Unraveling the Role of Ctla-4 in Intestinal Immune Homeostasis: Insights from a novel Zebrafish Model of Inflammatory Bowel Disease
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eLife assessment
This study focuses on the role of a T-cell-specific receptor, ctla-4, in a new zebrafish model of IBD-like phenotype. Although implicated in IBD diseases, the function of ctla-4 has been hard to study in mice as the KO is lethal. Ctla-4 mutant zebrafish exhibited significant intestinal inflammation and dysbiosis, mirroring the pathology of inflammatory bowel disease (IBD) in mammals, providing a new valuable model to the field of IBD research. However, although many of the results are solid, the methods as provided are incomplete, without information on methods for many data panels.
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Abstract
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 homolog 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, our findings demonstrate the pivotal role of Ctla-4 in maintaining intestinal homeostasis. Additionally, they offer substantial evidence linking CTLA-4 to IBD and establish a novel zebrafish model for investigating both the pathogenesis and potential treatments.
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eLife assessment
This study focuses on the role of a T-cell-specific receptor, ctla-4, in a new zebrafish model of IBD-like phenotype. Although implicated in IBD diseases, the function of ctla-4 has been hard to study in mice as the KO is lethal. Ctla-4 mutant zebrafish exhibited significant intestinal inflammation and dysbiosis, mirroring the pathology of inflammatory bowel disease (IBD) in mammals, providing a new valuable model to the field of IBD research. However, although many of the results are solid, the methods as provided are incomplete, without information on methods for many data panels.
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Reviewer #1 (Public review):
"Unraveling the Role of Ctla-4 in Intestinal Immune Homeostasis: Insights from a novel Zebrafish Model of Inflammatory Bowel Disease" suggests the identification of the zebrafish homolog of ctla-4 and generates a 14bp deletion/early stop codon mutation that is viable. This mutant exhibits an IBD-like phenotype, including decreased intestinal length, abnormal intestinal folds, decreased goblet cells, abnormal cell junctions between epithelial cells, increased inflammation, and alterations in microbial diversity. Bulk and single-cell RNA-seq show upregulation of immune and inflammatory response genes in this mutant (especially in neutrophils, B cells, and macrophages) and downregulation of genes involved in adhesion and tight junctions in mutant enterocytes. The work suggests that the makeup of immune cells …
Reviewer #1 (Public review):
"Unraveling the Role of Ctla-4 in Intestinal Immune Homeostasis: Insights from a novel Zebrafish Model of Inflammatory Bowel Disease" suggests the identification of the zebrafish homolog of ctla-4 and generates a 14bp deletion/early stop codon mutation that is viable. This mutant exhibits an IBD-like phenotype, including decreased intestinal length, abnormal intestinal folds, decreased goblet cells, abnormal cell junctions between epithelial cells, increased inflammation, and alterations in microbial diversity. Bulk and single-cell RNA-seq show upregulation of immune and inflammatory response genes in this mutant (especially in neutrophils, B cells, and macrophages) and downregulation of genes involved in adhesion and tight junctions in mutant enterocytes. The work suggests that the makeup of immune cells within the intestine is altered in these mutants, potentially due to changes in lymphocyte proliferation. Introduction of recombinant soluble Ctla-4-Ig to mutant zebrafish rescued body weight, histological phenotypes, and gene expression of several pro-inflammatory genes, suggesting a potential future therapeutic route.
Strengths:
- Generation of a useful new mutant.
- The demonstration of an IBD-like phenotype in this mutant is extremely comprehensive.
- Demonstrated gene expression differences provide mechanistic insight into how this mutation leads to IBD-like symptoms.
- Demonstration of rescue with a soluble protein suggests exciting future therapeutic potential.
- The manuscript is mostly well organized and well written.
Weaknesses:
- Given the sequence similarity between CTLA-4 and its related receptor CD28, and the difference in subcellular localization of this protein vs. human CTLA-4, some confusion remains about which gene is mutated in this manuscript (CD28 or CTLA-4/CD152).
- Some conclusions made from scRNAseq data (e.g. increased apoptosis, changes in immune cell numbers) could potentially result from dissociation artifacts and would be stronger with validation staining.
- The Methods section is woefully incomplete and describes fewer than half of the experiments performed in this manuscript.
