The role of CXCR2-ligands for neutrophil mobilization from the bone marrow to peripheral sites

骨髓是先天免疫系统细胞的主要储备，尤其是中性粒细胞。从这里，这些细胞可以通过生理刺激或外周炎症的作用迅速动员到外周。但是它们也会造成严重的损伤，例如在软骨破坏疾病如风湿性关节炎的初始阶段。然而，在动员或软骨破坏过程中，骨髓腔内单个中性粒细胞的行为以及外周炎症对这一过程的影响尚不清楚。利用新开发的活体双光子显微镜方法，我们在携带EGFP中性粒细胞特异性等位基因的小鼠中发现，造血细胞因子G-CSF是主要的生理动员剂之一，它通过快速诱导胫骨骨髓腔内单个中性粒细胞的运动而起作用。在分子水平上，我们发现趋化因子受体CXCR2的配体CXCL1 （KC）和CXCL2 （MIP-2）在骨髓巨核细胞中的表达增加。此外，通过特异性抗体阻断CXCR2可以阻断G-CSF对中性粒细胞的动员。在一项平行研究中，我们使用了一种新开发的小鼠模型来选择性地在皮肤中过表达IL17A。这导致了一种与人类牛皮癣高度相似的表型，伴随着大量中性粒细胞浸润到皮肤中，同时骨髓中有强烈的中性粒细胞。我们假设骨和皮肤中CXCL1和2的调节在骨髓中嗜中性粒细胞频率的增加及其向皮肤的募集中也起着重要作用。为了研究这些概念，我们提出了一种新的小鼠模型，其中CXCL1和cxcl2的等位基因在同一动物中是有条件的。此外，荧光蛋白Tomato将作为基因敲入CXCL1基因座，作为启动子活性的报告基因。给这些小鼠注射G-CSF或在皮肤中诱导IL17A过表达，将显示哪些细胞和哪些动力产生CXCL1/2来响应这些不同的刺激。此外，将这些小鼠与巨核细胞或角化细胞特异性的Cre系杂交，将允许以细胞类型特异性的方式删除cxcr2配体。这将使我们能够澄清，CXCR2配体在骨髓常驻细胞或外周细胞中的生理性表达是否对中性粒细胞被系统性G-CSF或皮肤源性IL17A募集起决定性作用。最后，我们将使用皮肤特异性il17a过表达的小鼠模型来研究骨髓中中性粒细胞过量产生介导的骨质流失和关节炎症，这是银屑病皮肤病的两个众所周知的实体。总的来说，这项研究应该为我们提供了以前没有的关于骨髓和外周中性粒细胞的生理和病理生理以及cxcr2配体在这些过程中的作用的知识，从而为细胞骨免疫学提供了一个非常重要的机制。

The bone marrow is a major reserve for cells of the innate immune system, especially neutrophil granulocytes. From here these cells can be rapidly mobilized to the periphery by action of physiologic stimuli or peripheral inflammation. But they can also cause severe damage, e.g. in the initiation phase of cartilage-destructive diseases such as rheumatoid arthritis. However, how individual neutrophils behave within the bone marrow cavity during the process of mobilization or cartilage destruction and which impact peripheral inflammation has on the process is little understood. Using a newly developed intravital 2-photon microscopy approach in mice carrying a neutrophil-specific allele of EGFP we have shown that one of the major physiologic mobilizers, the hematopoietic cytokine G-CSF, acts by rapidly inducing the motility of individual neutrophils within the marrow cavity of the tibia. On a molecular level we found the increased expression of CXCL1 (KC) and CXCL2 (MIP-2), ligands for the chemokine receptor CXCR2, by marrow-resident megakaryocytes. Further, a blockade of CXCR2 by a specific antibody could block neutrophil mobilization by G-CSF. In a parallel study we have used a newly developed mouse model for the overexpression of IL17A selectively in the skin. This led to a phenotype highly reminiscent of human psoriasis, which was accompanied by massive infiltration of neutrophils into the skin together with strong neutrophilia in the bone marrow. We assume that also here the modulation of CXCL1 and 2 in the bone and also the skin play a major role for the increased frequency of neutrophils in the bone marrow and their recruitment to the skin. To investigate these concepts we propose to generate a novel mouse model in which the alleles for CXCL1 and 2 are made conditional within the same animal. In addition, the fluorescent protein Tomato will be engineered as a knockin into the CXCL1 locus to function as a reporter for promoter activity. Injecting these mice with G-CSF or inducing IL17A overexpression in the skin will show, which cells and with which kinetic produce CXCL1/2 in response to these diverse stimuli. Furthermore, the crossing of these mice to either megakaryocyte- or keratinocyte-specific Cre lines will allow deleting the CXCR2-ligands in a cell type specific manner. This will enable us to clarify, whether physiologic expression of CXCR2 ligands in either bone marrow-resident or peripheral cells is decisive for neutrophil recruitment by systemic G-CSF or skin-derived IL17A. Finally, we will use the mouse model of skin-specific IL17A-overexpression to study bone loss and joint inflammation mediated by excess production of neutrophils in the bone marrow, which are two well known entities accompanying psoriatic skin disease. Collectively this study should provide us with a previously not available knowledge on the physiology and pathophysiology of neutrophils in the bone marrow and the periphery and the role of CXCR2-ligands for these processes, thus a very central mechanism of cellular osteoimmunology.