Molecular characterization, expression analysis and function identification of Pf_TNF-α and its two receptors Pf_TNFR1 and Pf_TNFR2 in yellow catfish (Pelteobagrus fulvidraco)

Abstract

Inflammation is a common manifestation of body immunity and mediates a cascade of cytokines. Tumor necrosis factor-α (TNF-α), as a multi-effect cytokine, plays an important role in the inflammatory response by interacting with its receptor (TNFR). In this study, Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 genes were cloned from yellow catfish (Pelteobagrus fulvidraco), and bioinformatics analyses showed that the three genes were conserved and possessed similar sequence characteristics as those of other vertebrates. The qPCR results showed that Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 mRNAs were constitutively expressed in 14 tissues and the lymphocytes of four tissues from healthy adults. The mRNA expression levels of Pf_TNF-α and Pf_TNFR1 genes were significantly up-regulated in the spleen, liver, trunk kidney, head kidney and gill after Edwardsiella ictaluri infection, while the mRNA expression of Pf_TNFR2 was significantly up-regulated in the spleen, and down-regulated in the liver and gill. In the isolated peripheral blood leukocytes (PBLs) of yellow catfish, the expression of Pf_TNF-α mRNA was notably up-regulated and the two Pf_TNFR transcripts were distinctly down-regulated after stimulation with lipopolysaccharides (LPS), peptidoglycan (PGN), polyinosinic-polycytidylic acid (Poly I:C) and phytohaemagglutinin (PHA). After stimulated by recombinant (r) Pf_sTNF protein, the mRNA expressions of various inflammatory factors genes were up-regulated in the PBLs. Meanwhile, rPf_sTNF promoted the phagocytic activity of leukocytes, whereas the activity mediated by rPf_sTNF could be inhibited by rPf_TNFR1CRD2/3 and rPf_TNFR2CRD2/3. The up-regulation of TNF-α and IL-1β mRNAs expression triggered by rPf_sTNF could be inhibited by MAPK inhibitor (VX-702) and NF-κB inhibitor (PDTC). rPf_sTNF induced the expression of FADD mRNA in PBLs and increased the apoptotic rate of PBLs, and inhibiting the NF-κB and MAPK signal pathways could enhance the apoptosis of PBLs. The results indicate that Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 play important roles in the immune response of yellow catfish to bacterial invasion.

Keywords: Functional analysis; Gene expression; Pelteobagrus fulvidraco; Pf_TNF-α; Pf_TNFR1; Pf_TNFR2.