Understanding Gut Inflammation Through RNA Sequencing

By Dr. Chinta SidharthanReviewed by Bethan DaviesNov 29 2024

The human gastrointestinal tract is essential for nutrient absorption and immune defense, but chronic conditions like inflammatory bowel disease and celiac disease disrupt its cellular processes and tissue integrity.

In a recent study published in Nature, an international team of scientists systematically integrated single-cell ribonucleic acid (RNA) sequencing data from 25 datasets to create a comprehensive atlas of gastrointestinal cell types in health and disease, spanning 1.6 million cells.

By comparing healthy and diseased states, the researchers identified a novel inflammatory epithelial cell type, which they termed INFLAREs, which arises from stem cell-derived metaplasia. The study explored how these cells contribute to inflammation and tissue remodeling in chronic gastrointestinal diseases.

Background

The gastrointestinal tract is a complex, multi-organ system critical for nutrient absorption and immune function. Globally, diseases like ulcerative colitis and Crohn's disease affect millions, presenting major health challenges.

Recent advances in single-cell transcriptomics have revolutionized our understanding of gastrointestinal tissues, uncovering cellular composition and dynamics in both health and disease. While previous studies have mapped changes in specific regions or cell types, these efforts lack a comprehensive, integrated atlas of the entire gastrointestinal system.

Metaplasia—a process where one cell type transforms into another atypical type—is frequently observed in chronic inflammation. Although pyloric metaplasia has been documented histologically in inflammatory conditions, its cellular origins and functional significance remain poorly understood.

Research Overview

This study employed large-scale single-cell RNA sequencing to analyze 385 samples from 189 healthy individuals, integrating data from 25 datasets to create a comprehensive map of gastrointestinal tract tissues. To ensure the data's reliability, the researchers developed a quality control pipeline, scAutoQC, which filtered out low-quality cells and standardized processing.

By integrating datasets using single-cell variational inference, the team generated a reference atlas of approximately 1.1 million cells, categorized into 136 cell types. This atlas served as a baseline for comparison with disease contexts.

Datasets from inflammatory bowel disease, celiac disease, and gastrointestinal cancers were linked to the healthy reference, expanding the resource to 1.6 million cells. This integration enabled detailed projections of inflammatory cells onto the atlas, revealing how cell composition and gene expression are altered in disease states.

Focusing on epithelial metaplasia, the researchers conducted trajectory analysis to trace the origins of metaplastic cells, which they named INFLAREs. Pseudotime analysis provided further insights into the transcriptional changes driving the development of these cells.

Validation of these findings in diseased tissues was achieved through immunohistochemistry and single-molecule fluorescence in situ hybridization, confirming the presence of INFLAREs in chronic inflammation.

To uncover broader mechanisms of cellular interaction, the researchers used computational analyses to study chemokine signaling and immune cell recruitment, shedding light on inflammatory pathways. Bulk RNA sequencing further validated the prevalence of INFLAREs in samples from chronic disease cases, reinforcing their significance.

By integrating advanced computational and experimental techniques, this study offers a comprehensive view of cellular changes across the gastrointestinal tract. It not only maps stem cell-driven metaplasia but also provides crucial insights into the inflammatory mechanisms driving chronic gastrointestinal diseases.

Major Findings

The study revealed that inflammation-induced alterations in gastrointestinal stem cells generated a distinct metaplastic lineage, termed INFLAREs. These cells, characterized by the expression of the mucin protein-encoding gene MUC6, were predominantly identified in patients with inflammatory bowel disease and celiac disease.

Transcriptional analysis showed that INFLAREs shared similarities with healthy mucous gland neck cells of the stomach but exhibited distinct inflammatory gene signatures. Further investigation demonstrated that INFLAREs originated from LGR5+ epithelial stem cells, undergoing altered differentiation trajectories driven by inflammatory signals. These cells retained stem-like properties, as evidenced by their expression of stemness and proliferation markers.

In addition to their stem-like features, INFLAREs exhibited unique immune functions. They produced chemokines such as CXCL16, CXCL2, and CXCL5, which recruited immune cells, including T cells and neutrophils, to sites of inflammation. Cell-cell communication analyses highlighted interactions between INFLAREs and venous endothelial cells, suggesting a role in facilitating immune cell trafficking.

Comparative analysis with healthy tissues further demonstrated that INFLAREs upregulated MHC class II genes, enabling interactions with CD4+ T cells. This antigen-presenting capability indicated that INFLAREs contributed to the perpetuation of inflammation. Immunohistochemical validation confirmed their localization at the base of intestinal crypts in diseased tissues but not in healthy controls.

The findings suggested that INFLAREs played a dual role in disease. On one hand, they promoted mucosal healing by secreting mucus and antimicrobial peptides. On the other, they drove chronic inflammation by recruiting and activating immune cells.

This duality linked stem cell-driven metaplasia to persistent tissue inflammation and identified potential pathways for therapeutic targeting.

Conclusions

Overall, the study provided a comprehensive atlas of gastrointestinal cell types, revealing how stem cell-derived INFLAREs contribute to inflammation in chronic intestinal diseases. These findings highlighted the dual role of metaplastic cells in promoting mucosal repair and exacerbating inflammation.

Furthermore, the results stressed the importance of targeting metaplastic pathways to mitigate disease progression and offered a valuable resource for future research into gastrointestinal health and disease.