Negative regulation of interferon regulatory factor 6 (IRF6) in interferon and NF-κB signalling pathways of common carp (Cyprinus carpio L.)

Abstract

Background: Interferon (IFN) regulatory factors (IRFs) is a kind of transcription factors, which play an important role in regulating the expression of type I IFN and related genes. In mammals, IRF6 is not relevant with IFN expression, while zebrafish IRF6 was reported to be a positive regulator of IFN expression and could be phosphorylated by both MyD88 and TBK1. However, the role of IRF6 in the immune response and IFN transcription of common carp is unknown.

Results: In the present study, the cDNA of IRF6 gene (CcIRF6) was cloned from common carp using RACE technique, with a total length of 1905 bp, encoding 471 amino acid residues, which possesses two functional domains of DBD and IAD. Similarity analysis showed that CcIRF6 had more than 50% similarity with IRFs of other vertebrates, and had the highest similarity with grass carp and zebrafish, among which the DBD domain was much more conserved. The phylogenetic analysis showed that CcIRF6 is in the branch of Osteichthyes and has the closest relationship with grass carp. In healthy common carp, the CcIRF6 was expressed in all the examined tissues, with the highest level in the oral epithelium, and the lowest level in the head kidney. After intraperitoneal injection of poly(I:C) or Aeromonas hydrophila, the expression of CcIRF6 increased in spleen, head kidney, foregut and hindgut of common carp. Moreover, poly(I:C), LPS, PGN and flagellin induced the expression of CcIRF6 in peripheral leukocytes and head kidney leukocytes of common carp in vitro. In EPC cells, CcIRF6 inhibited the expression of some IFN-related genes and pro-inflammatory cytokines, and dual luciferase reporter assay showed that CcIRF6 reduced the activity of IFN and NF-κB reporter genes.

Conclusions: The present study suggests that CcIRF6 is involved in the antiviral and antibacterial immune response of common carp, and negatively regulate the expression of IFN and NF-κB signalling pathways, which provides a theoretical basis for the study and prevention of fish disease pathogenesis.

Keywords: Aeromonas hydrophila; Common carp (Cyprinus carpio L.); Interferon; Interferon regulatory factor 6 (IRF6); NF-κB; Poly(I:C).

Fig. 1

Multiple alignments (a) and phylogenetic analysis (b) of IRF6 protein sequences in different species. Identical residues are indicated by (*). CcIRF6 is marked with solid diamond (◆). The GenBank accession numbers of the genes are listed in Table 1

Fig. 2

Tissue specific expression of CcIRF6 under normal physiological condition. CcIRF6 expressions in the liver, spleen, head kidney, gills, skin, foregut, hindgut, buccal epithelium, and muscle were determined by Real-time PCR. The expression levels were normalized using the 40S ribosomal protein S11 mRNA. (n = 4, mean ± SD)

Fig. 3

Expression analysis of CcIRF6 in response to poly(I:C) challenge in vivo. Total RNA was extracted from liver (a), spleen (b), head kidney (c), skin (d), foregut (e) and hindgut (f) at 0, 3, 6, 12, 24, 48 and 72 h post injection for Real-time PCR. The expression was normalized to the 40S ribosomal protein S11. (n = 3, mean ± SD, *P < 0.05)

Fig. 4

Expression analysis of CcIRF6 in response to A. hydrophila challenge in vivo. Total RNA was extracted from spleen (a), head kidney (b), foregut (c) and hindgut (d) at 0, 3, 6, 12, 24, 48 and 72 h post injection for Real-time PCR. The expression was normalized to the 40S ribosomal protein S11. (n = 3, mean ± SD, *P < 0.05)

Fig. 5

Expression levels of CcIRF6 in the PBLs induced by poly(I:C), LPS, PGN and flagellin. The expression was normalized using the 40S ribosomal protein S11. (n = 3, mean ± SD, *P < 0.05)

Fig. 6

Expression levels of CcIRF6 in the HKLs induced by poly(I:C), LPS, PGN and flagellin. The expression was normalized using the 40S ribosomal protein S11. (n = 3, mean ± SD, *P < 0.05)

Fig. 7

Effect of CcIRF6 on the expression of IFN-stimulated genes and inflammatory cytokines. The expression levels of the PKR (a), ISG15 (b), Viperin (c), IRF3 (d), TNF-α (e) and IL-10 (f) genes in CcIRF6-overexpressed EPC cells were detected by real-time PCR and normalized to β-actin (n = 3, mean ± SD, *P < 0.05)

Fig. 8

CcIRF6 negatively regulates the IFN and NF-κB pathways. 293 T cells were cotransfected with three IFNs or an NF-κB reporter gene, CcIRF6 and/or TRIF expression plasmids. Luciferase activity was measured after 48 h and determined against Renilla luciferase activity (n = 3, mean ± SD, *P < 0.05)