1. Immunology and Inflammation
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IL17 factors are early regulators in the gut epithelium during inflammatory response to Vibrio in the sea urchin larva

  1. Katherine M Buckley  Is a corresponding author
  2. Eric Chun Hei Ho
  3. Taku Hibino
  4. Catherine S Schrankel
  5. Nicholas W Schuh
  6. Guizhi Wang
  7. Jonathan P Rast  Is a corresponding author
  1. University of Toronto, Canada
  2. Sunnybrook Research Institute, Canada
Research Article
Cite this article as: eLife 2017;6:e23481 doi: 10.7554/eLife.23481
7 figures, 1 table and 3 additional files

Figures

Sea urchin larvae exhibit changes in cell behavior and gene expression following exposure to specific bacterial strains.

(a,b) The larval cellular immune response includes pigment cell migration to the gut epithelium. An uninfected control larva (a), and a larva exposed to V.d. for 24 hr (b) are shown. The red color of pigment cells is a consequence of echinochrome A, a naphthoquinone that is encapsulated in large granules. Under typical laboratory conditions, pigment cells localize to the outer ectoderm. In response to certain bacterial isolates, these cells migrate through the blastocoel to interact with the gut epithelium (Ch Ho et al., 2016). (c) Genes are activated with a variety of kinetics that varies among functional classes. RNA-Seq was performed on larvae collected at 0, 6, 12, and 24 hr of exposure to V.d. Differentially expressed transcripts (RPKM ≥3, fold-change in expression ≥2; 3238 total transcripts) were hierarchically clustered with average linkages to identify similarly temporally regulated genes using Gene Cluster 3.0. High expression is indicated in dark blue; low expression is shown in white. (d) A subset of IL17 genes is upregulated early in infection. Transcript levels are shown for 16,920 genes that are expressed in larvae during the infection time course. Expression levels (RPKM) are shown for uninfected larvae (x-axis) and larvae exposed to V.d. for 6 hr (y-axis). Most genes were not strongly differentially expressed at these two time points (gray). The 127 genes that exhibit ≥3-fold changes in expression levels between 0 and 6 hr of infection are shown in black. The genes within the SpIL17 subfamilies are indicated (SpIL17-1, red; SpIL17-4, green). The dashed box is enlarged in the inset. (e) Mapped reads were used to identify a novel SpIL17 transcript. RNA-Seq reads were mapped to the S. purpuratus genome. Genomic regions that contained mapped reads but no known genes were investigated for domains that are associated with immunity (Scaffold1325:145,000–160,000 from S. purpuratus genome, v3.1 is shown). The number of reads normalized to library size is plotted for each time point as a stacked bar graph. Reads that map to the positive strand are shown with positive values; negative values indicate reads that map on the negative strand. The exons of the experimentally confirmed SpIL17-4a transcript sequences (SpIL17-4a and SpIL17-4a´) are indicated in black. The location of the IL17 domain encoded by the transcripts is shown in gray.

https://doi.org/10.7554/eLife.23481.003
Figure 2 with 1 supplement
Phylogenetic analysis of the sea urchin IL17 sequences defines 10 subfamilies.

Predicted amino acid sequences of the 30 IL17 proteins from S. purpuratus (purple lines) and 14 IL17 sequences from L. variegatus (green lines) were aligned and used in a phylogenetic analysis. The neighbor-joining tree is shown to scale and was constructed in MEGA6.0 using Poisson corrected evolutionary distances, a gamma distribution model for rate variation among sites and complete deletion of alignment positions containing gaps (Tamura et al., 2013). Asterisks indicate branches with bootstrap values greater than 75 and based on 500 replicates. Each of the 10 major clades was recovered in phylogenetic trees constructed using maximum likelihood and maximum parsimony methods as well as variable model parameters using the neighbor-joining method (data not shown). Groups are indicated by brackets. Subfamilies expressed in the larval immune response are shown in blue; the family that is expressed in adult immune cells is indicated in red.

https://doi.org/10.7554/eLife.23481.004
Figure 2—figure supplement 1
Phylogenetic analysis of the echinoderm IL17 sequences.

Predicted IL17 genes were identified from the genome sequences of S. purpuratus (Sp; purple), L. variegatus (Lv; green), E. tribuloides (Et; brown), and P. miniata (Pm; red). Coordinates for each of the genes are shown in Supplementary file 2. Amino acid sequences were used in a phylogenetic analysis in MEGA6.0. The tree shown was constructed using Neighbor-joining methods using Poisson corrected evolutionary distances and complete deletion of alignment positions that contain gaps. Bootstrap values (based on 1000 replicates) are shown for nodes with >50% support. Groups as defined in S. purpuratus are indicated in bold.

https://doi.org/10.7554/eLife.23481.005
Figure 3 with 3 supplements
Gene structure and diversity of the SpIL17-1 and -4 genes.

