Causal relationships between immune cells and childhood asthma and the mediating role of inflammatory proteins

doi:10.12307/2026.484

Abstract

Abstract: BACKGROUND: Asthma is one of the most prevalent chronic respiratory diseases in children. Existing studies have indicated significant associations among immune cells, inflammatory proteins, and childhood asthma, but the specific causal relationships remain unclear.
OBJECTIVE: To investigate the causal relationship between immune cells and childhood asthma mediated by inflammatory proteins.
METHODS: Utilizing data from the largest genome-wide association studies on immune cells, inflammatory proteins, and childhood asthma, we performed bidirectional two-sample Mendelian randomization analyses to explore the causal links between these variables. The inverse variance weighted method served as the primary analytical approach. Cochran’s Q test, MR-Egger intercept test, and MR-PRESSO were employed to assess heterogeneity, outliers, and horizontal pleiotropy, respectively. Mediation analysis was conducted to determine whether inflammatory proteins mediate the effect of immune cells on childhood asthma. Key inflammatory mediators were detected in an established asthmatic rat model using quantitative real-time PCR and western blot assay.
RESULTS AND CONCLUSION: (1) Inverse variance weighted analyses identified significant causal associations of 40 immune-cell phenotypes and 7 inflammatory proteins with childhood asthma (P < 0.05), but there was no evidence of reverse causality. (2) Mediation analysis revealed that interleukin-33 could play a mediating role between two types of immune cells (T cell %leukocyte and CD86 on myeloid dendritic cells) and childhood asthma, with mediating ratios of 12.7% and 9.3%, respectively. (3) Animal experiments verified that compared with the normal control group, the mRNA and protein levels of interleukin-33 in the lung tissue of rats in the asthma model group were significantly increased (P < 0.01). (4) Protein-protein interaction networks revealed that interleukin-33 and molecules such as interleukin-1 receptor-like 1 formed a pivotal hub in inflammation regulation, and drug screening revealed the potential of targeted therapies like Astegolimab. (5) By integrating genetics with experimental validation, this study establishes interleukin-33 as the pivotal inflammatory mediator linking immune-cell dysregulation to childhood asthma onset. Leveraging bioinformatic pipelines and algorithmic frameworks developed by international multi-omics platforms, we provide a new avenue for refining variant-interpretation workflows tailored to the Chinese population, improving the efficiency of asthma-susceptibility-gene identification, and elucidating the biological basis of childhood asthma for the development of early-intervention therapies.

Key words: childhood asthma, Mendelian randomization, immune cells, inflammatory proteins, mediation analysis, inverse variance weighted, sensitivity analysis

CLC Number:

Gong Jiaxuan, Lin Lin, Wang Chun, Zhang Xingxing, Xiong Ying. Causal relationships between immune cells and childhood asthma and the mediating role of inflammatory proteins[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(33): 8761-8777.

