A pattern recognition receptor CgTLR3 involves in regulating the proliferation of haemocytes in oyster Crassostrea gigas

Abstract

Toll-like receptors (TLRs) are expressed on various immune cells as key elements of innate and adaptive immunity, and they also play significant roles in regulating cell proliferation and differentiation. In the present study, the binding activity of CgTLR3 to PAMPs and CgMyD88-2, and its role in mediating the proliferation of haemocytes was investigated. The recombinant proteins of the extracellular six LRR domains (rCgTLR3-LRR) and intracellular TIR domain (rCgTLR3-TIR) of CgTLR3 were obtained respectively. rCgTLR3-LRR exhibited binding activity to lipopolysaccharide (LPS), peptidoglycan (PGN), mannan (MAN) and Poly (I:C), with the highest affinity for LPS. While rCgTLR3-TIR displayed binding activity to the recombinant protein of rCgMyD88-2, with KD value of 7.22 × 10−7 M. The CgTLR3 mRNA and protein were detected in three subpopulations of oyster haemocytes, and they were mainly concentrated in granulocytes, which was 7.27-fold (p < 0.05) of that in semi-granulocytes and 8.51-fold (p < 0.01) of that in agranulocytes. The percentage of CgTLR3 positive cells (FITC+ haemocytes) in granulocytes was 4.45-fold (p < 0.01) and 2.57-fold (p < 0.05) of that in agranulocytes and semi-granulocytes, respectively. After Vibrio splendidus stimulation, the mRNA expression level of CgTLR3 in haemocytes significantly upregulated at 6 h and 12 h, which was 2.93-fold (p < 0.05) and 4.15-fold (p < 0.05) of that in the control group. After the expression of CgTLR3 was inhibited by the injection of si-CgTLR3, the expression levels of transcription factors associated with hematopoiesis (CgGATA, CgRunx), cell cycle-related genes (CgPCNA, CgCDC-45, CgCDK-2), the agranulocyte marker CgCD-9, the granulocyte marker CgAATase, and the inflammatory factor CgIL17-1 significantly decreased (p < 0.05) after the V. splendidus stimulation, which were 0.43-fold, 0.83-fold, 0.48-fold, 0.44-fold, 0.53-fold, 0.7-fold, 0.62-fold, and 0.47-fold of that in NC + V. s group in vivo, respectively. Meanwhile, the percentage of EdU+ haemocytes in si-CgTLR3+V. s group was significantly reduced by 0.54-fold (p < 0.05) compared to the control group (2.7%). These results collectively indicated that CgTLR3 was involved in modulating the proliferation of haemocytes by regulating the expression of proliferation-related genes and inflammatory factor in oyster C. gigas.