Immunomodulatory role of vasoactive intestinal peptide and ghrelin in Oncorhynchus mykiss

Abstract

Neuropeptides are a group of peptides derived from precursor proteins synthesized in neuronal and nonneuronal cells. The classical functions of neuropeptides have been extensively studied in mammals, including neuromodulation in the central nervous system, molecular signaling in the peripheral nervous system, and immunomodulation associated mainly with anti-inflammatory activity. In contrast, in teleosts, studies of the immunomodulatory function of these neuropeptides are limited. In Oncorhynchus mykiss, vasoactive intestinal peptide (VIP) mRNA sequences have not been cloned, and the role of VIP in modulating the immune system has not been studied. Furthermore, in relation to other neuropeptides with possible immunomodulatory function, such as ghrelin, there are also few studies. Therefore, in this work, we performed molecular cloning, identification, and phylogenetic analysis of three VIP precursor sequences (prepro-VIP1, VIP2 and VIP3) in rainbow trout. In addition, the immunomodulatory function of both neuropeptides was evaluated in an in vitro model using the VIP1 sequence identified in this work and a ghrelin sequence already studied in O. mykiss. The results suggest that the prepro-VIP2 sequence has the lowest percentage of identity with respect to the other homologous sequences and is more closely related to mammalian orthologous sequences. VIP1 induces significant expression of both pro-inflammatory (IFN-γ, IL-1β) and anti-inflammatory (IL-10 and TGF-β) cytokines, whereas ghrelin only induces significant expression of proinflammatory cytokines such as IL-6 and TNF-α.

Keywords: Cytokines; Ghrelin; Immune system; Neuropeptide; Oncorhynchus mykiss; RTS11 cell line; Vasoactive intestinal peptide.

Fig. 1

Amplification of mature mRNA sequences of rainbow trout VIPs. Conventional PCR products were obtained with the expected sizes for VIP1, VIP2 and VIP3. M: 100 bp DNA Ladder (New England Biolabs). HK: head kidney. G: gut. C: PCR control without cDNA template (no template control). Images of complete gels are shown in Supplementary Material 1.

Fig. 2

Multiple alignments of prepro-VIP mature proteins from O. mykiss and other representative species. In the alignment, sequences corresponding to the signal peptide, PHI peptide and VIP peptide are framed. In addition, “*” indicates positions with a single fully conserved residue, “:” indicates conservation between residues with highly similar properties, and “.” indicates conservation between residues with weakly similar properties. Gaps (indicated with “-“) were considered to achieve complete alignment of all sequences. The sequences of the other teleosts and mammals correspond to S. salar (NCBI Reference Sequence: XP_014010319.1), P. hypophthalmus (XP_026795265.1), D. rerio (NP_001108025.2), T. rubripes (XP_011601347.1), S. aurata (XP_030296899.1), E. lanceolatus (XP_033468632.1), H. hippoglossus (XP_034463973.1), P. olivaceus (XP_019946092.1), H. sapiens (NP_003372.1) and R. norvegicus (NP_446443.1).

Fig. 3

Phylogenetic analysis of VIP nucleotide sequences of O. mykiss and other representative species. A phylogenetic tree was constructed using the neighbor-joining method. All ambiguous positions were removed for each sequence pair (pairwise deletion option). The numbers on the branch indicate the frequencies for 1000 bootstrap analyses. The sequences of the other teleosts and mammals correspond to S. salar (NCBI Reference Sequence: XM_014154844.2), P. hypophthalmus (XM_026939464.2), D. rerio (NM_001114553.3), T. rubripes (XM_011603045.2), S. aurata (XM_030441039.1), E. lanceolatus (XM_033612741.1), H. hippoglossus (XM_034608082.1), P. olivaceus (XM_020090533.1), H. sapiens (NM_003381.4) and R. norvegicus (NM_053991.1).

Fig. 4

Stimulation of RTS11 cells with neuropeptides and analysis of relative cytokine expression. RTS11 cells were stimulated with ghrelin (GHR) and VIP1 at concentrations of 20 nM each for 4 and 8 h. The relative expression of the cytokines IFN-γ (A) IL-6 (B), IL-1β (C), IL-8 (D), TNF-α (E), IL-10 (F) and TGF-β (G), normalized to EF1-α gene expression, was evaluated. Statistical analyses were performed comparing each condition with the control using ordinary one-way ANOVA with Dunnett's multiple comparisons tests. * = P < 0.05; ** = P < 0.01; *** = P < 0.001.