Research Article

Protein crystallization promotes type 2 immunity and is reversible by antibody treatment

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Science  24 May 2019:
Vol. 364, Issue 6442, eaaw4295
DOI: 10.1126/science.aaw4295

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A crystal-clear ingredient for allergy?

Charcot-Leyden crystals (CLCs) are formed from the eosinophil granule protein galectin-10 (Gal10) and found in severe eosinophil-associated diseases like asthma and chronic rhinosinusitis. Whether CLCs actively contribute to disease pathogenesis is unknown. Persson et al. found that lab-grown Gal10 crystals are biosimilar to CLCs (see the Perspective by Allen and Sutherland). When given to mice, the crystals acted as a type 2 adjuvant, mimicking many of the features of human asthma. In contrast, a Gal10 mutein unable to crystallize had no effect. Antibodies against epitopes crucial for Gal10 autocrystallization could dissolve both in vitro–generated Gal10 crystals and patient-derived CLCs. Furthermore, these anti-Gal10 antibodies reversed the effects of Gal10 crystals in a humanized mouse model of asthma, suggesting a potential therapeutic approach for crystallopathies more broadly.

Science, this issue p. eaaw4295; see also p. 738

Structured Abstract

INTRODUCTION

Spontaneous protein crystallization 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-1β (IL-1β) 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 antibody-binding epitope on Gal10 was situated at Tyr69, a residue we had identified as a critical crystal-packing 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.

Dissolving CLCs with antibodies.

CLCs that are abundant in airways in type 2 immunity can be dissolved by using antibodies that target key residues of the crystal-packing interface on Gal10. This strategy leads to the resolution of key asthma-like features in mice. mAb, monoclonal antibody.

Abstract

Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.

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