Introduction

Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, refers to a group of chronic relapsing inflammation disorders affecting the gastrointestinal tract, that have been increasing in prevalence worldwide [1]. The precise etiology of IBD has yet to be fully elucidated. Conventional epidemiological studies have indicated that IBD tends to run in families and is linked to genetic factors [2, 3]. However, research also suggested that susceptibility gene patterns differ significantly among various geographic populations. Current evidence points towards a complicated interaction involving host genetics, disrupted intestinal microbiota, environmental triggers, and abnormal immune responses [4–6]. Advances in genomic sequencing techniques have allowed for the identification of genetic variants associated with an increased risk of developing IBD. Among these, mutations in immune-related genes have received particular attention. Research on humans with Crohn’s disease and mouse models of IBD has shown that genetically susceptible individuals exhibit defects in intracellular pattern-recognition receptors (PRRS), such as toll-like receptors (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), which are responsible for initiating innate immune responses to eliminate harmful bacteria [7, 8]. Genetic variations in the tumor necrosis factor ligand superfamily member 15 (TNFSF15) and interleukin 23 receptor (IL23R) genes, both involved in suppressing inflammation, have been associated with an increased risk of developing Crohn’s disease [9, 10].

Cytotoxic T-cell associated antigen-4 (CTLA-4), also known as CD152, is one of the most well-established immune checkpoint molecules expressed predominantly on T cells. It primarily regulates the early stages of T-cell activation by attenuating downstream signaling of the T cell receptor (TCR) [11–13]. Specifically, CTLA-4 has a much higher affinity for CD80 and CD86 ligands compared to the co-stimulatory receptor CD28 [14, 15]. By outcompeting CD28 for ligand binding, CTLA-4 provides an inhibitory signal that impacts immunological synapse formation and inhibits T-cell proliferation and activation [13, 16]. As expected, links between polymorphic alleles of CTLA4 and IBD have been reported in multiple studies [17–19]. Moreover, CTLA-4 is an intriguing target for novel immune checkpoint blockade therapies in cancer treatment, while intestinal inflammation is a common side effect in these clinical trials [20, 21]. Establishing a direct causal relationship between CTLA-4 and IBD has been challenging due to difficulties in finding appropriate models. Early lethality was observed in CTLA-4-deficient mice, adding to the complexity of this issue. Zebrafish is a powerful model system for immunological and biomedical research, due to its versatility and high degree of conservation in innate and adaptive immunities. In our current study, we identified the Ctla-4 homology in zebrafish and successfully developed an adult vertebrate model with homozygous knockout of the ctla-4 gene for the first time. These ctla-4-deficient (ctla-4^-/-) zebrafish survive but exhibit attenuated growth and weight loss. Notably, ctla-4 deficiency leads to an IBD-like phenotype in zebrafish characterized by altered intestinal epithelial cells morphology, abnormal inflammatory response, defects in microbial stratification and composition. Mechanistically, Ctla-4 exerts its inhibitory function by competing with Cd28 for binding to Cd80/86. These findings establish the ctla-4 knockout zebrafish as an innovative platform to elucidate CTLA-4 immunobiology, model human IBD, and develop novel therapeutic modalities.

Results

Identification of zebrafish Ctla-4

Through a homology search in the NCBI database, we identified the ctla-4 gene on zebrafish chromosome 9 (Fig. S1A-C). Zebrafish Ctla-4 protein contains an N-terminal signal peptide, a single IgV-like extracellular domain, a transmembrane region, and a cytoplasmic tail (Fig. 1A). A ¹¹³LFPPPY¹¹⁸ motif is present in the ectodomain of Ctla-4, similar to the MYPPPY motif, a binding site for CD80/CD86 found in mammals (Fig. 1A). Thus, we reasoned that the ¹³LFPPPY¹¹⁸ motif may be a potential binding site for Cd80/86 in zebrafish. The tyrosine-based YVKM motif in the cytoplasmic domain in mammals, which is involved in internalization and signaling inhibition, is absent in Ctla-4. By contrast, a unique tyrosine-based ²⁰⁶YVKF²⁰⁹ motif was found in the C-terminus of Ctla-4 molecule, which is relatively conserved in different fish species (Fig. 1A). The IgV-like domain of Ctla-4 was characterized by two-layer β-sandwich and was conserved between zebrafish and humans (Fig. 1B). Ctla-4 exists as a dimer, and unlike the intracellular localization of CTLA-4 in mammals, Ctla-4 was located on the cell membrane (Fig. 1C and D). Moreover, Ctla-4 was mainly expressed on the T cells, including the Cd4⁺ T cells and Cd8⁺ T cells (Fig. 1E and F).

