Novel Humanized Mouse Model for Steroid‐Resistant Asthma

Humanized mice are widely used to mimic human pathological conditions. We previously established a human asthma model using humanized human (h) IL-3/hGM-CSF/hIL-5 transgenic NOD/Shi-scid-IL2rγnull (NOG) mice, which supported the differentiation of human eosinophils, basophils, mast cells, T cells, and B cells, as well as limited numbers of natural killer (NK) cells and type 2 innate lymphoid cells (ILC2) [1]. Approximately 10%–15% of patients with asthma cannot achieve disease control despite receiving high-dose inhaled glucocorticoids plus long-acting β2-agonists or oral corticosteroids. In steroid-resistant asthma, chronic eosinophilic airway inflammation persists and remains unresponsive to corticosteroid treatment. Therefore, targeting eosinophils, particularly with biologics such as anti-IL-5 and IL-5 receptor (IL-5Rα) antibodies, can suppress steroid-resistant eosinophilic inflammation [2]. However, the mechanisms underlying persistent steroid-resistant asthma remain unclear due to difficulties in direct human lung analyses. First, we established a human steroid-resistant asthma model using our previously reported humanized mice [1]. We intratracheally administered recombinant human (rh) IL-33 (rhIL-33) and thymic stromal lymphopoietin (rhTSLP) to humanized mice. We also administered dexamethasone (Dexa) intraperitoneally over days 0–3 (Figure 1A; details in the Data S1). Human eosinophil infiltration in bronchoalveolar lavage fluid (BALF) increased more with the rhIL-33 treatment compared with the PBS control group. Dexa significantly suppressed rhIL-33-induced human eosinophil infiltration, yet did not significantly suppress rhIL-33 + rhTSLP-induced human eosinophil infiltration and human eosinophil-derived neurotoxin (EDN) levels—a marker for human eosinophil activation—compared with rhIL-33 (Figure 1B,C). Dexa did not suppress rhIL-33 + rhTSLP-induced airway inflammation, goblet cell hyperplasia, type 2 cytokine production, or airway hyperresponsiveness (AHR) (Figure 1D–F). These data suggest that rhTSLP counteracts steroid suppression of IL-33-induced human asthma phenotype. To confirm the role of intratracheally administered rhTSLP, we injected an anti-TSLP monoclonal antibody (Details in Data S1) subcutaneously into rhIL-33 plus rhTSLP-administered mice and measured human eosinophils in BALF (Figure S1A). The anti-TSLP monoclonal antibody inhibited rhIL-33 + rhTSLP-induced human eosinophilia (Figure S1B). As previously reported [3], TSLP itself may also have contributed to the prolonged eosinophil survival in our setting. Benralizumab, an anti-IL-5Rα monoclonal antibody, can treat steroid-resistant asthma [2]. We verified the efficacy of benralizumab in suppressing rhIL-33 + rhTSLP-induced human eosinophilic inflammations. We administered benralizumab intraperitoneally to our steroid-resistant human asthma model (Figure 2A, details in Data S1). Benralizumab suppressed rhIL-33 + rhTSLP-induced human eosinophilic inflammation, hEDN production in BALFs, and airway inflammation in lung tissues (Figure 2B,C). However, it did not suppress goblet cell hyperplasia, type 2 cytokines, or AHR (Figure 2D–F), which was supported by the findings that AHR correlates with airway TSLP in asthma independent of eosinophilic inflammation [4]. Our study demonstrated that benralizumab, while effectively depleting eosinophils, did not reduce IL-5 or IL-13 levels in BALF. Although mast cells and basophils, major sources of IL-5 and IL-13, express IL-5Rα [5], Benralizumab did not reduce IL-5 or IL-13 levels in BALF. As previously reported, benralizumab does not deplete mast cells or basophils [6], which may explain the persistent type 2 cytokine production. Furthermore, benralizumab did not inhibit AHR, which is consistent with previous findings that mast cell–airway smooth muscle interactions contribute to the pathogenesis of AHR [7]. These data suggest that benralizumab directly suppresses eosinophil survival, while mast cells and basophils remain unaffected. Benralizumab reportedly reduces eosinophil levels through NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) activity [2]. Eosinophils remained in BALF even after benralizumab administration. Since our humanized mouse model had few NK cells, ADCC activity may have been insufficient. Our model has several limitations. Murine eosinophils are not entirely excluded in our humanized mice, and their pulmonary structure cells remain murine-derived, preventing accurate replication of the human asthma phenotype. ILC2s are known to play a critical role in steroid-resistant asthma [8]. However, their number was extremely low in our humanized mouse model, making it technically difficult to perform reliable analyses. As a result, the underlying mechanisms of steroid resistance and the mechanism of action of biologics in this model remain insufficiently understood. Further improvements to this humanized mouse model are needed to overcome these limitations. Despite these limitations, we established a model of steroid-resistant asthma using humanized mice. This model has potential as a preclinical model for developing novel therapeutics. S.Y., S.M., R.I., and Y.O. conceived and designed the study. S.Y., S.M., S.T., Y.K., K.M., and R.I. conducted experiments. S.Y., S.M., S.T., Y.K., and R.I. developed and maintained the mice. S.Y., S.M., S.T., Y.K., K.M., R.I., Y.O., and Y.G. participated in the analysis and discussions of the results. S.Y. and S.M. wrote the paper. We sincerely appreciate the support and cooperation of everyone who contributed to the completion of this study. We are incredibly grateful to Kaori Soda and Ikuko Takeshita for their efforts in data collection, analysis, and technical assistance. Additionally, we extend our deep gratitude to Dr. Jian Huang for kindly providing the anti-TSLP monoclonal antibody used in this research. The authors, except Okayama Y, declare no relevant conflicts of interest. Okayama Y recently was a speaker and/or adviser for and/or has received research funding from AbbVie, Animal Allergy Clinical Laboratories Inc., AstraZeneca, Kyorin, Nissan Chemical Co., Novartis, Ono Pharma, Sanofi, Seikagaku Co., Thermo Fisher Scientific K.K., and Torii Pharmaceutical Co. Ltd. Gon Y has received research funding from AstraZeneca K.K. Research data are not shared. Figure S1: Anti-TSLP monoclonal antibody suppresses steroid-resistant asthma using humanized mice. Data S1: all70012-sup-0002-Supinfo.docx. 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