Targeting T cell glutamine metabolism in severe asthma

总结 哮喘是一种使人衰弱的气道炎症性疾病，在世界范围内发病率日益增加。较高的 在女性中，肥胖和代谢疾病会加重哮喘。潜在的哮喘是激活 先天免疫和T细胞驱动炎症。严重哮喘的气道炎症部分是由 效应CD 4 T细胞（包括Th 2和Th 17）和抑制性调节（Treg）T细胞之间的不平衡。 这种不平衡在肥胖女性中尤其严重，并导致嗜酸性粒细胞和/或中性粒细胞增加 在气道中，增加气道高反应性，并增加粘液产生。现在很清楚，T细胞 代谢在调节免疫功能和每个T细胞的不同代谢程序中起着关键作用 亚群可以影响炎症并在哮喘中发挥作用。除了糖酵解需要T细胞外， 我们的数据显示谷氨酰胺代谢在支持哮喘中的炎性T细胞中起关键作用。 来自严重哮喘供体的支气管肺泡液的代谢组学数据和T细胞的质量细胞术 显示了高浓度的多巴胺分解虽然体外和肺中产生IL 17的细胞具有 最高表达的代谢标志物，Th 17和Th 2细胞都依赖谷氨酰胺进行分化， 细胞因子产生。谷氨酰胺代谢通过转运蛋白ASCT 2的摄取来调节， 谷氨酰胺酶（GLS）依赖性转化为谷氨酸和α-酮戊二酸，支持线粒体电子 运输和调节控制染色质和基因表达的表观遗传标记。我们现在已经展示了 通过抑制、基因靶向和体内原代T细胞CRISPR筛选，ASCT 2 和GLS是Th 17和Th 2细胞所必需的。这些基因是调节性T细胞和阻断谷氨酰胺 相反，摄取可以增强Treg分化。重要的是，我们还在动物模型中表明，高脂肪饮食 喂养的雌性小鼠选择性地增加了Th 17细胞的频率。这种影响是性别特异性的，如高脂肪饮食 喂养雌性而非雄性的小鼠产生了IL 17的增加，我们表明雌激素受体 (ERα）在女性中起关键作用，以提高CD 4 T细胞糖酵解和线粒体代谢。我们假设 气道过敏原暴露和炎性细胞因子诱导T细胞谷氨酰胺代谢，驱动Th 2， Th 17细胞对肥胖女性哮喘发病率和严重程度的影响为了验证这个假设，我们将： (1)确定GLS和ASCT 2依赖的谷氨酰胺代谢调节细胞凋亡的作用和机制。 过敏性气道炎症混合模型中Th 2、Th 17和Treg细胞的平衡，和（2）测试是否肥胖- 女性哮喘严重程度的诱发性加重依赖于T细胞谷氨酰胺代谢的增强， 动物模型和来自健康和严重哮喘正常体重的外周血和切除的肺， 肥胖捐赠者这些研究将建立代谢机制，如果抑制谷氨酰胺摄取或 通过ASCT 2或GLS的代谢可以改变Th 17和Th 2细胞的平衡，转而有利于Treg并保护 以帮助缓解在患有代谢疾病的女性中观察到的这种健康差异。

SUMMARY Asthma is a debilitating disease of airway inflammation with increasing worldwide occurrence. With higher incidence in females, asthma is exacerbated by obesity and metabolic disease. Underlying asthma is activation of innate immune and T cells that drive inflammation. Airway inflammation in severe asthma is caused in part by an imbalance between effector CD4 T cells (including Th2 and Th17) and suppressive regulatory (Treg) T cells. This imbalance is particularly heightened in obese females and leads to increased eosinophils and/or neutrophils in the airway, increased airway hyperresponsiveness, and increased mucus production. It is now clear that T cell metabolism plays a critical role to regulate immune function and the distinct metabolic programs of each T cell subset can influence inflammation and play roles in asthma. In addition to a requirement of T cells for glycolysis, our data show that glutamine metabolism plays a key role to support inflammatory T cells in asthma. Metabolomics data from bronchoalveolar fluid and mass cytometry of T cells from donors with severe asthma showed markers of elevated glutaminolysis. While IL17 producing cells both in vitro and from lungs had the highest expression of metabolic markers, both Th17 and Th2 cells relied on glutamine for differentiation and cytokine production. Glutamine metabolism is regulated through uptake by the transporter ASCT2 and Glutaminase (GLS)-dependent conversion to glutamate and α-ketoglutarate that supports mitochondrial electron transport and regulates epigenetic marks that control chromatin and gene expression. We have now shown through inhibition, gene targeting, and in vivo primary T cell CRISPR screening in lung inflammation that ASCT2 and GLS are essential for Th17 and Th2 cells. These genes are dispensable for Treg and blocking glutamine uptake can instead enhance Treg differentiation. Importantly, we also show in an animal model that high fat diet fed female mice have selectively increased frequencies of Th17 cells. This effect is sex-specific, as high fat diet fed female, but not male, mice developed increased IL17 production and we show that the Estrogen Receptor (ERα) plays a key role in females to elevate CD4 T cell glycolysis and mitochondrial metabolism. We hypothesize that airway allergen exposure and inflammatory cytokines induce T cell glutamine metabolism, driving Th2 and Th17 cells that contribute to asthma incidence and severity in obese females. To test this hypothesis, we will: (1) Determine the role and mechanism of GLS and ASCT2-dependent glutamine metabolism to regulate the balance of Th2, Th17 and Treg cells in a mixed model of allergic airway inflammation and (2) Test if obesity- induced exacerbation of asthma severity in females is dependent on enhanced T cell glutamine metabolism in animal models and from peripheral blood and excised lungs of healthy and severe asthma normal weight or obese donors. These studies will establish metabolic mechanisms and if inhibiting glutamine uptake or metabolism through ASCT2 or GLS can shift the balance of Th17 and Th2 cells to instead favor Treg and protect against asthma to help alleviate this health disparity observed in females with metabolic disease.