Fig. 1

Ctla-4 deficiency induces inflammatory bowel disease (IBD)-like phenotype

To further investigate the function of Ctla-4, we generated a ctla-4^-/- zebrafish line with a 14-base deletion in the second exon of the ctla-4 gene (Fig. 2A-C). The zebrafish appeared grossly normal in appearance; however, the body weight and size were significantly reduced compared with those of wild-type zebrafish (Fig. 2D and E). Anatomically, the ctla-4^-/- zebrafish were featured by intestine shortening and splenomegaly, suggesting the occurrence of chronic inflammation in the intestines (Fig. 2F and G). For clarification, we first performed histological analysis on the anterior, mid, and posterior intestine segments using H&E staining. Compared with the wild-type zebrafish, the ctla-4^-/- fish exhibited significant epithelial hyperplasia in the anterior intestine segment, accompanied with a small amount of mucosal inflammatory cell infiltration (Fig. 2H). This result indicates an enhanced regeneration of the intestine, which is typically observed in cases of intestinal inflammation [22]. A noteworthy goblet cell loss which marks the severity of intestinal inflammation was also observed in ctla-4^-/- anterior intestine, as quantified through PAS staining (Fig. 2I and J). Besides, a small amount of lymphocytic infiltration and mild epithelial damage occurred in the mid intestine segment of ctla-4^-/- zebrafish (Fig. 2H). In posterior intestine of ctla-4^-/- fish, the intestinal villi were markedly shortened, the epithelial barrier showed severely disrupted, and the intestinal wall became thinner, wherein the mucosal and transmural inflammatory cells were significantly infiltrated (Fig. 2H). Consistent with these observations, ultrastructure analysis revealed that the epithelial cells of posterior intestine in ctla-4^-/- zebrafish exhibited alteration in tight junction, the loss of adhesion junctions and desmosomes, and disruption of microvilli (Fig. 2K). All these results strongly indicate that Ctla-4 plays a crucial role in preserving intestinal homeostasis in zebrafish. The intestinal phenotype resulting from Ctla-4 deficiency was similar to IBD in mammals.

