Electrostatic forces and M cell uptake at mucosal surfaces

描述（由申请人提供）：粘膜上皮是抵抗侵入性微生物的主要屏障，因此了解粘膜免疫监视中涉及的物理化学力对于了解发病机制与免疫力可能至关重要。覆盖在淋巴组织上的粘膜M细胞是免疫监视的中心，其利用主动摄取粘膜病原体的机制。具有讽刺意味的是，从沙门氏菌到炭疽等许多侵入性病原体也擅长使用M细胞穿过上皮屏障。这是如何吸收 调解？最近，我们发现气道腔液体中的低离子强度显著增强了M细胞对颗粒的摄取。低离子强度应增加远程静电相互作用，并增加带负电荷的上皮细胞的带负电荷的颗粒的排斥;奇怪的是，我们发现了一个矛盾的增加在体内M细胞顶端膜的摄取。这一发现引起了我们的工作假设：与邻近的粘膜上皮细胞相比，M细胞是静电优化的相互作用与微粒悬浮液作为一个新兴的性质，其光滑的顶膜。表面电荷的差异在肠上皮细胞和M细胞之间的边界处建立静电场，其有助于将带负电荷的颗粒吸引到M细胞顶端膜。我们提出了两个目的来验证这一假设：（1）我们将使用细胞培养模型和改进的切向流室来模拟具有不同表面电荷特性的上皮细胞顶端表面的颗粒结合。我们将比较荧光微生物和PLGA微粒产生不同的表面zeta电位，以评估结合肠上皮细胞与"M细胞样"光滑顶端膜。(2)使用M细胞缺陷的小鼠模型，我们将研究颗粒表面电荷对肠内摄取的影响，以及静电力对M细胞依赖性与非依赖性颗粒向免疫组织摄取的影响。理解这些静电力在肠道中的作用对于确定M细胞粘膜免疫监视的机制将是重要的。此外，预计这些力量在沙门氏菌等侵入性微生物的发病机制中很重要。最后，我们的研究将有助于确定设计粘膜疫苗递送系统的要求，无论是为M细胞靶向摄取还是为非特异性粘附和M细胞非依赖性摄取而设计。 公共卫生相关性：粘膜表面的静电力和M细胞摄取叙述：粘膜上皮细胞是抵抗侵入性微生物的主要屏障，因此了解粘膜免疫监视中涉及的物理化学力可能对了解发病机制与免疫力至关重要。覆盖在淋巴组织上的粘膜M细胞是免疫监视的中心，其利用主动摄取粘膜病原体的机制。具有讽刺意味的是，从沙门氏菌到炭疽等许多侵入性病原体也擅长使用M细胞穿过上皮屏障。最近，我们发现的证据表明，颗粒之间的电荷相互作用，如细菌和肠上皮细胞可能是一个主要因素，在M细胞摄取。细菌和肠上皮细胞之间的电荷相互作用可能受到M细胞表面特征的调节，M细胞比邻近的肠上皮细胞更光滑。也就是说，M细胞和肠上皮之间的局部差异可能会产生电荷效应，将颗粒驱动到M细胞膜上。如果这是真的，这将是一个惊人的和令人惊讶的例子，说明细胞分化的生物过程如何改变局部的物理化学性质。这些效应似乎是为了与具有病毒和细菌特异性电荷特性的颗粒进行最佳相互作用而设计的，并且似乎可以补充用于检测病原体的先天免疫受体阵列。我们建议使用定义明确的细胞培养模型来模拟M细胞电荷特性，以及具有特定M细胞缺陷的动物模型来测试这些电荷效应。 理解这些力量在肠道中的作用对于确定M细胞粘膜免疫监视的机制将是重要的。此外，预计这些力量在沙门氏菌等侵入性微生物的发病机制中很重要。最后，我们的研究将有助于确定设计粘膜疫苗递送系统的要求，无论是为M细胞靶向摄取还是为非特异性粘附和M细胞非依赖性摄取而设计。

DESCRIPTION (provided by applicant): The mucosal epithelium is a primary barrier against invasive microbes, so understanding the physicochemical forces involved in mucosal immune surveillance may be critical to understanding pathogenesis versus immunity. Mucosal M cells overlying lymphoid tissues are central to immune surveillance, using mechanisms for active uptake of mucosal pathogens. Ironically, many invasive pathogens ranging from Salmonella to Anthrax are also adept at using M cells for entry across the epithelial barrier. How is this uptake mediated? Recently, we showed that low ionic strength in the liquid of the airway lumen significantly enhanced particle uptake by M cells. Low ionic strength should increase long-range electrostatic interactions and increase the repulsion of negatively charged particles from negatively charged epithelium; curiously, we found a paradoxically increased uptake at M cell apical membranes in vivo. This finding has given rise to our working hypothesis: in contrast to neighboring mucosal epithelial cells, the M cell is electrostatically optimized for interaction wit microparticles in suspension as an emergent property of its smooth apical membrane. The differences in surface charge establishes an electrostatic field at the boundary between enterocytes and M cells that helps draw negatively charged particles to the M cell apical membrane. We propose two aims to test this hypothesis: (1) We will use cell culture models and a modified tangential flow chamber to model the binding of particles at the apical surface of the epithelial cells with different surface charge characteristics. We will compare fluorescent microbes and PLGA microparticles produced with different surface zeta potentials to assess binding to enterocyte versus "M cell-like" smooth apical membranes. (2) Using mouse models of M cell deficiency, we will study the effect of particle surface charge on uptake in the intestine, and the effect of electrostatic forces on M cell- dependent versus -independent particle uptake to immune tissues. An understanding of the effect of these electrostatic forces in the intestine will be important in defining the mechanisms of M cell mucosal immune surveillance. In addition, these forces would be expected to be important in the pathogenesis of invasive microbes such as Salmonella. Finally, our studies will help define requirements for designing mucosal vaccine delivery systems, whether designed for M cell targeted uptake or for nonspecific adhesion and M cell-independent uptake. PUBLIC HEALTH RELEVANCE: Electrostatic forces and M cell uptake at mucosal surfaces Narrative: The mucosal epithelium is a primary barrier against invasive microbes, so understanding the physicochemical forces involved in mucosal immune surveillance may be critical to understanding pathogenesis versus immunity. Mucosal M cells overlying lymphoid tissues are central to immune surveillance, using mechanisms for active uptake of mucosal pathogens. Ironically, many invasive pathogens ranging from Salmonella to Anthrax are also adept at using M cells for entry across the epithelial barrier. Recently, we discovered evidence that charge interactions between particles such as bacteria and the intestinal epithelium may be a major factor in M cell uptake. The charge interactions between bacteria and intestinal epithelium may be modulated by the surface features of M cells, which are smoother than the neighboring intestinal epithelium. That is, the local differences between M cells and intestinal epithelium may set up charge effects that drive particles to the M cell membrane. If true, this would be a striking and surprising example of how biological processes of cellular differentiation can modify local physico-chemical properties. These effects appear to be designed for optimal interactions with particles with charge properties specific to viruses and bacteria, and would appear to complement the array of innate immune receptors for detecting pathogens. We propose to test these charge effects using well-defined cell culture models to mimic M cell charge properties, and animal models with specific M cell defects. An understanding of the effect of these forces in the intestine will be important in defining the mechanisms of M cell mucosal immune surveillance. In addition, these forces would be expected to be important in the pathogenesis of invasive microbes such as Salmonella. Finally, our studies will help define requirements for designing mucosal vaccine delivery systems, whether designed for M cell targeted uptake or for nonspecific adhesion and M cell-independent uptake.