Regulation of TWIK-related potassium channel-1 (Trek1) restitutes intestinal epithelial barrier function

Abstract

The disruption of epithelial barrier integrity is an important factor in the pathogenesis of various immune disorders. However, the restitution of the compromised barrier functions is difficult. This study investigates the regulation of TWIK-related potassium channel-1 (Trek1) in the restitution of intestinal epithelial barrier functions. The human colon epithelial cell line T84 was cultured in monolayers and used to observe epithelial barrier functions in vitro. An intestinal allergy mouse model was created. Cytokine levels were determined by enzyme-linked immunosorbent assay and western blotting. The results showed that Trek1 deficiency induced T84 monolayer barrier disruption. Allergic responses markedly suppressed the expression of Trek1 in the intestinal epithelia via activating the mitogen-activated protein kinase pathways and increasing the expression of histone deacetylase-1. The inhibition of histone deacetylase-1 by sodium butyrate or the administration of a butyrate-producing probiotic (Clostridium butyricum) restored the intestinal epithelial barrier functions and markedly enhanced the effect of antigen-specific immunotherapy. The data suggest that Trek1 is required for the maintenance of intestinal epithelial barrier integrity. Allergic responses induce an insufficiency of Trek1 expression in the intestinal epithelia. Trek1 expression facilitates the restoration of intestinal epithelial barrier functions in an allergic environment.

Figure 1

Trek1 maintains T84 monolayer barrier functions. (a) T84 cells were treated with medium, Trek1 shRNA (shRNA), or control shRNA (cshRNA). The immune blots indicate the levels of the Trek1 protein in the T84 cell extracts 0 h–7 days after shRNA transduction. (b) Electrophysiological assessment of the membrane currents of single T84 cells (10 cells/group) from wild-type and Trek1-null (Trek1-n) T84 cells in the presence or absence of the Trek1 inhibitor spadin (1 µM). (c) The curves show the summarized current density. (d and e) T84 cells were cultured in Transwells. Upon reaching confluence, the T84 cells were treated with medium, Trek1 shRNA, or control shRNA. (d) The TER was recorded from 0 h to 7 days as denoted on the X axis. The bars indicate the TER. (e) HRP flux was performed with the T84 monolayers at time points as denoted on the X axis of the bar graphs. Each Transwell was only used for one permeability experiment. The test period ran for 2 h. (f) Confluent T84 monolayers were treated in Transwells as denoted on the X axis. FITC-dextran (10 mg/ml; 1, MW=4000; 2, MW=70 000) was added to the apical chambers. Samples were collected from the basal chambers 6 h later and analyzed by a fluorometer at 518 nm. The bars indicate the fluorescence intensity. The data are presented as mean±s.d. *P<0.01, compared with the medium group. The data are representative of three independent experiments. HRP, horseradish peroxidase; TER, transepithelial resistance; Trek1, TWIK-related potassium channel-1.

Figure 2

Allergic response suppresses Trek1 production by intestinal epithelial cells. BALB/c mice were sensitized to OVA. The serum and intestinal segments were collected and analyzed for the allergic response in the intestine. A-b, the bars indicate the serum levels of Th2 cytokines (a) and specific IgE (sIgE; b). c, the bars indicate the frequency of mast cells (MC) and eosinophils (Eo) in the intestinal tissue. d, the bars indicate the frequency of CD4^× T cell proliferation in response to exposure to specific antigens in the culture. e, the immune blots indicate the protein levels of Trek1 in the intestinal mucosa. f-h, the representative confocal images show the expression of Trek1 (in green) in the intestinal epithelia (the insert is an enlarged image field showing Trek1 staining). The nuclei were stained with propidiumidodide (in red) for morphological view. The data of bars are presented as mean ± SD. *, p<0.01, compared with saline group. Each group consists of 6 mice. Specimens from individual mice were processed separately. The data are a representative of 6 independent experiments.

Figure 3

Allergic mediators suppress Trek1 expression in T84 cells. T84 cells were exposed to agents as denoted on the X axis of C (p38 inhi=p38 inhibitor). The cells were collected 72 h later and analyzed by qRT-PCR, western blotting and methylation-specific PCR. (a) The bars indicate the mRNA levels of Trek1. (b) The immune blots indicate the protein levels of Trek1. The table above the blots shows the integrated density of the immune blots. (c) The bars indicate the unmethylated Trek1 gene DNA levels. Concentrations of reagents: IL-4=20 ng/ml, IL-5=30 ng/ml, IL-13=50 ng/ml, mMCP-1=20 ng/ml and TNF-α=10 ng/ml. *P<0.01, compared with the saline group. The data are presented as mean±s.d., and are representative of three independent experiments. mMCP-1, mouse mast cell protease-1; TNF, tumor necrosis factor; Trek1, TWIK-related potassium channel-1.

Figure 4

Increases in HDAC1 are associated with allergic mediator-suppressed Trek1 expression in T84 cells. T84 cells were treated with allergic mediators in the same procedures described in Figure 3. (a and b) The data show the levels of HDAC1 expression. (c and d) In the presence of HDAC inhibitor Trichostatin A (1 µM), the data show the effect of the allergic mediators on the levels of Trek1. The data are presented as mean±s.d. *P<0.01, compared with saline group. The data are representative of three independent experiments. Trek1, TWIK-related potassium channel-1.

Figure 5

Recovery of Trek1 facilitates the inhibitory effect of SIT on allergic responses. The treatment of allergic mice is denoted on the X axis. The bars indicate the serum Th2 cytokines (a), serum-specific IgE (b), frequency of mast cells and eosinophils in the intestinal mucosa (c) and the frequency of CD4⁺ T-cell proliferation after exposure to a specific antigen in the culture (d). (e and f) The epithelial specimens were collected from the mouse small intestine by scraping. The protein extracts were analyzed by western blot. The immune blots indicate the protein levels of Trek1 (e) and HDAC1 (f). SB, or CB or spadin was given i.p. prior to the exposure to specific antigens. Each group consists of six mice. Samples from individual mice were processed separately. The data are presented as mean±s.d. *P<0.01, compared with the saline group (a–d) or naive mice (e and f). ^#P<0.05, compared with the SIT group. The data are representative of six independent experiments. CB, C. Butyricum (0.3 ml of 10⁹ CB/ml); SB, sodium butyrate (0.5 ml of a 50 mM sodium butyrate in PBS); PBS, phosphate-buffered saline; SIT, specific immunotherapy; Spadin, Trek1 inhibitor (0.1 ml of 10 µM); Trek1, TWIK-related potassium channel-1.

Figure 6

Recovery of Trek1 restores intestinal epithelial barrier function. Mice were treated with the same procedures as those described in Figure 5. The small intestine segments were excised at the time of sacrifice and assessed in Ussing chambers. The bars indicate the levels of Isc (a), G (b) and HRP flux (c). The data are presented as mean±s.d. *P<0.01, compared with the saline group. ^#P<0.05, compared with the SIT group. The data are representative of six independent experiments. HRP, horseradish peroxidase; SIT, specific immunotherapy; Trek1, TWIK-related potassium channel-1.