Molecular and immunological characterizations of interleukin-11 in large yellow croaker (Larimichthys crocea).

Interleukin (IL)-11 is a multifunctional cytokine that exerts a series of important immunomodulatory effects and exists in many tissues and cells. A 1106-bp nucleotide sequence representing the complete cDNA of IL-11 was obtained from large yellow croaker (Larimichthys crocea), containing an open reading frame (ORF) of 603 bp encoding for 200 amino acids (aa). The predicted LcIL-11 protein included a 12aa signal peptide and a conserved IL-11 domain. The polypeptide sequence identities between LcIL-11 and its counterparts in mammals and other fish are from 84% to 92% with known fish IL-11a and 22%-27% with fish IL-11b. LcIL-11 mRNA existed in most tissues with the most predominant expression in the gill. After immune challenge, the expression levels of LcIL-11 were induced largely in vivo and in vitro, with the peak-value of 32 times as much as the control in the liver at 24 h after Vibrio parahaemolyticus injection (p < 0.05) and the greatest value of 13.9 times as much as the control in LCK cells at 12 h after poly I:C stimulation (p < 0.05). Furthermore, the overexpression vector pcDNA3.1-LcIL-11 was constructed and transfected to LCK cells. Our results showed that the transcriptional expression levels of tumor necrosis factor (TNF)-α and myxovirus resistant protein (Mx) significantly up-regulated in LCK cells after LcIL-11 overexpression (p < 0.05). However, no significant changes of IL-1ß, janus kinase (JAK)2 and signal transducers and activators of transcription (STAT)5 was detected. Our finding indicated that LcIL-11 might enhance TNF-α and antiviral protein Mx expression in large yellow croaker.

Cloning and functional characterization of IRAK4 in large yellow croaker (Larimichthys crocea) that associates with MyD88 but impairs NF-κB activation.

As crucial signaling transducer in Toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling pathway, IL-1R-associated kinase 4 (IRAK4) mediates downstream signaling cascades and plays important roles in innate and adaptive immune responses. In the present study, an IRAK4 orthologue was characterized from large yellow croaker (Larimichthys crocea), named Lc-IRAK4, with a conservative N-terminal death domain and a C-terminal protein kinase domain. The genome of Lc-IRAK4 is structured into eleven exons and ten introns. Expression analysis indicated that Lc-IRAK4 was widely expressed in tested tissues, with the highest level in liver and weakest in muscle. Additionally, in the spleen, liver tissues and blood, it could be induced by poly I:C and LPS stimulation, but not be induced by Vibrio parahemolyticus infection. Fluorescence microscopy assays revealed that Lc-IRAK4 localized in the cytoplasm in HEK 293T cells. It was also determined that Lc-IRAK4 could interact with MyD88, whereas MyD88-mediated NF-κB activation was significantly impaired when co-transfected the two in HEK 293T cells. These findings collectively indicated that although Lc-IRAK4 was evolutionarily conserved in vertebrates, the exact function especially the signaling transduction mediated by IRAK4 in fish immune response was different from that in mammals, which impaired MyD88-mediated NF-κB activation.

Identification and expression profiles of ERK2 and ERK5 in large yellow croaker (Larimichthys crocea) after temperature stress and immune challenge.

Fish is highly affected by many environmental stresses such as temperature and invasive infection. The extracellular signal-regulated kinase (ERK) pathway, part of the mitogen-activated protein kinase (MAPK) family, is found to act as crucial mediators for cell differentiation, proliferation and cell response to various stresses. In the present study, ERK2 (LcERK2) and ERK5 (LcERK2) were cloned and characterized from large yellow croaker, Larimichthys crocea. The full length cDNA sequence of LcERK2 was of 1910 bp, including an ORF of 1110bp encoding a polypeptide of 369 amino acids. The full length cDNA sequence of LcERK5 was of 3720bp, including an ORF of 3375bp encoding a polypeptide of 1124 amino acids. Multiple alignments showed that both LcERK2 and LcERK5 contained highly conserved TEY motif and S_TKc domain in MAPK family and the unique catalytic and active structures of ERK2 and ERK5. Subcellular localization revealed that both LcERK2 and LcERK5 expressed in the cytoplasm and cell nucleus. The expression of LcERK2 and LcERK5 were detected in most tissues of large yellow croaker, with the most predominant expression of LcERK2 in brain and LcERK5 in gill, and the weakest expression of LcERK2 in liver and LcERK5 in intestine, respectively. The expression levels of LcERK2 and LcERK5 after temperature stress and poly I:C and flagellin challenge were investigated in LCK (L. crocea kidney) cells. After temperature stress, significant down-regulations of LcERK2 transcripts were detected after 10 °C stress (p < 0.05) whereas LcERK2 transcripts increased significantly after 35 °C stress (p < 0.05). However, significant down-regulations of LcERK5 expression were detected at most time points after both cold and heat stress (p < 0.05). However, significant up-regulations of LcERK2 and LcERK5 transcripts were found after immune challenge (p < 0.05). Our results showed that LcERK2 transcripts enhanced after heat stress and both LcERK2 and LcERK5 transcripts could be induced by immune challenge. These findings indicated that LcERK2 might be more important in fish response to high temperature stress and both LcERK2 and LcERK5 might play an important role in fish immune response.

Characterization and expression analysis of Toll-like receptor 2 gene in large yellow croaker, Larimichthys crocea.

Toll-like receptor 2 (TLR2) plays an important role in innate immune responses. Here we describe the isolation and characterization of the full-length cDNA sequence of toll-like receptor 2 in large yellow croaker Larimichthys crocea (LcTLR2). The LcTLR2 cDNA contains a 5'-terminal untranslated region (5'-UTR) of 135 bp, an open reading frame (ORF) of 2478 bp encoding a polypeptide of 825 amino acid residues and a 3'-UTR of 50 bp. Subcellular localization analysis suggested that the LcTLR2-pEGFP was mainly expressed in cytoplasm. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis revealed a broad expression of LcTLR2 in most examined tissues, with the most predominant expression in blood, followed by spleen, and the weakest expression in stomach. The expression levels of LcTLR2 after injection with Vibrio parahaemolyticus, Lipopolysaccharides (LPS) and poly inosinic:cytidylic (polyI:C) were investigated in spleen, head-kidney and liver. Our results showed that LcTLR2 transcripts increased significantly after all the three immune challenges (p < 0.05). However, compared with polyI:C and LPS, higher expression levels of LcTLR2 were induced in all examined tissues after V. parahaemolyticus stimulation. In addition, the expression levels of LcTLR2 after flagellin, polyI:C, peptidoglycan (PGN) and LPS challenge in LCK were investigated, our findings showed that high LcTLR2 transcripts were induced after flagellin and PGN stimulation, suggesting that LcTLR2 might play a vital role in fish defense against bacterial infection. Furthermore, compared with LPS, flagellin and peptidoglycan might play an important role in LcTLR2 induction in large yellow croaker.