Stricturing Crohn's Disease Single-Cell RNA Sequencing Reveals Fibroblast Heterogeneity and Intercellular Interactions.

BACKGROUND & AIMS: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full-thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. METHODS: We performed scRNAseq of 13 fresh full-thickness CD resections containing noninvolved, inflamed nonstrictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next-generation sequencing, proteomics, and animal models. RESULTS: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and up-regulated, and its profibrotic function was validated using NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and knock-out and antibody-mediated CDH11 blockade in experimental colitis. CONCLUSIONS: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and open potential therapeutic developments. This work has been posted as a preprint on Biorxiv under doi: 10.1101/2023.04.03.534781.

Single-cell isolation from full-thickness human intestinal tissue resections for single-cell RNA sequencing.

Single-cell isolation techniques allow the investigation of physical and functional relationships between individual cells within a complex cell population. Here, we present a protocol for single-cell isolation from full-thickness intestinal tissue resections. We describe steps for pre-processing specimens, isolation of lamina propria and muscular layers, and red blood cell lysis. We then detail fixation of isolated cells and assessment of cell quality. The resulting cell suspension can be subjected to RNA sequencing on the 10× Chromium platform. For complete details on the use and execution of this protocol, please refer to Mukherjee et al.1.

Identification of a broadly fibrogenic macrophage subset induced by type 3 inflammation.

Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in fibrosis progression and resolution. Identifying key macrophage populations found in human fibrotic tissues could lead to new treatments for fibrosis. Here, we used human liver and lung single-cell RNA sequencing datasets to identify a subset of CD9+TREM2+ macrophages that express SPP1, GPNMB, FABP5, and CD63. In both human and murine hepatic and pulmonary fibrosis, these macrophages were enriched at the outside edges of scarring and adjacent to activated mesenchymal cells. Neutrophils expressing MMP9, which participates in the activation of TGF-ß1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-ß1 drive the differentiation of human monocytes into macrophages expressing scar-associated markers. Such differentiated cells could degrade collagen IV but not collagen I and promote TGF-ß1-induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-ß1 reduced scar-associated macrophage expansion and hepatic or pulmonary fibrosis. Our work identifies a highly specific macrophage population to which we assign a profibrotic role across species and tissues. It further provides a strategy for unbiased discovery, triage, and preclinical validation of therapeutic targets based on this fibrogenic macrophage population.

Stricturing Crohn's disease single-cell RNA sequencing reveals fibroblast heterogeneity and intercellular interactions.

Background: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding it's pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. Methods: We performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. Results: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. Conclusion: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments.

Cell type transcriptome atlas for the planarian Schmidtea mediterranea.

The transcriptome of a cell dictates its unique cell type biology. We used single-cell RNA sequencing to determine the transcriptomes for essentially every cell type of a complete animal: the regenerative planarian Schmidtea mediterranea. Planarians contain a diverse array of cell types, possess lineage progenitors for differentiated cells (including pluripotent stem cells), and constitutively express positional information, making them ideal for this undertaking. We generated data for 66,783 cells, defining transcriptomes for known and many previously unknown planarian cell types and for putative transition states between stem and differentiated cells. We also uncovered regionally expressed genes in muscle, which harbors positional information. Identifying the transcriptomes for potentially all cell types for many organisms should be readily attainable and represents a powerful approach to metazoan biology.

foxF-1 Controls Specification of Non-body Wall Muscle and Phagocytic Cells in Planarians.

Planarians are flatworms capable of regenerating any missing body part in a process requiring stem cells and positional information. Muscle is a major source of planarian positional information and consists of several types of fibers with distinct regulatory roles in regeneration. The transcriptional regulatory programs used to specify different muscle fibers are poorly characterized. Using single-cell RNA sequencing, we define the transcriptomes of planarian dorsal-ventral muscle (DVM), intestinal muscle (IM), and pharynx muscle. This analysis identifies foxF-1, which encodes a broadly conserved Fox-family transcription factor, as a master transcriptional regulator of all non-body wall muscle. The transcription factors encoded by nk4 and gata4/5/6-2 specify two different subsets of DVM, lateral and medial, respectively, whereas gata4/5/6-3 specifies IM. These muscle types all express planarian patterning genes. Both lateral and medial DVM are required for medial-lateral patterning in regeneration, whereas medial DVM and IM have a role in maintaining and regenerating intestine morphology. In addition to the role in muscle, foxF-1 is required for the specification of multiple cell types with transcriptome similarities, including high expression levels of cathepsin genes. These cells include pigment cells, glia, and several other cells with unknown function. cathepsin+ cells phagocytose E. coli, suggesting these are phagocytic cells. In conclusion, we describe a regulatory program for planarian muscle cell subsets and phagocytic cells, both driven by foxF-1. FoxF proteins specify different mesoderm-derived tissues in other organisms, suggesting that FoxF regulates formation of an ancient and broadly conserved subset of mesoderm derivatives in the Bilateria.

Neoblast specialization in regeneration of the planarian Schmidtea mediterranea.

Planarians can regenerate any missing body part in a process requiring dividing cells called neoblasts. Historically, neoblasts have largely been considered a homogeneous stem cell population. Most studies, however, analyzed neoblasts at the population rather than the single-cell level, leaving the degree of heterogeneity in this population unresolved. We combined RNA sequencing of neoblasts from wounded planarians with expression screening and identified 33 transcription factors transcribed in specific differentiated cells and in small fractions of neoblasts during regeneration. Many neoblast subsets expressing distinct tissue-associated transcription factors were present, suggesting candidate specification into many lineages. Consistent with this possibility, klf, pax3/7, and FoxA were required for the differentiation of cintillo-expressing sensory neurons, dopamine-ß-hydroxylase-expressing neurons, and the pharynx, respectively. Together, these results suggest that specification of cell fate for most-to-all regenerative lineages occurs within neoblasts, with regenerative cells of blastemas being generated from a highly heterogeneous collection of lineage-specified neoblasts.