Reactive oxygen species inhibits Listeria monocytogenes invasion into HepG2 epithelial cells

Abstract Listeria monocytogenes (Lm) can colonize human gastrointestinal tract and subsequently cross the intestinal barrier. Reactive oxygen species (ROS) are produced by NADPH oxidase. However, the role of ROS in bacterial invasion remains to be less understood. Herein, we investigated the impact of ROS on Lm invasion to HepG2 using NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), as well as the ROS scavenger, N‐acetyl cysteine (NAC). Our results showed that inhibiting ROS increased the invasive capability of Lm. Moreover, after Lm infection, inflammatory cytokines such as tumor necrosis factor alpha (TNF‐α) and interleukin 1beta (IL‐1β) in HepG2 were significantly upregulated. However, after inhibiting ROS, the expression levels of TNF‐α and IL‐1β were downregulated, indicating a failure of host cells to activate the immune mechanism. Taken together, ROS in Lm might be as a signal for host cells to sense Lm invasion and then stimulate cells to activate the immune mechanism.

Only a few studies have evaluated the exact relationship between ROS and Lm invasion. Thus, we investigated the role of ROS during Lm invasion using DPI and NAC to induce ROS in Lm. HepG2 cells were used, and inflammatory cytokines, TNFα and IL-1β, in cells were also detected. were cultured in liquid medium of brain-heart infusion (BHI) (Beijing Land Bridge Technology, Beijing, China) supplemented with 50% glycerol and 1.5% agar, and the cultures were stored at −80°C. The strain was incubated in 50 ml of BHI and shaken overnight under 37°C with 100 r/min shaking. A 1 ml aliquot of cultures was transferred into 50 ml of fresh BHI and shaken at 130 r/min and 37°C to obtain an optical density at 600 nm of 0.3 with a microplate reader (Molecular Devices, California, and USA).

| Determination of Lm viability
Bacterial suspensions with an optical density at 600 nm of 0.3 were statically incubated with DPI of 0, 0.1, 0.5 1, 2 μmol/L at 37°C for 30 min. Then, the samples were centrifuged at 2,057 g for 10 min, washed with PBS for three times, and finally resuspended in fresh BHI. Aliquots of 200 μl samples were added per well, and absorbance was measured at 600 nm with a microplate reader. The viability of Lm was estimated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoli um bromide (MTT) (Beyotime Institute of Biotechnology, Shanghai, China) with some modifications recommended as per (Schillaci et al., 2008). 20 μl of 1 mg/ml MTT was added per well. The plates were placed statically and then incubated at 37°C for 4 hr. The supernatant was separated from the medium by centrifugation at 2,057 g for 10 min. Insoluble purple formazan was dissolved in 150 μl of DMSO.
Bacterial viability was assessed by absorbance at OD 570 nm.
For treatment of Lm with NAC or DPI, the suspensions at an optical density of 0.3 at 600 nm were mixed with NAC or DPI at different concentrations (0, 0.1, 0.5, 1, 2 mmol/L) and incubated at 37°C for 14 hr under static condition. 200 μl samples were added per well, and absorbance at 600 nm was measured with a microplate reader. 20 μl of 1 mg/ml MTT was added per well.

| Lm invasion assays
HepG2 cells in DMEM (Gibco) were inoculated with bacterial suspension (1 × 10 7 CFU/ml) to obtain a multiplicity of infection of 1:100 for 1.5 hr at 37°C in the presence of 5% CO 2 . After infection, extracellular bacteria were abrogated by gentamicin (500 μg/ml) in PBS (0.01 mol/L, pH 7.2) for 1 hr. HepG2 cells were washed thrice with PBS to remove no adherent bacteria. Invasive bacteria were harvested after HepG2 lysis using a lysis solution (1% Triton X-100; Sigma). The concentration of invasive bacteria was determined by measuring optical density of the bacterial suspension via plate counting. The experiment was performed in five independent experiments.

| qPCR of TNFα and IL-1β
After Lm invasion, the RNA of HepG2 cells was extracted using Trizol (TaKaRa, Japan). qPCR was performed using the PrimeScript™ First-Strand cDNA Synthesis Kit (TaKaRa, Japan). The primer pairs shown in Table 1 were synthetized by Sangon Biotech (Shanghai, China), and SYBR GREEN real-time quantitative PCR was performed with the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). The relative quantification values of the genes were calculated using the comparative threshold cycle (△△CT) method.

| Statistical analysis
All experiments in this study were performed in n = 5 independent experiments. Data analysis and graphical evaluations were conducted using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA).

| DPI and NAC significantly downregulate ROS without affecting Lm proliferation and viability
To explore whether or not DPI and NAC affect the growth of Lm, EGDe and ATCC 43251 Lm strains were treated with different concentrations of DPI or NAC, and the optical density of the bacterial suspension was tested in 96-well plates. Figure 1a and d shows that the OD 600 nm of wild strain EGDe and ATCC 43251 suspensions did not significantly differ among the different DPI or NAC concentrations. Lm viability was also tested with an MTT assay. As shown in is more sensitive to the DPI than NAC, and EGDe is more sensitive to NAC.

| Lm invasion is enhanced following ROS suppression by DPI or NAC
In order to evaluate the role of Lm-produced ROS in host invasion, Lm was treated with a series of concentrations of DPI and NAC prior to infection of HepG2 cells. As shown in Figure 2b  F I G U R E 2 Invasion efficiency of Lm on HepG2 cells increased when ROS were inhibited by DPI or NAC. Confocal micrograph of 0 (a) or 1 (b) μmol/L DPI-treated Lm (ATCC 43251); confocal micrograph of 0 (c) or 0.1 (d) mmol/L NAC-treated EGDe; Lm, cell nucleus, and cytomembrane were stained as red, blue, and green, respectively, in this immunofluorescence assay. The invasiveness of NAC-treated ATCC 43251 (e) or EGDe (f) and DPI-treated ATCC 43251 (g) or EGDe (h) was estimated by plate counting. Data are mean ± SEM, *p < .05, **p < .01. DPI, diphenyleneiodonium chloride; Lm, Listeria monocytogenes; NAC, N-acetyl cysteine; ROC, reactive oxygen species

| Influence of Lm invasion on the ROS production of HepG2 cells
Reactive oxygen species production by HepG2 cells was evaluated to explore the changes after Lm invasion. HepG2 cells infected with 0.5 μmol/L DPI-treated EGDe showed the lowest ROS production than cells infected with untreated EGDe (Figure 3b). The same tendency was showed between Lm ROS and HepG2 cells, and ROS levels were also lower in DPI-treated Lm compared to NAC-treated Lm. (Figure 1c and f). Although the relationship between Lm and HepG2 ROS levels is unclear from these experiments, our results do suggest the important role of ROS in Lm invasion.   Our findings also raise questions regarding the use of antioxidants in over-the-counter medications and supplements. As this study suggests ROS enhances Lm invasion, evaluating whether antioxidants promote an increased risk of certain microbial infections warrants future investigation.

CO N FLI C T O F I NTE R E S T
We confirm that there is no conflict of interest exists for this manuscript.