MIF: a pro-inflammatory cytokine as a novel target to reduce bladder inflammation

描述(申请人提供)：实验证据支持巨噬细胞移动抑制因子(MIF)在膀胱炎中的促炎作用，但MIF如何介导膀胱炎尚不清楚。长期目标是了解MIF介导的膀胱炎的机制，目的是改变这些过程，以减少或消除下尿路慢性疼痛患者的膀胱炎。这项建议的总体目标是确定特定的尿路上皮受体对MIF释放和MIF介导的信号转导和膀胱炎的贡献。中心假设涉及MIF的炎症有两个组成部分：1)MIF释放增加；2)MIF受体上调，继而通过MIF/受体相互作用增强和/或维持炎症。这一假设是基于申请者实验室产生的初步数据提出的。这些研究的基本原理是，通过增加我们对膀胱中MIF上游和下游的信号事件的了解，将确定潜在的治疗靶点。这一假说将通过以下特定目的得到验证：1)确定尿路上皮蛋白水解酶激活受体(PAR)在尿路上皮MIF释放新机制中的作用：使用对不同PAR受体具有特异性的激动剂诱导尿路上皮细胞(人、体外和体内，使用小鼠)释放MIF，以确定哪些受体参与MIF释放。为了确定MIF在体内PAR介导的膀胱炎中的作用，我们将通过滴注特定的PAR激动剂同时拮抗MIF来诱导小鼠的膀胱炎。将测量膀胱的生理、组织和分子变化。2)明确尿路上皮MIF受体(CD74；CXCR4)在MIF信号和膀胱炎中的作用：MIF受体阻断剂将用于检测MIF介导的信号在小鼠膀胱炎(环磷酰胺)模型中的变化。这些实验还将通过测量膀胱的生理、组织学和分子变化来确定MIF受体阻断在介导膀胱炎中的作用。3)检测临床条件下膀胱炎患者尿MIF及其受体水平：尿路感染、放射性膀胱炎和PBS/IC(Hunner溃疡与非Hunner溃疡)患者均可检测尿液MIF水平。PBS/IC患者尿路上皮MIF、CD74和CXCR4免疫组织化学染色，以及PBS/IC患者逼尿肌细胞MIF介导的细胞因子产生情况。这项建议是创新的，因为它关注的是MIF，一种新的膀胱炎症介质。这项研究具有重要意义，因为深入了解MIF在膀胱炎症过程中的释放机制，以及MIF与尿路上皮MIF受体结合激活的信号通路，有望识别下尿路炎症的新治疗靶点。

DESCRIPTION (provided by applicant): Experimental evidence supports the pro-inflammatory effect of Macrophage Migration Inhibitory Factor (MIF) in bladder inflammation, but how MIF mediates bladder inflammation is poorly understood. The long-term goal is to understand the mechanisms of MIF-mediated bladder inflammation with the aim of altering these processes to reduce or abolish bladder inflammation in patients with chronic painful conditions of the lower urinary tract. The overall objective of this proposal is to determine the contribution of specific urothelial receptors to MIF release and to MIF-mediated signaling and bladder inflammation. The central hypothesis is that there are two components to inflammation involving MIF: 1) increased release of MIF; and 2) upregulation of MIF receptors and subsequent increased signaling via MIF/receptor interactions that enhances and/or maintains inflammation. This hypothesis has been formulated based on preliminary data generated in the applicants' laboratory. The rationale for propos- ing these studies is that by increasing our understanding of signaling events both upstream and downstream of MIF in the bladder will identify potential therapeutic targets. The hypothesis will be tested by the following specific aims: 1) Determine the contribution of urothelial Protease Activated Receptors (PAR) to a novel mechanism of urothelial MIF release: Agonists with specificity for different PAR receptors will be used to induce MIF release from urothelial cells (both human, in vitro and in vivo, using mice) to determine which receptors are involved in MIF release. To determine the contribution of MIF to PAR-mediated bladder inflammation in vivo, bladder inflammation will be induced in mice by instilling specific PAR agonists while simultaneously antagonizing MIF. Physiological, histological and molecular changes in the bladder will be measured. 2) Identify the contribution of urothelial MIF receptors (CD74; CXCR4) to MIF signaling and cystitis: MIF receptor blockade will be used to examine changes in MIF-mediated signaling during a well-established model of cystitis (cyclophosphamide) in mice. These experiments will also determine the effect of MIF receptor blockade on mediating cystitis by measuring physiological, histological and molecular changes in the bladder. 3) Determine MIF and MIF receptor levels in clinical conditions with bladder inflammation: Urinary MIF levels will be determined in UTI, radiation cystitis and PBS/IC (Hunner ulcer vs non-Hunner ulcer) patients. MIF, CD74 and CXCR4 urothelial immunostaining will be examined in PBS/IC (Hunner ulcer vs non-Hunner ulcer) and MIF-mediated cytokine production in human detrusor smooth muscle cells from PBS/IC patients will be examined. This proposal is innovative because it focuses on MIF, a novel mediator of bladder inflammation. The pro- posed research is significant because a greater understanding of the mechanisms of MIF release during bladder inflammation and of the signaling pathways activated by MIF binding to urothelial MIF receptors is expected to result in the identification of novel therapeutic targets for inflammatory conditions of the lower urinary tract.