Fig. 2

Molecular mechanisms of Ctla-4 deficiency-induced IBD-like phenotype

To explore the potential molecular mechanisms of Ctla-4 deficiency-induced IBD-like phenotype, we performed transcriptome profiling analysis of intestines from wild-type and ctla-4^-/- zebrafish. We identified a total of 1,140 differentially expressed genes (DEGs), among which 714 genes were up-regulated, and 426 genes were down-regulated in ctla-4^-/- zebrafish (Fig. 3A and B). GO enrichment analysis showed that DEGs or up-regulated genes in the top 10 enriched biological processes were associated with the immune response and inflammatory response (Fig. 3C and D). Moreover, the KEGG enrichment analyses indicated that the up-regulated DEGs are primarily involved in the process of cytokine-cytokine receptor interaction and cell adhesion molecules, which are also related to inflammation (Fig. S3A); however, the down-regulated DEGs were significantly enriched in the process of metabolism (Fig. S3B). The intestines of ctla-4^-/- zebrafish showed significant upregulation of il1b, tnfa, myeloid-specific peroxidase (mpx), matrix metallopeptidase 9 (mmp9), chemokine (C-X-C motif) ligand 8a (cxcl8a), and il13, a Th2-typic cytokine crucial for ulcerative colitis in mammals [23]. In contrast, il10, a potent anti-inflammatory cytokine, was markedly down-regulated in Ctla-4-deficient intestines (Fig. 3E). The transcriptional change of these genes was confirmed by RT-qPCR (Fig. 3F). By constructing the PPI network, we found that il1b was a major cytokine that played a hub role in promoting the bowel inflammation of ctla-4^-/- zebrafish (Fig. 3G). Moreover, Gene set enrichment analysis (GSEA) showed that genes involved in the lymphocyte chemotaxis, positive regulation of ERK1 and ERK2 cascade, Calcium and MAPK signaling pathways were positively enriched in ctla-4^-/- zebrafish intestines, implying a sensitized or activated state of lymphocytes due to the absence of Ctla-4 (Fig. S3C and D). Notably, biological processes related to neutrophil activation and chemotaxis were significantly enriched (Fig. 3C and D). Studies have shown that neutrophils can induce histopathological effects through releasing matrix metalloproteinases (MMPs), neutrophil elastase, and myeloperoxidase (MPO) [24]. To confirm the association between neutrophils and Ctla-4-deficient intestinal inflammation, the MPO level was examined. As a support, MPO activity was markedly increased in the intestines and peripheral blood of ctla-4^-/- zebrafish (Fig. 3H). Besides, a number of biological markers or susceptibility genes of IBD observed in mammals, including c-reactive protein 6 (crp6), crp7, MMPs, haptoglobin, il23r, insulin-like growth factor binding protein 1 a (igfbp1a), cAMP responsive element modulator b (cremb) and lymphocyte specific protein 1 b (lsp1b), were highly expressed in the ctla-4^-/- zebrafish (Fig. 3I and J) [9, 25, 26], suggesting the presence of a conserved molecular network underlying IBD pathogenesis between ctla-4^-/- zebrafish and mammalian models.

Fig. 3

Cellular mechanisms of Ctla-4 deficiency-induced IBD-like phenotype

To investigate the cellular mechanisms underlying the IBD-like phenotype induced by Ctla-4 deficiency, we performed scRNA-seq analysis on intestines from wild-type and ctla-4^-/- zebrafish using the 10× Genomics Chromium platform. We obtained nine discrete clusters from 7,539 cells of wild-type and ctla-4^-/- zebrafish (Fig. 4A). These clusters of cells were classified as enterocytes, enteroendocrine cells, smooth muscle cells, neutrophils, macrophages, B cells, and a group of T/NK/ILC-like cells based on their co-expression of lineage marker genes (Fig. 4B and C, Fig. S4A and B). Due to severe epithelial disruption and inflammatory cell infiltration in ctla-4^-/- zebrafish intestines, we focus on the pathological process and immune reactions in enterocytes and immune cell populations. KEGG analysis showed that apoptotic pathway was highly enriched in enterocytes of ctla4^-/- zebrafish, suggesting that aberrant apoptosis contributes to the epithelial injury (Fig. S4C). Additionally, genes functionally involved in the formation of tight and adhesion junctions, such as oclna, cdh1, pcdh1b and cldn15a, were significantly down-regulated in enterocytes of ctla-4^-/- zebrafish (Fig. 4D), consistent with the pathological observation under electron microscope. Furthermore, inflammation-related genes and pathways were significantly up-regulated and enriched in neutrophils, B cells, and macrophages of ctla-4^-/- zebrafish, suggesting active inflammatory responses (Fig. 4E-G, Fig. S4D). By sub-clustering analysis, six subpopulations were classified from T/NK/ILC-like cell groups based on their expression with a set of marker genes. These subpopulations include Cd8⁺ T cells, ILC3-like cells, maturing Ccr7^high T cells, NKT cells, and two groups of Th2 cells (Fig. 5A-C; Fig. S5A). The abundances of NKT and two subsets of Th2 cells were significantly increased in the intestines of ctla-4^-/- zebrafish (Fig. 5D-F). These cells exhibited high expression of il13, a key contributor to intestinal inflammation in mammals (Fig. 5G and H) [23]. Specifically, the second subset of Th2 cells was seldom observed in the intestine of wild-type zebrafish, indicating their unique role in the pathogenesis of IBD-like phenotype in ctla-4^-/- zebrafish (Fig. 5D-F). KEGG analysis of up-regulated genes from ctla-4^-/- NKT and Th2 cells indicated that Ctla-4 deficiency is positively associated with the inflammatory cytokine-cytokine receptor interaction, PPAR, calcium and MAPK signaling pathways, cellular adhesion and mucosal immune responses (Fig. 5I and J; Fig. S5B), suggesting high activity of NKT and Th2 cells in inflamed intestines [27, 28]. Although the abundance of Cd8⁺ T cells was not significantly changed in Ctla-4-deficient intestines, the inflammatory genes and pathways were up-regulated and enriched in the subset of T cells (Fig. S5C and D). Notably, the proportion of ILC3-like cells was decreased, and they highly expressed il17a/f1 and il17a/f3 in the Ctla-4-deficient intestines (Fig. 5D-F, and K). Investigations have consistently reported a substantial decline in the population of ILC3s within the inflamed intestines and IL-17A-secreting ILC3s play a significant role in the development of intestinal inflammation [29–34]. Thus, the reduced ILC3-like cells and increased expression of il17a/f1 and il17a/f3 may be responsible for intestinal inflammation induced by Ctla-4 deficiency.

