Supplementary Material for: Ingestion of Casein Hydrolysate Induces Oral Tolerance and Suppresses Subsequent Epicutaneous Sensitization and Development of Anaphylaxis Reaction to Casein in Mice

<b><i>Background:</i></b> Casein is the most dominant causal allergen in cow’s milk allergy (CMA). Casein hydrolysates are frequently applied in infant formulas for children with a risk or history of CMA. However, there is limited information on the oral tolerance-inducing ability of casein hydrolysates. <b><i>Objectives:</i></b> The aim of this study was to investigate whether the ingestion of casein hydrolysate induces tolerance to casein, ultimately preventing subsequent epicutaneous sensitization and development of an anaphylaxis reaction. <b><i>Methods:</i></b> BALB/c mice were orally administered casein or a casein hydrolysate (CNH) via the drinking water and were then epicutaneously sensitized by repeated exposure of casein on tape-stripped skin. Sensitization was assessed by basophil activation tests, the serum levels of casein-specific antibodies, and cytokine production from casein-stimulated spleen and mesenteric lymph node (MLN) cells. Occurrence of an anaphylaxis reaction was evaluated by measuring rectal temperature and the plasma level of mouse mast cell protease-1 (mMCP-1) after oral casein challenges. The T cell population in the spleen and MLN was assessed by flow cytometry. Intestinal mast cells and basophils were analyzed histologically. <b><i>Results:</i></b> Sensitization and anaphylaxis reaction to casein were significantly suppressed in casein- or CNH-fed mice compared to controls. Prior ingestion of casein or CNH had no effect on the population of regulatory T cells and activated T cells in lymphoid tissues. Intestinal basophils increased by the epicutaneous sensitization of casein, which was suppressed in casein- or CNH-fed mice. Although the increase in the plasma level of mMCP-1 after oral challenge was suppressed in casein- or CNH-fed mice, there was no change in the number of intestinal mast cells. <b><i>Conclusion:</i></b> Prior ingestion of casein or CNH induced oral tolerance and suppressed subsequent epicutaneous sensitization and development of systemic anaphylaxis to casein.