Direct IL-4 and IL-13 effects on pulmonary smooth muscle in allergic airway disea

描述（由申请人提供）：小鼠变应性气道疾病的发生取决于IL- 4r1介导的Stat6被IL-4和IL- 13激活，在疾病的效应期，后者比前者更重要。我们已经评估了IL-4和IL-13参与过敏性气道疾病的细胞类型，并探索了IL-13发挥更大作用的机制。结果表明：1)吸入IL-4和IL-13可通过直接作用于气道平滑肌细胞，分别独立诱导小鼠气道高反应性（AHR）；2) AHR的诱导独立于嗜酸性粒细胞增多和杯状细胞增生；3) IL-4和IL-13诱导AHR需要通过2型IL-4R信号传导；4) IL-4而非IL-13诱导IFN-3和IL-10分泌增加，从而抑制变应性气道疾病；5) IL-4通过1型IL-4R信号通路抑制IL-13在肺中诱导的部分基因的表达；6) IL-13抑制miR-1的表达，miR-1是一种在肌肉中表达的microRNA，可抑制促进分化、增殖和反应性的多种基因的翻译。其他研究者同期的研究表明：1)IL-13可以通过直接作用于肺上皮诱导AHR，但也可以在肺上皮不能直接响应该细胞因子的小鼠中诱导AHR；2) 2型IL-4R与IL-13的结合方式不同，与IL-4的结合具有更大的亲和力。综上所述，这些观察结果表明了我们的3个中心假设：1)IL-4和IL-13对AHR的诱导完全可以通过它们对平滑肌和气道上皮的作用来解释；2) IL-4/IL-13诱导的平滑肌基因转录和翻译的变化有助于这些细胞因子对气道反应性和肌肉重塑的影响；3)吸入IL-13比IL-4更能增加小鼠AHR，这是由于IL-4激活1型IL-4受体（IL- 4r），从而产生IFN-3和IL-10，而不是由于IL-4与IL-13通过2型IL- 4r或IL- 13r12信号传导的差异。这些假设将通过4个具体目标进行检验：1。通过从平滑肌细胞和上皮细胞中删除IL-4R1，确定是否可以阻止IL-13诱导AHR。2. 比较肺过表达IL-4或IL-13和在平滑肌或肺上皮上选择性表达IL-4R1的小鼠慢性气道重塑的发展。3. 评估miR-1下调对气道反应性的影响，鉴定IL-4和IL-13对气道平滑肌细胞上调或下调的基因。4. 确定IL-4诱导IFN-3和/或IL-10是否有助于IL-13对AHR的更强诱导。拟议的实验将使用转基因小鼠、重组细胞因子、单克隆抗体、细胞转移系统、体内细胞因子产生测定、流式细胞术和3种不同的技术来测量气道反应性，以验证我们的假设。结果将提高对哮喘发病机制的理解，提供一个可用于调节人类哮喘中平滑肌依赖性AHR的药物体内试验的模型，并提供指导选择IL-13或IL-4R拮抗剂作为潜在哮喘治疗药物的信息。

DESCRIPTION (provided by applicant): Development of allergic airway disease in mice depends on IL-4R1-mediated activation of Stat6 by IL-4 and IL- 13, with the latter cytokine being more important than the former during the effector phase of disease. We have evaluated the cell types through which IL-4 and IL-13 contribute to allergic airway disease and explored mechanisms responsible for the greater effects of IL-13. Results show that: 1) inhaled IL-4 and IL-13 can each independently induce airway hyperresponsiveness (AHR) in mice through direct effects on airway smooth muscle cells; 2) AHR is induced independently of eosinophilia and goblet cell hyperplasia; 3) induction of AHR by IL-4 and IL-13 requires signaling through the type 2 IL-4R; 4) IL-4, but not IL-13, induces increased secretion of IFN-3 and IL-10, cytokines that can suppress allergic airway disease; 5) IL-4 signaling through the type 1 IL-4R suppresses the expression of some of the genes induced in the lungs by IL-13; and 6) IL-13 suppresses expression of miR-1, a microRNA expressed in muscle that suppresses translation of multiple genes that promote differentiation, proliferation and responsiveness. Concurrent studies by other investigators have shown that: 1) IL-13 can induce AHR through direct effects on pulmonary epithelium, but also induces AHR in mice whose pulmonary epithelium cannot directly respond to this cytokine; and 2) the type 2 IL-4R binds IL-13 differently and with greater affinity than it binds IL-4. Taken together, these observations suggest our 3 central hypotheses: 1) induction of AHR by IL-4 and IL-13 is completely accounted for by their effects on smooth muscle and airway epithelium; 2) IL-4/IL-13-induced changes in smooth muscle gene transcription and translation contribute to the effects of these cytokines on airway responsiveness and muscle remodeling and 3) the greater ability of inhaled IL-13 than IL-4 to increase AHR in mice results from IL-4 activation of the type 1 IL-4 receptor (IL-4R), with consequent production of IFN-3 and IL-10, rather than from differences in IL-4 vs. IL- 13 signaling through the type 2 IL-4R or IL-13R12. These hypotheses will be tested by 4 specific aims: 1. Determine whether IL-13 induction of AHR is prevented by deleting IL-4R1 from both smooth muscle and epithelial cells. 2. Compare development of chronic airway remodeling in mice that overexpress IL-4 or IL-13 in their lungs and selectively express IL-4R1 on smooth muscle or pulmonary epithelium. 3. Evaluate the effects of miR-1 downregulation on airway responsiveness and Identify genes that are up- or downregulated by IL-4 and IL-13 on airway smooth muscle cells. 4. Determine whether induction of IFN-3 and/or IL-10 by IL-4 contributes to the stronger induction of AHR by IL-13. Proposed experiments will use transgenic mice, recombinant cytokines, monoclonal antibodies, cell transfer systems, assays of in vivo cytokine production, flow cytometry and 3 different techniques for measuring airway responsiveness to test our hypothesis. Results should improve understanding of asthma pathogenesis, provide a model that can be used for in vivo tests of agents that may regulate smooth muscle-dependent AHR in human asthma and provide information that should guide the choice of IL-13 vs. IL-4R antagonists as potential asthma therapeutics. PUBLIC HEALTH RELEVANCE: Asthma, a chronic lung disease characterized by airway smooth muscle hyperplasia and hyperresponsiveness, affects more than 7% of Americans and has been increasing in incidence for the past 50 years. Two cytokines, IL-4 and IL-13, induce acute and chronic changes in pulmonary structure and function that contribute to airway hyperresponsiveness. However, mechanisms responsible for IL-4- and IL-13-induced changes in smooth muscle structure and function that contribute to airway hyperresponsiveness are poorly understood, as are mechanisms that account for differences in the effects of IL-4 vs. IL-13. Proposed studies with a mouse model in which airway hyperresponsiveness is induced solely by direct IL-4 and IL-13 effects on smooth muscle should help to elucidate these mechanisms and may reveal new approaches for asthma therapy.