The Evolution and Diversity of Interleukin-17 Highlight an Expansion in Marine Invertebrates and Its Conserved Role in Mucosal Immunity

Abstract

The interleukin-17 (IL-17) family consists of proinflammatory cytokines conserved during evolution. A comparative genomics approach was applied to examine IL-17 throughout evolution from poriferans to higher vertebrates. Cnidaria was highlighted as the most ancient diverged phylum, and several evolutionary patterns were revealed. Large expansions of the IL-17 repertoire were observed in marine molluscs and echinoderm species. We further studied this expansion in filter-fed Mytilus galloprovincialis, which is a bivalve with a highly effective innate immune system supported by a variable pangenome. We recovered 379 unique IL-17 sequences and 96 receptors from individual genomes that were classified into 23 and 6 isoforms after phylogenetic analyses. Mussel IL-17 isoforms were conserved among individuals and shared between closely related Mytilidae species. Certain isoforms were specifically implicated in the response to a waterborne infection with Vibrio splendidus in mussel gills. The involvement of IL-17 in mucosal immune responses could be conserved in higher vertebrates from these ancestral lineages.

Keywords: IL-17; comparative genomics; comparative immunology; cytokines; evolution; interleukin; mucosal immunity; mussel.

Figure 1

Phylogenetic analysis of the 379 unique IL-17 sequences obtained from the 16 mussel genomes. The 379 unique IL-17 sequences retrieved from 16 mussel genomes were submitted to phylogenetic analysis with Bayesian phylogenetic inference using the Jukes and Cantor (JC) evolution model. The 23 different IL-17 clusters or isoforms obtained are indicated with ellipses that englobe all their unique variants. The figure shows branch posterior probabilities. It can be seen how isoforms IL17-3 and IL17-16 would be diverging from IL17-17, since the branch of the latter contains both isoforms. The distinction between these isoforms was maintained due to the homology criterion and to expression evidence.

Figure 2

Phylogenetic analysis of the 96 unique IL-17R sequences obtained from the 16 mussel genomes. The 96 unique IL-17R sequences retrieved from 16 mussel genomes were submitted to phylogenetic analysis with Bayesian phylogenetic inference using the GTR+G evolution model. The 6 different IL-17 clusters or isoforms obtained are indicated. The figure shows branch posterior probabilities.

Figure 3

Phylogenetic analysis of the IL-17 families found in Mytilidae species. Homologous sequences to the M. galloprovincialis IL-17 isoforms were found in several Mytilidae species and submitted to neighbor joining analysis using the Jukes–Cantor substitution model. Clustering was based on isoform homologs, and species are indicated with colors.

Figure 4

Comparative genomics analysis of IL-17 gene families throughout evolution. IL-17 repertoires were analyzed in several species from different phyla and clustered with an 80% homology criterion. The evolutionary cladogram is presented, along with the extension of the IL-17 family from each species.

Figure 5

IL-17 genes often appear in tandem repetitions in the genomes of marine invertebrates with expanded IL-17 repertoires. Examples of several scaffolds with IL-17 tandem duplications are presented for these species. IL-17s are indicated with the name of the cluster to which they belong in our analysis. Tandem repetitions can occur with genes from the same or different isoforms. Neighbor genes are represented with their functional annotations.

Figure 6

Expression of the different mussel IL-17 isoforms in the gill transcriptome against a waterborne bacterial infection (A) and in the hemocyte transcriptome against a bacterial infection injected into the muscle (B). It is shown that the isoforms expressed in each transcriptome changed completely. Colored pie charts represent the diversity of expressed IL-17 isoforms in each individual mussel from the control (C1, C2, C3) and infected conditions (I1, I2, I3). The white pie charts below represent, for each experiment, the weight that the expression values of each of the upper graphs have (calculated by the sum of the expression values of every IL-17 form for each of the individual samples). Clear upregulation of IL-17 expression is seen in the gill transcriptome against infection but not in the hemocyte transcriptome. Expression of IL-17 receptors in the same gill (C) and hemocyte (D) transcriptomes. Regarding the IL-17 receptors, it can be seen that the expressed isoforms also varied between transcriptomes.

Figure 7

Normalized expression by qPCR of IL-17 isoforms expressed in the gill (A) and in the adductor muscle hemocytes of the same individual mussels (B) after a 24-hour waterborne infection with Vibrio splendidus, as well as the expression of the receptors (C). In gills, upregulation of the expressed IL-17 isoforms occurred, whereas in hemocytes, there was downregulation. Receptors did not show the antagonistic regulation between hemocytes and gills seen with the IL-17 genes, but instead, the expression of each receptor was correlated in both gill and hemocyte samples.