Periodic Reporting for period 2 - ASTHMACRYSTALCLEAR (Role of protein crystallization in type 2 immunity and asthma)
Okres sprawozdawczy: 2020-02-01 do 2021-07-31
Asthma and chronic rhinosinusitis are very common diseases that affect at least 20% of people in Europe.New treatments for these disorders are urgently needed. A few yers ago, we noticed that the airway secretions in these diseases (the mucus that patients sneeze or cough up) very often contain protein crystals This is an odd observation, since spontaneous protein crystallization is a very rare event in nature. This led us to a further investigation what these protein crystals might mean. Eosinophilic inflammation such as seen in the airways in asthma, chronic rhinosinusitis and helminth infection has long been known to be accompanied by accumulation of large amounts of extracellular Charcot-Leyden crystals. These are made of Galectin-10, a protein of unknown function produced by eosinophils, hallmark cells of type 2 immunity. In mice, eosinophilic inflammation is also accompanied by protein crystal build up, composed of the chitinase-like proteins Ym1 and Ym2, produced by alternatively activated macrophages. In this project we challenge the current view that these crystals are just markers of eosinophil demise or macrophages activation. We hypothesized that protein crystallization serves an active role in immunoregulation of type 2 immunity. On the one hand, crystallization might turn a harmless protein into a danger signal. On the other hand, crystallization might sequester and eliminate the physiological function of soluble Galectin-10 and Ym1, or prolong it via slow release elution. For full understanding, we therefore needed to understand the function of the proteins in a soluble and crystalline state. Our program at the frontline of immunology, molecular structural biology and clinical science combines innovative tool creation and integrative research to investigate the structure, function, and physiology of galectin-10 and related protein crystals. We chose to study asthma as the crystallizing proteins are abundantly present in human and murine disease. There is still a large medical need for novel therapies that could benefit patients with chronic steroid-resistant disease, and are alternatives to eosinophil-depleting antibodies whose long term effects are unknown.
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.
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.
We have gone considerably beyond the state of the art in both structural biology and immunology
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.
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.