Crohn’s disease is a chronic inflammatory condition that primarily affects the gastrointestinal tract, particularly the ileum and colon. A significant complication of this disease is the formation of fistulae, which are abnormal tunnels that connect the intestine to the skin or other organs. These fistulae can cause substantial morbidity, significantly impacting patients' quality of life and often requiring surgical intervention. Despite the prevalence and clinical importance of fistulae in Crohn’s disease, the molecular mechanisms underlying their formation and persistence remain poorly understood. This study aims to address this gap by providing a detailed molecular and cellular map of intestinal fistulae across various anatomical locations, identifying the key cell types and molecular pathways involved in their development and maintenance.
The research team constructed a high-resolution spatial atlas of 68 intestinal fistulae, combining multiple advanced techniques such as spatial transcriptomics (ST), single-cell RNA sequencing (scRNA-seq), and multiplexed immunofluorescence. This multi-omics approach allowed for a comprehensive understanding of the molecular and cellular heterogeneity associated with fistulae. The findings reveal that the formation of fistulae involves a complex interplay between different cell types, including epithelial, immune, and stromal cells, as well as the extracellular matrix (ECM). The study highlights the existence of distinct fibroblast subtypes, termed fistula-associated stromal (FAS) cells, which are organized in concentric layers within the fistula tract. These layers are associated with different functional states, ranging from active proliferation and migration to quiescence and fibrosis. The presence of these distinct layers suggests a spatially organized process of tissue remodelling and fibrosis, which may be critical in determining the progression and persistence of fistulae.
One of the key observations is the abnormal zonation of growth factors and morphogens within the fistula tract. These molecular signals are crucial for tissue development and repair, but their disruption in the context of fistulae may contribute to the abnormal tunnel formation and the chronic nature of the condition. The study identifies a specific subset of fibroblasts, FAS cells, which are enriched in the fistula tracts and exhibit a unique set of molecular signatures. These include the upregulation of genes associated with fibrosis, tissue remodelling, and immune modulation. The presence of these FAS cells in different layers of the fistula tract suggests a dynamic process where fibroblasts adapt to the local microenvironment, contributing to the formation and stabilization of the fistula.
The research also explores the role of FAS cells in the epithelialization of fistula tracts. Epithelialization is a critical process in wound healing, and in the context of fistulae, it can both promote healing and contribute to the persistence of the condition. The study reveals that FAS cells adjacent to epithelial cells upregulate genes associated with epithelial regeneration, such as *WNT5A* and *NRG1*. These findings suggest that FAS cells may play a dual role in the disease process—facilitating the repair of the epithelial layer while also contributing to the structural reprogramming that maintains the fistula.
The spatial distribution of FAS cells is further analyzed in relation to immune cells, particularly neutrophils and macrophages. These immune cells are found in the granulation tissue lining the fistula tracts and are associated with the expression of cytokines and chemokines that drive inflammation and tissue remodelling. The interaction between FAS cells and these immune cells is likely a key driver of the persistent inflammatory environment observed in Crohn’s fistulae. The study also identifies a unique subset of FAS cells, termed FAS-LAZ (lesion adjacent zone), which are positioned at the interface between the epithelial and fibroblast-rich regions. These cells express markers associated with tissue repair and ECM remodelling, indicating their potential role in promoting the healing of the fistula tract.
In addition to their role in tissue repair, FAS cells are also implicated in the fibrotic processes that contribute to the stabilization of the fistula. Fibrotic FAS-FOZ (fibrotic outer zone) cells are found in deeper layers of the tract and are associated with the deposition of dense collagen and the activation of fibrotic pathways. The study highlights the importance of these fibrotic processes in the persistence of the fistula, as the formation of dense ECM structures may reinforce the tract and prevent its resolution. This finding underscores the need for therapies that can modulate fibroblast activity to prevent excessive fibrosis while preserving their regenerative functions.
