The Role of Neutrophil Mechanosensing During Oropharyngeal Candidiasis

项目摘要/摘要 中性粒细胞是对感染部位的第一反应，是抗真菌反应的基础。 这包括口咽部念珠菌病，血液中的中性粒细胞渗入感染组织。 并移动去联系入侵的假丝酵母。在这个过程中，中性粒细胞不仅对 组织微环境的生化特性以及物理特性。口腔是 其复合组织的机械性能差异很大，从相对柔软和有弹性的舌头 和软腭到相对中等硬度的结构，如食道到刚性结构，如 就像是硬的味觉。Reichner实验室和其他实验室已经表明，人类中性粒细胞对 其微环境的力学特征包括衬底硬度。他们报告说，人类 与软性基质相比，中性粒细胞在刚性基质上的运动速度更慢，方向性更强。 矩阵。牵引力的量化和映射表明，在较硬的矩阵上产生更大的力 大部分的力产生在牢房的后部。关于这些发现的一个显著差距是，我们没有 关于病原体或其成分PAMP如何影响机械敏感反应的信息 中性粒细胞与整体运动和对不同硬度的基质产生牵引力有关。这 是重要的，因为它是诱导中性粒细胞进入受感染组织执行 抗菌活性，与口腔念珠菌感染作为OPC的原因高度相关。这个 在这个建议中要检验的最重要的假设是念珠菌的存在影响机械敏感性 人中性粒细胞的特性。在目标1中，我们将使用可调弹性水凝胶来确定底物如何 僵硬调节中性粒细胞抗念珠菌效应反应，进而调节念珠菌PAMPβ- 葡聚糖，影响牵引力的产生。 此外，组织是高度受限的三维空间，因此与2D不同 通常用于体外研究的组织培养底物。同样，引用了Reichner和他的同事之前的工作， 禁闭的物理性质对中性粒细胞的运动和牵引力有很大影响。例如, 尽管已知中性粒细胞需要整合素在2D表面上黏附和迁移，但整合素成为 对于进入高度受限的3D间隙后的迁移来说是必不可少的。要研究……的敏感性 为了将中性粒细胞限制在物理范围内，我们开发了一种双水凝胶压缩装置，并表明 限制是中性粒细胞转向整合素非依赖性迁移和产生的物理触发因素 牵引力。因此，尽管先前的工作已经显示了对中性粒细胞整合素结合的机械敏感效应 在运动性方面，对于限制是否代表组织内的机械属性，我们一无所知 调节抗念珠菌效应器功能(目标2)。

Project Summary / Abstract Neutrophils are the first responders to sites of infection and are fundamental to the anti-fungal response. This includes conditions of oropharyngeal candidiasis, where blood neutrophils extravasate into infected tissues and locomote to contact invading Candida. During this process, neutrophils are highly sensitive not only to the biochemical properties of the tissue microenvironment but the physical properties as well. The oral cavity is highly variant in the mechanical properties of its composite tissues, from the relatively soft and elastic tongue and soft palate to structures of relatively intermediate stiffness such as the esophagus to rigid structures such as the hard palate. The Reichner lab and others have shown that human neutrophils are highly sensitive to the mechanical features of their microenvironment including substrate stiffness. They have reported that human neutrophils move more slowly and with greater directionality when placed on stiff matrices as compared to soft matrices. Quantification and mapping of tractional forces show greater force generation on stiffer matrices with most of the force produced at the rear of the cell. A significant gap regarding these findings is that we have no information regarding how a pathogen, or its component PAMPs, affect the mechanosensitive response of neutrophils with regard to overall motility and the production of traction on matrices of varying stiffnesses. This is significant because it is the pathogen that induces neutrophil entry into an infected tissue to execute antimicrobial activity, and Candida infection is highly relevant in the oral cavity as a cause of OPC. The overarching hypothesis to be tested in this proposal is that the presence of candida affects the mechanosensitive properties of the human neutrophil. In Aim 1, we will use tunable elasticity hydrogels to determine how substrate stiffness regulates the neutrophil anti-Candida effector responses and, in turn, how the candida PAMP beta- glucan, affects the generation of traction force. Additionally, tissues are highly confined 3-dimensional spaces and, as such, are different than the 2D tissue culture substrates typically used for in vitro studies. Again, citing prior work from Reichner and colleagues, the physical property of confinement has a significant affect on neutrophil motility and traction. For example, whereas neutrophils are known to require integrins for adhesion and migration on 2D surfaces, integrins become dispensable for migration following entry into the highly confined 3D interstitial space. To study the sensitivity of neutrophils to physical confinement, we developed a double hydrogel compression device and have shown that confinement is the physical trigger for neutrophils to switch to integrin-independent migration and generation of traction. Therefore, although prior work has shown mechanosensitive effects on neutrophil integrin engagement in motility, nothing is known about whether confinement represents a mechanical property within tissues that regulates the anti-Candida effector functions (Aim 2).