Periodic Reporting for period 2 - ASTHMACRYSTALCLEAR (Role of protein crystallization in type 2 immunity and asthma)
Reporting period: 2020-02-01 to 2021-07-31
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.
1. Structural biology. Currently structural biology approaches heavily rely on use of recombinant proteins that need to be highly purified and crystallised in vitro, using techniques of recombinant protein engineering. We have shown that it is possible to equally get high quality protein crystal structure from in vivo grown crystals, and that the crystal structure generated completely resembles the in vitro engineered crystals. We have also provided as one of the first times in history, a detailed mutational analysis of the crystal packing interface of three autocrystalizing proteins, allowing us to draw some common ground rules into the process of autocrystallization.
2. For immunology we have shown that protein crystals are much more common than was anticipated, and that crystals do not promote neutrophilic inflammation, but rather boost type 2 immunity. Crossing boundaries, we found that these crystals can even be solubilised using therapeutic antibodies that target the key crystal packing epitope amino acid residues.
3. For medicine in general, our studies show that it is possible to use antibioses to sollublize immobilised protein aggregates. This could have ramifications into neurodegenerative and cardiodegenerative disease, where protein aggregates are common.