Elucidation of the Interleukin 12 Production Mechanism during Intracellular Bacterial Infection in Amberjack, Seriola dumerili

Abstract

Intracellular bacterial infections affect all vertebrates. Cultured fish are particularly vulnerable because no effective protection measures have been established since such infections emerged approximately 50 years ago. As in other vertebrates, the induction of cell-mediated immunity (CMI) plays an important role in protecting fish against infection. However, details of the mechanism of CMI induction in fish have not been clarified. In the present study, we focused on the production of interleukin 12 (IL-12), an important factor in CMI induction in fish. Using several different approaches, we investigated IL-12 regulation in amberjack (Seriola dumerili), the species most vulnerable to intracellular bacterial disease. The results of promoter assays and transcription factor gene expression analyses showed that the expression of interferon regulatory factor-1 (IRF-1) and activator protein-1 (AP-1) is necessary for IL-12 production. Phagocytosis of living cells (LCs) of Nocardia seriolae bacteria induced IL-12 production in neutrophils, accompanied by IRF-1 and AP-1 gene expression. Bacteria in which the exported repetitive protein (Erp)-like gene was deleted (Δerp-L) could not establish intracellular parasitism or induce IRF-1 and AP-1 expression or IL-12 production, despite being phagocytosed by neutrophils. These data suggest that IL-12 production is regulated by (i) two transcription factors, IRF-1 and AP-1, (ii) phagocytosis of LCs by neutrophils, and (iii) one or more cell components of LCs. Our results enhance the understanding of the immune response to intracellular bacterial infections in vertebrates and could facilitate the discovery of new agents to prevent intracellular bacterial disease.

Keywords: cell-mediated immunity; fish pathogens; interleukin 12; intracellular bacteria.

FIG 1

Transcription factors predicted to control IL-12p35a gene expression, as determined using a promoter assay. Preincubated GAKS cells cotransfected with expression and control plasmids were stimulated with medium only, LPS, or rgIFN-γ1-1 for 6 h. Data represent mean values ± SE from five independent experiments after normalization for transfection efficiency. Luciferase activity is reported as fold difference from induction in GAKS cells stimulated with medium only. Asterisks indicate significant differences (*, P < 0.05, one-way ANOVA followed by Tukey’s post hoc test). RLU, relative light units.

FIG 2

IL-12 production and transcription factor gene expression in amberjack leukocytes stimulated with N. seriolae FKCs or LCs, measured using ELISA (A) and real-time PCR analysis (B to L). (A) Isolated amberjack leukocytes (1 × 10⁷) were stimulated with medium only, 1 × 10⁷ CFU of N. seriolae FKCs, or N. seriolae LCs. (B to L) Real-time PCR analysis of amberjack spleen leukocytes showing mRNA expression of AP-1-related genes, including c-Fos1 (B), c-Fos2 (C), c-Fos3 (D), c-Jun1 (E), and c-Jun2 (F) genes, the IRF-1 gene (G), and NF-κB-related genes, including RelA (H), RelB (I), c-Rel (J), p100 (K), and p105 (L) genes. Isolated leukocytes were stimulated with medium only, 1 × 10⁷ CFU of N. seriolae FKCs, or N. seriolae LCs. Expression level of each of the above-mentioned genes in each group is presented as the ratio of its expression to that of the EF-1α gene. Fold change in expression of each gene was calculated based on its expression in cells stimulated for 6 h with medium only (n = 4). Asterisks indicate significant differences (*, P < 0.05, and **, P < 0.01, one-way ANOVA followed by Tukey’s post hoc test).

FIG 3

(A and B) Isolation and identification of amberjack leukocyte populations using flow cytometry (A) and May-Grunwald-Giemsa staining (B). SS, side scatter; FS, forward scatter; Lin, linear amplification. (B) Arrowheads indicate representative cell morphology in each fraction. Images were acquired at 2,000 × magnification. (C) Each isolated amberjack cell was cultured with N. seriolae. Real-time PCR analysis of cells showing mRNA expression of IL-12p35a. Expression levels of IL-12p35a are presented as the ratio of expression to that of the EF-1a gene. Fold change in expression of genes was calculated based on expression in cells stimulated with medium only (n = 4). An asterisk indicates a significant difference (*, P < 0.05).

FIG 4

Effect of phagocytosis on IL-12 production. Isolated neutrophils (1 × 10⁶) were preincubated with or without cytochalasin D for 6 h. Preincubated neutrophils were cocultured with SYBR gold-labeled N. seriolae LCs (1 × 10⁷ CFU). (A and B) The numbers of SYBR gold-positive (SYBR Gold+) neutrophils were determined using flow cytometry (A), and the concentrations of IL-12 in culture supernatants were determined by ELISA (B). Data represent mean values ± SE from four independent experiments. Asterisks indicate significant differences (*, P < 0.05, and **, P < 0.01, Student’s t test). (C to H) Expression of IL-12p35a gene (C), AP-1-related genes, including c-Fos2 (D) and c-Jun2 (E) genes, the IRF-1 gene (F), and NF-κB-related genes, including RelB (G) and p100 (H) genes, in cultured cells was examined using real-time PCR analysis. Expression level of each of the above-mentioned genes in each group is presented as the ratio of its expression to that of the EF-1α gene. Fold change in expression of each gene was calculated based on its expression in cells stimulated with medium only (n = 4). Asterisks indicate significant differences (*, P < 0.05, and **, P < 0.01, Student’s t test).

FIG 5

Comparison of IL-12 production by neutrophils stimulated with N. seriolae LCs, FKCs, and Δerp-L mutant cells. Isolated neutrophils (1 × 10⁶) were stimulated with 1 × 10⁷ CFU of SYBR gold-labeled N. seriolae LCs, FKCs, or Δerp-L mutant cells. (A and B) The numbers of SYBR gold-positive neutrophils were determined by flow cytometry (A), and the concentrations of IL-12 in culture supernatants were determined by ELISA (B). Data represent the mean values ± SE from four independent experiments. Asterisks indicate significant differences (*, P < 0.05, one-way ANOVA followed by Tukey’s post hoc test). (C to G) Expression of the IL-12p35a gene (C), AP-1-related genes, including c-Fos2 (D) and c-Jun2 (E) genes, the IRF-1 gene (F), and NF-κB-related genes, including RelB (G) and p100 (H) genes, in cultured cells was examined by real-time PCR analysis. Expression level of each of these genes in each group is presented as the ratio of its expression to that of the EF-1α gene. Fold change in expression of each gene was calculated based on its expression in cells stimulated with medium only (n = 4). Asterisks indicate significant differences (*, P < 0.05, one-way ANOVA followed by Tukey’s post hoc test).