Corneal Lymphatics & Adaptive Immunity

 描述（由申请人提供）：单纯疱疹病毒1型（HSV-1）诱导无血管角膜中的新血管形成，包括分别称为血管生成和淋巴管生成的血液和淋巴成分的生成。临床上，中央角膜的血管生成对视力具有不利影响，并且在许多情况下，对移植后角膜植入的成功具有不利影响。以前，我们已经发现HSV-1在角膜感染后诱导淋巴管生成，这需要局部（即，上皮细胞）产生血管内皮生长因子（VEGF）A，通过VEGF受体2（VEGFR 2）介导其作用。此外，我们发现HSV-1的即刻早期基因编码蛋白ICP 4通过Sp1和（可能）EGR 1启动子位点驱动VEGF A基因的表达。与这些发现一致，角膜淋巴管生成不通过Toll样受体起作用，也不需要MAP激酶诱导途径。这是该病原体独有的新观察结果。本申请中包括的新获得的数据表明，HSV-1感染后的角膜新血管形成在感染后第10天至第30天（pi）之间在病毒清除角膜后以更稳健的速度进行。在此期间，我们已经鉴定了除VEGF A外的几种促血管生成因子，包括白细胞介素（IL）-6， 肝细胞生长因子（HGF）和基质金属蛋白酶9（MMP 9），其上调并在感染后第14天达到峰值。我们还鉴定了包括嗜中性粒细胞、巨噬细胞、炎性单核细胞和T细胞在内的细胞，它们在HSV-1感染后角膜的整个稳健的新血管形成期驻留并维持在角膜中。另外的初步结果表明IL-6、HGF和炎性单核细胞与感染后10-30天之间病毒清除后角膜的新血管形成的进展最密切相关。因此，我们建议检验IL-6和HGF在清除HSV-1后促进角膜淋巴管发育和维持的假设（目的1）。此外，初步结果表明，HSV-1感染后角膜中VEGF A表达的减少和淋巴管的损失显著降低了对病原体的适应性免疫应答，导致三叉神经节中更高的病毒产量。我们假设适应性免疫应答的降低是由于引流淋巴结中功能性抗原呈递细胞的丧失（目的2）。总的来说，拟议的实验设计将使我们能够表征角膜淋巴管生成相对于HSV-1感染的预期结果的识别额外的促血管生成因子和途径作为候选目标的治疗干预。

 DESCRIPTION (provided by applicant): Herpes simplex virus type 1 (HSV-1) induces neovascularization in the avascular cornea including the genesis of blood and lymphatic components referred to as hemangiogenesis and lymphangiogenesis respectively. Clinically, angiogenesis of the central cornea has a detrimental impact on visual acuity and in many instances, the success of corneal engraftment following transplantation. Previously, we have found HSV-1 induces lymphangiogenesis following cornea infection that requires the local (i.e., epithelial cell) production of vascular endothelial growth factor (VEGF) A mediating its effects through VEGF receptor 2 (VEGFR2). Moreover, we found the immediate early gene-encoded protein ICP4 of HSV-1 drives expression of the VEGF A gene through Sp1 and (perhaps) EGR1 promoter sites. Consistent with these findings, corneal lymphangiogenesis does not act through toll-like receptors or require a MAP kinase pathway of induction. This is a novel observation unique to the pathogen. Newly acquired data included in this application demonstrates corneal neovascularization following HSV-1 infection proceeds at much more robust pace after the virus clears the cornea between day 10 and day 30 post infection (pi). During this time period, we have identified several pro- angiogenic factors in addition to VEGF A including interleukin (IL)-6, hepatocyte growth factor (HGF), and matrix metalloproteinase 9 (MMP9) that are up regulated and peak at day 14 pi. We have also identified cells including neutrophils, macrophages, inflammatory monocytes, and T cells that reside and are maintained in the cornea throughout the robust neovascularization period of the cornea post HSV-1 infection. Additional preliminary results suggest IL-6, HGF, and inflammatory monocytes are most closely associated with the progress in neovascularization of the cornea post virus clearance between days 10-30 pi. As such, we propose to test the hypothesis that IL-6 and HGF facilitate the development and maintain corneal lymphatic vessels upon clearance of HSV-1 (aim 1). In addition, preliminary results suggest the reduction in VEGF A expression and loss of lymphatic vessels in the cornea post HSV-1 infection significantly reduces the adaptive immune response to the pathogen resulting in a higher viral yield in the trigeminal ganglion. We hypothesize the reduced adaptive immune response is due to a loss of functional antigen presenting cells in the draining lymph node (aim 2). Collectively, the proposed experimental design will allow us to characterize corneal lymphangiogenesis relative to HSV-1 infection with the anticipated outcome of the identification of additional pro-angiogenic factors and pathways as candidate targets for therapeutic intervention.