The effect of kiwifruit (Actinidia deliciosa) cysteine protease actinidin on the occludin tight junction network in T84 intestinal epithelial cells

Highlights

• Actinidin is a kiwifruit-specific allergen with cysteine protease properties.

• We investigated if actinidin has any effect on the tight junction protein occludin.

• Actinidin exerted direct proteolytic cleavage of occludin in T84 intestinal cells.

• Disruption of the epithelial barrier led to a leakage of kiwifruit allergens.

• Opening of tight junctions may contribute to sensitization in kiwifruit allergy.

Abstract

Actinidin, a kiwifruit cysteine protease, is a marker allergen for genuine sensitization to this food allergen source. Inhalatory cysteine proteases have the capacity for disruption of tight junctions (TJs) enhancing the permeability of the bronchial epithelium. No such properties have been reported for allergenic food proteases so far. The aim was to determine the effect of actinidin on the integrity of T84 monolayers by evaluating its action on the TJ protein occludin. Immunoblot and immunofluorescence were employed for the detection of occludin protein alterations. Gene expression was evaluated by RT-PCR. Breach of occludin network was assessed by measuring transepithelial resistance, blue dextran leakage and passage of allergens from the apical to basolateral compartment. Actinidin exerted direct proteolytic cleavage of occludin; no alteration of occludin gene expression was detected. There was a reduction of occludin staining upon actinidin treatment as a consequence of its degradation and dispersion within the membrane. There was an increase in permeability of the T84 monolayer resulting in reduced transepithelial resistance, blue dextran leakage and passage of allergens actinidin and thaumatin-like protein from the apical to basolateral compartment. Opening of TJs by actinidin may increase intestinal permeability and contribute to the process of sensitization in kiwifruit allergy.

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.