TLR21 is involved in the NF-κB and IFN-β pathways in largemouth bass (Micropterus salmoides) and interacts with TRIF but not with the Myd88 adaptor

• Published in: Fish & shellfish immunology Vol. 151; p. 109734
• Main Authors: Gao, Fengying, Dong, Junjian, Li, Jiaxin, Zhu, Zhilin, Zhang, Hetong, Sun, Chengfei, Ye, Xing
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.08.2024
• Subjects: Adaptor Proteins, Vesicular Transport - chemistry; Adaptor Proteins, Vesicular Transport - genetics; Adaptor Proteins, Vesicular Transport - immunology; Adaptor Proteins, Vesicular Transport - metabolism; Amino Acid Sequence; Animals; Base Sequence; Bass - genetics; Bass - immunology; Fish Diseases - immunology; Fish Proteins - chemistry; Fish Proteins - genetics; Fish Proteins - immunology; Gene Expression Profiling - veterinary; Gene Expression Regulation - immunology; IFN-β; Immunity, Innate - genetics; Largemouth bass (Micropterus salmoides); Myd88; Myeloid Differentiation Factor 88 - chemistry; Myeloid Differentiation Factor 88 - genetics; Myeloid Differentiation Factor 88 - immunology; Myeloid Differentiation Factor 88 - metabolism; NF-kappa B - genetics; NF-kappa B - immunology; NF-kappa B - metabolism; NF-κB; Phylogeny; Sequence Alignment - veterinary; Signal Transduction - immunology; TLR21; Toll-Like Receptors - chemistry; Toll-Like Receptors - genetics; Toll-Like Receptors - immunology; Toll-Like Receptors - metabolism; TRIF; NF-κB; TLR21; IFN-β; TRIF; Largemouth bass (Micropterus salmoides); Myd88
• ISSN: 1050-4648; 1095-9947; 1095-9947
• DOI: 10.1016/j.fsi.2024.109734

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-β (IFN-β) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-β (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion. •The cDNA sequence of TLR21 was identified from largemouth bass (Micropterus salmoides).•Stimulation with Nocardia seriolae and PolyI:C can change the gene expression levels of TLR21.•TLR21 was mainly present in the cytoplasm of 293T cells.•TLR21 overexpression significantly upregulated NF-κB and IFN-β activity.•TLR21 did not interact with the Myd88 protein but interact with TRIF adaptor.