Fig. 4

Fig. 5

Decreased microbiota diversity in ctla-4^-/- zebrafish intestines

The intestinal microbiota plays a crucial role in host functions such as nutrient acquisition, metabolism, epithelial cell development and immunity. Notably, lower microbiota diversity has consistently been observed in patients with IBD phenotype [35, 36], making it a valuable indicator of host health. Therefore, we further analyzed whether microbes are involved in the Ctla-4-deficiency induced intestinal inflammation. The results revealed a significantly higher number of amplicon sequence variants (ASVs) in wild-type zebrafish intestines, with 730 ASVs unique to the wild-type group and 276 ASVs exclusively found in ctla-4^-/- group (Fig. 6A). Furthermore, the Shannon index and the Simpson index indicated a decreased microbial diversity in ctla-4^-/- zebrafish intestines (Fig. 6B) and the Principal Coordinate Analysis (PCoA) using Bray Curtis distance revealed a significant separation in the microbial composition between ctla-4^-/- group and the wild-type group (Fig. 6C). To gain insights into the microbial community composition, we analyzed the identified microbial populations at the class and family level. Alphaproteobacteria and Gammaproteobacteria were found to be the most prevalent classes. Relative to wild-type group, Ctla-4 deficiency resulted in a significant reduction in Alphaproteobacteria abundance. However, the Gammaproteobacteria, one of the main classes of Gamma-negative pathogenic bacteria expanded under inflammation conditions, was increased, although the change did not reach statistical significance (Fig. 6D and E) [37]. In addition, we observed a decreased relative abundance of short-chain fatty acids (SCFAs)-producing Bacilli and Verrucomicrobiae, the latter of which contributes to glucose homeostasis and intestinal health (Fig. 6F and G) [38, 39]. Notably, the family-level analysis revealed a notable enrichment of Enterobacteriaceae, overgrowing under host inflammatory conditions, and the Shewanellaceae, serving as the most important secondary or opportunistic pathogens, in ctla-4^-/- zebrafish (Fig. 6H and I). To identify differentially abundant bacterial taxa between the wild-type and ctla-4^-/-zebrafish, we conducted Linear discriminant analysis (LDA) effect size (LEfSe). The results showed that several potentially opportunistic pathogens, including Gammaproteobacteria, Enterobacterales, and Aeromonadales were found to be overrepresented in ctla-4^-/- zebrafish (Fig. 6J). In contrast, Actinobacteriota, Cetobacterium, and Planctomycetota (Planctomycetes) were more abundant in the wild-type zebrafish. These findings strongly indicated an association between Ctla-4 deficiency-induced gut inflammation and dysbiosis, as characterized by decreased microbial diversity, loss of potentially beneficial bacteria, and expansion of pathobionts.

