Investigating the CDC42 pathway as a novel pathway for pediatric non-atopic obesity-related asthma

摘要 非特应性或Th 2低哮喘现在被认为是儿童哮喘的一个主要亚组。肥胖相关 哮喘是儿童非特应性哮喘中最常见的一种， 负担，肺功能恶化，对药物缺乏反应/耐药性。因此，迫切需要 研究非特应性哮喘的免疫生物学，以确定新的治疗靶点。我们和其他人已经 先前报道的肥胖哮喘儿童外周血中的非特应性免疫应答， TH 1/TH 2比值升高，TNF、IL-6、IFNγ和IP-10升高，与肺功能相关 肥胖相关哮喘的缺陷。使用RNA-Seq，我们探索了非特应性反应的生物学， 刺激肥胖哮喘CD 4+（TH）细胞，发现几种基因（DOCK 5，VAV 2， CDC 42 EP 4、PAK 3、MLK 3和PLD 1）在细胞分裂周期42（CDC 42）途径中的作用。更高的CDC 42 EP 4和 肥胖哮喘儿童DOCK 5基因表达与气道阻塞加重相关磷酸化 p38，MLK 3的下游，与哮喘中的类固醇抵抗有关，在刺激性肥胖中， 哮喘TH细胞小干扰RNA（siRNA）介导的TH细胞中CDC 42沉默导致IFNγ和IFN γ水平降低， TNF，而不是IL-4基因表达。总之，这些结果表明，CDC 42途径在细胞凋亡中的新作用。 肥胖相关性哮喘中非特应性炎症根据这些观察，我们假设， 肥胖哮喘患者，非TH 2 TH细胞中CDC 42通路的上调，该细胞在 气道和CDC 42调节的信号通路的激活，有助于类固醇抵抗， 疾病负担。为了验证我们的假设，我们将鉴定具有CDC 42通路上调的非TH 2 TH细胞， 并量化肥胖哮喘患者中CDC 42调节的信号通路的激活。我们将调查 50例非特应性肥胖哮喘患者外周血中非TH 2 TH细胞的富集， 非特应性正常体重哮喘患者，50名肥胖非哮喘患者和50名健康对照。富集 将在20名肥胖哮喘患者的亚组中研究气道中的非TH 2 TH细胞，并与20名 正常体重的哮喘患者在非TH 2 TH细胞中响应于 地塞米松将提供细胞是类固醇抗性的证据，并且在以下情况下获得类固醇敏感性： CDC 42和/或CDC 42调节的信号通路抑制将支持CDC 42在类固醇抗性中的作用 非特应性哮喘为了确定肥胖的贡献，以及肥胖以外的因素，我们将比较 在非特应性肥胖哮喘患者和肥胖非哮喘患者中的发现。最后，将CDC 42激活与 疾病负担，我们将确定一个预测CDC 42激活的生化标志，并研究其 儿童非特应性肥胖相关哮喘的疾病负担。这些研究将证实 CDC 42通路在非特应性哮喘的免疫生物学和疾病负担中的作用，并将确定非特应性哮喘的 TH 2 TH细胞和/或信号通路中的蛋白质作为肥胖相关哮喘的新治疗靶点

ABSTRACT Non-atopic or Th2-low asthma is now recognized as a major subgroup of pediatric asthma. Obesity-related asthma, the most commonly reported form of pediatric non-atopic asthma, is associated with high disease burden, worse lung function, and lack of response/resistance to medications. Thus, there is an urgent need to investigate the immunobiology of non-atopic asthma to identify novel therapeutic targets. We and others have previously reported non-atopic immune responses in peripheral blood from obese asthmatic children, with elevated TH1/ TH2 ratio and increased TNF, IL-6, IFNγ, and IP-10 that correlated with pulmonary function deficits in obesity-related asthma. Using RNA-Seq, we probed the biology of non-atopic responses in un- stimulated obese asthmatic CD4+ (TH) cells and found upregulation of several genes (DOCK5, VAV2, CDC42EP4, PAK3, MLK3 and PLD1) in the Cell Division Cycle 42 (CDC42) pathway. Higher CDC42EP4 and DOCK5 gene expression correlated with worse airway obstruction in obese asthmatic children. Phosphorylated p38, downstream of MLK3, and linked with steroid resistance in asthma, was higher in stimulated obese asthmatic TH cells. Small interfering RNA (siRNA)-mediated CDC42 silencing in TH cells led to lower IFNγ and TNF, but not IL-4, gene expression. Together, these results suggest a novel role for the CDC42 pathway in non-atopic inflammation in obesity-related asthma. Based on these observations, we hypothesize that in obese asthmatics, upregulation of the CDC42 pathway in a non-TH2 TH cell, which is enriched in the airway, and activation of CDC42-regulated signaling pathways, contribute to steroid resistance and disease burden. To test our hypothesis, we will identify the non-TH2 TH cell with CDC42 pathway upregulation and quantify activation of CDC42-regulated signaling pathways in obese asthmatics. We will investigate enrichment of the non-TH2 TH cell in peripheral blood in 50 non-atopic obese asthmatics as compared to 50 non-atopic normal-weight asthmatics, 50 obese non-asthmatics, and 50 healthy controls. Enrichment of the non-TH2 TH cell in the airway will be investigated in a subset of 20 obese asthmatics and compared to 20 normal-weight asthmatics. Absent or attenuated cytokine suppression in the non-TH2 TH cell in response to dexamethasone will provide evidence that the cell is steroid resistant and gain of steroid sensitivity following CDC42 and/or CDC42-regulated signaling pathway inhibition will support a role of CDC42 in steroid resistance in non-atopic asthma. To identify the contribution of obesity, and of factors other than obesity, we will compare the findings in non-atopic obese asthmatics to obese non-asthmatics. Lastly, to link CDC42 activation with disease burden, we will identify a biochemical signature predictive of CDC42 activation, and investigate its contribution to disease burden in pediatric non-atopic obesity-related asthma. These studies will confirm a role of CDC42 pathway in the immunobiology and disease burden of non-atopic asthma, and will identify the non- TH2 TH cell and/or proteins in the signaling pathways as novel therapeutic targets for obesity-related asthma.