Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.

Clinical allergic airway disease is associated with persistent airway hyperreactivity and remodeling, but little is known about the mechanisms leading to these alterations. This paucity of information is related in part to the absence of chronic models of allergic airway disease. Herein we describe a model of persistent airway hyperreactivity, goblet cell hyperplasia, and subepithelial fibrosis that is initiated by the intratracheal introduction of Aspergillus fumigatus spores or conidia into the airways of mice previously sensitized to A. fumigatus. Similar persistent airway alterations were not observed in nonsensitized mice challenged with A. fumigatus conidia alone. A. fumigatus-sensitized mice exhibited significantly enhanced airway hyperresponsiveness to a methacholine challenge that was still present at 30 days after the conidia challenge. Eosinophils and lymphocytes were present in bronchoalveolar lavage (BAL) samples from A. fumigatus-sensitized mice at all times after conidia challenge. Compared with levels measured in A. fumigatus-sensitized mice immediately before conidia, significantly elevated interferon-␥ (IFN-␥) and transforming growth factor (TGF-␤) levels were present in whole lung homogenates up to 7 days after the conidia challenge. At day 30 after conidia challenge, significantly elevated levels of interleukin-4 (IL-4) and IL-13 were present in the A. fumigatussensitized mice. Histological analysis revealed profound goblet cell hyperplasia and airway fibrosis at days 30 after conidia, and the latter finding was confirmed by hydroxyproline measurements. Thus the

Allergic responses to Aspergillus species exacerbate asthma and cystic fibrosis. The natural defense against live Aspergillus fumigatus spores or conidia depends on the recruitment and activation of mononuclear and polymorphonuclear leukocytes, events that are dependent on chemotactic cytokines. In this study, we explored the relative contribution of the monocyte chemoattractant protein-1 receptor, CCR2, in the pulmonary response to A. fumigatus conidia. Following sensitization to soluble A. fumigatus Ags, mice lacking CCR2 due to targeted deletion were markedly more susceptible to the injurious effects of an intrapulmonary challenge with live conidia compared with mice that expressed CCR2 or CCR2+/+. CCR2−/− mice exhibited a major defect in the recruitment of polymorphonuclear cells, but these mice also had significantly more eosinophils and lymphocytes in bronchoalveolar lavage samples. CCR2−/− mice also had significant increases in serum levels of total IgE and whole lung levels of IL-5, IL-13, eotaxin, and RANTES compared with CCR2+/+ mice. Airway inflammation, hyper-responsiveness to spasmogens, and subepithelial fibrosis were significantly enhanced in CCR2−/− mice compared with CCR2+/+ mice after the conidia challenge. Thus, these findings demonstrate that CCR2 plays an important role in the immune response against A. fumigatus, thereby limiting the allergic airway inflammatory and remodeling responses to this fungus.

IL-13 and IL-4 are key contributors to the asthmatic phenotype. The temporal role of these cytokines in airway function, inflammation, and remodeling were assessed in a chronic murine model of Asperigillus fumigatus-induced allergic asthma. IL-13 and IL-4 protein levels were significantly elevated by 30 days after conidia challenge in A. fumigatus-sensitized mice. Furthermore, IL-13Rα1 mRNA expression was significantly elevated 7 days after conidia challenge and remained elevated until day 21. In contrast, IL-13Rα2 mRNA expression, although constitutively expressed in naive lung, was absent in the lungs of A. fumigatus-sensitized mice both before and after conidia challenge. Membrane-bound IL-4R mRNA expression was significantly elevated 7 days after conidia challenge; however, soluble IL-4R mRNA expression was increased 30 days after conidia challenge. Immunoneutralization of IL-13 between days 14 and 30 or days 30 and 38 after fungal sensitization and challenge significantly attenuated airway hyperresponsiveness, collagen deposition, and goblet cell hyperplasia at day 38 after conidia challenge; however, the effects of IL-4 immunoneutralization during the same time periods were not as marked. IFN-γ and IL-12 release after Aspergillus Ag restimulation was elevated from spleen cells isolated from mice treated with IL-4 anti-serum compared with IL-13 anti-serum or normal rabbit serum-treated mice. This study demonstrates a pronounced therapeutic effect of IL-13-immunoneutralization at extended time points following the induction of chronic asthma. Most importantly, these therapeutic effects were not reversed following cessation of treatment, and IL-13 anti-serum treatment did not alter the systemic immune response to Ag restimulation, unlike IL-4 immunoneutralization. Therefore, IL-13 provides an attractive therapeutic target in allergic asthma.

Asthmatic-like reactions characterized by elevated IgE, Th2 cytokines, C-C chemokines, eosinophilic inflammation, and persistent airway hyperresponsiveness follow pulmonary exposure to the spores or conidia from Aspergillus fumigatus fungus in sensitized individuals. In addition to these features, subepithelial fibrosis and goblet cell hyperplasia characterizes fungal-induced allergic airway disease in mice. Because lung concentrations of macrophage inflammatory protein-1α and RANTES were significantly elevated after A. fumigatus-sensitized mice received an intrapulmonary challenge with A. fumigatus spores or conidia, the present study addressed the role of their receptor, C-C chemokine receptor 1 (CCR1), in this model. A. fumigatus-sensitized CCR1 wild-type (+/+) and CCR1 knockout (−/−) mice exhibited similar increases in serum IgE and polymorphonuclear leukocyte numbers in the bronchoalveolar lavage. Airway hyperresponsiveness was prominent in both groups of mice at 30 days after an intrapulmonary challenge with A. fumigatus spores or conidia. However, whole lung levels of IFN-γ were significantly higher whereas IL-4, IL-13, and Th2-inducible chemokines such as C10, eotaxin, and macrophage-derived chemokine were significantly lower in whole lung samples from CCR1−/− mice compared with CCR1+/+ mice at 30 days after the conidia challenge. Likewise, significantly fewer goblet cells and less subepithelial fibrosis were observed around large airways in CCR1−/− mice at the same time after the conidia challenge. Thus, these findings demonstrate that CCR1 is a major contributor to the airway remodeling responses that arise from A. fumigatus-induced allergic airway disease.