Doxycycline Induces Mitophagy and Suppresses Production of Interferon-ß in IPEC-J2 Cells

Xing, Yang; Liqi, Zhu; Jian, Lin; Qinghua, Yu; Qian, Yang

The use of antibiotics has increased dramatically in recent years but is now forbidden for promoting animal growth in the European Union, because of the development of antibioticresistant bacteria (Gilbert, 2011; Du and Liu, 2012) . However, DOX remains the most widely used antibiotic as frontline therapy against bacterial infection (Authority and Authority, 2015) . Due to the similarity of bacterial and mitochondrial ribosomes, DOX exert effects in host cells (Kroon et al., 1984; Ryan et al., 1995) . The effects of DOX related to the mitochondria of individual organ systems differ because of different energy demands (Peters et al., 2011) . It was reported that plasma DOX concentrations could be 1 µg/ml after pigs were administered with DOX (Schmidt et al., 2009) . The gastrointestinal tract is the first target for the potential effects of doxycycline. The gastrointestinal tract FIGURE 5 | Mitophagy induced by DOX suppresses the antiviral innate immunity of IPEC-J2 cells. IPEC-J2 cells were pretreated with DOX for 24 h. Then, these cells were transfected with or without 500 ng/ml poly (I: C) for 24 h. RT-PCR for GAPDH was used as an internal control. RNA expression levels normalized relative to those of mock cells are shown. Relative IFN-β (A), IFIT1(C) and DDX58 (RIG-I) (D) mRNA level is shown. (B) IPEC-J2 cells were pretreated with or without 50 µg/ml DOX for 24 h and transfected with 500 ng/ml poly (I: C) for 12, 24, and 48 h. IFN-β mRNA expression levels were normalized to the GAPDH mRNA level in each sample. Data are the means ± standard deviations of three independent experiments. One-way analysis of variance; *P < 0.01; **P < 0.001. The cell culture supernatants were harvested 24 h post-infection (hpi) and assayed for the production of infectious virus by TCID50 assay on ST cells. Each data point represents the average titer derived from two independent TCID50 assays. Error bars represent standard errors. Data represent means ± standard deviations of three independent experiments. One-way analysis of variance; *P < 0.01; **P < 0.001. can be exposed to high concentrations of DOX after ingestion of feed or drinking water with 100-200 mg/kg doxycycline. The concentration of DOX used in the individual applications can be 100 µg/ml in intestinal lumen. Therefore, we selected three DOX doses in this study: 1, 50 and 100 (µg/ml). Our results demonstrate that DOX induced mitophagy in IPEC-J2 cells, consequently decreasing IFN-β production in IPEC-J2 cells transfected with poly (I: C).