Notch signaling in diabetic wounds

项目摘要/摘要 2型糖尿病(T2D)患者的伤口无法愈合是发病率增加和 死亡率。T2D患者伤口愈合失败是美国截肢最常见的原因 5年死亡率接近50%。因此，迫切需要了解伤口愈合情况。 T2D的缺陷，以开发靶向治疗。我们利用了遗传(db/db)和饮食(饮食- 诱导肥胖)小鼠T2D模型以及从T2D采集的人伤口组织和血液样本 探讨创面愈合受损的机制。我们公布的和初步的数据表明 创伤巨噬细胞(MφS)通过Notch信号与CD_4~+T细胞相互作用，决定CD_4~+T细胞 激活，最终调节伤口的炎症和愈合。我们和其他人已经确认一处伤口 CD4+Treg细胞表型对正常组织修复至关重要，而TH17表型促进过度 发炎，影响愈合。我们的初步数据证实TLR4信号在创伤Mφ中上调S Notch配体DLL4，然后与CD4+T细胞上的Notch 1和2受体相互作用，促进TH17 AS 反对特雷格分化。这些相互作用推动了糖尿病患者的过度炎症和病理愈合。 此外，DLL4-Notch通路可能通过表观遗传机制在糖尿病创面进一步上调 涉及组蛋白甲基转移酶MLL1。MLL1直接上调MφS的DLL4，并可间接增加 此外，MLL1还可直接上调φ细胞中Notch1和Notch2受体的表达。 CD4+T细胞，使它们更容易接受Notch的激活，并促进延长的TH17反应。这些 糖尿病创面的病理TH17表型随病情变化而变化。这些结果导致了我们 糖尿病创面中DLL4介导的Notch受体信号极化创面中的CD4+T细胞的假说 朝向Th17，并促进慢性炎症和不可愈合。此外，下游IL17A增加 信号转导增加了糖尿病组织中表皮和真皮细胞介导的炎症。我们的数据表明 创伤的Mφ/CD4+T细胞创伤表型可以通过Mφ靶向治疗(局部传递到 用MLL1抑制剂(S)或用抗DLL4抗体局部治疗。为了测试 我们的假设，我们将追求以下目标：目标1：考察直接(MLL1)和间接 (TLR4/MyD88)正常和糖尿病条件下伤口巨噬细胞中DLL4的调节。目标2： 正常和糖尿病时Notch受体激活在Treg/TH17分化中的作用 伤口修复。目的3：比较局部和Mφ靶向治疗以减少Notch信号转导，限制TH17 分化，促进愈合，并确定伤口中IL17A信号的细胞靶点。

PROJECT SUMMARY/ABSTRACT Non-healing wounds in patients with Type 2 Diabetes (T2D) are a major cause of increasing morbidity and mortality. Failure of wound healing in T2D patients represents the most common cause of amputation in the US with a 5-year mortality rate of nearly 50%. Thus, a critical need exists for understanding the wound healing defects in T2D in order to develop targeted therapies. We have utilized both genetic (db/db) and dietary (diet- induced obese) murine models of T2D as well as human wound tissue and blood samples collected from T2D patients to explore mechanisms of impaired wound healing. Our published and preliminary data demonstrate that interactions between wound macrophages (Mφs), and CD4+T cells, via Notch signaling, dictate CD4+T cell activation to ultimately regulate inflammation and healing in wounds. We and others have identified that a wound CD4+Treg cell phenotype is critical for normal tissue repair, while a TH17 phenotype promotes excess inflammation and impairs healing. Our preliminary data identify TLR4 signaling in wound Mφs upregulates the Notch ligand, DLL4, which then interacts with the Notch 1 and 2 receptors on CD4+T cells to promote TH17 as opposed to Treg differentiation. These interactions drive excess inflammation and pathologic healing in diabetes. Additionally, the DLL4-Notch pathway may be further upregulated in diabetic wounds via epigenetic mechanisms involving MLL1, a histone methyltransferase. MLL1 directly upregulates DLL4 in Mφs and can indirectly increase DLL4 via TLR4 signaling in Mφs. In addition, MLL1 can directly increase Notch 1 and 2 receptor expression in CD4+T cells, making them more receptive to Notch activation and promoting a prolonged TH17 response. These changes then dictate the pathologic TH17 phenotype in diabetic wounds. These results have led to our hypothesis that DLL4-mediated Notch receptor signaling in diabetic wounds polarizes CD4+T cells in the wound towards TH17 and promotes chronic inflammation and non-healing. Further, increased downstream IL17A signaling increases epidermal and dermal cell mediated inflammation in diabetic tissue. Our data suggest that wound Mφ/CD4+T cell wound phenotypes may be restored via Mφ-targeted treatment (delivered locally to the wound via engineered nanoparticles) with MLL1 inhibitor(s) or local treatment with anti-DLL4 antibodies. To test our hypotheses, we will pursue the following aims: Aim 1: To examine the direct (MLL1) and indirect (TLR4/MyD88) regulation of DLL4 in wound macrophages during normal and diabetic conditions. Aim 2: To identify the role of Notch receptor activation on Treg/TH17 differentiation during normal and diabetic wound repair. Aim 3: To compare local and Mφ-targeted therapies to reduce Notch signaling, limit TH17 differentiation, improve healing and identify the cellular targets for IL17A signaling in wounds.