PMID- 37593738 OWN - NLM STAT- MEDLINE DCOM- 20230821 LR - 20230823 IS - 1664-3224 (Electronic) IS - 1664-3224 (Linking) VI - 14 DP - 2023 TI - Molecular characterization, gene expression and functional analysis of goldfish (Carassius auratus L.) macrophage colony stimulating factor 2. PG - 1235370 LID - 10.3389/fimmu.2023.1235370 [doi] LID - 1235370 AB - BACKGROUND: Macrophage colony-stimulating factor 2 (MCSF-2) is an important cytokine that controls how cells of the monocyte/macrophage lineage proliferate, differentiate, and survive in vertebrates. Two isoforms of MCSF have been identified in fish, each exhibiting distinct gene organization and expression patterns. In this study, we investigated a goldfish MCSF-2 gene in terms of its immunomodulatory and functional properties. METHODS: In this study, goldfish were acclimated for 3 weeks and sedated with TMS prior to handling. Two groups of fish were used for infection experiments, and tissues from healthy goldfish were collected for RNA isolation. cDNA synthesis was performed, and primers were designed based on transcriptome database sequences. Analysis of gfMCSF-2 sequences, including nucleotide and amino acid analysis, molecular mass prediction, and signal peptide prediction, was conducted. Real-time quantitative PCR (qPCR) was used to analyze gene expression levels, while goldfish head kidney leukocytes (HKLs) were isolated using standard protocols. The expression of gfMCSF-2 in activated HKLs was investigated, and recombinant goldfish MCSF-2 was expressed and purified. Western blot analysis, cell proliferation assays, and flow cytometric analysis of HKLs were performed. Gene expression analysis of transcription factors and pro-inflammatory cytokines in goldfish head kidney leukocytes exposed to rgMCSF-2 was conducted. Statistical analysis using one-way ANOVA and Dunnett's post hoc test was applied. RESULTS: We performed a comparative analysis of MCSF-1 and MCSF-2 at the protein and nucleotide levels using the Needleman-Wunsch algorithm. The results revealed significant differences between the two sequences, supporting the notion that they represent distinct genes rather than isoforms of the same gene. Sequence alignment demonstrated high sequence identity with MCSF-2 homologs from fish species, particularly C. carpio, which was supported by phylogenetic analysis. Expression analysis in various goldfish tissues demonstrated differential expression levels, with the spleen exhibiting the highest expression. In goldfish head kidney leukocytes, gfMCSF-2 expression was modulated by chemical stimuli and bacterial infection, with upregulation observed in response to lipopolysaccharide (LPS) and live Aeromonas hydrophila. Recombinant gfMCSF-2 (rgMCSF-2) was successfully expressed and purified, showing the ability to stimulate cell proliferation in HKLs. Flow cytometric analysis revealed that rgMCSF-2 induced differentiation of sorted leukocytes at a specific concentration. Moreover, rgMCSF-2 treatment upregulated TNFalpha and IL-1beta mRNA levels and influenced the expression of transcription factors, such as MafB, GATA2, and cMyb, in a time-dependent manner. CONCLUSION: Collectively, by elucidating the effects of rgMCSF-2 on cell proliferation, differentiation, and the modulation of pro-inflammatory cytokines and transcription factors, our findings provided a comprehensive understanding of the potential mechanisms underlying gfMCSF-2-mediated immune regulation. These results contribute to the fundamental knowledge of MCSF-2 in teleosts and establish a foundation for further investigations on the role of gfMCSF-2 in fish immune responses. CI - Copyright (c) 2023 Gouife, Ban, Yue, Jiang and Xie. FAU - Gouife, Moussa AU - Gouife M AD - School of Marine Sciences, Ningbo University, Ningbo, Zhejiang, China. FAU - Ban, Ziqi AU - Ban Z AD - School of Marine Sciences, Ningbo University, Ningbo, Zhejiang, China. FAU - Yue, Xinyuan AU - Yue X AD - School of Marine Sciences, Ningbo University, Ningbo, Zhejiang, China. FAU - Jiang, Jianhu AU - Jiang J AD - Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture, Zhejiang Institule of Freshwater Fisheries, Huzhou, Zhejiang, China. FAU - Xie, Jiasong AU - Xie J AD - School of Marine Sciences, Ningbo University, Ningbo, Zhejiang, China. AD - Key Laboratory of Aquacultural Biotechnology, Ministry of Education, Ningbo University, Ningbo, China. AD - National Engineering Research Laboratory of Marine Biotechnology and Engineering, Ningbo University, Ningbo, Zhejiang, China. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20230801 PL - Switzerland TA - Front Immunol JT - Frontiers in immunology JID - 101560960 RN - 81627-83-0 (Macrophage Colony-Stimulating Factor) RN - 0 (Cytokines) SB - IM MH - Animals MH - *Macrophage Colony-Stimulating Factor MH - *Goldfish/genetics MH - Phylogeny MH - Cytokines/genetics MH - Gene Expression PMC - PMC10431942 OTO - NOTNLM OT - cytokine OT - fish OT - hematopoiesis OT - macrophage colony-stimulating factor 2 OT - progenitors COIS- The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. EDAT- 2023/08/18 06:42 MHDA- 2023/08/21 06:42 PMCR- 2023/01/01 CRDT- 2023/08/18 03:59 PHST- 2023/06/06 00:00 [received] PHST- 2023/07/13 00:00 [accepted] PHST- 2023/08/21 06:42 [medline] PHST- 2023/08/18 06:42 [pubmed] PHST- 2023/08/18 03:59 [entrez] PHST- 2023/01/01 00:00 [pmc-release] AID - 10.3389/fimmu.2023.1235370 [doi] PST - epublish SO - Front Immunol. 2023 Aug 1;14:1235370. doi: 10.3389/fimmu.2023.1235370. eCollection 2023.