The Role of Heparan Sulfate in the B cell response

描述（由申请方提供）：硫酸乙酰肝素影响许多细胞过程，包括粘附、运动、配体-受体相互作用和增殖。B细胞是适应性免疫系统的主要组成部分，在适应性免疫系统中，它们有助于消除和保护免受入侵的病原体。B细胞定位、细胞因子/趋化因子应答和细胞间相互作用对B细胞应答的启动和维持至关重要。我们最近观察到硫酸乙酰肝素在B细胞上显著上调。事实上，尽管初始B细胞不表达硫酸乙酰肝素，但从病毒感染的小鼠或注射聚I：C的小鼠中分离的B细胞表达硫酸乙酰肝素。重要的是，当使用干扰素受体缺陷小鼠时，这种表型被消除，表明在该过程中需要干扰素。因此，单独用干扰素离体处理的B细胞表达高水平的硫酸乙酰肝素。有趣的是，我们已经表明干扰素处理的B细胞对B细胞特异性细胞因子APRIL更敏感，APRIL是一种与硫酸乙酰肝素表达增加相关的表型。总之，这些实验表明，在病毒感染时，干扰素诱导B细胞上的硫酸乙酰肝素表达，使这些细胞对其环境更具反应性，并可能影响B细胞抗体反应。为了验证这一假设，我们产生了B细胞上硫酸乙酰肝素表达缺陷的小鼠（EXT 1-/-小鼠），并表明它们的B细胞发育没有改变。有趣的是，使用2-光子显微镜产生的初步数据表明，B细胞运动性的影响，在干扰素的存在下，硫酸乙酰肝素依赖的方式。 使用流感作为感染模型，我们已经证明了在EXT 1-/-小鼠中胸腺依赖性和非依赖性抗体应答都增加。尽管产生了这种增加的应答，但EXT 1-/-小鼠中的病毒限制不受影响。该建议旨在进一步表征体液应答期间B细胞上硫酸乙酰肝素表达的作用。由于我们体内感染数据的初步性质，我们的目标是确认和进一步我们的初步发现。为此，我们将用一系列感染剂量的流感病毒感染小鼠，并通过监测宿主健康、病毒限制、应答B细胞亚群和产生的抗体来评估抗体应答的质量。这将通过测量病毒滴度、抗体滴度、应答B细胞群以及宿主发病率和死亡率来实现。为了解决观察结果，EXT 1-/-小鼠产生扩大的抗体反应，但不能更有效地限制流感，我们将评估体液反应的特异性和效率。这将通过确定EXT 1-/-抗体的亲和力和病毒中和能力来完成。最后，我们将评估从流感免疫EXT 1-/-小鼠分离的血清抗体的保护作用。 我们希望揭示EXT 1-/-抗体反应的其他改变，这将进一步支持我们的假设，即IFN介导的B细胞上HS的诱导作为一种新的免疫调节形式。基于此，我们认为这项工作可能有助于开发疫苗，其中有效的B细胞反应是必要的。 公共卫生相关性：我们在这里所做的工作可能有助于理解对传染病的抗体反应。此外，对该主题的理解可能会促进疫苗的开发。

DESCRIPTION (provided by applicant): Heparan sulfate influences many cellular processes including adhesion, motility, ligand-receptor interaction, and proliferation. B cells account for a major component of the adaptive immune system, in which they aid in the elimination of, and protection against, invading pathogens. Crucial to the initiation and maintenance of a B cell response is B cell localization, cytokine/chemokine responsiveness, and cell-to-cell interaction. We have recently observed that heparan sulfate is drastically upregulated on B-cells. Indeed, whereas naive B-cells do not express heparan sulfate, B-cells isolated from mice infected with viruses, or mice injected with poly I:C, express heparan sulfate. Importantly this phenotype is abolished when interferon receptor deficient mice are used, indicating a requirement for interferon in this process. Accordingly, B-cells treated ex-vivo with interferon alone express high levels of heparan sulfate. Interestingly, we have shown that interferon treated B-cells are more responsive to the B cell-specific cytokine APRIL, a phenotype associated with increased heparan sulfate-expression. Altogether, these experiments suggest that upon viral infection, interferon induces heparan sulfate expression on B-cells, rendering these cells more responsive to their environment, and potentially affecting the B-cell antibody response. To test this hypothesis, we have generated mice that are deficient for heparan sulfate expression on B-cells (EXT1-/- mice) and have shown that their B- cell development is not altered. Interestingly, preliminary data generated using 2-photon microscopy indicates that B-cell motility was affected in the presence of interferon in a heparan sulfate-dependent manner. Using influenza as an infectious model, we have demonstrated that both the thymus -dependent and - independent antibody responses are increased in EXT1-/- mice. Despite generating this increased response, viral restriction in EXT1-/- mice is unaffected. This proposal aims to further characterize the role of heparan sulfate expression on B cells during a humoral response. Due to the preliminary nature of our in vivo infection data, we aim to confirm and further our initial findings. To this end, we will infect mice with a range of infectious doses of influenza virus and assess the quality of the antibody response by monitoring host health, viral restriction, responding B cell subsets, and the antibodies produced. This will be accomplished by measuring viral titers, antibody titers, responding B cell populations, and host morbidity and mortality. To address the observations that EXT1-/- mice generate an expanded antibody response, yet are unable to more efficiently restrict influenza, we will assess the specificity and efficiency of the humoral response. This will be done by determining the affinity and the virus-neutralizing capabilities of EXT1-/- antibodies. Finally, we will assess the protective effect of serum antibodies isolated from influenza- immune EXT1-/- mice. We hope to reveal additional alterations in the EXT1-/- antibody response that will further support our hypothesis that IFN-mediated induction of HS on B cells serves as a novel form of immune regulation. Based on this, we believe that this work may aid in the development of vaccines in which an effective B cell response is necessary. PUBLIC HEALTH RELEVANCE: The work we prose here may help in understanding of the antibody response against infectious disease. Furthermore, understanding of the topic may enhance vaccine development.