Molecular characterization of an IL-1β gene from the large yellow croaker (Larimichthys crocea) and its effect on fish defense against Vibrio alginolyticus infection

Abstract

Interleukin 1β (IL-1β), the first interleukin to be characterized, plays a key role in regulating the immune response. In this study, we determined the cDNA and genomic DNA sequences of the IL-1β gene from the large yellow croaker, Larimichthys crocea. Phylogenetic analysis indicated that the IL-1β (LcIL-1β) gene was most closely related to that of European seabass (Dicentrarchus labrax), sharing 67.8% amino acid identity. In healthy large yellow croaker, LcIL-1β transcription was detected in all tested tissues, with the highest level found in the head kidney. Upon Vibrio alginolyticus infection, LcIL-1β transcription in all tested tissues was significantly upregulated. Intraperitoneal injection of recombinant LcIL-1β (rLcIL-1β) improved the survival rate and reduced the tissue bacterial load after V. alginolyticus infection. In addition, rLcIL-1β induced monocytes/macrophages (MO/MΦ) chemotaxis and increased phagocytosis and bactericidal activity in vitro. These results suggest that LcIL-1β plays an important role in the large yellow croaker immune response against V. alginolyticus.

Keywords: Interleukin 1β; Large yellow croaker; Monocytes/ macrophages; Survival rate; Vibrio alginolyticus.

Figure 1

Multiple alignment of the LcIL-1β amino acid sequence with that of other related animal IL-1β sequences Similar residues are marked with gray shading and identical residues with black shading. The ICE site in humans is indicated with an arrow. Two conserved cysteine residues are marked by “▼”, and the IL-1 family signature is lined above the alignment. Accession numbers of sequences are provided in Table 1.

Figure 2

Phylogenetic tree analysis of IL-1β amino acid sequences of large yellow croaker and some related fish using the neighbor-joining method The values at the forks indicate the percentage of trees in which this grouping occurred after bootstrapping the data (1 000 replicates; shown only when >60%). Scale bar shows number of substitutions per base. Accession numbers of sequences are provided in Table 1.

Figure 3

QPCR analysis of the relative mRNA expression of LcIL-1β in different tissues of healthy (A) and V. alginolyticus-challenged large yellow croaker (B-I) A: tissue expression profile of LcIL-1β in healthy fish. L: liver; HK: head kidney; S: spleen; TK: trunk kidney; B: brain; H: heart; G: gill; I: intestine. Results from three fish are expressed as means±SEM. *: P<0.05 versus PBS group.

Figure 4

Prokaryotic expression, purification and refolding of rLcIL-1β A: Bacterial lysates were electrophoresed on 12% SDS-PAGE gels. Lane M: protein marker; 1: E. coli BL21 (DE3) transformed with pET28a after IPTG induction; 2: E. coli BL21 (DE3) transformed with pET28a-LcIL-1β before IPTG induction; 3: E. coli BL21 (DE3) transformed with pET28a-LcIL-1β after IPTG induction. B: His affinity chromatography purification of rLcIL-1β using His Trap^TM FF Crude column. C: Refolding of rLcIL-1β using urea gradient gel filtration on a Superdex 75 column. D: SDS-PAGE analysis of peaks in B and C. Lane M: protein marker; Lane 1: peak 1; 2: peak 2; 3: peak 3.

Figure 5

Effect of different doses of rLcIL-1β on the survival rate of V. alginolyticus-infected large yellow croaker Fish (20 in each group) were i.p.injected with V. alginolyticus (6.5×10⁴ CFU/g). At 0.5 hpi, 0.001, 0.01 or 0.1 µg/g rLcIL-1β was i.p.injected into fish, respectively. The control group received an equal volume of PBS. Fish were monitored for signs of sickness and mortality every 24 h for 9 days. Group survival rates for each treatment were analyzed by the Log-rank test. *: P<0.05 versus PBS-treated group.

Figure 6

Effect of different doses of rLcIL-1β on bacterial loads in immune tissues and blood of large yellow croaker Fish (6 in each group) were i.p.-injected with V. alginolyticus (6.5×10⁴ CFU/g) and received 0.001, 0.01 or 0.1 µg/g of rLcIL-1β at 0.5 hpi, respectively. The control group received an equal volume of PBS. Fish were euthanized at 72 h post treatment of rLcIL-1β. Liver, spleen, kidney, and blood samples were collected. Homogenates and blood were cultured on TCBS agar plates. Colony numbers were normalized to volume (0.1 mL for blood) and tissue weight (0.1 g for liver, spleen and kidney). *: P<0.05 versus PBS-treated group.

Figure 7

Effect of rLcIL-1β on MO/MΦ chemotaxis, phagocytosis and bactericidal activity A: Dose-response relationship of refolded rLcIL-1β to attract large yellow croaker MO/MΦ. Denatured rLcIL-1β and BSA were used as controls. B: Fluorescence images of phagocytosis of FITC-DH5α in MO/MΦ treated with rLcIL-1β. Histogram represents mean fluorescence intensity (MFI) percentage of bacteria engulfed by cells. Magnification ratio: 400X. C: Plates display survival V. alginolyticus from MO/MΦ treated with rLcIL-1β. Histogram demonstrates effects of rLcIL-1β on bacterial killing. Data are representative of at least three independent experiments. *: P<0.05 versus PBS-treated group.