R848 Is Involved in the Antibacterial Immune Response of Golden Pompano (Trachinotus ovatus) Through TLR7/8-MyD88-NF-κB-Signaling Pathway

Abstract

R848 is an imidazoquinoline compound that is a specific activator of toll-like receptor (TLR) 7/8 and is often used in immunological research in mammals and teleosts. However, the immune responses initiated by R848 through the TLR7/8 pathway in response to bacterial infection remain largely unexplored in teleosts. In the current study, we investigated the antibacterial response and the participating signaling pathway initiated by R848 in golden pompano (Trachinotus ovatus). We found that R848 could stimulate the proliferation of head kidney lymphocytes (HKLs) in a dose-dependent manner, enhance the survival rate of HKLs, and inhibit the replication of bacteria in vivo. However, these effects induced by R848 were significantly reduced when chloroquine (CQ) was used to blocked endosomal acidification. Additionally, an in vivo study showed that R848 strengthened the antibacterial immunity of fish through a TLR7/8 and Myd88-dependent signaling pathway. A cellular experiment showed that Pepinh-MYD (a Myd88 inhibitor) significantly reduced the R848-mediated proliferation and survival of HKLs. Luciferase activity analysis showed that R848 enhanced the nuclear factor kappa B (NF-κB) activity, whereas this activity was reduced when CQ and Pepinh-MYD were present. Additionally, when an NF-κB inhibitor was present, the R848-mediated pro-proliferative and pro-survival effects on HKLs were significantly diminished. An in vivo study showed that knockdown of TLR7, TLR8, and Myd88 expression in golden pompano via siRNA following injection of R848 resulted in increased bacterial dissemination and colonization in fish tissues compared to that of fish injection of R848 alone, suggesting that R848-induced antibacterial immunity was significantly reduced. In conclusion, these results indicate that R848 plays an essential role in the antibacterial immunity of golden pompano via the TLR7/8-Myd88-NF-κB- signaling pathway.

Keywords: Myd88; NF-κB; R848; TLR7/8; antibacterial immune response.

Figure 1

Effect of R848 and chloroquine (CQ) on bacterial infection. Golden pompano administered with R848, CQ, R848 plus CQ, or PBS (control) were infected with Edwardsiella tarda, and the amount of bacteria in the liver (A), spleen (B), and kidney (C) were determined at different time points. All data are shown as the means ± SD (N = 5), and the statistical significance is indicated. *P < 0.05, **P < 0.01.

Figure 2

Effect of R848 on the proliferation of HKLs. (A) Golden pompano head kidney lymphocytes (HKLs) were treated with different concentrations of R848, and then the proliferation of HKLs was examined by CCK8 assay. (B) Golden pompano HKLs were treated with or without (control) 5 µg/ml of chloroquine (CQ), 8 µg/ml R848 plus 5 µg/ml of CQ, or 8 µg/ml R848, and then examined for proliferation by CCK8 assay. Data are presented as means ± SEM (N = 3). N, the number of times the experiment was performed. **P < 0.01.

Figure 3

Effect of R848 and chloroquine (CQ) on the apoptosis of HKLs. (A) Golden pompano HKLs treated with or without (control) 8 µg/ml R848, 5 µg/ml CQ, or 8 µg/ml R848 plus 5 µg/ml CQ, and then the apoptosis rates of these cells were determined by the flow cytometry. (B) The percentages of apoptotic cells are represented with statistic bar charts. Data are presented as means ± SD (N = 3). N, the number of times the experiment was performed. **P < 0.01.

Figure 4

R848-induced cellular activity through Myd88-signaling pathway. Golden pompano HKLs were treated with PBS (control), R848, Pepinh-Control, Pepinh-MYD, R848 plus Pepinh-Control, or R848 plus Pepinh-MYD. The cells were then determined for proliferation (A), apoptosis (B), and statistic bar chart of the apoptosis percentage (C). Data in are presented as means ± SEM (N = 3), and (C) are presented as means ± SD (N = 3). **P < 0.01. Data in (B) are one representative of three independent experiments.

Figure 5

Effect of R848 on NF-κB activity. GPS cells containing NF-κB-RE-luc2P reporter were treated with or without (control) R848, chloroquine (CQ), or R848 plus CQ and then analyzed for luciferase activity (A). GPS cells containing NF-κB-RE-luc2P reporter were transfected with siTLR7, siTLR8, siTLR7 plus siTLR8, siRNA-C, or PBS (control) and then treated with R848. The luciferase activity in the cells was then determined (B). GPS cells containing NF-κB-RE-luc2P reporter were treated with R848 in the presence or absence of Pepinh-Control or Pepinh-MYD; the control cells were treated with PBS. The luciferase activity in the cells was then determined (C). Data are expressed as means ± SEM (N = 3). N, the number of times the experiment was performed. **P < 0.01.

Figure 6

Effect of BAY-11-7082 on R848-induced cellular activity. (A) Golden pompano HKLs were treated with PBS (control), R848, BAY-11-7082, or R848 plus BAY-11-7082. The cells were then determined for proliferation (A), apoptosis (B), and statistic bar chart of the apoptosis percentage (C). Data in (A) are presented as means ± SEM (N = 3), and (C) are presented as means ± SD (N = 3). **P < 0.01. Data in (B) are one representative of three independent experiments.

Figure 7

Effect of R848 on bacterial infection through TLR7/8-signaling pathway. Golden pompano administered with R848, R848 plus siRNA-C, R848 plus siTLR7, R848 plus siTLR8, R848 plus siTLR7 and siTLR8, or PBS (control) were infected with Edwardsiella tarda, and the amount of bacteria in the liver (A), spleen (B), and kidney (C) were determined at different time points. All data are shown as the means ± SD (N = 5), and the statistical significance is indicated. **P < 0.01.

Figure 8

Effect of R848 on bacterial infection through Myd88-signaling pathway. Golden pompano administered with R848, R848 plus siRNA-C, R848 plus siMyd88, or PBS (control) were infected with Edwardsiella tarda, and the amount of bacteria in the liver (A), spleen (B), and kidney (C) were determined at different time points. All data are shown as the means ± SD (N = 5), and the statistical significance is indicated. *P < 0.05, **P < 0.01.

Figure 9

Effect of R848 on the expression of immune genes through TLR7/8-Myd88-signaling pathway. Expression of golden pompano TLR7/8 and Myd88 were knocked down in golden pompano via siRNA technology. Then, fish were treated with or without (control) R848. The expression profiles of immune genes were investigated in head kidney by qRT-PCR. The internal reference gene was B2M. Values are shown as means ± SD (N=5), and the statistical significance is indicated. *P < 0.05, **P < 0.01.