The primary regulators of the innate immune response to implanted biomaterials are macrophages, which change phenotype over time to regulate multiple phases of the tissue repair process. Immunomodulatory biomaterials that target macrophage phenotype are a promising approach for promoting tissue repair. Although expression of multiple markers has been widely used to characterize macrophage phenotype, the complexity of the macrophage response to biomaterials makes interpretation difficult. The aim of this study was to put forth an objective method to characterize macrophage phenotype with respect to specific biological processes or standard phenotypes of interest. We investigated the utility of gene set analyses to analyze macrophages as they respond to model biomaterials in comparison to "reference" M1 and M2a macrophage phenotypes. Primary human macrophages were seeded onto crosslinked collagen scaffolds with or without adsorption of the proinflammatory cytokine interferon-gamma (IFNg). Gene expression of a custom-curated panel of 48 genes, representing the M1 and M2a gene signatures as well as other genes important for angiogenesis and tissue repair, was quantified using NanoString on days 3, 5, and 8 of culture. A dataset of phenotype controls, consisting of M0, M1, and M2a macrophages, was used as a source of comparison and to validate the methods of characterization. Gene expression of M1 and M2a markers showed mixed upregulation and downregulation by macrophages seeded on collagen and IFNg-adsorbed collagen scaffolds, highlighting the need for more holistic analyses. Euclidean distance measurements to the reference phenotypes were unable to resolve differences between groups. In contrast, rotation gene set testing with and without gene weighting based on the genes' ability to differentiate between M1, M2a, and M0 controls, followed by gene set variation analysis, showed that collagen scaffolds inhibited the classic M1 phenotype without promoting a classic M2a phenotype, and that IFNg-adsorbed collagen scaffolds promoted the M1 phenotype and inhibited the M2a phenotype. In summary, this work demonstrates a powerful, objective methodology for characterizing the macrophage response to biomaterials in comparison to reference macrophage phenotypes. With the addition of more macrophage phenotypes with defined gene expression signatures, this method could prove beneficial for characterizing complex hybrid phenotypes.

中文翻译：

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通过基因组分析表征巨噬细胞对免疫调节生物材料的反应。

对植入的生物材料的先天免疫反应的主要调节剂是巨噬细胞，其随时间改变表型以调节组织修复过程的多个阶段。靶向巨噬细胞表型的免疫调节生物材料是促进组织修复的有前途的方法。尽管多种标记物的表达已被广泛用于表征巨噬细胞表型，但是巨噬细胞对生物材料反应的复杂性使解释变得困难。这项研究的目的是提出一种客观的方法来表征有关特定的生物学过程或感兴趣的标准表型的巨噬细胞表型。我们调查了基因组分析的实用程序来分析巨噬细胞，因为它们与“参考” M1和M2a巨噬细胞表型相比对模型生物材料有反应。将原代人巨噬细胞接种在具有或不具有促炎细胞因子干扰素-γ（IFNg）吸附的交联胶原蛋白支架上。在培养的第3天，第5天和第8天使用NanoString量化了48个定制定制基因组的基因表达，这些基因代表M1和M2a基因签名以及对血管生成和组织修复重要的其他基因。由M0，M1和M2a巨噬细胞组成的表型对照数据集被用作比较的来源并验证了表征方法。M1和M2a标记的基因表达表明，接种在胶原蛋白和IFNg吸附的胶原蛋白支架上的巨噬细胞混合了上调和下调，这突出表明需要进行更全面的分析。到参考表型的欧式距离测量无法解决组之间的差异。相比之下，根据基因区分M1，M2a和M0对照的能力，在有无基因加权的情况下进行轮换基因集测试，然后进行基因集变异分析，结果表明胶原蛋白支架抑制了经典M1表型，而没有促进经典M2a表型，而IFNg吸附的胶原蛋白支架可促进M1表型并抑制M2a表型。总而言之，这项工作展示了一种强大的，客观的方法，与参考巨噬细胞表型相比，它可以表征巨噬细胞对生物材料的反应。随着更多具有定义的基因表达特征的巨噬细胞表型的出现，