tallization rarely occurs in vivo. When it does, crystals are generated, which sustain long-term protein storage or enable the slow release of proteins. In 1853, Charcot reported extracellular bipyramidal crystals in the airways of asthmatics, an observation also made by Leyden in 1872. Charcot-Leyden crystals (CLCs) have since been described mostly in eosinophil-rich inflammatory lesions. They have become a hallmark of eosinophil death and can persist in tissues for months. CLCs are composed of galectin-10 (Gal10), one of the most abundant proteins in human eosinophils. Recent studies suggest that Gal10 is released from the cytoplasm of activated eosinophils. However, whether Gal10 can have a functional role in airway disease and type 2 immunity in vivo after a phase transition to a crystalline state is unknown. RATIONALE: To test the hypothesis that CLCs stimulate immunity in the lung, we produced recombinant Gal10 crystals that were structurally and biochemically similar to CLCs obtained from patients with rhinosinusitis and asthma. Additionally, we engineered Gal10 muteins that selectively lost the ability to crystallize. Using these tools, we studied immune responses in mouse models of asthma. To complement these experiments in mice, we studied Gal10 expression in human samples and developed antibodies that bind and dissolve CLCs. RESULTS: CLCs were abundantly present in the airways of chronic rhinosinusitis patients and correlated with the degree of eosinophil extracellular trap formation. Biosimilar crystalline Gal10 injected into the airways of naïve mice induced an innate immune response, rich in neutrophils and monocytes, and led to the uptake of crystals by dendritic cells (DCs). Soluble Gal10 muteins carrying a mutation of Tyr69 to glutamic acid were unable to crystallize and were immunologically inert. Simultaneous injection of CLCs with innocuous ovalbumin (OVA) resulted in DC uptake and T helper type 2 cell priming, together with airway eosinophilia and immunoglobulin G1 (IgG1) responses. Mechanistically, these effects were accompanied by NLRP3 inflammasome activation and interleukin-1b (IL-1b) release. However, the observed response to CLCs in vivo could occur independently of the NLRP3 inflammasome. In an effort to develop new therapeutic opportunities against this type of crystallopathy, we generated antibodies against crystalline Gal10. The epicenter of each crystal-dissolving antibodybinding epitope on Gal10 was situated at Tyr69, a residue we had identified as a critical crystalpacking hotspot. These antibodies rapidly dissolved preexisting CLCs in vitro and in the native mucus environment of patients. Crystal-dissolving antibodies suppressed airway inflammation, goblet-cell metaplasia, bronchial hyperreactivity, and IgE synthesis induced by CLC and house dust mite inhalation in a humanized mouse model. CONCLUSION: Our results demonstrate that CLCs are more than just markers of eosinophilic inflammation. Rather, Gal10 is released by activated eosinophils and undergoes a phase transition to a crystalline state that actively promotes key features of asthma. Antibodies can rapidly dissolve CLCs abundantly present in the native mucus of patients and resolve key features of CLC crystallopathy in a preclinical model. Although protein crystallization is a rare event, we establish Charcot-Leyden crystallopathy as a druggable trait in patients with airway disease and provide a rationale for how antibodies can dissolve protein crystals.▪

Additional Metadata
Persistent URL dx.doi.org/10.1126/science.aaw4295, hdl.handle.net/1765/117135
Journal Science
Persson, E.K., Verstraete, M, Heyndrickx, I., Gevaert, E., Aegerter, H., Percier, J.M., … Lambrecht, B.N.M. (2019). Protein crystallization promotes type 2 immunity and is reversible by antibody treatment. Science, 364(6442), 751–75+. doi:10.1126/science.aaw4295