Molecular and cellular mechanisms in food anaphylaxis

摘要 食物过敏是通过与肥大细胞(MC)结合的IgE抗体进行抗原交联而介导的，通常 与肠道生物失调有关。IgE本身对食物的敏感度不足以引发食物过敏反应。 肠道MC负荷控制肠道通透性和全身抗原吸收，是一种重要的 行列式。鸟嘌呤核苷酸交换因子DOCK8对STAT3的激活、IL-22和IL-2的表达起重要作用。 17生产。由于食物过敏和过敏反应是DOCK8缺陷患者的主要特征，DOCK8 缺乏为确定食物过敏的潜在机制提供了一个有吸引力的疾病模型。 我们发现DOCK8缺乏症患者血清类胰蛋白酶水平升高。Dock8-/-小鼠表现出夸大的IgE- 介导性过敏反应为口服，但不是腹腔注射。抗原挑战，以及肠道MC扩张，这是我们展示的 既不是MC内细胞固有的DOCK8缺陷的结果，也不依赖于IgE。小鼠带有 Dock8-/-小鼠T细胞DOCK8选择性缺陷，两者表现出相似的肠道生物失调T Dock8-/-小鼠小肠(SI)固有层(LP)细胞IL22和IL17表达减少 IL-4、IL-13表达增加。DOCK8缺乏的SI上皮细胞表达减少 抗菌肽(AMP)基因，但丛生细胞数量增加和IL25表达增加。治疗 使用sIL-22或口服抗生素鸡尾酒可使肠道MC扩张正常化，这表明 由17型细胞因子缺乏引起的生物失调。IL-25和IL-4阻断降低肠道MC负荷和被动 Dock8-/-小鼠出现口服过敏反应，提示IL-25在促进2型细胞因子产生中起关键作用 通过ILC2和Th2细胞，以及作用于MC的IL-4。重要的是，粪便的无菌(GF)WT接受者 来自传统饲养的Dock8-/-小鼠的微生物区系移植(FMT)具有显著的肠道 MC负荷与接受常规提高的WT对照的FMT的GF WT受者的比较。因此，我们提出的 研究探讨DOCK8缺乏症导致肠道MC负荷增加的机制 从而促进过敏反应与经典食物过敏患者有关。 我们将检验这样的假设：1)由于缺陷导致17型细胞因子的产生减少 DOCK8缺乏症中STAT3的激活导致肠道生物失调，2)生物失调导致肠丛生 细胞产生IL-25，它驱动ILC2和Th2细胞产生2型细胞因子，以及3)IL-4，在 反过来，推动肠道MC的扩张和激活，从而促进对摄入的抗原的过敏反应。 我们将利用转基因小鼠，并利用我们接触DOCK8缺陷患者和 以前很好地描述了典型食物过敏儿童的粪便样本，以验证我们的假设。 拟议的研究将勾勒出免疫机制对维持肠道的重要性 微生物群调节肠道MC的扩张和激活，从而调节食物过敏的严重程度，从而 确定预防和治疗食物过敏的新策略。

Abstract Food allergy is mediated by antigen crosslinking of IgE antibodies bound to mast cells (MC) and is often associated with intestinal dysbiosis. IgE sensitization to food is not by itself sufficient to drive food anaphylaxis. Intestinal MC load, which controls intestinal permeability and systemic antigen absorption, is an important determinant. The guanine nucleotide exchange factor, DOCK8, is important for STAT3 activation, IL-22, and IL- 17 production. As food allergy and anaphylaxis are cardinal features of DOCK8 deficient patients, DOCK8 deficiency offers an attractive disease model for identifying mechanisms underlying food allergy. We find elevated serum tryptase levels in DOCK8 deficient patients. Dock8-/- mice exhibit exaggerated IgE- mediated anaphylaxis to oral, but not i.p. antigen challenge, as well as intestinal MC expansion, which we show is neither a consequence of a cell intrinsic DOCK8 deficiency within MCs, nor dependent on IgE. Mice with selective deficiency in DOCK8 in T cells phenocopy Dock8-/- mice, and both exhibit similar intestinal dysbiosis T cells in the small intestine (SI) lamina propria (LP) of Dock8-/- mice have decreased expression of Il22 and Il17 and increased expression of Il4 and Il13. DOCK8 deficient SI epithelium has decreased expression of antimicrobial peptide (AMP) genes, but increased numbers of tuft cells and elevated Il25 expression. Treatment with either sIL-22 or an oral antibiotic cocktail normalizes intestinal MC expansion, suggesting a critical role for dysbiosis driven by type 17 cytokine deficiency. IL-25 and IL-4 blockade reduce intestinal MC load and passive oral anaphylaxis in Dock8-/- mice, suggesting a critical role for IL-25, which promotes type 2 cytokine production by ILC2s and Th2 cells, and for IL-4, which acts on MCs. Importantly, germ free (GF) WT recipients of fecal microbiota transplantation (FMT) from conventionally raised Dock8-/- mice have significantly greater intestinal MC load compared to GF WT recipients of FMT from conventionally raised WT controls. Thus, our proposed studies investigate the mechanisms by which dysbiosis in DOCK8 deficiency may increase intestinal MC load and thereby promote anaphylaxis are relevant to patients with classic food allergy. We will test the hypotheses that 1) reduced production of type 17 cytokines due to defective STAT3 activation in DOCK8 deficiency results in intestinal dysbiosis, 2) dysbiosis causes intestinal tuft cells to produce IL-25, which drives type 2 cytokine production by ILC2s and Th2 cells, and 3) IL-4, in turn, drives intestinal MC expansion and activation, thereby promoting anaphylaxis to ingested antigen. We will make use of transgenic mice and take advantage of our access to DOCK8 deficient patients and previously well-characterized fecal samples from children with classic food allergy to test our hypotheses. The proposed studies will delineate how immune mechanisms important for maintaining the intestinal microbiome regulate intestinal MC expansion and activation, and thereby the severity of food allergy, thus identifying novel strategies for the prevention and treatment of food allergy.