內科“不治喘”，可以終結？_風聞

科學家解開了治療哮喘和氣管炎症的百年之謎

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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.

據比利時佛蘭德斯生物技術研究所（the Flanders Institute for Biotechnology, VIB）2019年5月23日提供的消息，來自比利時VIB、根特大學(Ghent University)、根特大學醫院(Ghent University Hospital)和生物技術公司Argenx的研究小組，合作研究解開了一個關於哮喘中是否存在蛋白質晶體的百年謎題。正常情況下，蛋白質不會在體內結晶，但在某些情況下確實會發生這一過程。Charcot-Leyden晶體是由蛋白質半乳凝素-10（galectin-10, 簡稱Gal10）構成的，早在1853年就在哮喘患者的呼吸道中發現。 

然而，科學家在很大程度上忽略了這些晶體，它們與疾病的實際聯繫仍然未知。比利時的研究小組現在已經證實，這種晶體在呼吸道粘液中含量非常豐富，可以刺激免疫系統，促進哮喘患者呼吸道中常見的炎症和粘液分泌的改變。學界和公司的科學家們還共同開發了抗體，可以溶解這些晶體，以減少哮喘的關鍵特徵。這種抗體可能是逆轉蛋白質晶體、治療哮喘和其他呼吸道慢性炎症疾病的一流療法。這項研究發表在著名的《科學》（Science）雜誌上—— Emma K. Persson, Kenneth Verstraete, Ines Heyndrickx, Elien Gevaert，Helena Aegerter, Jean-Michel Percier, Kim Deswarte, Koen H. G. Verschuere, Ann Dansercoer, Delphine Gras, Pascal Chanez, Claus Bachert,  Amanda Gonçalves, Hanne Van Gorp, Hans De Haard, Christophe Blanchetot, Michael Saunders, Hamida Hammad, Savvas N. Savvides, Bart N. Lambrecht. Protein crystallization promotes type 2 immunity and is reversible by antibody treatment. Science, 24 May 2019, Vol. 364, Issue 6442, eaaw4295. DOI: 10.1126/science.aaw4295

晶體和疾病

1853年，巴黎著名的薩爾佩替耶病院（Salpêtrière Hospital in Paris）的珍-馬丁·夏科特（Jean-Martin Charcot）報告了他在哮喘患者痰中觀察到的雙錐體晶體的詳細草圖，恩斯特·馮·萊登(Ernst von Leyden)在1872年也做了同樣的觀察。這些晶體沉積物在醫學界被廣泛稱為夏科特-萊登晶體(Charcot-Leyden crystals, CLCs)。

從那時起，它們被描述為廣泛的慢性過敏和炎症性疾病，如哮喘（asthma）、支氣管炎（bronchitis）、過敏性鼻炎（allergic rhinitis）和鼻竇炎（rhinosinusitis）。然而，直到最近幾十年，科學家們才確認CLCs的內容是由Gal10組成的，最終解決了持續近一個半世紀的爭論。Gal10是嗜酸細胞中最豐富的蛋白質之一，它能幫助人體產生炎症反應。令人驚訝的是，Gal10在嗜酸細胞中仍然是可溶性的，它只有在作為免疫防禦的一部分被釋放後才會形成晶體。Gal10的功能也仍然難以捉摸。

這些晶體會造成傷害嗎?

由Emma Persson, Kenneth Verstraete和Ines Heyndrickx領導的研究小組開始測試一個長期懸而未決的假設:CLCs是否刺激肺部免疫，並導致過度炎症反應最終導致疾病呢?

Bart Lambrecht教授(VIB-UGent)説:“每一位醫生都在醫學培訓中學習了關於夏科特-萊登晶體的知識，每個人都把這種晶體與嗜酸性粒細胞的存在聯繫起來。它們經常出現在哮喘患者的痰中，尤其是那些患有嚴重疾病的患者。然而，沒有人真正知道這些晶體在做什麼，以及它們為什麼會在那裏。與痛風類似，尿酸結晶會引起非常痛苦的關節炎症，我們推斷夏科特-萊登結晶也可能對哮喘患者的肺部造成傷害。”

晶體轉換為溶液

科學家克服了許多技術上的挑戰來驗證這個想法。為了研究目的，他們必須在實驗室中找到一種方法來產生數百萬的Gal10晶體，並確定這些晶體與病人體內發現的CLCs相同。研究人員使用病人獲得的晶體來確定Gal10的三維結構，並將其精確到原子尺度。這提供了一個長期尋求的答案，證實實驗產生的CLCs與病人來源的CLCs相同。

Savvas Savvides教授(VIB-UGent)説:“這是生化和醫學史上第一次在原子分辨率下研究病人來源的蛋白質晶體。這種微小的晶體只有幾微米大小(大約為千分之一毫米)，卻經歷了從醫院手術室開始，到歐洲同步加速器輻射設備的專業x射線束線的艱苦而嚴酷的實驗過程，並最終存活了下來，這是完全不同尋常的。最重要的是，他們獲得了數據，這些數據形成了蛋白質分子內部美麗的三維結構。”

研究人員發現，只有當Gal10處於晶體狀態時，它才會引發完全成熟的免疫反應。在溶液中，Gal10是無害的。最重要的是，以夏科特-萊登晶體形式存在的Gal10晶體誘發了哮喘的主要特徵，包括產生改變的粘液，這對大多數哮喘患者來説是一個大問題。因此，該研究已經取得了重大突破，得出了非常清晰的結論。

尋找解決方案

然後研究干預小細胞肺癌的形成是否會成為哮喘患者的治療選擇。這就是總部位於根特的生物技術公司Argenx介入的原因。聯合研究小組開發出了能特異性對抗小細胞肺癌的抗體。值得注意的是，抗體能夠在實驗室的皮氏培養皿中在幾分鐘內溶解小細胞肺癌，在病人的黏液中(同樣在體外)也能在幾小時內溶解小細胞肺癌。在哮喘小鼠模型中使用這些抗體可顯着減少肺部炎症、肺功能改變和粘液生成。Savvas Savvides教授説:“這就像‘現在你看到了，現在你沒有’展示分子魔術。我花了25年的時間學習和苦於如何為結構生物學培育蛋白質晶體，突然間，我看到蛋白質晶體實時溶解。最重要的是，我們還能看到這些抗體是如何通過決定它們與抗原的晶體結構來發揮神奇的作用的。”Bart Lambrecht教授説:“我們的研究結果出乎意料，同時也非常清晰。抗體能迅速溶解患者粘液中大量存在的CLCs，這讓我完全震驚。雖然還需要更多的測試，但小鼠模型中的數據表明，使用這些抗體可能是減少哮喘患者肺部過度炎症和粘液積聚的非常有效的方法。由於目前還沒有針對氣道黏液積聚的藥物，這可能會改變治療這種疾病的遊戲規則。” 更多信息請注意瀏覽原文或者相關報道。

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.

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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