Figures/Tables 15

2.1 免疫细胞与儿童哮喘的孟德尔随机化分析结果通过两样本孟德尔随机化方法对免疫细胞和儿童哮喘的关联进行分析，结果显示有40种免疫细胞与儿童哮喘存在因果关系。在已鉴定的免疫细胞中，CD24+ CD27+ AC (CD24阳性CD27阳性B细胞的绝对计数)、Memory B cell %lymphocyte(记忆B细胞占淋巴细胞总数的百分比)、Im MDSC AC (未成熟髓源性抑制细胞的绝对计数)、CD33br HLA DR+ CD14- AC (CD33高表达HLA DR阳性CD14阴性细胞的绝对计数)、Mo MDSC AC (单核细胞样髓源性抑制细胞的绝对计数)、Basophil %CD33dim HLA DR- CD66b- (CD33弱表达HLA DR阴性CD66b阴性细胞占嗜碱性粒细胞总数的百分比)、TD DN (CD4-CD8-) %DN [终末分化双阴性(CD4阴性CD8阴性)T细胞占双阴性T细胞总数的百分比]、CD14+ CD16- monocyte% monocyte (CD14阳性CD16阴性经典单核细胞占单核细胞总数的百分比)、T cell %leukocyte (T细胞占白细胞总数的百分比)、BAFF-R on transitional (过渡型B细胞表面BAFF受体的表达水平)、CD24 on transitional (过渡型B细胞表面CD24的表达水平)、CD38 on CD3- CD19- (CD3阴性CD19阴性细胞表面CD38的表达水平)、CD86 on myeloid DC (髓样树突状细胞表面CD86的表达水平)、CD33 on CD14+ monocyte (CD14阳性单核细胞表面CD33的表达水平)、CD33 on CD33dim HLA DR+ CD11b- (CD33弱表达HLA DR阳性CD11b阴性细胞表面CD33的表达水平)、CD33 on Gr MDSC (粒细胞样髓源性抑制细胞表面CD33的表达水平)、CD33 on CD66b++ myeloid cell (CD66b高表达髓样细胞表面CD33的表达水平)、CD33 on basophil (嗜碱性粒细胞表面CD33的表达水平)、CX3CR1 on CD14- CD16- (CD14阴性CD16阴性单核细胞表面CX3CR1的表达水平) 共19种免疫细胞与儿童哮喘风险增加有关。IgD+ CD38dim %B cell (IgD阳性CD38弱表达B细胞占B细胞总数的百分比)、IgD+ CD38dim %lymphocyte (IgD阳性CD38弱表达B细胞占淋巴细胞总数的百分比)、CD39+ activated Treg AC (CD39阳性活化调节性T细胞的绝对计数)、TD CD4+ %T cell (终末分化CD4阳性T细胞占T细胞总数的百分比)、TD CD8br %T cell (终末分化CD8强阳性T细胞占T细胞总数的百分比)、EM DN (CD4-CD8-) %DN[效应记忆型双阴性(CD4阴性CD8阴性)T细胞占双阴性T细胞总数的百分比]、HLA DR+ NK AC (HLA DR阳性自然杀伤细胞的绝对计数)、HLA DR+ NK %NK (HLA DR阳性自然杀伤细胞占自然杀伤细胞总数的百分比)、HLA DR+ NK %CD3- lymphocyte (HLA DR阳性自然杀伤细胞占CD3阴性淋巴细胞总数的百分比)、CD19 on CD20- CD38- (CD20阴性CD38阴性B细胞表面CD19的表达水平)、CD62L on CD62L+ DC (CD62L阳性树突状细胞表面CD62L的表达水平)、CD3 on resting Treg (静息调节性T细胞表面CD3的表达水平)、CD28 on CD45RA+ CD4+ (CD45RA阳性CD4阳性T细胞表面CD28的表达水平)、CD28 on resting Treg (静息调节性T细胞表面CD28的表达水平)、CD45 on HLA DR+ CD8br (HLA DR阳性CD8高表达T细胞表面CD45的表达水平)、PD-L1 on CD14- CD16+ monocyte (CD14阴性CD16阳性单核细胞表面PD-L1的表达水平)、CD8 on HLA DR+ CD8br (HLA DR阳性CD8高表达T细胞表面CD8的表达水平)、SSC-A on CD4+ (CD4阳性细胞侧向散射光面积)、SSC-A on HLA DR+ T cell (HLA DR阳性T细胞侧向散射光面积)、HLA DR on myeloid DC (髓样树突状细胞表面HLA DR的表达水平)、HLA DR on DC (树突状细胞表面HLA DR的表达水平) 共21种免疫细胞与儿童哮喘风险降低有关。结果见图2。"

