OBJECTIVES:

The objective of this study was to investigate how mast cell tryptase may influence intestinal permeability and tight junction (TJ) proteinsin vitroand explore translation to irritable bowel syndrome (IBS).

METHODS:

We investigated the effect of:(1) tryptase on Caco-2 monolayers,(2) mast cell degranulation in a Caco-2/human mast cell-1 (HMC-1) co-culture model,(3) mast cell degranulation±tryptase inhibition with nafamostat mesilate (NM). Epithelial integrity was assessed by transepithelial resistance (TER), permeability to fluorescein isothiocyanate (FITC)-dextran and transmission electron microscopy (TEM). The expression of junctional proteins zonula occludens-1 (ZO-1), junctional adhesion molecule-A (JAM-A), claudin-1 (CLD-1), CLD-2, CLD-3, occludin and E-cadherin was determined by western blot analysis and immunofluorescence confocal microscopy. Based on thein vitroresults, we further assessed JAM-A expression in biopsy tissue (cecum) from 34 IBS patients, 12 controls, and 8 inflammatory controls using immunofluorescence confocal microscopy and explored associations between JAM-A and IBS symptoms.

RESULTS:

ptase disrupted epithelial integrity in Caco-2 monolayers as shown by a significant decrease in TER, an increase in permeability to FITC-dextran, and a decrease in the expression of junctional proteins JAM-A, CLD-1, and ZO-1 within 24 h. Correspondingly, in the Caco-2/HMC-1 co-culture model we showed a significant decrease in TER, an increase in permeability to FITC-dextran, and the presence of open TJs (TEM) in response to mast cell degranulation within 24 h. In this co-culture model, mast cell degranulation significantly decreased JAM-A and CLD-1 protein expression at 24 h. Tryptase inhibition (NM) significantly reduced the effect of mast cell degranulation on the junctional protein JAM-A, TER, and FITC-dextran flux. In IBS, epithelial JAM-A protein expression was significantly reduced in IBS tissue compared with controls. Lower JAM-A expression was associated with more severe abdominal pain (r s=− 0.69, P= 0.018) and longer duration of symptoms (r s=− 0.7, P= 0.012) in IBS-alternating subtype.

CONCLUSIONS:

uced JAM-A expressionin vitroappears to contribute to the underlying mechanisms of altered epithelial integrity in response to tryptase released from degranulating mast cells. In IBS, JAM-A expression was significantly reduced in the cecal epithelium and associated with abdominal pain severity. JAM-A may provide new insights into the underlying mechanisms in IBS.