A soluble TLR5 is involved in the flagellin-MyD88-mediated immune response via regulation rather than activation in large yellow croaker (Larimichthys crocea)

Toll-like receptor 5 (TLR5) plays a crucial role in the immune response through recognizing bacterial flagellin. Some teleosts possess two forms of TLR5, including a canonical membrane TLR5 (TLR5M) ortholog and a piscine soluble TLR5 (TLR5S). In this report, the full-length cDNA sequences of Larimichthys crocea TLR5M (LcTLR5M) and TLR5S (LcTLR5S) were identified. The predicted 885-aa-LcTLR5M protein contained a 20-aa signal peptide, followed by 12 leucine-rich repeats (LRRs), a transmembrane (TM) region, and a cytoplasmic Toll/Interleukin-1 receptor homology (TIR) domain while the predicted 642-aa-LcTLR5S only contained 13 LRRs. The LcTLR5M transcripts were detected in most tissues examined, with the highest expression in heart and the lowest in stomach. The expression of LcTLR5S was high in liver whereas low in other examined tissues. Both LcTLR5M and LcTLR5S transcripts could be induced by immune challenge. Subcellular localization revealed that LcTLR5M existed on the cell membrane while LcTLR5S expressed in the cytoplasm. Furthermore, to investigate the role of LcTLR5S in downstream signaling transduction, a LcTLR5S-TIR chimera was constructed by fusing the ORF of LcTLR5S with TM and TIR domains from LcTLR5M. A dual-luciferase reporter assay revealed that the TIR domain is essential in the flagellin induced MyD88-mediated-TNFα activation but not in -NF-κB activation. However, the flagellin-LcTLR5M-MyD88-mediated NF-κB and TNFα activation was largely suppressed by LcTLR5S. These findings suggested that the flagellin-LcTLR5M/LcTLR5S mediated immune activation was MyD88-dependent, and the role of the TIR-domain might differ between NF-κB and TNFα signaling transduction.