Cochran’Q 统计量检验表明已筛选的40种免疫细胞的单核苷酸多态性中未发现异质性的证据(P > 0.05)(表3)，MR-Egger截距检验和MR-PRESSO检验两项测试均未显示潜在的水平多效性(P > 0.05)(表4)。留一法敏感性分析结果显示，逐一剔除各单核苷酸多态性后，总体效应估计值保持稳定，结果具有较好的稳健性(图3)。Steiger检验结果显示，40种免疫细胞的因果关系方向为TRUE，且P值均 < 0.05，表明该暴露因素对结局产生影响，确保了方向性(表5)。散点图分析显示，逆方差加权法、MR-Egger、加权中位数法、加权模式法及简单模式法共5种统计方法的因果效应方向呈现一致性，未检测到异常值(图4)。漏斗图检验结果表明，绝大多数单核苷酸多态性的效应值集中分布于对称区域，分布形态紧密且无显著离群值，提示因果效应估计具备较好的对称性和稳健性(图5)。 2.2 炎症蛋白与儿童哮喘的孟德尔随机化分析结果将炎症蛋白作为暴露因素，儿童哮喘作为结局因素，以逆方差加权法为主要分析方法，经过一系列筛选后，发现7种炎症蛋白与儿童哮喘存在显著相关性。其中，Delta/Notch样表皮生长因子相关受体、白细胞介素33、骨保护素、沉默信息调节因子2是儿童哮喘的危险因素，CUB结构域包含蛋白1、白细胞介素2受体亚基、血管内皮生长因子A是儿童哮喘的保护因素(图6)。敏感性分析结果表明，已筛选的7种炎症蛋白与儿童哮喘均不存在异质性(PCochran’Q > 0.05)和水平多效性(PMR-Egger > 0.05和PMR-PRESSO > 0.05)(表6，7)。留一法敏感性检验表明，当逐一排除单个单核苷酸多态性时，因果效应的整体估计值未发生显著偏移(图7)。散点图和漏斗图未识别到异常值和工具变量的选择偏倚(图8，9)。Steiger检验结果未发现反向因果关系的证据(表8)。 2.3 中介孟德尔随机化分析结果为了探索儿童哮喘发展和进展的潜在机制，利用中介分析以鉴定介导从免疫细胞到儿童哮喘致病途径的炎症蛋白。以逆方差加权法方法作为主要方法进行双样本孟德尔随机化分析，探究免疫细胞与炎症蛋白之间的因果关系。分析过程同上述过程一致，显著性水平为P < 0.05。结果确定了5种免疫细胞与炎症蛋白之间存在因果关系。具体而言，IgD+ CD38dim %lymphocyte (IgD阳性CD38弱表达B细胞占淋巴细胞总数的百分比)与Delta/Notch样表皮生长因子相关受体、T cell %leukocyte (T细胞占白细胞总数的百分比)与白细胞介素33、CD86 on myeloid DC (髓样树突状细胞表面CD86的表达水平)与白细胞介素33呈正相关，CD14+ CD16- monocyte %monocyte (CD14阳性CD16阴性经典单核细胞占单核细胞总数的百分比)与白细胞介素33、CD28 on resting Treg (静息调节性T细胞表面CD28的表达水平)与沉默信息调节因子2呈负相关(表9)。随后，中介效应模型显示，白细胞介素33在T cell %leukocyte (T细胞占白细胞总数的百分比)、CD86 on myeloid DC (髓样树突状细胞表面CD86的表达水平)与儿童哮喘的因果关系中起到了介导作用(图10)。其中，白细胞介素33在T cell %leukocyte (T细胞占白细胞总数的百分比)到儿童哮喘路径中发挥中介作用的比例为12.7%，中介效应为0.010；白细胞介素33在CD86 on myeloid DC(髓样树突状细胞表面CD86的表达水平)到儿童哮喘路径中发挥中介作用的比例为9.3%，中介效应为0.008(表10)。 2.4 哮喘小鼠肺组织中白细胞介素33基因和蛋白表达的实验验证结果 RT-qPCR检测结果显示，与空白对照组相比，哮喘模型组大鼠肺组织中白细胞介素33 mRNA表达水平显著升高(P < 0.01)(图11A)。Western blot检测结果显示，与空白对照组相比，哮喘模型组大鼠肺组织中白细胞介素33蛋白表达水平显著升高(P < 0.01)。综合以上结果表明，白细胞介素33可能在哮喘的发病机制中发挥实际作用，并且白细胞介素33表达与疾病状态相关(图11B)。 2.5 蛋白质-蛋白质网络相互作用分析和药物-基因相互作用分析结果针对分子靶点炎症因子白细胞介素33构建的蛋白质-蛋白质相互作用网络分析显示，白细胞介素33作为核心节点与白细胞介素1受体样1 (IL1RL1)、白细胞介素1受体辅助蛋白(IL1RAP)、白细胞介素1受体1型(IL1R1)、半胱天冬酶1 (CASP1)、髓样分化初级反应蛋白88(MYD88)、细胞因子受体样因子2(CRLF2)、白细胞介素5 (IL5)、白细胞介素13 (IL13)、白细"

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