(a,b) Coding sequence is shown in the colored boxes; non-coding sequence is in white boxes. Untranslated regions have been verified using RACE PCR and through analysis of the RNA-Seq data. The genomic structure of all the SpIL17 genes is shown in Figure 3—figure supplement 1. (a) The SpIL17-1 genes are arrayed in a tandem cluster. The eight SpIL17-1 genes (light gray) and the adjacent SpIL17-10a gene (black) are located on a single scaffold in a 59.8 kb region (Scaffold1147; Genbank KN912785). The SpIL17-1 genes are encoded in two exons, the first of which includes the methionine and a single amino acid. The entire region is located on BAC clone R3-17F18, which was used to construct a GFP reporter for gene spIL17-1d. The position of the GFP in this reporter construct is indicated. (b) The spIL17-4a gene encodes two transcripts that initiate from distinct TSS. The nucleotide and translated amino acid sequences are shown for each of the initial exons. (c) The sea urchin and human IL17 proteins share key cysteine residues. The amino acid sequences of the IL17 domains of a member of each of the SpIL17 subfamilies as well as the six human IL17 factors are shown. The conserved cysteine residues implicated in forming the cysteine knot are highlighted in dark gray with white text. Positions in which the SpIL17 proteins have a cysteine that corresponds to a conserved serine in vertebrates are shaded light gray. Additional conserved cysteine residues are indicated in bold.

https://doi.org/10.7554/eLife.23481.007
Figure 3—figure supplement 1
Genomic organization of the S. purpuratus and L. variegatus IL17 genes.

Coding sequence is shown as colored boxes (according to the groups defined in Figure 1; the color scheme is indicated below the scaffolds). Untranslated regions and predicted pseudogenes are shown as white boxes. Scaffolds and genes are shown to scale except for large intergenic regions, which are abbreviated by brackets (total size is indicated in kb). Scaffolds from S. purpuratus are shown in black; L. variegatus scaffolds are in gray. The red asterisk indicates the location of two additional transcripts: a transcript that encodes a protein with an SGNH_hydrolase domain (SPU_005467) and a predicted non-coding RNA (ncRNA; Genbank XR_973245.1). Analysis of the RNA-Seq data indicates that these two transcripts are expressed at low levels in larvae, but expression levels do not change in the course of infection (data not shown).

https://doi.org/10.7554/eLife.23481.008
Figure 3—figure supplement 2
The SpIL17 sequences within subfamilies are highly conserved.

The average percent identities of the proteins encoded by the SpIL17 genes within (boxes are outlined in black) and among subfamilies (see Figure 2) are shown. When only a single sequence is present within a subfamily, the within group identities cannot be calculated (indicated as blank boxes). Protein identities were also calculated against the six human IL17 (HsIL17) proteins (IL17A-F).

https://doi.org/10.7554/eLife.23481.009
Figure 3—figure supplement 3
Diversity of the IL17 proteins.

The proteins encoded by the SpIL17 genes exhibit greater conservation within the IL17 domain. The diversity of each position within the alignment of SpIL17 protein sequences was calculated as a measure of entropy (Shannon, 1948). The average entropy over a 15 amino acid sliding window is shown (blue line). The position of the IL17 domain is highlight in gray. The average entropy of the N-terminal sequence and the C-terminal IL17 domain is shown as a dashed gray line.

https://doi.org/10.7554/eLife.23481.010
Figure 4 with 1 supplement
Expression of the SpIL17 factors in response to bacterial infection.

(a). Genes within two SpIL17 subfamilies are quickly upregulated in response to bacteria. Expression of the SpIL17-1 (red bars) and SpIL17-4 (blue bars) genes was measured by RT-qPCR. Relative expression values are normalized to the level of expression in uninfected larvae (0 hr). Non-normalized data with error bars are shown in Figure 4—figure supplement 1. Oligonucleotides used in the RT-qPCR reaction anneal to all the SpIL17-1 genes and both of the SpIL17-4 transcripts (Supplementary file 1). (b,c). The two SpIL17-4a transcripts are both expressed during infection. Transcript levels of the SpIL17-4a (dark green) and −4a´ (light green) transcripts were measured are shown as expression relative to 18S transcripts (b) and as the proportion of total SpIL17-4 (blue) as measured with primers located in the shared exons 3 and 4 (c; see gene structure in Figure 2b). Only time points with significant SpIL17-4 transcript levels are shown in (c). (d) Activation of the SpIL17 genes precedes transcriptional changes in many other genes. Transcript prevalence was measured using RT-qPCR for genes that are either known to be involved in immune response in either sea urchins or other organisms. Expression values are log transformed and centered on the mean values for each gene.

