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. 2019 Feb 14;9(1):2030.
doi: 10.1038/s41598-018-38322-8.

VSports注册入口 - Modulation of Intestinal Epithelial Permeability by Plasma from Patients with Crohn's Disease in a Three-dimensional Cell Culture Model

Pan Xu  1   2 , Elhaseen Elamin  1   2 , Montserrat Elizalde  1   2 , Paul P H A Bours  1 , Marieke J Pierik  1   2 , Ad A M Masclee  1   2 , Daisy M A E Jonkers  3   4
Affiliations

V体育ios版 - Modulation of Intestinal Epithelial Permeability by Plasma from Patients with Crohn's Disease in a Three-dimensional Cell Culture Model

V体育2025版 - Pan Xu et al. Sci Rep. .

Abstract

Intestinal epithelial barrier is affected by multiple factors, such as tumour necrosis factor-α (TNF-α). Plasma concentration of TNF-α is higher in patients with Crohn's disease (CD) than healthy controls (HC) and correlates positively with disease activity VSports手机版. This study aimed to determine the effect of plasma from active, inactive CD patients on intestinal barrier function and to investigate the underlying mechanism. Plasma samples were collected from CD patients and HC. 3D Caco-2 cysts were treated with plasma or TNF-α, with or without pre-incubation of adalimumab (a monoclonal antibody that antagonizes TNF-α) or JNK inhibitor SP600125. The results demonstrated that exposure of the cysts to plasma from CD patients resulted in enhanced paracellular permeability in a disease activity-dependent manner. Compared to HC, active CD plasma decreased ZO-1 and OCCLUDIN expression on mRNA and protein levels, and led to an increased JNK phosphorylation. Pre-incubation with adalimumab or SP600125 ameliorated TJ disruption and barrier dysfunction induced by plasma from CD patients. These results indicate that plasma from CD patients is able to induce epithelial barrier disruption, in part through TNF-α induced TJs modulation. The data also demonstrate an involvement of MAPK pathway, in particular the JNK isoform, in CD patient plasma-induced barrier dysfunction. .

