Department of Pathology and Medical Biology, Laboratory of Experimental Pulmonology and Inflammation Research (EXPIRE), University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
Groningen Research Institute for Asthma and COPD (GRIAC), University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
Methods Mol Biol. 2021;2223:295-335. doi: 10.1007/978-1-0716-1001-5_20.
Allergic asthma is characterized by airway hyperresponsiveness, remodeling, and reversible airway obstruction. This is associated with an eosinophilic inflammation of the airways, caused by inhaled allergens such as house dust mite or grass pollen. The inhaled allergens trigger a type-2 inflammatory response with the involvement of innate lymphoid cells (ILC2) and Th2 cells, resulting in high immunoglobulin E (IgE) antibody production by B cells and mucus production by airway epithelial cells. As a consequence of the IgE production, subsequent allergen reexposure results in a classic allergic response with distinct early and late phases, both resulting in bronchoconstriction and shortness of breath. Allergen-specific immunotherapy (AIT) is the only treatment that is capable of modifying the immunological process underlying allergic responses including allergic asthma. Both subcutaneous AIT (SCIT) as well as sublingual AIT (SLIT) have shown clinical efficacy in long-term suppression of the allergic response. Although AIT treatments are very successful for rhinitis, application in asthma is hampered by variable efficacy, long duration of treatment, and risk of severe side effects. A more profound understanding of the mechanisms by which AIT induces tolerance to allergens in sensitized individuals is needed to be able to improve its efficacy. Mouse models have been very valuable in preclinical research for characterizing the mechanisms of desensitization in AIT and evaluating novel approaches to improve its efficacy. Here, we present a rapid and reproducible mouse model for allergen-specific immunotherapy. In this model, mice are sensitized with two injections of allergen adsorbed to aluminum hydroxide, followed by subcutaneous injections (SCIT) or sublingual administrations (SLIT) of allergen extracts as an immunotherapy treatment. Finally, mice are challenged by intranasal allergen administrations. We will also describe the protocols as well as the most important readout parameters for the measurements of invasive lung function, serum immunoglobulin levels, isolation of bronchoalveolar lavage fluid (BALF), and preparation of cytospin slides. Moreover, we describe how to perform ex vivo restimulation of lung single-cell suspensions with allergens, flow cytometry for identification of relevant immune cell populations, and ELISAs and Luminex assays for assessment of the cytokine concentrations in BALF and lung tissue.
过敏性哮喘的特征是气道高反应性、重塑和可逆转的气道阻塞。这与气道嗜酸粒细胞炎症有关,由吸入性过敏原如屋尘螨或草花粉引起。吸入性过敏原引发 2 型炎症反应,涉及固有淋巴细胞 (ILC2) 和 Th2 细胞,导致 B 细胞产生高免疫球蛋白 E (IgE) 抗体和气道上皮细胞产生黏液。由于 IgE 的产生,随后的过敏原再暴露导致经典的过敏反应,具有明显的早期和晚期阶段,两者都导致支气管收缩和呼吸急促。过敏原特异性免疫疗法 (AIT) 是唯一能够改变过敏反应(包括过敏性哮喘)背后免疫过程的治疗方法。皮下免疫疗法 (SCIT) 和舌下免疫疗法 (SLIT) 都已显示出在长期抑制过敏反应方面的临床疗效。尽管 AIT 治疗对鼻炎非常有效,但在哮喘中的应用受到疗效不一、治疗时间长和严重副作用风险的阻碍。需要更深入地了解 AIT 如何诱导致敏个体对过敏原产生耐受的机制,才能提高其疗效。小鼠模型在过敏免疫疗法的脱敏机制的临床前研究中非常有价值,并评估了提高其疗效的新方法。在这里,我们提出了一种快速且可重复的过敏原特异性免疫疗法小鼠模型。在该模型中,通过将过敏原吸附到氢氧化铝上进行两次注射来致敏小鼠,然后进行皮下注射 (SCIT) 或舌下给药 (SLIT) 过敏原提取物作为免疫治疗。最后,通过鼻内过敏原给药对小鼠进行挑战。我们还将描述协议以及用于测量侵袭性肺功能、血清免疫球蛋白水平、支气管肺泡灌洗液 (BALF) 分离和细胞涂片制备的最重要的读数参数。此外,我们描述了如何对肺单细胞悬液进行过敏原的离体再刺激、流式细胞术鉴定相关免疫细胞群以及 ELISA 和 Luminex 测定评估 BALF 和肺组织中的细胞因子浓度。
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