The effect of kiwifruit (Actinidia deliciosa) cysteine protease actinidin on the occludin tight junction network in T84 intestinal epithelial cells
Graphical abstract
Introduction
Together with its growing popularity kiwifruit (Actinidia deliciosa) has become an important cause of plant food allergy both in children and adults (Lucas et al., 2004, Popovic et al., 2013). Although the molecular basis of kiwifruit allergy has been associated with eleven IUIS kiwifruit proteins (www.allergen.org), the most abundant is a papain-like cysteine protease actinidin (Act d 1, EC 3.4.22.14) which constitutes up to 50% of soluble proteins in mature kiwifruit. Actinidin is the major IgE reactive protein (Aleman et al., 2004, Bublin et al., 2004, Pastorello et al., 1996) and a kiwifruit-specific allergen since no crossreactivity with birch or grass pollen has been observed (Pastorello et al., 1996). A link between IgE levels to actinidin and anaphylaxis has been found in some clinical subgroups of kiwifruit allergic patients (Aleman et al., 2004, Palacin et al., 2008).
Enzymatic activity of allergens has been proposed as a contributing factor to their sensitization potential. This idea is supported by the observation that the cysteine protease activity of major house dust mite allergens (Der p 1, Der p 3, Der p 6, Der p 9) has a pro-allergenic adjuvant effect by disruption of tight junctions, (Shakib et al., 1998, Wan et al., 1999, Robinson et al., 1997), inducing pulmonary epithelial cell detachment (Tomee et al., 1998) and production of proinflammatory cytokines (King et al., 1998, Tomee et al., 1998), resulting in the enhancement of bronchial epithelium permeability. No such properties have been reported for allergenic food proteases so far.
The physico-chemical properties of most food allergens confer stability to the proteolytic and acidic conditions in the digestive tract, which increases the probability of reaching the intestinal mucosa, where absorption and interaction with the immune system may occur (Bublin et al., 2008). The intestinal tract is covered by a continuous monolayer of intestinal epithelial cells responsible for maintaining the physical and functional barrier between the intestinal lumen and the underlying lamina propria (Groschwitz and Hogan, 2009). In the intestine, epithelial cells are connected to one another by a transmembrane protein complex of tight junctions (TJs), which govern the passive passage of ions, solutes, peptides and proteins from the lumen to the mucosa via the paracellular route (Sander et al., 2005, Camilleri et al., 2012). Various ethiological factors can cause defects in TJ proteins and increase intestinal epithelial permeability leading to disorders such as Crohn’s disease, celiac disease, inflammatory bowel disease, food allergy, and acute pancreatitis (Catalioto et al., 2011). Proteolytic activity is also a factor contributing to intestinal barrier disruption resulting in increased amounts of antigen crossing into the lamina propria, driving further immune responses and sustaining the inflammatory process (Biancheri et al., 2013, Steck et al., 2011). The change of intestinal permeability is regarded as a risk factor for the development of food allergy as potential food allergens need to come into direct contact with the antigen-presenting cells in the submucosa of the intestine in order to induce sensitization (Chahine and Bahna, 2010, Groschwitz and Hogan, 2009, Liu et al., 2011). Although it has been shown that food allergens can permeate the intestinal barrier directly via intestinal epithelial cells (Perrier and Corthésy, 2011), the paracellular passage of food allergens has been less studied. Elucidation of the molecular mechanism responsible for epithelial barrier irregularities is of crucial importance for designing new therapeutic strategies for food allergy.
In the present study the direct action of kiwifruit allergen actinidin on T84 intestinal monolayer integrity was investigated by evaluating its effect on the tight junction protein occludin and by assesing monolayer barrier properties and the paracellular leakage of allergens.
Section snippets
Cell culture
T84 colorectal adenocarcinoma cells (ATCC, Manassas, VA, USA) were grown and maintained in a 1:1 (vol/vol) mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/F12, Sigma–Aldrich, St. Louis, USA), supplemented with 10% fetal bovine serum and penicillin (100 IU)/streptomycin (100 μg/mL) (Sigma–Aldrich, St. Louis, USA) at 37 °C in 5% CO2 humidified atmosphere. Cells were passaged using 0.25% trypsin solution in phosphate-buffered saline (PBS) without calcium and magnesium every
Actinidin induced a decrease of TEER and BD leakage
The effects of actinidin’s proteolytic activity on the permeability of the T84 monolayer were assessed by evaluating the TEER change and blue dextran leakage. Control cells maintained the basal TEER or showed a statistically non-significant increase up to 105 ± 4% of pretreatment TEER over the 4 h period (Fig. 1A). After 1 h of actinidin treatment the TEER was reduced to 89 ± 2% of control (p < 0.05), and after 4 h this effect was more pronounced with the decrease to 77 ± 3% of control TEER (p < 0.001). The
Discussion
In this study, we report that actinidin, a prominent cysteine protease from kiwifruit, disrupted the epithelial barrier function of human intestinal T84 cells by degrading the tight junction protein occludin. Exposure of T84 cells to biologically active actinidin significantly decreased the transepithelial resistance across the intestinal epithelial monolayers. Disruption of the epithelial integrity was confirmed by detecting Blue Dextran 2000 in basolateral samples after apical exposure of T84
Conclusions
The detected impairment of the T84 epithelial barrier observed in this study was a result of actinidin’s direct protease-dependent action on the transmembrane tight junction protein occludin. The breach of the epithelial barrier caused by actinidin led to a nonselective paracellular transport of allergens and may be an important factor for sensitization in kiwifruit allergy.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Transparency Document
Acknowledgement
This research was supported by the Ministry of Education Science and Technological Development of the Republic of Serbia, Grant No. 172049.
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