Single-cell transcriptional analysis reveals ILC-like cells in zebrafish

Abstract

Innate lymphoid cells (ILCs) are important mediators of the immune response and homeostasis in barrier tissues of mammals. However, the existence and function of ILCs in other vertebrates are poorly understood. Here, we use single-cell RNA sequencing to generate a comprehensive atlas of zebrafish lymphocytes during tissue homeostasis and after immune challenge. We profiled 14,080 individual cells from the gut of wild-type zebrafish, as well as of rag1-deficient zebrafish that lack T and B cells, and discovered populations of ILC-like cells. We uncovered a rorc-positive subset of ILCs that could express cytokines associated with type 1, 2, and 3 responses upon immune challenge. Specifically, these ILC-like cells expressed il22 and tnfa after exposure to inactivated bacteria or il13 after exposure to helminth extract. Cytokine-producing ILC-like cells express a specific repertoire of novel immune-type receptors, likely involved in recognition of environmental cues. We identified additional novel markers of zebrafish ILCs and generated a cloud repository for their in-depth exploration.

Figure 1. Rag1^-/- zebrafish have cytokine producing cells in the gut.

A. Representative FACS plots showing the percentage of cells in the lymphocytes’ gate (as defined by FSC/SSC gating) in the gut of wild-type zebrafish (left) and rag1-/-mutant (right). B. Percentage of cells in the lymphocytes’ gate within 50 000 recorded events in the gut of wild-type and rag1^-/-mutant zebrafish. Bars represent the geometric mean ± 95% confidence interval to estimate total number of lymphocytes. Mann-Whitney test. C. qPCR expression of T cells associated markers (cd3z, trac) and lymphocytes’ markers (il7r, lck) in mutant and wild-type zebrafish. Bars represent the geometric mean ± 95% confidence interval to estimate fold changes. Mann-Whitney test. D. Scheme of short-term inflammation experiment. E. qPCR expression of immune type 1 (ifng1-1, ifng1-2), immune type 2 (il4, il13) and immune type 3 (il17a/f3, il22) signature cytokines in the gut of the wild-type (rag1^+/+) and mutant (rag1^-/-) zebrafish following six hours challenge with V. anguillarumor A. simplex. Bars represent the geometric mean ± 95% confidence interval to estimate fold changes. One-way ANOVA test.

Figure 2. Analysis of the lck+cells, collected from the gut of rag1^-/- zebrafish.

2D projection of tSNE analysis of 10x RNAseq data showing heterogeneity of innate lymphoid cells. Dotplots show the level of expression of marker genes and percentage of cells per cluster that express the gene of interest.

Figure 3. Analysis of the lck+cells collected from the gut of wild-type zebrafish.

A. 2D projection of tSNE analysis of 10x RNAseq data showing heterogeneity of innate and adaptive lymphocytes’ pool. B. Dotplot shows the level of expression of signature genes and percentage of cells per cluster that express the gene of interest.

Figure 4. Integrated analysis of PBS - V. anguillarum- A. simplex-injected rag1^-/- zebrafish.

A. Dotplot with the expression level of selected marker genes in each of the clusters. The size of the dots indicates the percentage of cells within the cluster that express the gene of interest; each cluster contains cells from three different conditions. B. Volcano plot showing the top 20 differentially expressed genes between nitr+rorc+(Cluster 7) and nitr-rorc+(Cluster 13) cells originated from rag1^-/- PBS-injected zebrafish using aligned dataset.

Figure 5. Genotype clustering of cells from the rag1^-/- PBS-,V. anguillarum- and A. simplex-injected zebrafish.

The first three principal components were used and the clusters were generated using the probabilistic PCA algorithm. Individual 1 - violet colour; Individual 2 - green colour; Individual 3 - red colour; Undetermined - light violet colour. Bar plots showing frequency (log scaled) of different donor within the cell type clusters under different challenge conditions.