Paredes-Juarez Genaro A, de Haan Bart J, Faas Marijke M, de Vos Paul
Department of Pathology and Medical Biology, Section of Immunoendocrinology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, EA11, 9700 RB Groningen, The Netherlands.
Materials (Basel). 2014 Mar 12;7(3):2087-2103. doi: 10.3390/ma7032087.
Alginates are widely used in tissue engineering technologies, e.g., in cell encapsulation, in drug delivery and various immobilization procedures. The success rates of these studies are highly variable due to different degrees of tissue response. A cause for this variation in success is, among other factors, its content of inflammatory components. There is an urgent need for a technology to test the inflammatory capacity of alginates. Recently, it has been shown that pathogen-associated molecular patterns (PAMPs) in alginate are potent immunostimulatories. In this article, we present the design and evaluation of a technology platform to assess (i) the immunostimulatory capacity of alginate or its contaminants, (ii) where in the purification process PAMPs are removed, and (iii) which Toll-like receptors (TLRs) and ligands are involved. A THP1 cell-line expressing pattern recognition receptors (PRRs) and the co-signaling molecules CD14 and MD2 was used to assess immune activation of alginates during the different steps of purification of alginate. To determine if this activation was mediated by TLRs, a THP1-defMyD88 cell-line was applied. This cell-line possesses a non-functional MyD88 coupling protein, necessary for activating NF-κB via TLRs. To identify the specific TLRs being activated by the PAMPs, we use different human embryonic kidney (HEK) cell-line that expresses only one specific TLR. Finally, specific enzyme-linked immunosorbent assays (ELISAs) were applied to identify the specific PAMP. By applying this three-step procedure, we can screen alginate in a manner, which is both labor and cost efficient. The efficacy of the platform was evaluated with an alginate that did not pass our quality control. We demonstrate that this alginate was immunostimulatory, even after purification due to reintroduction of the TLR5 activating flagellin. In addition, we tested two commercially available purified alginates. Our experiments show that these commercial alginates contained peptidoglycan, lipoteichoic acid, flagellin, and even lipopolysaccharides (LPS). The platform presented here can be used to evaluate the efficacy of purification procedures in removing PAMPs from alginates in a cost-efficient manner.
藻酸盐广泛应用于组织工程技术中，例如细胞封装、药物递送及各种固定化程序。由于组织反应程度不同，这些研究的成功率差异很大。造成这种成功率差异的原因之一，除其他因素外，是其炎症成分的含量。迫切需要一种技术来测试藻酸盐的炎症能力。最近研究表明，藻酸盐中的病原体相关分子模式（PAMPs）是强效免疫刺激剂。在本文中，我们介绍了一个技术平台的设计与评估，该平台用于评估：（i）藻酸盐或其污染物的免疫刺激能力；（ii）在纯化过程中PAMPs是在何处被去除的；（iii）涉及哪些Toll样受体（TLRs）和配体。利用表达模式识别受体（PRRs）以及共信号分子CD14和MD2的THP1细胞系，来评估藻酸盐在不同纯化步骤中的免疫激活情况。为确定这种激活是否由TLRs介导，应用了THP1-defMyD88细胞系。该细胞系具有无功能的MyD88偶联蛋白，而通过TLRs激活NF-κB需要该蛋白。为识别被PAMPs激活的特定TLRs，我们使用了仅表达一种特定TLR的不同人胚肾（HEK）细胞系。最后，应用特异性酶联免疫吸附测定（ELISA）来识别特定的PAMP。通过应用这三步程序，我们能够以一种既节省人力又经济高效的方式筛选藻酸盐。使用一种未通过我们质量控制的藻酸盐对该平台的功效进行了评估。我们证明，即使经过纯化，由于重新引入了激活TLR5的鞭毛蛋白，这种藻酸盐仍具有免疫刺激作用。此外，我们测试了两种市售的纯化藻酸盐。我们的实验表明，这些市售藻酸盐含有肽聚糖、脂磷壁酸、鞭毛蛋白，甚至脂多糖（LPS）。本文介绍的平台可用于以经济高效的方式评估从藻酸盐中去除PAMPs的纯化程序的功效。
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