Identifying Epithelial Endocytotic Mechanisms of the Peanut Allergens Ara h 1 and Ara h 2

Abstract

Background: Peanuts are still one of the highest contributors to anaphylactic deaths after ingestion of a food allergen. At the molecular level, interactions between peanut allergens and the intestinal epithelium are largely unexplored. Previous findings by our research group demonstrated that the major peanut allergens, i.e., Ara h 1, Ara h 2, Ara h 3, and Ara h 6, were able to cross the Caco-2 human cell culture model of the intestinal epithelium. This research broadened our investigation to identify the mechanisms by which the Caco-2 monolayers uptake peanut allergens, specifically by endocytosis. Here, we aim to increase our understanding of allergen-epithelial interactions and, more broadly, the pathway from allergen to allergy. Methods: The human Caco-2 cell culture model was exposed to peanut extract and a combination of confocal microscopy and inhibition studies were used to identify the endocytotic mechanisms of peanut allergens in intestinal epithelia. Results: Our findings demonstrate that the peanut allergens Ara h 1 and Ara h 2 are transported through intestinal epithelia initially via early endosomes using multiple endocytotic mechanisms. From there, they are then transported to late endosomes and ultimately to lysosomes. Conclusions: These novel findings provide insight into the allergen-epithelial interactions of peanut allergens with the intestinal epithelium. Consequently, this opens the possibility of the use of these endocytotic pathways as targets for inhibitors in therapeutic development and preventative measures for peanut allergy in the future.

Applying the adverse outcome pathway (AOP) for food sensitization to support in vitro testing strategies

“…In addition, TEER values were restored to their original levels, demonstrating the absence of any permanent damage on the monolayer caused by egg digests tested (Grootaert et al, 2017). In line with this, previous studies also reported that purified proteins from wheat (ω5-gliadin and LTP 1) (Bodinier et al, 2007), peach (Pru p 3 and LTP 1) (Tordesillas et al, 2013), and peanut (Ara h 1 and Ara h 2) (Price, Ackland, & Suphioglu, 2017) were able to cross Caco-2 monolayers without compromising cell monolayer integrity.…”

“…Important to mention here is that many of these inhibitors (e.g. filipin, cytochalasinD and monodansylcadaverine) also affect viability as well as TEER values (Price et al, 2017). Little information with regard to the importance of various mechanisms of endocytosis is yet available for food allergens.…”

Detection of the Peanut Allergens Ara h 2 and Ara h 6 in Human Breast Milk: Development of 2 Sensitive and Specific Sandwich ELISA Assays

“…Previous investigations using a Caco-2 human cell culture model of the intestinal epithelium showed that the major allergens Ara h 1 and Ara h 2, as well as Ara h 3 and Ara h 6, can cross via these cells [32]. Price et al [33] recently specified the endocytic mechanisms by which Ara h 1 and Ara h 2 pass the Caco-2 cells. …”

Recent developments and highlights in food allergy

“…11 Taking advantage of endocytotic inhibitors, Ara h 1 was proposed to utilize a combination of macropinocytosis and clathrin-mediated endocytosis for transportation, whereas Ara h 2 was suggested to utilize macropinocytosis and caveolae-mediated endocyotosis. 11 In a human organoid and a mouse-based approach, an IL-13-driven, CD38 cyclic adenosine diphosphate ribose-dependent process has been identified, which is responsible for the transfer of food allergens to lamina propria mucosal mast cells. 12 As suppression of these mechanisms resulted in the abrogation of food-induced allergic responses, these exciting data may add to knowledge of the benefit of blocking IL-13/IL-4 pathways.…”