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Reviewer #2 (Public review):
Summary:
The authors aimed to elucidate the role of Ctla-4 in maintaining intestinal immune homeostasis by using a novel Ctla-4-deficient zebrafish model. This study addresses the challenge of linking CTLA-4 to inflammatory bowel disease (IBD) due to the early lethality of CTLA-4 knockout mice. Four lines of evidence were shown to show that Ctla-4-deficient zebrafish exhibited hallmarks of IBD in mammals:
(1) impaired epithelial integrity and infiltration of inflammatory cells;
(2) enrichment of inflammation-related pathways and the imbalance between pro- and anti-inflammatory cytokines;
(3) abnormal composition of immune cell populations; and
(4) reduced diversity and altered microbiota composition. By employing various molecular and cellular analyses, the authors established ctla-4-deficient zebrafish as a …Reviewer #2 (Public review):
Summary:
The authors aimed to elucidate the role of Ctla-4 in maintaining intestinal immune homeostasis by using a novel Ctla-4-deficient zebrafish model. This study addresses the challenge of linking CTLA-4 to inflammatory bowel disease (IBD) due to the early lethality of CTLA-4 knockout mice. Four lines of evidence were shown to show that Ctla-4-deficient zebrafish exhibited hallmarks of IBD in mammals:
(1) impaired epithelial integrity and infiltration of inflammatory cells;
(2) enrichment of inflammation-related pathways and the imbalance between pro- and anti-inflammatory cytokines;
(3) abnormal composition of immune cell populations; and
(4) reduced diversity and altered microbiota composition. By employing various molecular and cellular analyses, the authors established ctla-4-deficient zebrafish as a convincing model of human IBD.Strengths:
The characterization of the mutant phenotype is very thorough, from anatomical to histological and molecular levels. The finding effectively established ctla-4 mutants as a novel zebrafish model for investigating human IBD. Evidence from the histopathological and transcriptome analysis was very strong and supported a severe interruption of immune system homeostasis in the zebrafish intestine. Additional characterization using sCtla-4-Ig further probed the molecular mechanism of the inflammatory response and provided a potential treatment plan for targeting Ctla-4 in IBD models.
Weaknesses:
Since CTLA-4 is one of the most well-established immune checkpoint molecules, it is not clear whether the ctla-4 mutant zebrafish exhibits inflammatory phenotypes in other tissues than the intestine. Although the evidence for intestinal phenotypes is clear and similar to human IBD, it can be ambiguous whether the mutant is a specific model for IBD, or abnormal immune response in general.
To probe the molecular mechanism of Ctla-4, the authors used a spectrum of antibodies that target Ctla-4 or its receptors. The phenotype assayed was lymphocyte proliferation, while it was the composition rather than the number of in immune cell number that was observed to be different in the scRNASeq assay. Although sCtla-4 has an effect of alleviating the IBD-like phenotypes, I found this explanation a bit oversimplified.
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Reviewer #3 (Public review):
Summary:
The current study on the mutant zebrafish for IBD modeling is worth trying. The author provided lots of evidence, including histopathological observation, gut microflora, as well as intestinal tissue or mucosa cells' transcriptomic data. The multi-omic study has demonstrated the enteritis pathology at multi levels in zebrafish model. However, poor writing of methods and insufficient discussion of current findings were the main defects.
Strengths:
The important immune checkpoint of Treg cells was knocked out in zebrafish, and the enteritis was found then. It could be a substitution of the mouse knockout model to investigate the molecular mechanism of gut disease.
Weaknesses:
(1) The use of the English language requires further editing.
(2) The background of this study has not been introduced …
Reviewer #3 (Public review):
Summary:
The current study on the mutant zebrafish for IBD modeling is worth trying. The author provided lots of evidence, including histopathological observation, gut microflora, as well as intestinal tissue or mucosa cells' transcriptomic data. The multi-omic study has demonstrated the enteritis pathology at multi levels in zebrafish model. However, poor writing of methods and insufficient discussion of current findings were the main defects.
Strengths:
The important immune checkpoint of Treg cells was knocked out in zebrafish, and the enteritis was found then. It could be a substitution of the mouse knockout model to investigate the molecular mechanism of gut disease.
Weaknesses:
(1) The use of the English language requires further editing.
(2) The background of this study has not been introduced sufficiently.
(3) The medical concepts were overstated for immune cell populations.
(4) A lot of methods were not provided.
(5) The age of fish varied a lot in this study.
(6) The pathological index can't reflect the detailed changes in intestinal mucosa.
(7) A lot of findings reflected by the current were not discussed.
(8) The structuring of the text is poor and lacks good logic.
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