Increase in the tight junction protein claudin-1 in intestinal inflammation

Abstract

Background and aims: Studies have shown a decrease in key tight junction (TJ) proteins such as ZO-1 and occludin in both inflammatory bowel disease (IBD) and experimental models of inflammation. Our group has also shown an increase in claudin-1 in experimental colitis.

Methods: IEC-18 cells were treated with increasing doses of tumor necrosis factor alpha (TNFα). The TJ was assessed by transepithelial resistance (TER), permeability, Western blot, PCR, and immunofluorescence. Mucosal samples from patients with ulcerative colitis (UC), Crohn's disease (CD), and without IBD (normal) were assayed for TJ proteins occludin and claudin-1 by Western blot and a ratio of claudin-1 to occludin (C:O) was calculated.

Results: IEC-18 cells had increased permeability, decreased TER and an increase in claudin-1 with TNFα treatment. In human specimens, there was a decrease in occludin and an increase in claudin-1 leading to a significant increase in the C:O ratio in diseased UC colon compared to non-diseased UC colon (P < 0.001) and normal colon (P < 0.01). In CD, the C:O ratio was similar in all CD tissue irrespective of disease status.

Conclusions: Treatment of IEC-18 cells with TNFα, a key inflammatory cytokine in IBD, led to a significant increase in claudin-1 expression. There was a significant increase in the C:O ratio in diseased colon in UC compared to the healthy appearing UC colon and normal controls. The C:O ratio was unchanged in CD despite presence or abscence of gross disease. This suggests that there may be an underlying difference in the TJ between UC and CD.

Fig. 1

Induction of Claudin-1 by TNFα. IEC-18 monolayers were treated on the basolateral side of the monolayer with increasing doses of TNFα. a Sample Western blot showing increase in claudin-1 with TNFα treatment. Actin confirms equivalent lane loading. b Graphical summary of IEC-18 Western blots showing mean claudin-1 expression ± standard error. There was a significant increase in claudin-1 by Western blot starting at 50 ng/ml of TNFα, n = 4. *P <0.05 versus 0 ng/ml, **P <0.01 versus 0 ng/ml. Claudin-1 is expressed as a ratio to actin and control. c PCR for Claudin-1 in IEC-18 cells. There is minimal detectable claudin-1 at baseline. With 5 ng/ml of TNFα there is a marked increase in claudin-1 transcription and an even further increase at 50 ng/ml

Fig. 2

TNFα decreases occludin expression in IEC-18 cells: IEC-18 monolayers were treated on the basolateral side of the monolayer with increasing doses of TNFα. a Sample Western blot showing decrease in occludin with TNFα treatment. Actin confirms equivalent lane loading. b Graphical summary of IEC-18 Western blots showing mean occludin expression ± standard error. There was a significant decrease in occludin by Western Blot starting at 5 ng/ml of TNFα, n = 4. *P <0.01 versus 0 ng/ml, **P <0.001 versus 0 ng/ml. Occludin is expressed as a ratio to actin, and untreated cells

Fig. 3

TNFα alters barrier function in IEC-18 cells. a Change in TER from day 0 to day 2 was calculated for TNFα-treated and -untreated monolayers. There was a decrease in TER with increasing dose of TNFα. Values are mean ± standard error, n = 13. b There was an increase in permeability of the IEC-18 cell monolayer to 70,000 RITC dextran with increasing doses of TNFα applied to the basolateral side of the monolayer. There was a significant increase in permeability with 50, 100, and 200 ng/ml of TNFα. This increase corresponds to the decrease in TER. Values are mean of 4 experiments ± standard error. *P <0.05 versus untreated monolayer, **P <0.01 versus untreated monolayer

Fig. 4

Claudin:occludin ratio distinguishes active UC from inactive and UC from CD. Western blot was performed on the mucosa of operative specimens from patients with CD, UC, and non-inflammatory diseased. a Western blot for occludin and claudin-1 in representative UC patients. In UC patients with healthy appearing mucosa (lane 0), there was a dark band showing a large amount of occludin present, but minimal claudin-1 leading to a low claudin-1 to occludin ratio. In diseased appearing UC mucosa (lane 1), the opposite was seen, with minimal to no occludin and a dark band at claudin-1. This results in a significantly higher claudin-1 to occludin ratio. Caco-2 cells are shown as a positive control for both proteins. b Graphical summary showing the mean C:O ratio in all samples segregated by disease and tissue state. There is a significant increase in the C:O ratio in diseased UC tissue compared to healthy UC and ND (**P <0.001, and ***P <0.01, respectively). There is no difference in the C:O ratio amongst any of the CD tissue regardless of disease state. There is also a significant increase in the C:O ratio in diseased UC tissue compared to healthy and diseased CD tissue. Values are mean ± standard error. *P <0.01 versus disease UC [1]. n ≥ 11 for ND, n ≥ 9 for CD, n ≥ 16 for UC