Roles of the Src-Family Kinases LynA and LynB in Macrophage Inflammatory Signaling

摘要 自身免疫性疾病青少年特发性关节炎（JIA），导致关节，眼睛和骨骼损伤， 慢性疼痛是美国最常见的儿童慢性疾病。JIA患者关节液 含有大量的巨噬细胞免疫细胞，并能驱动巨噬细胞分泌炎症因子 这种疾病目前针对JIA的疗法阻断了这种炎症信号，但不能区分疾病- 相关的炎症信号和对感染的正常免疫反应。因此，压制- 抗微生物免疫是这些现有疗法的严重副作用。我们发现了一个信号机器人- 巨噬细胞通过蛋白LynA进行过敏性炎症信号传导，但 病原体识别并不严格要求。我们现在也产生了第一个LynA-/-小鼠，并提出 测试LynA调节途径是否有希望作为一种新的治疗靶点， 炎症巨噬细胞信号传导而不抑制免疫系统。我们的长期目标是 开发专门针对病理性巨噬细胞的新治疗方法。为了支持多样性 为了在我们的实验室完成使命，并增加我们资助的研究的深度，我们的目标是测试LynA在孤立的 巨噬细胞和小鼠炎性关节炎的其他模型。具体而言，我们建议： 检测LynA和LynB基因敲除在K/BxN遗传模型和SKG小鼠模型中的作用。 炎性关节炎：我们将用现有的敲除模型繁殖这些菌株，并评估疾病进展- 锡永。我们还将获得用于后续目标的组织。我们预测，LynA-/-小鼠将有更少的 比LynB-/-小鼠更严重的疾病。B）定量上述模型中自身抗体和细胞因子的产生： 将对上述小鼠血清中的IgG、IgM和炎性细胞因子水平进行额外分析， 自身免疫性疾病，这将为自身免疫性疾病的独特病理特征和细胞驱动因素提供线索， 放松。基于我们最近发表的工作，我们预测LynA-/-小鼠将有更多的巨噬细胞/单核细胞， 细胞驱动的分布，而LynB-/-小鼠将更多的树突状细胞驱动。我们预测雌性老鼠 比雄性小鼠产生更多的自身抗体。C）测试骨髓细胞的极化和B细胞的活化 我们假设，与小鼠一样，JIA影响关节的炎症诱导了关节炎。 人类巨噬细胞中的LynA上调。为了补充我们的细胞因子分析，我们将使用流式细胞术， qRT-PCR表征骨髓细胞极化和B细胞活化这将有助于连接细胞因子谱 疾病的细胞驱动因素。它还可以揭示驱动自身免疫病理学的最相关的途径。 这个项目将受益于我们在巨噬细胞信号传导方面的专业知识和中心内的密切合作 明尼苏达大学的免疫学博士这些研究将确定靶向LynA的可行性， 为JIA和其他巨噬细胞驱动的炎症性疾病开发更安全、有针对性的治疗方案。

ABSTRACT The autoimmune disease juvenile idiopathic arthritis (JIA), which causes joint, eye, and bone damage and chronic pain, is the most common chronic childhood disease in the United States. Joint fluid from JIA patients contains large numbers of macrophage immune cells, and macrophage secretion of inflammatory factors drives the disease. Current therapies for JIA block this inflammatory signaling but do not discriminate between disease- associated inflammatory signaling and the normal immune response to infection. Therefore, suppression of an- timicrobial immunity is a severe side effect of these existing therapies. We have discovered a signaling mecha- nism through the protein LynA that is required for hypersensitive inflammatory signaling by macrophages, but is not strictly required for pathogen recognition. We have now also generated the first LynA-/- mice and propose to test whether the LynA regulatory pathway has promise as a novel therapeutic target, to specifically suppress inflammatory macrophage signaling without suppressing the immune system generally. Our long-term goal is to develop new therapeutic approaches targeted specifically to pathological macrophages. To support the diversity mission in our lab and increase the depth of our funded investigation, we aim to test the role of LynA in isolated macrophages and in additional models of mouse inflammatory arthritis. More specifically, we propose to: A) Examine the effect of LynA and LynB knockout in the K/BxN genetic model and the SKG model of murine in- flammatory arthritis: We will breed these strains with our existing knockout moels and assess disease progres- sion. We will also obtain tissues to be used in subsequent Aims. We predict that LynA-/- mice will have less severe disease than LynB-/- mice. B) Quantify autoantibody and cytokine production in the above models: We will perform additional profiling of IgG, IgM, and inflammatory cytokine levels in sera from the above mice with autoimmune disease, which will yield clues to unique pathological profiles and cell drivers of autoimmune dis- ease. Based on our recently published work, we predict that LynA-/- mice will have a more macrophage/mono- cyte-driven profile, whereas LynB-/- mice will be more dendritic-cell driven. We predict that female mice will have more autoantibody production than male mice. C) Test polarization of myeloid cells and activation of B cells taken from arthritic mice above: We hypothesize that, as in mice, inflammation in JIA-affected joints induces LynA upregulation in human macrophages. To complement our cytokine profiling, we will use flow cytometry and qRT-PCR to characterize myeloid-cell polarization and B-cell activation This will help connect cytokine profiles to cellular drivers of disease. It may also reveal the most relevant pathways driving the autoimmune pathology. This project will benefit from our expertise in macrophage signaling and close collaborations within the Center for Immunology at the University of Minnesota. These studies will establish the feasibility of targeting LynA to develop safer, targeted treatment options for JIA and other macrophage-driven inflammatory diseases.