Fig. 6

Inhibitory role of Ctla-4 in T cell activation

Given that Ctla-4 is primarily expressed on T cells (Fig. 1E-F), and its absence has been shown to result in intestinal immune dysregulation, indicating a crucial role of this molecule as a conserved immune checkpoint in T cell inhibition. For clarification, the lymphocyte proliferation was examined by a series of blockade/inhibition assays using anti-Ctla-4 Ab, recombinant soluble Ctla-4-Ig (sCtla-4-Ig), sCd28-Ig, and sCd80/86 proteins in a PHA-stimulating and mixed lymphocyte reaction (MLR) model (Fig. S6 A-C). Results showed that the lymphocyte proliferation was more pronounced in the absence of Ctla-4, and the supplementation of sCtla-4-Ig inhibited cell proliferation, suggesting the inhibitory function of Ctla-4 in T cells (Fig. 7A and B). Consistent with these results, blockade of Ctla-4 by administering anti-Ctla-4 Ab significantly promoted the proliferation of lymphocytes from wild-type zebrafish (Fig. 7C). Moreover, administration of sCd28-Ig into the co-cultures also inhibited the proliferation of lymphocytes from ctla-4^-/- zebrafish (Fig. 7D). Besides, the addition of Cd80/86 significantly promoted the expansion of Ctla-4-deficient lymphocytes (Fig. 7E). Based on these results, we concluded that the presence of Ctla-4 obstructs the Cd80/86-Cd28 mediated costimulatory signaling, consequently impeding cell proliferation. To further investigate the relationship between Cd28, Ctla-4 and Cd80/86, we assessed the interaction between these molecules in vitro. Our bioinformatics analysis by AlphaFold2, flow cytometry and Co-IP assays demonstrated the interaction between Cd28/Ctla-4 and Cd80/86 (Fig. 7F-H, Fig. S6D). Moreover, using a microscale thermophoresis assay, we found that Ctla-4 has higher affinity for Cd80/86 than Cd28, as indicated by a lower equilibrium association constant value (K_D = 0.50 ± 0.25 μM vs. K_D = 2.64 ± 0.45 μM) (Fig. 7I). These results suggested that Ctla-4 could exert its inhibitory function by competing with Cd28 for binding Cd80/86.

Fig. 7

sCtla-4-Ig alleviates IBD-like phenotype

As described above, engagement of Cd80/86 by sCtla-4-Ig effectively suppressed T cell activation in vitro (Fig. 7B), indicating that sCtla-4-Ig holds promise as a potential intervention for IBD-like phenotype. This is supported by the observation that zebrafish treated with sCtla-4-Ig exhibited obvious body weight restoration compared to those treated with the IgG isotype control (Fig. 8A). To provide further evidence, histological analysis was performed on the posterior intestine, which is known to experience severe tissue destruction under Ctla-4 deficient conditions. As expected, Ctla-4-Ig treatment resulted in a significant decrease in lymphocyte infiltration and an improvement in the epithelial barrier (Fig. 8B). Moreover, Ctla-4-Ig treatment significantly reduced the expression of pro-inflammatory genes, including il13, tnfa, mpx, mmp9, and cxcl8a, as well as igfbp1a, cremb, and lsp1a, which are susceptibility genes for IBD observed in mammals (Fig. 8C and D). These findings demonstrate that the supplementation of Ctla-4-Ig alleviates intestinal inflammation in Ctla-4-deficient zebrafish, highlighting its potential as a therapeutic intervention for CTLA-4 deficiency-induced IBD in mammals.