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Effects of plasma from patients with Crohn’s disease (CD) on paracellular permeability in 3D Caco-2 cysts. (a) Representative Caco-2 cysts with luminal and basal compartments indicated. The bar indicates 100 µm (b) Representative images showing the intraluminal accumulation of FITC-dextran-4 (FITC-D4, green) in the cysts treated with plasma (37.5% v/v) from healthy controls (HC), active or inactive CD patients, or EGTA (2 mM, positive control) for 24 hours. Images were captured from the middle of the cysts using confocal microscopy. The bar indicates 50 µm. (c) The mean fluorescence intensity of FITC-D4, expressed as the ratio of the luminal (L) over the basal (BL) compartment, after the abovementioned plasma exposure. The L/BL ratio of EGTA exposure was set to 1. (d) Effect of the abovementioned plasma treatments on LDH release in Caco-2 cysts. Triton X-100 was used as positive control to induce maximum LDH leakage. Data are reported as percentage of maximum LDH release. All graphs are expressed as means ± SEM of three independent experiments with 6 subjects per group and at least 8 cysts per subject. *P < 0.05; **P < 0.01; ***P < 0.001 by one-way ANOVA and Tukey’s post-hoc test.
Figure 2
Figure 2
Effects of plasma from patients with Crohn’s disease (CD) on OCCLUDIN and ZO-1 in 3D Caco-2 cysts. mRNA levels (a), confocal microscopy analyses of OCCLUDIN and ZO-1 (b–d) in Caco-2 cysts that were treated with plasma (37.5% v/v) from healthy controls (HC), active or inactive CD patients for 24 hours. Images in (b) were captured from the middle cross-section of the cysts. Bar indicates 50 µm. Quantified fluorescence intensities in (b) are shown in (c). Images in (d) were captured from the top surface of the cysts. Bar indicates 20 µm. All graphs represent the results of three replicate experiments. Data expressed as means ± SEM with 6 subjects per group and at least 8 cysts per subject. **P < 0.01; ***P < 0.001 by one-way ANOVA and Tukey’s post-hoc test.
Figure 3
Figure 3
Effects of TNF-α and adalimumab on paracellular permeability, OCCLUDIN and ZO-1 in 3D Caco-2 cysts. Caco-2 cysts were exposed at the basal side to medium only (Ctrl), TNF-α (25 pg/mL) with or without pre-incubation of adalimumab (20 μg/mL) for 24 hours. (a) Representative images showing the intraluminal accumulation of FITC-dextran-4 (FITC-D4, green) in the Caco-2 cysts. Bar indicates 50 μm. (b) The mean FITC-D4 intensity of Caco-2 cysts measured and expressed as the L/BL ratio of the luminal (L) over the basal (BL) compartment. (c) mRNA levels of OCCLUDIN and ZO-1 in 3D Caco-2 cysts. (d–f) Confocal microscopy analyses of ZO-1 (green), OCCLUDIN (red) and nuclei (blue). Images in (d) were captured from the middle cross-section of 3D cysts. Bar indicates 50 μm. Images in (e) were captured from the top surface of 3D cysts. Bar indicates 20 μm. Quantified fluorescence intensities in (d) are shown in (f). All graphs represent the results of three replicate experiments. Data expressed as means ± SEM with at least 8 cysts per treatment. *P < 0.05; **P < 0.01; ***P < 0.001 by one-way ANOVA and Tukey’s post-hoc test.
Figure 4
Figure 4
Pre-treatment of adalimumab attenuates the effects of plasma from Crohn’s disease (CD) patients on paracellular permeability, OCCLUDIN and ZO-1 expression of 3D Caco-2 cysts. Cysts were exposed at the basal side to plasma (37.5% v/v) from healthy controls (HC) or active CD patients with or without pre-incubation of adalimumab (10 or 20 μg/mL) for 24 hours. (a) Representative images showing the intraluminal accumulation of FITC-D4 (green) in Caco-2 cysts. Bar indicates 50 μm. (b) The mean fluorescence intensity of FITC-D4 measured and expressed as the L/BL ratio of the luminal (L) over the basal (BL) compartment. Representative images (c), and fluorescence intensities (d–e) from confocal microscopy analyses of ZO-1, OCCLUDIN in 3D Caco-2 cysts, Images were captured from the middle cross-section of the cysts. Bar indicates 50 μm. All graphs represent the results of three replicate experiments. Data expressed as means ± SEM with at least 8 cysts per subject (6 subjects per group) or per treatment. *P < 0.05; **P < 0.01; ***P < 0.001 by one-way ANOVA and Tukey’s post-hoc test.
Figure 5
Figure 5
MAPK signalling mediates CD plasma induced disruption of barrier function in 3D Caco-2 cysts. (a–e) Phosphorylation levels of ERK1/2, p38 and JNK in Caco-2 cysts that were exposed to plasma from healthy control (HC), active or inactive CD patients, with or without pre-treatment of JNK inhibitor SP600125 (100 nmol/L). (f,g) Representative images showing the intraluminal accumulation of FITC-D4 (green) in Caco-2 cysts (f), and the mean fluorescence intensity of FITC-D4 measured and expressed as the L/BL ratio of the luminal (L) over the basal (BL) compartment (g) in 3D Caco-2 cysts that were exposed to plasma from healthy control (HC) or active CD, with or without pre-treatment of JNK inhibitor SP600125. Bar indicates 50 μm. (h) Graphical summary illustrating how plasma from CD active patients promotes barrier dysfunction. Data are expressed as means ± SEM of at least 8 cysts per condition, with 6 subjects per group. **P < 0.01; ***P < 0.001 by one-way ANOVA and Tukey’s post-hoc test.
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