Ctla-4 deficiency induces an inflammatory bowel disease-like phenotype in a zebrafish model [bulkRNA-seq]

Inflammatory bowel disease (IBD) is a chronic and relapsing immune-mediated disorder characterized by intestinal inflammation and epithelial injury. The underlying causes of IBD are not fully understood, but genetic factors have implicated in genome-wide association studies, including CTLA-4, an essential negative regulator of T cell activation. However, establishing a direct link between CTLA-4 and IBD has been challenging due to the early lethality of CTLA-4 knockout mice. In this study, we identified zebrafish Ctla-4 ortholog and investigated its role in maintaining intestinal immune homeostasis by generating a Ctla-4-deficient (ctla-4-/-) zebrafish line. These mutant zebrafish exhibit reduced weight, along with impaired epithelial barrier integrity and lymphocytic infiltration in their intestines. Transcriptomics analysis revealed upregulation of inflammation-related genes, disturbing immune system homeostasis. Moreover, single-cell RNA-sequencing analysis indicated increased Th2 cells and interleukin 13 expression, along with decreased innate lymphoid cells and upregulated proinflammatory cytokines. Additionally, Ctla-4-deficient zebrafish exhibited reduced diversity and an altered composition of the intestinal microbiota. All these phenotypes closely resemble those found in mammalian IBD. Lastly, supplementation with Ctla-4-Ig successfully alleviated intestinal inflammation in these mutants. Altogether, these findings offer substantial evidence linking CTLA-4 to IBD and establish a new model for investigating pathogenesis and potential treatments. Overall design: Total RNAs were isolated from wild-type or ctla-4-/- intestines (three biological replicates) using TRIzol reagent following the manufacturer's instructions (Takara). cDNA libraries were constructed using NEB Next Ultra Directional RNA Library Prep Kit (NEB), and sequencing was performed according to the Illumina Hiseq2500 standard protocol at LC Bio (Hangzhou, China).