Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, GRJ-5-504, Boston, MA 02114, USA.
Integr Biol (Camb). 2012 Feb;4(2):220-7. doi: 10.1039/c2ib00089j. Epub 2011 Dec 23.
The number of life-threatening fungal infections has risen in immunocompromised patients, and identification of the rules that govern an appropriate immune response is essential to develop better diagnostics and targeted therapeutics. The outer cell wall component on pathogenic fungi consists of β-1,3-glucan, and Dectin-1, a pattern recognition receptor present on the cell surface of innate immune cells, binds specifically to this carbohydrate. A barrier in understanding the exact immunological response to pathogen-derived carbohydrate epitopes is the presence of multiple types of carbohydrate moieties on fungal cell walls. To dissect the immunological mechanisms used to recognize pathogens, a system of "fungal like particles" was developed that consisted of polystyrene beads, which mimicked the three dimensional shape of the fungus, coated covalently with purified β-1,3-glucan derived from Saccharomyces cerevisiae. The morphology of the β-1,3-glucan layer was examined by immunofluorescence, flow cytometery, and immuno-transmission electron microscopy. The covalent linkages of the β-1,3-glucan to the polystyrene surface were stable after subjecting the beads to detergents. By pre-treating β-1,3-glucan beads with laminarinase, a specific β-1,3-gluconase, the reactivity of the anti-β-1,3-glucan antibody was abrogated in comparison to treatment with proteinase K indicating that the coating of these beads was predominantly β-1,3-glucan. TNF-α was also measured by stimulating bone-marrow derived macrophages with the β-1,3-glucan beads, and showed a dose dependent response compared to soluble β-glucan, insoluble β-1,3-glucan, uncoated beads, and soluble β-1,3-glucan mixed with uncoated beads. Finally, β-1,3-glucan beads were incubated with GFP-Dectin-1 expressing macrophages and imaged using confocal microscopy. β-1,3-beads were taken up within minutes and retained Dectin-1 recruitment to the phagosome as compared to uncoated beads. These data describe a unique fungal-like particle system that will permit immunologists to probe the critical steps in early recognition of pathogen-derived fungal carbohydrate antigens by innate immune cells.
免疫功能低下患者中危及生命的真菌感染数量有所增加，因此确定控制适当免疫反应的规则对于开发更好的诊断方法和靶向治疗方法至关重要。致病性真菌的外壳细胞壁成分由β-1,3-葡聚糖和 Dectin-1 组成，Dectin-1 是先天免疫细胞表面存在的一种模式识别受体，特异性结合这种碳水化合物。理解对病原体衍生碳水化合物表位的确切免疫反应的一个障碍是真菌细胞壁上存在多种类型的碳水化合物部分。为了剖析用于识别病原体的免疫学机制，开发了一种“真菌样颗粒”系统，该系统由聚苯乙烯珠组成，这些珠子模拟真菌的三维形状，通过共价键与来自酿酒酵母的纯化β-1,3-葡聚糖结合。通过免疫荧光、流式细胞术和免疫透射电子显微镜检查β-1,3-葡聚糖层的形态。将珠子用去污剂处理后，β-1,3-葡聚糖与聚苯乙烯表面的共价键仍然稳定。通过用特定的β-1,3-葡聚糖酶（几丁质酶）预处理β-1,3-葡聚糖珠，与用蛋白酶 K 处理相比，抗β-1,3-葡聚糖抗体的反应性被阻断，表明这些珠子的涂层主要是β-1,3-葡聚糖。通过用β-1,3-葡聚糖珠刺激骨髓来源的巨噬细胞，还测量了 TNF-α，与可溶性β-葡聚糖、不溶性β-1,3-葡聚糖、未涂层珠和与未涂层珠混合的可溶性β-1,3-葡聚糖相比，显示出剂量依赖性反应。最后，将β-1,3-葡聚糖珠与表达 GFP-Dectin-1 的巨噬细胞孵育，并使用共聚焦显微镜进行成像。与未涂层珠相比，β-1,3-葡聚糖珠在几分钟内被摄取，并保留了 Dectin-1 向吞噬体的募集。这些数据描述了一种独特的真菌样颗粒系统，该系统将允许免疫学家探测先天免疫细胞对病原体衍生的真菌碳水化合物抗原的早期识别的关键步骤。
