Objective

Cysteinyl leukotrienes (CysLTs), a group of inflammatory lipid mediators, are found elevated in obese-asthmatic patients. Leukotriene D₄ (LTD₄), a representative CysLT, is implicated in promoting lung inflammation and remodelling in allergic asthma, but its role in non-allergic asthma, especially in obese-asthmatic patients, is not known. Here, using primary human small airway epithelial cells (SAECs) we have investigated the mechanism of LTD₄-induced inflammation and remodelling and assessed high proneness of obese mice to develop asthma upon challenge with allergen ovalbumin (OVA).

Methods

Primary human small airway epithelial cells (SAECs) were stimulated with different concentrations of LTD₄ for different time intervals and various inflammatory markers were measured through cytokine array, membrane-based ELISA and Western blotting. An air–liquid interface (ALI) model of SAECs was used to study the effects of LTD₄-induced remodelling in SAECs using Western blotting, H&E staining and PAS staining. Further, OVA-based murine model was used to examine the propensity of high-fat diet (HFD)-fed obese mice to develop asthma symptoms by studying the infiltration of inflammatory cells (assessed by bronchioalveolar lavage (BAL) cytology) and airway remodelling (assessed by histopathology) upon allergen exposure.

Results

The human primary small airway epithelial cells (SAECs) treated with LTD₄ showed significant alterations in the levels of inflammatory markers such as GM-CSF, TNF-α, IL-1β, EGF and eotaxin in dose- and time-dependent manner. Further, LTD₄ enhanced the activation of inflammasomes as evidenced by increased levels of NALP3, cleaved caspase-1 and IL-1β. LTD₄ also enhanced inflammation by increasing the expression of COX-2 in SAECs. The airway remodelling markers Vimentin and Muc5AC were found elevated in ALI culture of SAECs when stimulated with LTD₄, as it also increased TGF-β levels and activation of Smad2/3 phosphorylation in SAECs. Last, sensitization and challenge of HFD-fed obese mice with OVA showed increased infiltration of inflammatory cells in BAL and enhanced levels of remodeling phenotypes like loss of cilia, mucus cell metaplasia and collagen deposition in mice lung tissues.

Conclusion

The results suggest that LTD₄ could induce inflammatory response in human airway epithelial cell by activating NALP3 inflammasome. LTD₄ could further promote airway epithelial cells’ remodelling through TGF-β/smad2/3-mediated pathway. Our in vivo results suggested that obesity predisposed the OVA challenged mice to develop lung inflammation and remodelling akin to asthma-like phenotypes during obesity.

中文翻译：

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半胱氨酰白三烯 D 4 (LTD 4 ) 促进气道上皮细胞炎症和重塑

 客观的

半胱氨酰白三烯 (CysLT) 是一组炎症脂质介质，在肥胖哮喘患者中被发现升高。白三烯D ₄ (LTD ₄ )是一种代表性的CysLT，与促进过敏性哮喘中的肺部炎症和重塑有关，但其在非过敏性哮喘、尤其是肥胖哮喘患者中的作用尚不清楚。在这里，我们使用原代人小气道上皮细胞 (SAEC) 研究了 LTD ₄诱导的炎症和重塑的机制，并评估了肥胖小鼠在受到过敏原卵清蛋白 (OVA) 攻击后发生哮喘的高倾向性。

 方法

用不同浓度的LTD ₄刺激原代人小气道上皮细胞(SAEC) 不同的时间间隔，并通过细胞因子阵列、基于膜的ELISA 和Western blotting 测量各种炎症标志物。 SAEC 的气液界面 (ALI) 模型用于通过蛋白质印迹、H&E 染色和 PAS 染色研究 LTD ₄诱导的 SAEC 重塑的影响。此外，基于OVA的小鼠模型被用来通过研究炎症细胞的浸润（通过支气管肺泡灌洗（BAL）细胞学评估）和气道重塑来检查高脂饮食（HFD）喂养的肥胖小鼠出现哮喘症状的倾向。通过组织病理学评估）在过敏原暴露后。

 结果

用LTD ₄处理的人原代小气道上皮细胞（SAEC）显示出炎症标志物（例如GM-CSF、TNF-α、IL-1β、EGF和eotaxin）水平的显着变化，且呈剂量和时间依赖性。此外，LTD ₄增强了炎症小体的激活，NALP3、裂解的 caspase-1 和 IL-1β 水平的增加证明了这一点。 LTD ₄还通过增加 SAEC 中 COX-2 的表达来增强炎症。当用 LTD ₄刺激时，在 SAEC 的 ALI 培养物中发现气道重塑标记物 Vimentin 和 Muc5AC 升高，因为它还增加了 SAEC 中 TGF-β 水平和 Smad2/3 磷酸化的激活。最后，用 OVA 对 HFD 喂养的肥胖小鼠进行致敏和攻击，结果显示 BAL 中炎症细胞的浸润增加，并且重塑表型的水平提高，例如小鼠肺组织中纤毛损失、粘液细胞化生和胶原沉积。

 结论

结果提示LTD ₄可以通过激活NALP3炎症小体诱导人气道上皮细胞的炎症反应。 LTD ₄可以通过TGF-β/smad2/3介导的途径进一步促进气道上皮细胞的重塑。我们的体内结果表明，肥胖容易使 OVA 激发的小鼠在肥胖期间出现肺部炎症和类似于哮喘样表型的重塑。