Fig. 8

Discussion

As an essential negative regulator of T cell activation, dysfunction of CTLA-4 was implicated in various diseases in both humans and murine models [40–42]. Numerous previous studies have established the connection between CTLA-4 and autoimmune thyroiditis, Graves’ disease, myocarditis, pancreatitis, multiple sclerosis, rheumatoid arthritis, and type I diabetes [43–49]. However, the involvement of CTLA-4 in IBD has been understudied. Several investigations have reported that haploinsufficiency resulting from mutations in CTLA-4 in humans is associated with IBD, and genome-wide association studies (GWAS) have implicated CTLA-4 as a susceptibility gene for IBD [50–52]. Nevertheless, the exact contributions and mechanisms of CTLA-4 deficiency in the occurrence and pathology of IBD remain incompletely understood, primarily due to the lack of animal models attributable to the lethality of CTLA-4 knockout in mice. In this study, we identified the Ctla-4 homolog in zebrafish, and discovered that defect in Ctla-4 did not have a severe lethal effect, but did show a clear IBD-like phenotype. This makes zebrafish an attractive animal model for investigating the molecular and cellular mechanisms underlying Ctla-4 mediated IBD. Multiple lines of experimental evidence demonstrated the IBD-like phenotype in Ctla-4-deficienct zebrafish. These include the observed destruction of epithelial integrity and infiltration of inflammatory cells in the inflamed intestines during histopathological analysis, the enrichment of inflammation-related pathways and the imbalance between pro-inflammatory and anti-inflammatory cytokines identified through transcriptome profiling, the abnormal composition of immune cell populations revealed by single-cell RNA sequencing, as well as the reduced diversity and altered composition of intestinal microbiota. These observations collectively suggest that Ctla-4 may serve as a potential genetic determinant of the IBD-like phenotype, although further research is required to definitively identify the causative variant responsible for this association. Moreover, the establishment of zebrafish models provides a valuable tool for comprehending the underlying mechanisms of the disease’s pathophysiology.

A multi-omics study was conducted to investigate the mechanisms of Ctla-4-deficiency induced IBD. RNA-seq analysis demonstrated a significant upregulation of important inflammatory cytokines, such as il1b and tnfa in the Ctla-4-deficient intestines. This is consistent with studies showing that IL-1β and TNF-α act as crucial mediators in mammalian IBD models by disrupting epithelial junctions and inducing apoptosis of epithelial cells [53, 54]. Conversely, the key anti-inflammatory cytokines, such as il10, were downregulated. These findings highlight an imbalance between pro-inflammatory and anti-inflammatory cytokines in the intestines of Ctla-4-deficient fish. Consistently, the inflammatory signaling pathways associated with these upregulated cytokines, such as the ERK1/2 and MAPK pathways, were positively enriched in inflamed intestines. Single-cell RNA-seq analysis revealed the upregulation and enrichment of these inflammatory cytokines and pathways in neutrophils, macrophages, and B cells of inflamed intestines, indicating their active involvement in inflammatory responses and as major sources of inflammatory signals. Additionally, there was a marked increase in the abundance of Th2 subset cells in the inflamed intestines. These Th2 cells exhibited high expression of il13 and were significantly enriched in inflammatory signaling pathways, indicating their activated state. Previous research in mammals has identified IL-13 as the key effector Th2 cytokine in ulcerative colitis, directly causing damage to epithelial cells by affecting epithelial tight junctions, apoptosis, and cell restitution [23]. Therefore, upregulated Il13 from Th2 cells may be a significant contributor to epithelial injury in zebrafish model. Notably, the proportion of ILC3-like cells was downregulated in the inflamed intestines, consistent with recent studies reporting a substantial decline of ILC3 in IBD patients [29–31]. ILC3 is the most abundant type of ILCs in the intestines and plays a protective role in IBD in mammals by promoting epithelial cell proliferation and survival, as well as enhancing intestinal barrier function through the production of IL-22 [32, 34]. Thus, the marked decrease in ILC3-like cells may exacerbate intestinal inflammation and damage. Besides, abnormal activation of Th2 cells can lead to dysfunction in downstream B cells and mucosa-associated immunity, which is essential in maintaining the symbiotic bacterial homeostasis in intestines [38]. Thus, a potential correlation may exist between changes in Th2 cells and the observed alterations in the intestinal microbial community in Ctla-4-deficient zebrafish. Overall, our findings provided insights into the occurrence of IBD. However, gaining a comprehensive understanding of the complex interactions among these immune cells, intestinal epithelial cells, and the microbiome in IBD requires further exploration.

Materials and Methods

Experimental fish

Wild-type AB zebrafish (D rerio) of both sexes, 4-6 months of age with body weights ranging from 0.3 to 0.8 g and lengths of 3-4 cm, were reared in the laboratory in recirculating water at 26-28 ℃ under standard conditions as previously described [55]. All animal experiments were performed in accordance with legal regulations and were approved by the Research Ethics Committee of Zhejiang University.

