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。

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.