Table_3_Evaluating the effects of vitamin D Level on airway obstruction in two asthma endotypes in humans and in two mouse models with different intake of vitamin D during early-life.docx

IntroductionAsthma is primarily divided into two categories: type 2 (T2-high) and non-type 2 (T2-low). A relationship between asthma severity and vitamin D deficiency has been identified, but its impact on each asthma endotype remains unknown.
MethodsWe clinically examined the influence of vitamin D on patients with T2-high (n = 60) or T2-low asthma (n = 36) compared with controls (n = 40). Serum 25(OH)D levels, inflammatory cytokines and spirometry were measured. Mouse models were then used to further analyze the effects of vitamin D on both asthmatic endotypes. BALB/c mice were fed with vitamin D-deficient (LVD), -sufficient (NVD), or -supplemented diets (HVD) throughout lactation and offspring followed the same diet after weaning. Offspring were sensitized/challenged with ovalbumin (OVA) to establish “T2-high” asthma or OVA combined with ozone exposure (OVA + ozone) to induce “T2-low” asthma. Spirometry and serum, bronchoalveolar lavage fluid (BALF), and lung tissues were analyzed.
ResultsSerum 25(OH)D levels were decreased in asthmatic patients compared with controls. Patients with vitamin D deficiency (Lo) had varying degrees of elevation of the pro-inflammatory cytokines IL-5, IL-6, and IL-17A, decreased expression of the anti-inflammatory cytokine IL-10, and altered forced expiratory volume in the first second as a percentage of predicted value (FEV1%pred) in both asthmatic endotypes. Vitamin D status had a stronger correlation with FEV1%pred in T2-low asthma than T2-high asthma, and 25(OH)D level was only positively linked to maximal mid-expiratory flow as a percentage of predicted value (MMEF%pred) in the T2-low group. Inflammation, hyperresponsiveness, and airway resistance (RL) was increased in both asthma models compared with controls while vitamin D deficiency further increased airway inflammation and airway obstruction. These findings were particularly prominent in T2-low asthma.
DiscussionThe potential function and mechanisms of vitamin D and both asthma endotypes should be studied individually, and further analysis of the potential signaling pathways involved with vitamin D on T2-low asthma is warranted.

引言
哮喘主要分为两类：2型（T2-high）和非2型（T2-low）哮喘。目前已明确哮喘严重程度与维生素D缺乏存在关联，但该关联对不同哮喘内型（endotype）的影响尚不明确。

方法
本研究临床层面分析了维生素D对T2-high哮喘患者（n=60）、T2-low哮喘患者（n=36）的影响，并以健康对照者（n=40）作为参照。检测了受试者血清25(OH)D水平、炎症细胞因子水平及肺通气功能检测结果。随后通过小鼠模型进一步探究维生素D对两种哮喘内型的作用机制。实验中，BALB/c母鼠在整个泌乳期分别喂食维生素D缺乏（LVD）、充足（NVD）及过量（HVD）饲料，其子代小鼠在断奶后亦采用相同饲料喂养。子代小鼠通过卵清蛋白（OVA）致敏并激发以构建"T2-high"哮喘模型，或采用OVA联合臭氧暴露（OVA+臭氧）的方式诱导"T2-low"哮喘模型。后续对小鼠进行肺通气功能检测，并分析其血清、支气管肺泡灌洗液（BALF）及肺组织样本。

结果
与健康对照者相比，哮喘患者血清25(OH)D水平显著降低。在两种哮喘内型中，维生素D缺乏（Lo）患者的促炎细胞因子IL-5、IL-6及IL-17A均出现不同程度升高，抗炎细胞因子IL-10表达下调，且第一秒用力呼气容积占预计值百分比（FEV1%pred）出现异常。与T2-high哮喘相比，T2-low哮喘患者的维生素D状态与FEV1%pred的相关性更强；且仅在T2-low组中，血清25(OH)D水平与最大呼气中期流量占预计值百分比（MMEF%pred）呈正相关。与对照组相比，两种哮喘模型小鼠的炎症水平、气道高反应性及气道阻力（RL）均显著升高；而维生素D缺乏可进一步加重气道炎症与气道阻塞，该效应在T2-low哮喘模型中尤为显著。

讨论
未来应分别针对维生素D与两种哮喘内型的潜在功能及作用机制开展独立研究，且有必要进一步分析维生素D调控T2-low哮喘的潜在信号通路。