https://doi.org/10.7554/eLife.23481.011
Figure 4—figure supplement 1
Many genes are transcriptionally regulated in larvae responding to microbial perturbation of the gut.

Larvae were collected at 0, 2, 4, 6, 8, 12, and 24 hr of exposure to V.d. RNA isolated from the larvae was used in RT-qPCR assays. Reactions were performed in at least triplicate. Error bars indicate the standard deviation among the replicates. Relative expression is normalized to 18S expression.

https://doi.org/10.7554/eLife.23481.012
Figure 5 with 1 supplement
The SpIL17-1 and −4 genes are expressed in gut epithelial cells in response to bacterial challenge.

Expression of the SpIL17-1 (a, c, e–g) and −4 (b, d, h, i) genes was assessed using WMISH (a–d) and BAC-based GFP reporter constructs (e–i). White numbers shown in (a, b) indicate the number of positive larvae out of the total examined. Hours post-infection (hpi) with V.d. are indicated in yellow. Larval morphology is shown in c1 – i1 (b, blastocoel, yellow; hg, hindgut; mg, midgut; gut, green; skeleton, purple; blastocoelar immune cells, blue). White dashed lines shown in c2 – i2 indicate the location of the insets. Black scale bars indicate 50 µM; white bars indicate 20 µM.

https://doi.org/10.7554/eLife.23481.013
Figure 5—figure supplement 1
Transgene reporter constructs recapitulate endogenous SpIL17-1 expression.

Larvae transgenic for either the BAC reporter or GFP Construct (shown in a) were exposed with V.d. and used for RT-qPCR analysis. Expression values are normalized to the number of transgenes incorporated into the genomic DNA as described in Solek et al. (2013).

https://doi.org/10.7554/eLife.23481.014
The SpIL17-9 genes are expressed in adult coelomocytes.

Data from two independent experiments are shown (a,b). qPCR was used to measure transcript prevalence in coelomocytes collected from adult animals that were either injected with live V.d. (animals 1, 2, and 4) or sham injection controls (ASW only; animals 3 and 5). The treatment for each animal is indicated below the graphs. Expression levels for the SpIL17-9 genes (a1, b1) increase strongly by 3 hr of exposure to bacteria, and more slowly in response to injury. Expression of the effector genes 185/333 (a2, b2) serves as a marker of immune activation (Ghosh et al., 2010).

https://doi.org/10.7554/eLife.23481.015
Figure 7 with 4 supplements
Two IL17 receptors mediate IL17 signaling in the sea urchin.

(a) The sea urchin IL17 receptor sequences have similar domain architectures as those in vertebrates. The sea urchin receptors have conserved SEFIR domains (red). SpIL17R1 also has a TILL domain (blue). The structure of the protein encoded by the SpIL17R1 transcript in the presence of the splice-blocking MASO (MASOSplice) is also shown. This MASO interferes with splicing by binding to the donor splice site in exon 15 (see Figure 7—figure supplement 2). Consequently, a cryptic donor splice site in exon 14 is used, which introduces a frameshift and premature stop codon. The resulting truncated protein does not contain a transmembrane or SEFIR domain (indicated by white shapes). (b). Interfering with IL17 signaling affects the expression of downstream genes during immune challenge. Fertilized eggs were injected with the IL17R1 MASOSplice and grown to 10 dpf. Larvae were infected with V.d. and collected for RT-qPCR analysis. Complete data are shown in Figure 7—figure supplement 3.

https://doi.org/10.7554/eLife.23481.016
Figure 7—figure supplement 1
Phylogeny of SEFIR domain-containing proteins.

