**Background:** Ghrelin is a multifunctional peptide hormone initially discovered as a ligand for the growth hormone secretagogue receptor (GHSR). Beyond its well-known roles in regulating food intake and energy balance, ghrelin also acts as a cytokine that modulates immune processes across many vertebrate species. In rainbow trout (Oncorhynchus mykiss), two active ghrelin isoforms exist: the full-length 23-amino acid rt-Ghrl and a truncated 20-amino acid isoform (rt-desVRQ-Ghrl) generated by alternative splicing. While ghrelin's metabolic functions are well characterized, its direct immunomodulatory effects in fish—particularly the differential roles of these two isoforms—remain poorly understood. The head kidney in teleost fish is functionally equivalent to bone marrow in higher vertebrates, serving as the primary hematopoietic and lymphoid organ. This study aimed to evaluate the direct immunomodulatory actions of both ghrelin isoforms using primary head kidney cell cultures from rainbow trout.
**Methods:** Primary head kidney cells were isolated from 36 mature rainbow trout (1.5 years old, 1.6 ± 0.11 kg). Cells were cultured for 7 days, with nonadherent cells removed after 24 hours and adherent cells maintained for an additional 6 days to allow differentiation of myeloid and lymphoid cells. Prior to treatment, cells were serum-starved for 24 hours. Cells were then treated with rt-Ghrl (1.25 ng/μL) or rt-desVRQ-Ghrl (1.11 ng/μL) for 0, 2, 4, or 24 hours. Four independent replicates were performed per treatment and time point, totaling 112 cultures. Total RNA was extracted, DNase-treated, and purified. First-strand cDNA was synthesized from 4 μg RNA per 40 μL reaction. Expression of 20 immune-related genes was measured by real-time qPCR using SYBR Green, with elongation factor-1 alpha (ef1a) as the housekeeping gene and beta-actin (actb) for normalization confirmation. Relative expression was calculated using the Pfaffl method. Statistical analysis employed two-way ANOVA with time and treatment as independent variables, followed by Fisher's LSD post-hoc tests (p < 0.05). Hierarchical clustering was performed using the heatmapper web tool with average linkage and absolute Pearson correlation coefficients.
**Key Results:** Hierarchical clustering identified three major gene clusters. Cluster I divided into two subclades: Subclade I contained interferon-gamma (ifng), suppressor of cytokine signaling 1 (socs1), integrin b2 (cd18), and immunoglobulin M (igm)—all showing suppression at 2 hours followed by recovery or augmentation at 4–24 hours. Subclade II contained interferon regulatory factor 7 (irf7) and three antimicrobial peptide genes (beta-defensin 1/db1, hepcidin/hamp, liver-expressed antimicrobial peptide 2/leap2), which were inhibited at 4 hours by rt-Ghrl but elevated at 4 hours by rt-desVRQ-Ghrl. Cluster II comprised 10 genes (tnfa, il1b, il6, tgfb1, cxcl8, cxcl11, lyz, cox2, cd86, cath1) that showed the most divergent responses between isoforms—all were significantly induced by rt-Ghrl at 24 hours, while only il6 and cxcl11 showed >2-fold induction by rt-desVRQ-Ghrl at 24 hours. Nine of ten genes in Cluster II showed significant effects of treatment (p < 0.000), time (p < 0.000), and treatment × time interaction (p < 0.000). Cluster III contained mhc2 and cath2, which were massively upregulated at 2 hours by both isoforms but dropped below baseline by 24 hours. For mhc2 and cath2, rt-desVRQ-Ghrl showed greater overall effects (LS means 1.7 vs. 1.12) than rt-Ghrl. Statistical analyses confirmed that all genes except db1 (no treatment effect, p = 0.99) and tgfb1 (no treatment effect, p = 0.96) showed significant treatment effects.
**Clinical Implications:** This study provides direct evidence that ghrelin isoforms differentially modulate immune gene expression in rainbow trout head kidney cells, supporting ghrelin's role as an immunomodulatory cytokine in fish. The divergent expression patterns between rt-Ghrl and rt-desVRQ-Ghrl suggest these isoforms activate distinct signaling pathways—rt-Ghrl appears to promote pro-inflammatory gene expression at later time points (24 hours), while rt-desVRQ-Ghrl shows more transient or suppressive effects. The modulation of antimicrobial peptides (db1, hamp, leap2, cath1, cath2) by ghrelin isoforms suggests a potential mechanism for enhancing innate immunity. The early induction of mhc2 and cd86 (at 2–4 hours) indicates ghrelin may enhance antigen presentation processes. These findings have potential applications for improving disease resistance and immune function in aquaculture settings. However, further studies are needed to identify the full cohorts of responsive genes and clarify the differential signaling mechanisms by which these ghrelin analogs affect fish immunity.