In vivo and ex vivo inflammatory responses of the esophageal mucosa to food challenge in adults with eosinophilic esophagitis

To the Editor, Elimination diets without the causative foods induce histological and clinical remission in patients with eosinophilic esophagitis (EoE), an allergendriven type 2 inflammatory disease of the esophagus.1 However, current tests using skin or serum are poorly predictive of the causative foods,2 likely because the allergic inflammation may be restricted to the esophagus. We aimed to determine whether in vivo and ex vivo challenge of the esophageal mucosa with whole food extracts could yield clinically and immunologically relevant information about esophageal responses to specific foods. During endoscopy, the esophageal mucosa of 12 EoE patients was challenged by local injection of 3 common food triggers (cow's milk, wheat, and apple) and 3 foods based on patient's clinical history, and by local flush (i.e., spray) of a mixture of the 6 foods. Acute local responses were monitored for 20 min. Skin prick tests (SPT) and serum IgE measurements were also performed. Esophageal biopsies were exposed to foods in culture to analyze inflammatory mediator production, which was compared with 6 nonEoE controls. Methods are fully described in the Appendix S1. Patient characteristics are provided in Table 1. All patients were previously diagnosed with EoE and presented with the typical symptoms and endoscopic signs of EoE at time of endoscopy. Of the 11 patients that underwent injections, acute responses characterized by edema, erythema, or smooth muscle contraction (determined by the formation of a muscular ring) at the injection site were observed in 8 patients after injection with apple (n = 4), peanut (n = 4), wheat (n = 3), milk (n = 2), tomato (n = 1), egg (n = 1), and mango (n = 1) (Table 1; Figure 1A). In addition, after the end of the endoscopy, 4 patients experienced dysphagia, cramping retrosternal pain or burning sensation that was similar to pain occurring after ingesting those foods. Of the in total 17 foods that induced acute responses following local injections, 9 foods (53%) corresponded with patient's clinical history, 6 (35%) with SPT results and 6 (35%) with serum IgE results. The local flush with a mixture of foods also induced acute responses but, unlike the injections, these responses were barely notable and were observed in only 4 patients (Table S1). Our results confirm the observations of our previous study that esophageal food challenge can trigger local responses in adult EoE patients.3 However, there was no clear relation between foods that induced a response by mucosal injection, and SPT or serum IgE. The fact that the foods that induce mucosal responses do not necessarily show positive SPT and/or serum IgE results, and the fact that SPT and serum IgE are poorly predictive of the causative foods,2 indicate that local esophageal challenge may indeed be needed for a better prediction of the causative foods. Nonetheless, given the moderate responsiveness to challenge by flush, the clinical challenges associated with injections, and the invasiveness for patients as endoscopic challenge can induce short lasting but severe symptoms, both challenge tests will not likely become a useful test in clinical practice. In contrast, a less invasive biopsybased ex vivo food challenge test may be considered a promising tool for the identification of causative foods in EoE patients. Nonchallenged EoE esophageal biopsies maintained in culture for 24 h showed increased production of total IgE (13.7 vs. 0.1 ng/mg, p = .0002), IL5 (12.5 vs. 1.1 pg/mg, p = .0288), IL6 (29.8 vs. 1.5 ng/mg, p = .0047), IL8 (86.6 vs. 23.2 ng/ mg, p = .0069), IL13 (28.6 vs. 0.0 pg/mg, p = .0080), and MCP1 (659 vs. 112 pg/mg, p = .0320) compared with nonchallenged biopsies from controls (Figure 1B). Eotaxin, IL9, and IFNγ were below the detection limit. Analysis of protein levels based on peak eosinophil count did not provide additional insights (data not shown). Furthermore, when exposing biopsies to food in culture, an immunological response is triggered that may reflect the inflammatory cascade seen in EoE. Interestingly, IL5 levels were increased after ex vivo exposure to milk (89.8 vs. 12.5 pg/mg, p = .0195), and IL9 was increased after exposure to apple (132.3 vs. 0.0 pg/mg, p = .0039; Figure 1C). To our knowledge, we are the first to report foodspecific induction of IL5, an important factor in eosinophil trafficking,4 and IL9, a promotor of mast cell expansion and function,5 in the inflamed esophagus of EoE patients, highlighting a potential role for both cytokines in the allergenspecific immune response in EoE. Lastly, we used a machine learning approach6 to study whether the ex vivo challenge test can better discriminate clinically suspected (as provided in Table 1) from nonsuspected foods than the conventional SPT and serum IgE. Indeed, the ex vivo challenge test outperformed SPT/serum IgE with an AUC of 0.64 vs. 0.5 (Figure S1), evidencing sufficient discriminative scores.7 Performing food rechallenges based on the ex vivo results was beyond the scope of this study. This study has limitations. Our study was conducted in a small cohort, and the tested foods were not proven by elimination diets.


