Contextual specification of fibroblast-driven causalities in chronic intestinal inflammation and fibrosis)
Reporting period: 2022-06-01 to 2024-11-30
Inflammatory bowel disease (IBD) is a serious and long-lasting condition characterized by ongoing inflammation and tissue scarring in the intestines. The exact causes and critical pathways involved in IBD are not fully understood. Recent research has highlighted the importance of interactions between stromal cells (like fibroblasts) and immune cells in maintaining tissue balance. Our lab has provided strong evidence showing that fibroblasts, a type of stromal cell, exhibit different characteristics and play key roles in intestinal health and disease.
Our preliminary findings indicate that various subsets of activated fibroblasts are distributed differently throughout the intestines and collaborate with important inflammatory pathways to drive disease progression. However, many details about these interactions remain unclear. We propose a new theory suggesting that specific subsets of fibroblasts may initiate and worsen IBD, depending on their location and interactions with immune and other stromal cells.
To explore this theory, we will use advanced techniques to analyze individual cells over time and space in animal models of IBD. Our goals are to: (1) Map out the genetic and molecular programs that make fibroblasts diverse and understand how they interact with other cells in the intestines (2) Investigate where fibroblasts come from, how they move within the intestines, and how they change over time (3) Identify new pathways involving fibroblast subsets and explore how they interact with known inflammatory pathways (like TNF and interleukins) (4) Use clinical samples to confirm our findings in humans.
This research aims to unravel the complexities of chronic intestinal disorders like IBD, advance our understanding of immune-related diseases, and pave the way for new treatments targeting fibroblasts.
Our preliminary findings indicate that various subsets of activated fibroblasts are distributed differently throughout the intestines and collaborate with important inflammatory pathways to drive disease progression. However, many details about these interactions remain unclear. We propose a new theory suggesting that specific subsets of fibroblasts may initiate and worsen IBD, depending on their location and interactions with immune and other stromal cells.
To explore this theory, we will use advanced techniques to analyze individual cells over time and space in animal models of IBD. Our goals are to: (1) Map out the genetic and molecular programs that make fibroblasts diverse and understand how they interact with other cells in the intestines (2) Investigate where fibroblasts come from, how they move within the intestines, and how they change over time (3) Identify new pathways involving fibroblast subsets and explore how they interact with known inflammatory pathways (like TNF and interleukins) (4) Use clinical samples to confirm our findings in humans.
This research aims to unravel the complexities of chronic intestinal disorders like IBD, advance our understanding of immune-related diseases, and pave the way for new treatments targeting fibroblasts.
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
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
The prevalence of IBDs is increasing worldwide, and currently approximately 0.2% of the European population suffer from these chronic and life-threatening diseases. Still, current therapies, including the introduction of biologics, offer mainly maintenance treatments rather than cures. This project aims to produce fundamental and translational knowledge on mechanisms of chronicity and fibrosis in IBD, with focus on the well appreciated but still understudied role of intestinal fibroblasts, which should fill major gaps in current knowledge and provide new opportunities for fibroblast-targeted prognostic and therapeutic solutions. This new knowledge is expected to also provide clues as to the role of fibroblasts in immunopathologies beyond IBD, where fibroblast biology is also starting to be scrutinized, in hope for better understanding of tissue inflammation, immunity and fibrosis. Industrial interest for innovations in this new niche of prognostic / therapeutic opportunities is currently very high and the project can provide impact via new academic-industrial collaborations. Our lab and the host institute, BSRC Fleming, are leading biomedical research organizations in Greece, historically offering know-how of new technologies to several third-party organizations with limited access to these technologies. Thus, the project is expected to also boost productivity and impact of scientific research beyond its own boundaries.