Molecular cloning, expression analysis of interleukin 17D (cysteine knot cytokine) from Amphiprion clarkii and their functional characterization and NFκB pathway activation using FHM cells. Issue 126 (July 2022)

• Record Type: Journal Article
• Authors: Liyanage, D.S.; Omeka, W.K.M.; Nadarajapillai, Kishanthini; Lim, Chaehyeon; Yang, Hyerim; Choi, Ji Young; Kim, Kyong Min; Noh, Jae Koo; Jeong, Taehyug; Lee, Jehee
• Is Part Of: Fish & shellfish immunology. Issue 126(2022)
• Page Start: 217
• Page End: 226
• Publication Date: 2022-07
• Subjects: Interleukin 17D -- Antibacterial -- Gene expression analysis -- NFκB activation; Fishes -- Immunology -- Periodicals; Shellfish -- Immunology -- Periodicals; Poissons -- Immunologie -- Périodiques; Crustacés -- Immunologie -- Périodiques; 571.9617
• DOI: 10.1016/j.fsi.2022.05.047
• Languages: English
• ISSNs: 1050-4648

Abstract

Abstract: Interleukin 17D (IL-17D), a pro-inflammatory cytokine, is a signature cytokine of T helper 17 (Th17) cells. However, studies characterizing the functions of IL-17D in teleost are scarce. Therefore, we aimed to characterize the properties of IL-17D in Amphiprion clarkii . We performed spatial and temporal expression, AcIL-17D -mediated antibacterial and inflammatory gene expression, NFκB pathway-related gene expression analyses, and bacterial colony counting and cell protection assays. We found that AcIL-17D contains a 630 bp coding sequence and encodes 210 amino acids. The spatial expression analysis of AcIL-17D in 12 tissues showed ubiquitous expression, with the highest expression in the brain, followed by blood and skin. Temporal expression analysis of AcIL-17D in blood showed upregulated expression at 6 and 24 h (polyinosinic: polycytidylic acid and lipopolysaccharide), 12 h (all stimulants), and 48 h (polyinosinic: polycytidylic acid and Vibrio harveyi ). AcIL-17D expression in the blood gradually decreased at later hours in response to all the stimulants. After treatment of fathead minnow (FHM) cells with different recombinant AcIL-17D concentrations, the downstream gene expression analysis showed increased expression of antimicrobial genes in the FHM cells, namely [NK-Lysin ( NKL ), Hepcidin antimicrobial peptide-1 ( HAMP-1 ), Defensin-β ( DEFB1 )] and some inflammatory genes such as IL-1β, TNF-α, IL-11, and STAT3. Further nuclear factor κB (NFκB) subunits ( NFκB1, NFκB2, RelA, and Rel-B) showed upregulated gene expression at 12 and 24 h. The bacterial colony counting assay using FHM cells showed lower bacterial colony counts in rAcIL-17D-treated cells than in control. Furthermore, the Water-Soluble Tetrazolium Salt (WST -1) assay confirmed the ability of rAcIL-17D in the protection of FHM cells from bacterial infection and conducted the Hoechst 33342 staining upon treatment with rAcIL-17D and rMBP. Therefore, our findings provide important insights into the activation of IL-17D pathway genes in FHM cells, the protective role of AcIL-17D against bacterial infection, and host defense mechanisms in teleost. Graphical abstract: Image 1 Highlights: AcIL-17D can recruit the antimicrobial peptides and pro-inflammatory cytokines. AcIL-17D can reduce growth of bacteria by releasing several molecules. PcDNA3.1 (+) transfected FHM cells exhibited protection against bacteria. NFκB subunits (NFKB1, NFKB2, Rel-A and Rel-B) showed upregulated gene expression.