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Antimicrobial peptides (AMPs) are an essential part of the vertebrate innate immune system. Piscidins are a family of AMPs specific in fish. In our previous investigation, we identified four paralogous genes of piscidins in the orange-spotted grouper (Epinephelus coicodes), which exhibited distinct activities against bacteria, fungi, and parasitic ciliated protozoa. Piscidins demonstrated their capability to modulate the expression of diverse immune-related genes; however, their precise immunoregulatory functions remain largely unexplored. In this study, we examined the immunomodulatory properties of putative mature peptides derived from four E. coicodes piscidins (ecPis1S, ecPis2S, ecPis3S, and ecPis4S) in head kidney leukocytes (HKLs) or monocytes/macrophages (MO/MΦ)-like cells isolated from E. coicodes. Our data demonstrate that E. coicodes piscidins exhibit immunomodulatory activities supported by multiple lines of evidence. Firstly, all four piscidins displayed chemotactic activities towards HKLs, with the most potent chemotactic activity observed in ecPis2S. Secondly, stimulation with E. coicodes piscidins enhanced respiratory burst and phagocytic activity in MO/MФ-like cells, with ecPis3S showing the highest efficacy in increasing phagocytosis of MO/MΦ-like cells. Thirdly, mRNA expression levels of chemokine receptors, Toll-like receptors, T cell receptors, and proinflammatory cytokines were modulated to varying extents by the four piscidins in E. coicodes HKLs. Overall, our findings indicate that the immunological activities of these four paralogous piscidins from E. coicodes are exhibited in a paralog-specific and concentration-dependent manner, highlighting their distinct and versatile immunomodulatory properties. This study makes a significant contribution to the field of fish AMPs immunology by elucidating the novel mechanisms through which members of the piscidin family exert their immunomodulatory effects. Moreover, it provides valuable insights for further exploration of fish immunomodulating agents., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Piscidins are important components in protecting microbial infections in teleost. The present study purified and identified a truncated peptide, whose sequence was very close to that of putative mature peptide of epinecidin-1 (piscidin-1) in orange-spotted grouper (Epinephelus coioides), Epi-1 (also named as short form of ecPis-1, ecPis-1S). The immunomodulatory effects of ecPis-1S on splenic lymphocytes of orange-spotted grouper were explored in vitro. The transcriptome study was carried out by De novo transcriptome sequencing (RNA-Seq) in splenic lymphocytes of orange-spotted grouper. Regarding the profiles of gene expressions, 2994 genes were up-regulated and 2679 genes were down-regulated in the splenic lymphocytes stimulated by ecPis-1S. In the case of differential expression genes, 330 genes were involved in immune related pathways. Among them, 34 genes were involved in T cell receptor signaling pathway, 31 genes in natural killer cell mediated cytotoxicity and 23 genes in leukocyte transendothelial migration, respectively. Immune-related genes selected for qRT-PCR verification, such as interleukin-1β (il-1b), tumor necrosis factor α (tnfa), T cell antigen receptor (tcr), major histocompatibility complex class I (mhc I), and mhc II were significantly up-regulated by ecPis-1S (p < 0.05). ecPis-1S could significantly enhance the proliferation of splenic lymphocytes of orange-spotted grouper in vitro (p < 0.05). In addition, the result of qRT-PCR revealed that ecPis-1S also significantly up-regulated cell cycle-related genes, including cyclin A (cyca), cyclin-dependent kinase 2 (cdk2), cdk4, cell division cycle protein 6 (cdc6), and transforming growth factor β (tgfb) (p < 0.05), which suggested that ecPis-1S promoted the proliferation of lymphocytes by activating cell division cycle. In conclusion, the results indicated that the mature peptide of piscidin-1 in orange-spotted grouper could act as immune modulator and play an important role in regulation of the immune response in fish., (Copyright © 2018 Elsevier Ltd. All rights reserved.)