Gut Barrier Dysfunction-A Primary Defect in Twins with Crohn's Disease Predominantly Caused by Genetic Predisposition

Abstract

Background and aims: The aetiology of Crohn's disease is poorly understood. By investigating twin pairs discordant for Crohn's disease, we aimed to assess whether the dysregulated barrier represents a cause or a consequence of inflammation and to evaluate the impact of genetic predisposition on barrier function.

Methods: Ileal biopsies from 15 twin pairs discordant for Crohn's disease [monozygotic n = 9, dizygotic n = 6] and 10 external controls were mounted in Ussing chambers to assess paracellular permeability to 51Chromium [Cr]-EDTA and trancellular passage to non-pathogenic E. coli K-12. Experiments were performed with and without provocation with acetylsalicylic acid. Immunofluorescence and ELISA were used to quantify the expression level of tight junction proteins.

Results: Healthy co-twins and affected twins displayed increased 51Cr-EDTA permeability at 120 min, both with acetylsalicylic acid [p < 0.001] and without [p < 0.001] when compared with controls. A significant increase in 51Cr-EDTA flux was already seen at 20 min in healthy monozygotic co-twins compared with controls [p≤0.05] when stratified by zygosity, but not in healthy dizygotic co-twins. No difference in E. coli passage was observed between groups. Immunofluorescence of the tight junction proteins claudin-5 and tricellulin showed lower levels in healthy co-twins [p < 0.05] and affected twins [p < 0.05] compared with external controls, while ELISA only showed lower tricellulin in Crohn's disease twins [p < 0.05].

Conclusion: Our results suggest that barrier dysfunction is a primary defect in Crohn's disease, since changes were predominantly seen in healthy monozygotic co-twins. Passage of E. coli seems to be a consequence of inflammation, rather than representing a primary defect.

Figure 1.

Electrophysiology measurements at 120 min from the start of the Ussing chamber experiments showed: [A] A similar short-circuit current [ISC] in ileal biopsies from Crohn’s disease [CD] twins, healthy co-twins, and external controls. Provocation with acetylsalicylic acid [ASA] significantly increased ISC in all groups. [B] A similar transepithelial resistance [TER] in all groups, and significantly lower TER after ASA-provocation. The intergroup comparisons were not statistically significant.

Figure 2.

Paracellular permeability assessed by ⁵¹Chromium [Cr]-EDTA flux in ileal biopsies from healthy co-twins, twins with Crohn’s disease [CD], and external controls mounted in Ussing chambers. Twins were subdivided into monozygotic [filled squares] and dizygotic [filled circles]. Permeability to ⁵¹Cr-EDTA was measured after 20 and 120 min, with and without ASA provocation. [A] Comparisons between CD twins, healthy co-twins, and external controls. [B] Healthy co-twins stratified on zygosity and compared between mono- and dizygotic healthy co-twins, and external controls. Comparisons were done using the Mann–Whitney U test. Grey points represent values ±3 IQR outside the 1st and 3rd quartiles, which were judged as outliers and not included in the comparison.

Figure 2.

Paracellular permeability assessed by ⁵¹Chromium [Cr]-EDTA flux in ileal biopsies from healthy co-twins, twins with Crohn’s disease [CD], and external controls mounted in Ussing chambers. Twins were subdivided into monozygotic [filled squares] and dizygotic [filled circles]. Permeability to ⁵¹Cr-EDTA was measured after 20 and 120 min, with and without ASA provocation. [A] Comparisons between CD twins, healthy co-twins, and external controls. [B] Healthy co-twins stratified on zygosity and compared between mono- and dizygotic healthy co-twins, and external controls. Comparisons were done using the Mann–Whitney U test. Grey points represent values ±3 IQR outside the 1st and 3rd quartiles, which were judged as outliers and not included in the comparison.

Figure 3.

Bacterial passage assessed by E. coli K-12 flux in ileal biopsies from healthy co-twins, twins with Crohn’s disease [CD], and external controls mounted in Ussing chambers. Twins were subdivided into monozygotic [filled squares] and dizygotic [filled circles]. E. coli passage was measured after 20 and 120 min, with and without ASA provocation. Comparisons were done using the Mann–Whitney U test.

Figure 4.

Levels of the tight junction proteins claudin-1 [A], claudin-5 [B], and tricellulin [C] assessed by immunofluorescence in ileal biopsies. [D] Representative photographs of claudin-5 staining in one twin with Crohn’s disease [CD], the healthy co-twin, and the external control. The sealing tight junction protein claudin-5 [red] was mostly expressed on the apical side of the colonocytes; blue = staining of cell nucleus with DAPI. Comparisons were done using the Mann–Whitney U test.