The study also examines the role of developmental transcription factors in the function of FAS cells. Transcription factors such as *TWIST1*, *OSR2*, and *PRRX1* are found to be significantly upregulated in FAS cells, suggesting their involvement in the morphogenesis and structural reprogramming of the fistula tract. These transcription factors are known to play critical roles in tissue development and repair, and their dysregulation in the context of Crohn’s disease may contribute to the abnormal formation of fistulae. The study further demonstrates that the overexpression of *TWIST1* and *OSR2* in fibroblasts leads to distinct cellular phenotypes, with *TWIST1* promoting fibrotic activity and *OSR2* activating pathways associated with epithelialization and ECM degradation. These findings provide important insights into the molecular mechanisms underlying the pathogenic features of FAS cells in Crohn’s fistulae.
The research also investigates the potential role of FAS cells in the progression of ulcers to fistulae. It is hypothesized that fistulae may originate from the bases of ulcers, but the exact mechanism remains unclear. The study finds that FAS-like cells are present at the base of all ulcer samples, suggesting that they are not unique to ulcers that progress to fistulae but are a general feature of ulceration in Crohn’s disease. However, these cells exhibit distinct gene expression profiles in the context of fistulae, indicating that they undergo reprogramming to acquire fibrotic and invasive properties. This reprogramming may be driven by local inflammatory signals and the altered microenvironment of the fistula tract.
The analysis of the extracellular matrix (ECM) in the context of fistulae reveals significant differences in collagen structure and organization. The study identifies three distinct collagen-related regions within the fistula tract: a hypo-dense region near the lumen, a region of active ECM remodelling, and a dense fibrotic region deeper in the tract. These differences in collagen structure are associated with the distinct FAS subtypes, with FAS-LAZ and FAS-ALC cells being linked to the hypo-dense and active remodelling regions, respectively, and FAS-FOZ cells associated with the fibrotic outer zone. The presence of these distinct ECM features suggests that the fibroblast-driven remodelling of the ECM is a key component of the pathogenesis of Crohn’s fistulae.
In addition to the molecular and cellular characterization of FAS cells, the study also explores the potential for developing targeted therapies to address the aberrant fibroblast activity associated with fistulae. By identifying the specific molecular pathways and cellular niches that drive the formation and persistence of fistulae, the research provides a foundation for the development of interventions that can modulate these processes. For example, therapies that inhibit fibrotic pathways or promote epithelialization may be effective in reducing the severity of fistulae and improving patient outcomes.
The study further highlights the importance of spatial resolution in understanding the complex interplay between different cell types and molecular pathways in the context of fistulae. By using advanced spatial technologies such as the Visium and Xenium platforms, the researchers were able to capture the spatial organization of the fistula tract and its associated cellular and molecular features. This spatial resolution is critical for understanding how different cell populations interact within the tract and how these interactions contribute to the disease process.
The findings of this study have important implications for the development of model systems and interventions for Crohn’s fistulae. The identification of distinct FAS cell states and their associated molecular signatures provides a framework for understanding the pathogenesis of the condition and for designing targeted therapies. The study also emphasizes the need for further research into the molecular mechanisms that drive the transition from normal fibroblast function to the pathogenic features observed in FAS cells. This includes the role of developmental transcription factors, the influence of local inflammatory signals, and the potential for epigenetic reprogramming to alter fibroblast behavior.
Overall, this study represents a significant advancement in the understanding of Crohn’s fistulae. By combining scRNA-seq and spatial transcriptomics, the researchers have provided a detailed molecular and cellular map of the fistula tract, revealing the complex interplay between different cell types and molecular pathways. The identification of distinct FAS cell states and their functional roles in the disease process offers new insights into the mechanisms underlying the formation and persistence of fistulae. These findings will inform the development of targeted therapies that can modulate fibroblast activity and promote the resolution of fistulae while preserving their regenerative properties. The study also underscores the importance of spatial resolution in understanding the molecular and cellular heterogeneity of the disease and highlights the potential for future research to build on these findings to develop more effective treatments for Crohn’s fistulae.
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