In vivo and ex vivo inflammatory responses of the esophageal mucosa to food challenge in adults with eosinophilic esophagitis
To the Editor, Elimination diets without the causative foods induce histological and clinical remission in patients with eosinophilic esophagitis (EoE), an allergen-driven type 2 inflammatory disease of the esophagus. 1 However, current tests using skin or serum are poorly predictive of the causative foods, 2 likely because the allergic inflammation may be restricted to the esophagus. We aimed to determine whether in vivo and ex vivo challenge of the esophageal mucosa with whole food extracts could yield clinically and immunologically relevant information about esophageal responses to specific foods.
During endoscopy, the esophageal mucosa of 12 EoE patients was challenged by local injection of 3 common food triggers (cow's milk, wheat, and apple) and 3 foods based on patient's clinical history, and by local flush (i.e., spray) of a mixture of the 6 foods. Acute local responses were monitored for 20 min. Skin prick tests (SPT) and serum IgE measurements were also performed. Esophageal biopsies were exposed to foods in culture to analyze inflammatory mediator production, which was compared with 6 non-EoE controls. Methods are fully described in the Appendix S1. Patient characteristics are provided in Table 1.
All patients were previously diagnosed with EoE and presented with the typical symptoms and endoscopic signs of EoE at time of endoscopy. Of the 11 patients that underwent injections, acute responses characterized by edema, erythema, or smooth muscle contraction (determined by the formation of a muscular ring) at the injection site were observed in 8 patients after injection with apple (n = 4), peanut (n = 4), wheat (n = 3), milk (n = 2), tomato (n = 1), egg (n = 1), and mango (n = 1) (Table 1; Figure 1A). In addition, after the end of the endoscopy, 4 patients experienced dysphagia, cramping retrosternal pain or burning sensation that was similar to pain occurring after ingesting those foods. Of the in total 17 foods that induced acute responses following local injections, 9 foods (53%) corresponded with patient's clinical history, 6 (35%) with SPT results and 6 (35%) with serum IgE results. The local flush with a mixture of foods also induced acute responses but, unlike the injections, these responses were barely notable and were observed in only 4 patients (Table S1). Our results confirm the observations of our previous study that esophageal food challenge can trigger local responses in adult EoE patients. 3 However, there was no clear relation between foods that induced a response by mucosal injection, and SPT or  Figure 1B). Eotaxin, IL-9, and IFNγ were below the detection limit. Analysis of protein levels based on peak eosinophil count did not provide additional insights (data not shown).
Furthermore, when exposing biopsies to food in culture, an immunological response is triggered that may reflect the inflammatory cascade seen in EoE. Interestingly, IL-5 levels were increased after ex vivo exposure to milk (89.8 vs. 12.5 pg/mg, p = .0195), and IL-9 was increased after exposure to apple (132.3 vs. 0.0 pg/mg, p = .0039; Figure 1C). To our knowledge, we are the first to report food-specific induction of IL-5, an important factor in eosinophil trafficking, 4 and IL-9, a promotor of mast cell expansion and function, 5 in the inflamed esophagus of EoE patients, highlighting a potential role for both cytokines in the allergen-specific immune response in EoE.
Lastly, we used a machine learning approach 6 to study whether the ex vivo challenge test can better discriminate clinically suspected (as provided in Table 1) from non-suspected foods than the conventional SPT and serum IgE. Indeed, the ex vivo challenge test outperformed SPT/serum IgE with an AUC of 0.64 vs. 0.5 ( Figure S1), evidencing sufficient discriminative scores. 7 Performing food re-challenges based on the ex vivo results was beyond the scope of this study.
This study has limitations. Our study was conducted in a small cohort, and the tested foods were not proven by elimination diets.    .1292