Lucia Ramirez-Navarro

Crohn’s disease (CD) is a chronic inflammatory bowel disease exhibiting substantial heterogeneity in clinical presentation and response to therapy. To explore its molecular basis, we developed IBDverse, a large single-cell RNA sequencing (scRNA-seq) dataset of terminal ileal biopsies, profiling over 1.1 million cells from 111 patients with CD and 232 healthy controls. This resource integrates discovery and replication cohorts for the robust identification of CD-associated cell types, genes and pathways. We uncovered epithelial changes marked by interferon-driven upregulation of major histocompatibility complex class I molecules that persisted in progenitor cells after macroscopic inflammation resolution. ITGA4 ⁺ macrophages were identified as key inflammatory drivers, showing enriched JAK–STAT signaling and cytokine expression (interleukin-6 (IL-6), IL-12 and IL-23). Heritability analysis linked inflammatory monocytes and macrophages to CD susceptibility, implicating resident and recruited immune cells in pathogenesis. These findings establish a comprehensive cellular and molecular framework for CD, offering insights into disease mechanisms and therapeutic opportunities.

Most genetic variants associated with complex diseases lie in non-coding regions, complicating efforts to identify effector genes and relevant cell types. Here, we map cis-eQTLs across 2.2 million single cells from blood and intestinal biopsies of 421 individuals, including 125 with inflammatory bowel disease (IBD). Cell-type-level eQTLs were more distal to transcription start sites, enriched in enhancers, less likely to regulate the nearest gene, and over two-fold more likely to colocalise with IBD GWAS loci than eQTLs detected at tissue-level resolution. We nominate effector genes at over half of known IBD loci, including MAML2 , PSEN2, and ZMIZ1 in myeloid cells, implicating reduced Notch signalling in intestinal immune dysfunction. We also identify Wnt regulated genes, including MYC , in epithelial stem and progenitor cells, suggesting that impaired renewal contributes to barrier breakdown. Our results provide a mechanistic map linking genetic risk to specific genes and cell types in IBD, and a framework for effector gene discovery in complex disease.

Crohn’s disease (CD) is a heterogeneous disease characterized by chronic inflammation of the gastrointestinal tract driven by an aberrant immune response. To understand the immune mechanisms underlying mucosal inflammation, we performed single-cell RNA sequencing on paired terminal ileum biopsies and blood samples from 125 CD patients. Our findings reveal that the transcriptional profiles of immune cells are primarily shaped by the local tissue microenvironment, with mucosal immune cells undergoing profound inflammation-associated changes, while circulating immune cells largely fail to reflect these signatures. However, a subset of circulating T cells with a tissue-resident memory-like phenotype retained transcriptional hallmarks of mucosal inflammation, mirroring their ileal counterparts with enrichment in cytokine pathways, including interferon gamma and TNF receptor signaling. Altogether, our single-cell atlas provides a high-resolution resource for dissecting the mucosal and circulatory immune landscape of CD and its link to mucosal inflammation.

Summary: Crohn’s disease (CD) is a chronic inflammatory bowel disease exhibiting substantial heterogeneity in clinical presentation and response to therapy. To explore its molecular basis, we developed IBDverse, the largest single-cell RNA sequencing (scRNA-seq) dataset of terminal ileal biopsies, profiling over 1.1 million cells from 111 CD patients and 232 healthy controls. This resource integrates discovery and replication cohorts for robust identification of CD-associated cell types, genes, and pathways. We uncovered epithelial changes marked by interferon-driven MHC-I upregulation, persisting in progenitors after macroscopic inflammation resolution. ITGA4 ⁺ macrophages were identified as key inflammatory drivers, showing enriched JAK/STAT signaling and cytokine expression (IL-6, IL-12, IL-23). Heritability analysis linked inflammatory monocytes and macrophages to CD susceptibility, implicating resident and recruited immune cells in pathogenesis. These findings establish a comprehensive cellular and molecular framework for CD, offering new insights into disease mechanisms and therapeutic opportunities.

During activation, T cells undergo extensive gene expression changes that shape the properties of cells to exert their effector function. Understanding the regulation of this process could help explain how genetic variants predispose to immune diseases. Here, we mapped genetic effects on gene expression (expression quantitative trait loci (eQTLs)) using single-cell transcriptomics. We profiled 655,349 CD4 ⁺ T cells, capturing transcriptional states of unstimulated cells and three time points of cell activation in 119 healthy individuals. This identified 38 cell clusters, including transient clusters that were only present at individual time points of activation. We found 6,407 genes whose expression was correlated with genetic variation, of which 2,265 (35%) were dynamically regulated during activation. Furthermore, 127 genes were regulated by variants associated with immune-mediated diseases, with significant enrichment for dynamic effects. Our results emphasize the importance of studying context-specific gene expression regulation and provide insights into the mechanisms underlying genetic susceptibility to immune-mediated diseases.

Gene expression is controlled by the involvement of gene-proximal (promoters) and distal (enhancers) regulatory elements. Our previous results demonstrated that a subset of gene promoters, termed Epromoters, work as bona fide enhancers and regulate distal gene expression. Here, we hypothesized that Epromoters play a key role in the coordination of rapid gene induction during the inflammatory response. Using a high-throughput reporter assay we explored the function of Epromoters in response to type I interferon. We find that clusters of IFNa-induced genes are frequently associated with Epromoters and that these regulatory elements preferentially recruit the STAT1/2 and IRF transcription factors and distally regulate the activation of interferon-response genes. Consistently, we identified and validated the involvement of Epromoter-containing clusters in the regulation of LPS-stimulated macrophages. Our findings suggest that Epromoters function as a local hub recruiting the key TFs required for coordinated regulation of gene clusters during the inflammatory response.

Gene regulatory regions contain short and degenerated DNA sites recognized by transcription factors (TFs). When such regions harbor SNPs, the DNA motifs where TFs bind may be affected, thereby altering the transcriptional regulation of the target genes. Such regulatory SNPs have been implicated as causal variants in GWAS studies. In this study, we describe the application of the programs Var-tools designed to predict regulatory variants, and present four case studies to illustrate their usage and applications. In brief, Var-tools facilitate i) obtaining variation information, ii) interconversion of variation file formats, iii) retrieval of sequences surrounding variants, and iv) calculating the change on predicted TF affinity scores between alleles, using motif scanning approaches. Notably, the tools support the analysis of haplotypes. The tools are included within the well-maintained suite Regulatory Sequence Analysis Tools (RSAT, http://rsat.eu ), and accessible through a web interface that currently enables analysis of five metazoa and ten plant genomes. Vart-tools can also be used in command-line with any locally-installed Ensembl genome. Users can input personal collections of variants and motifs, providing flexibility in the analysis.