Exploring the Role of a Novel Interleukin-17 Homolog from Invertebrate Marine Mussel Mytilus coruscus in Innate Immune Response: Is Negative Regulation by Mc-Novel_miR_145 the Key?

Abstract

Interleukin-17 (IL-17) represents a class of proinflammatory cytokines involved in chronic inflammatory and degenerative disorders. Prior to this study, it was predicted that an IL-17 homolog could be targeted by Mc-novel_miR_145 to participate in the immune response of Mytilus coruscus. This study employed a variety of molecular and cell biology research methods to explore the association between Mc-novel_miR_145 and IL-17 homolog and their immunomodulatory effects. The bioinformatics prediction confirmed the affiliation of the IL-17 homolog with the mussel IL-17 family, followed by quantitative real-time PCR assays (qPCR) to demonstrate that McIL-17-3 was highly expressed in immune-associated tissues and responded to bacterial challenges. Results from luciferase reporter assays confirmed the potential of McIL-17-3 to activate downstream NF-κb and its targeting by Mc-novel_miR_145 in HEK293 cells. The study also produced McIL-17-3 antiserum and found that Mc-novel_miR_145 negatively regulates McIL-17-3 via western blotting and qPCR assays. Furthermore, flow cytometry analysis indicated that Mc-novel_miR_145 negatively regulated McIL-17-3 to alleviate LPS-induced apoptosis. Collectively, the current results showed that McIL-17-3 played an important role in molluscan immune defense against bacterial attack. Furthermore, McIL-17-3 was negatively regulated by Mc-novel_miR_145 to participate in LPS-induced apoptosis. Our findings provide new insights into noncoding RNA regulation in invertebrate models.

Keywords: Mytilus coruscus; apoptosis; innate immunity; interleukin-17; microRNA.

Figure 1

Molecular characterization of McIL-17-3. (A) Architecture analysis of conserved domains in McIL-17-3 using SMART. A conserved IL-17 domain was shown. (B) Phylogenetic analysis of McIL-17-3. The phylogenetic tree was constructed using MEGAX software with 2000 replications of bootstrapping using the neighbor-joining method. McIL-17-3 was labeled with a green triangle. Species included in the phylogenetic tree were all retrieved from the Genebank database, and accession numbers were also listed in the tree. Green triangle on behalf of McIL-17-3. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Figure 2

Multiple alignment of McIL-17-3 with other IL-17 family members. (A) The amino acid sequence of McIL-17-3 was aligned with that of other IL-17s retrieved from mussels and vertebrates, including mice and zebrafish. These cysteines, which form a canonical knot, were marked with green, with replaced amino acid residues marked with red. The cysteines knot is indicated by color lines, and blue meaning their presence in molluscs and red meaning their presence in vertebrates. Note: Only the second half of the sequence alignment is preserved for visualization. (B) The tertiary structure of McIL-17-3 protein was predicted using the Swiss model and pyMol software. These cysteines, which form a canonical knot, were marked. (C) A cartoon representation of the canonical cysteine-knot fold. Cysteine residues were indicated by filled circles; those present in IL-17 proteins were yellow, whereas the two missed were gray. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Figure 3

Expression profile analysis of McIL-17-3 transcripts. (A) Distribution of McIL-17-3 transcripts in common mussel tissues. (B) Temporal expression changes of McIL-17-3 transcripts in response to V. alginolyticus challenge. The results were expressed as mean ± SD (n = 3, * p < 0.05, ** p < 0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Figure 4

The activation of NF-κB reporter by McIL-17-3. The recombinant vector pEGFP-McIL-17-3 in three concentrations (0.1, 0.5, and 1.0 µg/well) was cotransfected into HEK293 cells using Lipo6000^TM for 24 h. The relative luciferase activities were calculated by normalizing to the pRL-TK value. The experimental results were expressed as fold changes by comparing the luciferase activities of recombinant vector-induced cells with those of empty vector-induced cells at the same concentration. Each value was shown as mean ± SD (n = 3), and bars with an asterisk symbol were significantly different (* p < 0.05, ** p < 0.01).

Figure 5

Mc-novel_miR_145 targeted McIL-17-3. (A) The McIL-17-3 3′ UTR sequence was inserted into the pmiR-RB-Report™ vector, respectively constructed wild-type and mutant plasmids. Mc-novel_miR_145 sequence and McIL-17-3-3′UTR target site and mutant site sequence were labeled with red markers. (B) McIL-17-3-3′ UTR-WT plasmid was co-transfected with Mc-novel_miR_145 mimic or Mc-novel_miR_145 inhibitor into HEK293 cells. (C) HEK293 cells were transfected with McIL-17-3-3′UTR-WT or the mutant type of McIL-17-3-3′UTR-MUT, together with Mc-novel_miR_145 or NC, for 24 h. The luciferase activity was measured using the dual-luciferase reporter assay system. (D) The concentration gradient experiments were conducted for Mc-novel_miR_145 transfection. All data are presented as the means ± SD from at least three independent triplicated experiments. **, p < 0.01, *, p < 0.05 versus the controls. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Figure 6

Mc-novel_miR_145 inhibited the expression of McIL-17-3 in M. coruscus hemocytes. (A) The expression of Mc-novel_miR_145 was assessed by qPCR in hemocytes transfected with 145 mimic, 145 inhibitor and their respective control. (B) After transfection for 24 h, the transcriptional levels of McIL-17-3 were determined by qPCR. (C) Recombinant expression, purification, and the antiserum preparation for McIL-17-3. Lane M, standard protein molecular weight marker. Lane 1, negative control (without induction). Lane 2, induced recombinant protein McIL-17-3. Lane 3, purified McIL-17-3. Lane 4, Western blot with anti-McIL-17-3 antibody in the hemocytes of M. coruscus. (D) After transfection for 24 h, the protein levels of McIL-17-3 were determined by Western blot. ** p < 0.01 versus the controls.

Figure 7

The hemocyte apoptotic rate was assessed by flow cytometry using propidium iodide (PI) and FITC-Annexin-V staining. (A) Flow cytometry quadrant diagram. The control group was represented by (a): hemocytes with LPS challenge for 24 h. The experimental group by (b–d) with pEGFP-McIL-17-3 vector, Mc-novel_miR_145 + pEGFP-McIL-17-3, and Mc-novel_miR_145-I + pEGFP-McIL-17-3 addition. The numbers in the lower right quadrant (FITC⁺/PI⁻) represented the percentage of total fluorescence positive for early apoptosis; and the right upper quadrant (FITC⁺/PI⁺), total fluorescence positive for late apoptosis. (B) Significance test of the apoptotic rate. The vertical bars represent the mean ± SD. (n = 3, p < 0.05 *).