Eosinophil:M2 Macrophage:CCL11 Axis in Experimental Colitis and Pediatric Cortico

描述(申请人提供)：皮质类固醇(CS)仍然是治疗儿童UC的主要药物；然而，20%的患者对CS治疗无效，需要进行二线治疗，并加强医疗管理或结肠切除术4。在最近的以患者为基础的研究中，我们证明CCL11水平与组织嗜酸性粒细胞数量相关，而组织嗜酸性粒细胞数量又与儿童UC的UC组织学严重程度指数(UCHIS)相关。在初步研究中，我们确定，在儿童UC诊断时，直肠乙状结肠嗜酸性粒细胞水平高与实现无类固醇缓解(SFR)的可能性降低有关。值得注意的是，高嗜酸性粒细胞水平与CCL11和一种称为钙保护素的低分子细胞内钙结合蛋白的水平呈正相关。在实验研究中，我们已经确定了实验性结肠炎中钙保护素、M衍生的CCL11和嗜酸性粒细胞之间的联系。重要的是，我们发现结肠MéS在体外表达钙保护素受体(RAGE)，而CaP可刺激M？S的p65激活和CCL11的表达。本应用的目的是进一步了解钙保护素在实验性结肠炎和儿童UC中对炎性M？来源的CCL11和嗜酸性炎症的相对贡献和治疗反应。我们的中心假设是，钙保护素诱导的M？S激活调节实验性结肠炎和儿童UC中CCL11依赖的结肠嗜酸细胞炎症，这一途径推动难治性疾病。我们将通过检测诊断时儿童UC中钙保护素、MéS、CCL11和嗜酸性粒细胞的关系以及这一途径与难治性疾病的关系来检验这一假说。我们将使用S100A9-/-、RAGE-/-、CCR2-/-、CCR2-/-/IL10-/-小鼠；使用DSS和NSAID诱导的IL10-/-结肠炎模型；产生骨髓来源的M？特异性嵌合小鼠并进行嗜酸性粒细胞趋化试验，以确定M？来源的CCL11在实验性结肠炎嗜酸细胞募集和组织病理学中的作用以及这一途径对CS治疗的敏感性。关于预期结果，Aim I中建议的研究有望确定钙保护素、CCL11+M？S和儿童UC中嗜酸性粒细胞之间的关联；这一途径对CS的敏感性；以及CCL11和嗜酸性粒细胞疾病作为一线治疗耐药指标的预测价值。目的研究实验性结肠炎大鼠结肠嗜酸性粒细胞炎症及对钙保护素/RAGE诱导的CCL11的组织病理学依赖性。目的III确定钙保护素/RAGE诱导巨噬细胞来源的CCL11和嗜酸性粒细胞趋化的能力以及对CS诱导的抑制的敏感性。证实钙保护素/M/CCL11/嗜酸性粒细胞轴参与了一线治疗在儿童UC中的耐药，这对于使用CCL11和嗜酸性粒细胞作为诊断一线治疗耐药的预后指标，以及可能使用针对嗜酸性粒细胞和嗜酸性粒细胞调节分子的治疗药物作为改善儿童UC和难治性UC治疗的方法具有重要的临床意义。 公共卫生相关性：儿童溃疡性结肠炎(UC)具有较高的皮质类固醇(CS)依赖率和频繁的复发，需要更积极的治疗，包括手术。我们已经确定了儿童UC患者诊断时的白细胞、嗜酸性粒细胞水平与CS无反应之间的联系。在这项应用中，我们将揭开UC中嗜酸性粒细胞运动和功能调节的机制，并确定嗜酸性粒细胞水平是否可以作为儿童UC CS反应性的潜在生物标志物。

DESCRIPTION (provided by applicant): Corticosteriods (CS) remains the mainstay of therapy for pediatric UC; however, 20% of patients fail to respond to CS therapy and require secondary-line therapies and escalation of medical management or colectomy4. In recent patient-based studies, we demonstrated that CCL11 levels correlated with tissue eosinophil numbers, which, in turn, correlated with the UC Histologic Index of Severity (UCHIS) in pediatric UC. In preliminary studies, we determined that high rectosigmoid eosinophil levels at the time of pediatric UC diagnosis is linked with reduced likelihood of achieving steroid-free remission (SFR). Notably, high eosinophil levels positively correlated with levels of CCL11 and a low molecular weight intracellular calcium binding protein called calprotectin. In experimental studies, we have identified a link between calprotectin, M¿-derived CCL11, and eosinophils in experimental colitis. Importantly, we show that colonic M¿s express the calprotectin receptor (RAGE); and calprotectin stimulates p65 activation and CCL11 expression in M¿s in vitro. The object of this application is to further understand the relative contribution of calprotectin to inflammatory M¿-derived CCL11 and eosinophilic inflammation in experimental colitis and pediatric UC and treatment responses. Our central hypothesis is that calprotectin-induced activation of M¿s regulates CCL11-dependent colonic eosinophilic inflammation in experimental colitis and pediatric UC and that this pathway drives refractory disease. We will test this hypothesis by examining the relationship between calprotectin, M¿s, CCL11, and eosinophils in pediatric UC at diagnosis and the relationship of this pathway to refractory disease. We will employ S100A9-/-, RAGE-/-, CCR2-/-, CCR2-/-/Il10-/- mice; use DSS- and NSAID-induced Il10-/- models of colitis; generate bone marrow-derived M¿-specific chimeric mice and perform eosinophil chemotaxis assays to define M¿-derived CCL11 in eosinophil recruitment and histopathology in experimental colitis and the sensitivity of this pathway to CS treatment. With respect to expected outcomes, the studies proposed in Aim I are expected to define the association between calprotecin, CCL11+ M¿s, and eosinophils in pediatric UC; the sensitivity of this pathway to CS; and the predictive value of CCL11 and eosinophils disease as an indicator of resistance to first-line therapies. Aim II is expected to delineate colonic eosinophilc inflammation and histopathology dependency on calprotectin/RAGE-induced M¿-derived CCL11 in experimental colitis. Aim III is expected to define the capacity of calprotectin/RAGE to induce M¿-derived CCL11 and eosinophil chemotaxis and sensitivity to CS-induced inhibition. Demonstration of calprotectin/M¿/CCL11/eosinophil axis involvement in resistance to first-line therapies in pediatric UC will have important clinical implications for both the usage of CCL11 and eosinophils as a prognostic indicator for resistance to first-line therapies at diagnosis and the possible usage of therapeutic agents targeting eosinophils and eosinophil regulatory molecules as an approach for improved treatment of pediatric UC and refractory UC. PUBLIC HEALTH RELEVANCE: Pediatric ulcerative colitis (UC) has a high rate of corticosteroid (CS) dependency and frequent relapse requiring more aggressive therapy including surgery. We have identified a link between the level of the white blood cell, eosinophil at diagnosis in pediatric UC, and unresponsiveness to CS. In this application, we will unravel the mechanisms involved in the regulation of eosinophil movement and function in UC and identify if eosinophil levels can be used as a potential biomarker for CS-responsiveness in pediatric UC.