SEFIR domains from IL17 receptors and Act1/CIKS molecules were collected and used in a phylogenetic analysis. The tree shown was constructed using Neighbor-joining methods using a Poisson substitution model and a Gamma distribution for variation among sites in MEGA6.086. Similar results were obtained using maximum parsimony and maximum likelihood methods (data not shown). Bootstrap values based on 500 replicates are indicated (* > 50; ** > 75). Act1 sequences are indicated in red; IL17 receptor sequences in shades of blue. Large clusters of sequences are condensed to colored boxes. Species included in these boxes are listed below. The IL17 receptor sequences from sea urchin species are shown in bold. A complete list of the sequences used in the analysis is shown in Supplementary file 3.

https://doi.org/10.7554/eLife.23481.017
Figure 7—figure supplement 2
The splice-blocking SpIL17R1 MASOS yields a transcript with a frameshift and premature stop codon.

Fertilized eggs were injected with either the splice-blocking MASO (MASOSpl.; gray sequences) or a control MASO (MASOCon.; black sequences). Exons 14 through 17 from SpIL17R1 transcripts were amplified, cloned and sequenced from larvae (10 dpf). The sequences are shown. The boundaries of exons 14-17 are indicated by black lines. As a consequence of the MASOS, a cryptic donor splice site is used (indicated in red) that results in a frameshift and premature stop codon (shown in green) that results in a truncated protein.

https://doi.org/10.7554/eLife.23481.018
Figure 7—figure supplement 3
The S. purpuratus genome encodes two IL17 receptors.

Gene structures are shown for the SpIL17-R1 (a) and SpIL17-R2 (b) genes. Sequences encoding the predicted transmembrane domains are shown in green; SEFIR domains are shown in red. Genes are shown to scale, except for large introns, which are abbreviated by brackets (total intron size is shown in kb). The binding sites for the SpIL17-R1 MASOs are indicated by red bars. The incorrect splice products that are generated in the presence of the MASOSplice are indicated by the dashed red line. (c) Expression levels of the SpIL17 receptors are regulated during embryogenesis. qPCR was used to measure the transcript prevalence for the SpIL17-R1 (blue) and SpIL17-R2 (red) genes. Error bars indicate deviation among replicates. Expression of SpIL17-R2 peaks at 48 hpf, whereas SpIL17-R1 expression increases during development into the larval stage (72 hpf). (d) Expression of the SpIL17-R1 transcript varies in response to exposure to V.d. (e) The SpIL17-R1 transcript is primarily expressed in the larval gut. WMISH was performed on uninfected larvae (10 dpf). (f) The SpIL17-R2 transcript is slightly upregulated at 2 hr of exposure to V.d., but returns to unexposed levels by 4 hr. (g) The MASOSplice specifically affects the expression of SpIL17-R1 exon 15. (h) Perturbation of IL17 signaling does not affect pigment cell migration during immune response.

https://doi.org/10.7554/eLife.23481.019
Figure 7—figure supplement 4
Effects of IL17R1 perturbation on downstream gene expression.

RT-qPCR was used to assess expression of genes involved in the larval immune response to bacteria. Larvae were exposed to either control MASO (gray), SpIL17-R1 MASOSplice (red) or MASOTranslation (blue). Error bars indicate deviation among replicates.

https://doi.org/10.7554/eLife.23481.020

Tables

Table 1

Numbers of IL17 genes by subfamily in echinoderm species.

https://doi.org/10.7554/eLife.23481.006
Echinodermata
EchinoideaAsteroidea
EuechinoideaCidaroidea
StrongylocentrotidaeToxopneustidae
SubfamilyS. purpuratusS. fragilis*
(5–7 myr)
M. franciscanus*a
(20 myr)
L. variegatus
(50 myr)
E. tribuloides
(268 myr)
P. miniata
(480 myr)
1118.610.0260
210.50.4110
310.50.9200
411.90.4130
523.33.5220
634.44.4230
712.97.8120
811.96.5120
9713.87.1100
1021.05.2100
Other----412
Total3038.647.0152212
  1. *Estimates are based on the number of best reciprocal blast hits using the SpIL17 sequences against the unassembled genomic trace sequences (Buckley and Rast, 2012).

  2. Estimated divergence times shown in million years from S. purpuratus (Thompson et al., 2015; Pisani et al., 2012; Biermann et al., 2003; Smith et al., 2006).

  3. See Figure 2—figure supplement 1 for the phylogenetic analysis of these genes.

Additional files

Supplementary file 1

Sequences of oligonucleotides used for qPCR, RACE, WMISH, and reporter BAC constructs.

https://doi.org/10.7554/eLife.23481.021
Supplementary file 2

Genomic coordinates for the echinoderm IL17 genes.

https://doi.org/10.7554/eLife.23481.022
Supplementary file 3

SEFIR domain-containing proteins.

https://doi.org/10.7554/eLife.23481.023

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