Generation of Ctla-4-deficient zebrafish

CRISPR/Cas9 system was used to knock out the ctla-4 gene. The targeting sequence 5’-CTCAGAGCCCTACTTCGCAA-3’ was designed by optimized CRISPR Design (http://crispr.mit.edu/) and synthesized by T7 RNA polymerase and purified by MEGAclear Kit (AM1908; Invitrogen) in vitro. Cas9 protein (500 ng/μl, A45220P; Thermo Fisher Scientific) and purified RNA (90 ng/μl) were coinjected into one cell-stage wild-type embryos. For genotyping, DNA fragment was amplified with primers (F: 5’ -TGTGACAGGAAAAGATGGAGAA-3’ and R: 5’-GATCAGATCCACTCCTCCAAAG-3’) and subjected to sequencing. The mutant alleles (-14 bp) were obtained and their offspring were used in experiments.

Histopathological analysis

The anterior, mid, and posterior intestines (n = 3 replicates) were fixed in 4% PFA overnight and embedded in paraffin. The tissues were cut into 4 μm sections and stained with Hematoxylin and eosin (H&E) for pathological analysis. To evaluate the severity of intestinal inflammation, histologic scores were determined according to the criteria supplied by previous publication [22]. Briefly, three independent parameters, including inflammation, extent, and epithelial changes, were assessed and scored as follows: the severity of inflammation (0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked); the level of inflammation extent (0 = none, 1 = mucosa, 2 = mucosa and submucosa, 3 = transmural), the degree of epithelial changes (0 = none, 1 = minimal hyperplasia, 2 = mild hyperplasia, minimal goblet cell loss, 3 = moderate hyperplasia, mild goblet cell loss, 4 = marked hyperplasia with moderate to marked goblet cell loss). Each parameter was calculated and summed to obtain the overall score. The sections were stained with Periodic Acid-Schiff (PAS) reagent to evaluate the number of goblet cells.

RNA-sequencing and bioinformatic analysis

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). The differentially expressed genes (DEGs) were identified with absolute Log₂ fold change > 1 and adjusted p-value < 0.05 by R package DESeq2. Gene Ontology (GO) enrichment and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed by the OmicStudio (http://www.omicstudio.cn/tool) tools. Gene-set enrichment analysis was performed using software GSEA (v4.1.0, https://www.gsea-msigdb.org/gsea/index.jsp), and the heatmap was generated using the R package ggplot2. For the protein-protein interaction (PPI) networks, the DEGs were retrieved in STRING (version 11.5, https://string-db.org/) database (combined score > 0.4), and the PPI network was visualized by Cytoscape software (version 3.9.1) [56]. The betweenness centrality (BC) was calculated using the CytoNCA plugin in Cytoscape software. The RNA-sequencing (RNA-seq) data in this study were deposited in the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) database.

Quantitative real-time PCR

Total RNA from intestines was extracted using TRIzol reagent (Takara Bio) and reverse transcribed into cDNAs according to the manufacturer’s protocol. PCR experiments were performed in a total volume of 10 μl by using an SYBR Premix Ex Taq kit (Takara Bio). The reaction mixtures were incubated for 2 min at 95℃, then subjected to 40 cycles of 15 s at 95℃, 15 s at 60℃, and 20 s at 72℃. Relative expression levels of the target genes were calculated using the 2^-ΔΔct method with β-actin for normalization. Each PCR trial was run in triplicate parallel reactions and repeated three times. The primers used in the experiments are listed in Supplemental Table 1.

