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.
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 in the airways of naïve mice induced an innate immune response, rich in neutrophils and monocytes and lead to uptake of crystals by dendritic cells. Soluble Gal10 muteins carrying a mutation to glutamic acid at position Tyr69 were unable to crystallize and were immunologically inert. Simultaneous injection of CLCs with harmless ovalbumin (OVA) resulted in DC uptake and Th2 priming, together with airway eosinophilia and IgG1 responses. Mechanistically, these effects were accompanied by NLRP3 inflammasome activation and IL-1beta release, yet the observed response to CLCs in vivo could occur independently of the NLRP3 inflammasome. In an effort to develop novel therapeutic opportunities against this type of crystallopathy, we generated antibodies against crystalline Gal10. The epicenter of each crystal-dissolving antibody-binding epitope on Gal10 situated at Tyr69, a residue we had identified as a critical crystal packing hotspot. These antibodies rapidly dissolved pre-existing 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 model. Our results therefore demonstrate that CLCs are more than just markers of eosinophilic inflammation. Gal10 is released by activated eosinophils and undergoes a phase transition to a crystalline state that actively promotes key features of asthma. Antibodies rapidly dissolve CLCs that are abundantly present in 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 drugable trait in patients with airway disease, and provide a rationale for how antibodies can dissolve protein crystals.
In another series of experiments, we have demonstrated that CLCs have the capacity to trigger neutrophil recruitment driven by airway epithelial cells. The CLCs trigger the epithelium to release neutrophil selective chemokine, and when the neutrophils arrive in the airways, the undergo intense activation and NETosis, leading to the release of extracellular DNA that can make the mucus very hard to snee up or cough up. We have similar data in murine models of the disease.
Finally, since mice to don produce Gal10, we created several new mouse tools to overexposes GAl10 from various promotors, and we have studied the importance of pseudo-CLCs, made up of the chitiinase like proteins Ym1 and Ym2 in various mouse models. These experiments show that protein crystallisation does serve a purpose in boosting inflammation and promoting mucus production.
