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

描述（由申请人提供）：实验证据支持巨噬细胞迁移抑制因子（MIF）在膀胱炎症中的促炎作用，但对MIF如何介导膀胱炎症知之甚少。长期目标是了解MIF介导的膀胱炎症的机制，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症。这项建议的总体目标是确定特定尿路上皮受体对MIF释放和MIF介导的信号传导和膀胱炎症的贡献。中心假设是炎症有两个组分涉及MIF：1）MIF的释放增加;和2）MIF受体的上调和随后通过MIF/受体相互作用增加的信号传导，其增强和/或维持炎症。该假设是基于申请人实验室中产生的初步数据而制定的。提出这些研究的理由是，通过增加我们对膀胱中MIF上游和下游的信号传导事件的理解，将确定潜在的治疗靶点。 1）确定尿路上皮蛋白酶激活受体（PAR）对尿路上皮MIF释放的新机制的贡献：将使用对不同PAR受体具有特异性的激动剂来诱导MIF从尿路上皮细胞（人，体外和体内，使用小鼠）释放，以确定哪些受体参与MIF释放。为了确定MIF对体内PAR介导的膀胱炎症的贡献，通过滴注特异性PAR激动剂同时拮抗MIF在小鼠中诱导膀胱炎症。将测量膀胱中的生理学、组织学和分子变化。2)确定尿路上皮MIF受体（CD 74; CXCR 4）对MIF信号传导和膀胱炎的贡献：将使用MIF受体阻断剂检查在小鼠膀胱炎（环磷酰胺）的良好建立模型期间MIF介导的信号传导的变化。这些实验还将通过测量膀胱中的生理学、组织学和分子变化来确定MIF受体阻断对介导膀胱炎的影响。3)测定膀胱炎症临床状况下的MIF和MIF受体水平：将测定UTI、放射性膀胱炎和PBS/IC（Hunner溃疡vs非Hunner溃疡）患者的尿MIF水平。将在PBS/IC中检查MIF、CD 74和CXCR 4尿路上皮免疫染色（Hunner溃疡vs非Hunner溃疡），并将检查来自PBS/IC患者的人逼尿肌平滑肌细胞中MIF介导的细胞因子产生。 该提案具有创新性，因为它关注的是膀胱炎症的新型介导因子MIF。所提出的研究是重要的，因为更好地理解膀胱炎症期间MIF释放的机制以及MIF与尿路上皮MIF受体结合激活的信号传导途径，预计将导致鉴定下尿路炎性疾病的新治疗靶点。

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.