Single-cell RNA-sequencing analysis

The intestines from wild-type and ctla-4^-/- zebrafish were washed by D-Hank’s and incubated with D-Hank’s containing EDTA (0.37 mg/mL) and DTT (0.14 mg/mL) for 20 min. The resulting supernatants were collected as fraction 1. The remaining tissues were subsequently digested with type Ⅳ collagenase (0.15 mg/mL) for 1 h at room temperature and the resulting supernatants were collected as fraction 2. Fractions 1 and 2 were combined and centrifuged at 350 g for 10 min. Cells were then washed with D-Hank’s and suspended in 40% percoll (percoll : FBS: L-15 medium = 4 : 1: 5) and passed through a 40-μm strainer. The cell suspension was carefully layered onto 63% percoll (percoll : FBS : L-15 medium = 6.3 : 1: 2.7) and centrifuged at 400 g for 30 min at room temperature. The cell layer of the interface was collected and washed with D-Hank’s at 400 g for 10 min. Cell quantity and viability were assessed using 0.4% trypan blue staining, revealing that over 90% of the cells were viable. Single-cell samples were submitted to the LC-Bio Technology Co., Ltd (Hangzhou, China) for 10× Genomics library preparation and data analysis assistance. Libraries were prepared using the Chromium^TM Controller and Chromium^TM Single Cell 3’ Library & Gel Bead Kit v2 (10× Genomics) according to the manufacturer’s protocol, and sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run, 150 bp) at a minimum depth of 20,000 reads per cell. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software and the data were aligned to Ensembl zebrafish genome assembly GRCz11. Quality control was performed using the Seurat. DoubletFinder R package was used to identify and filter the doublets (multiplets) [57]. The cells were removed if they expressed fewer than 500 unique genes, or > 60% mitochondrial reads. The number of cells after filtration in the current study was 3,263 in wild-type and 4,276 in ctla-4^-/- groups, respectively. Cells were grouped into an optimal number of clusters for de novo cell type discovery using Seurat’s FindNeighbors() and FindClusters() functions, graph-based clustering approach with visualization of cells being achieved through the use of t-SNE or UMAP plots [58]. The cell types were determined using a combination of marker genes identified from the literature and gene ontology. The marker genes were visualized by dot plot and t-SNE plots to reveal specific individual cell types.

In vivo administration of sCtla-4-Ig

An in vivo sCtla-4-Ig administration assay was conducted to evaluate the potential therapeutic effect of sCTLA-4-Ig on intervention of a ctla-4-deficiency induced IBD-like phenotype. For this procedure, the ctla-4^-/- zebrafish were i.p administered with recombinant soluble Ctla-4-Ig protein (sCtla-4-Ig, 2 μg/g body weight) every other day until day 14. Fish that received an equal amount of human IgG isotype were used as control. The dose of sCtla-4-Ig was chosen based on its ability to significantly inhibit the mRNA expression of il13 in Ctla-4-deficient zebrafish.

Statistical analysis

Statistical analysis and graphical presentation were performed with GraphPad Prism 8.0. All data were presented as the mean ± SD of each group. Statistical evaluation of differences was assessed using one-way ANOVA, followed by an unpaired two-tailed t-teat. Statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All experiments were replicated at least three times.

Data availability

All data generated or analyzed during this study are included in this article and its supplementary information files. The RNA-seq and scRNA-seq data for this study have been deposited in NCBI Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE255304 and GSE255303, respectively. The 16S rRNA sequencing data in this study have been deposited in the NCBI Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra/) with an accession number of BioProject PRJNA1073727.

Acknowledgements

We are grateful to Bio-ultrastructure analysis Lab of Analysis center of Agrobiology and environmental sciences, Zhejiang Univ in the TEM sample preparation and observation. We thank Hong Deng and Qiong Huang for advice and expertise in pathological analysis. We also thank She-long Zhang for two-photon laser confocal scanning microscope capture. This work was supported by grants from the National Natural Science Foundation of China (32173003), the National Key Research and Development Program of China (2018YFD0900503, 2018YFD0900505).

Author Contributions

L. L. Q. and J. Z. S. conceived and designed the experiments. L. L. Q. and C. B. H. performed the experiments. L. L. Q., C. B. H., Q. Z., Y. W., A. F. L., L. X. X., Y. C., and J. Z. S. analyzed the data. L. X. X., D. D. F., and J. Z. S. contributed reagents/materials/analysis tools. L. L. Q., Y. C., L. X. X. and J. Z. S. wrote the manuscript. All authors reviewed the paper and provided comments.

Conflict of Interest

The authors declare that they have no competing financial interests.

Ethical approval and consent to participate

All animal experiments were performed with the approval of the Ethics Committee for Animal Experimentation of Zhejiang University.

Supplemental Figures

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