FIGURE 8

Examination of the inhibitory role of TcpAh in zebrafish adaptive humoral immunity against infection by repressing CD80/86 expression. (A–C) Activation of zebrafish CD80/86 promoter in HEK293T cells transfected with CD80/86 luciferase reporter (CD80/86-Luc; 200 ng/mL), renilla luciferase reporter (15 ng/mL) and expression vectors for zebrafish TLR2 or TLR9 or TLR3 (20 ng/mL) in combination with MyD88 or TRIF (20 ng/mL) with or without TcpAh expression vector (250 ng/mL). After 24 h, the HEK293T cells were stimulated with PAM3 or CpG-ODN or Poly(I:C) for 12 h. Data are the average luciferase activity ± SD (**p < 0.01; ***p < 0.001). (D) Real-time PCR analysis for the expression of zebrafish cd80/86 in leukocytes, which were sorted from peripheral blood, spleen, and kidney tissues 2 days after i.p. stimulation with PBS, wild-type A. hydrophila, ΔtcpAh mutant, or ΔtcpAh mutant complemented with CP-TcpAh protein. Data are representative of three independent experiments as mean ± SD (*p < 0.05; **p < 0.01). (E) Flow cytometric analysis of CD80/86 expression level on MHC-II+ antigen-presenting cells (APCs) of each in vivo treatment group. Data are representative of three independent experiments as mean ± SD (*p < 0.05; **p < 0.01). (F) Proliferation of lymphocytes determined by CFSE dilution through flow cytometry under the indicated experimental treatment. (G) Proliferation of IgM+ B cells determined by flow cytometry under the indicated experimental treatment. Data are representative of three independent experiments as mean ± SD (*p < 0.05; **p < 0.01). (H) Real-time PCR analysis of the expression levels of zebrafish LCK and CD154 of each in vitro treatment group. Data are representative of three independent experiments as mean ± SD (*p < 0.05; **p < 0.01). (I) Examination of the inhibitory role of TcpAh in IgM production in response to A. hydrophila infection in each treatment group by ELISA. Data are representative of three independent experiments as mean ± SD (n = 20; *p < 0.05; **p < 0.01). (J) Examination of the inhibitory role of TcpAh in zebrafish defense against A. hydrophila infection. Zebrafish were infected with A. hydrophila ΔtcpAh or with wild-type A. hydrophila or with A. hydrophila ΔtcpAh complement with CP-TcpAh. Differences were analyzed using log-rank test (*p < 0.05; **p < 0.01). Group of fish injected with mock PBS was used as a negative control.