Macrophages play a key role in orchestrating the host immune response toward invading organisms or non-self molecules in the oral mucosa. Three-dimensional (3D) oral mucosal equivalents (OME) containing oral fibroblasts and keratinocytes are used extensively to mimic the human oral mucosa where they have been employed to examine innate immune responses to both bacterial and fungal pathogens as well as to biomaterials. Although the presence of immune cells is critical in generating an immune response, very few studies have incorporated leukocytes into OME, and to date, none have contained primary human macrophages. In this study, we report the generation of an immunocompetent OME to investigate immune responses toward bacterial challenge. Primary human monocyte-derived macrophages (MDM) were as responsive to bacterial lipopolysaccharide (LPS) challenge when cultured within a 3D hydrogel in terms of proinflammatory cytokine (IL-6, CXCL8, and TNF-α) gene expression and protein secretion compared with culture as two-dimensional monolayers. MDM were incorporated into a type 1 collagen hydrogel along with oral fibroblasts and the apical surface seeded with oral keratinocytes to generate an MDM-containing OME. Full-thickness MDM-OME displayed a stratified squamous epithelium and a fibroblast-populated connective tissue containing CD68-positive MDM that could be readily isolated to a single-cell population for further analysis by collagenase treatment followed by flow cytometry. When stimulated with LPS, MDM-OME responded with increased proinflammatory cytokine secretion, most notably for TNF-α that increased 12-fold when compared with OME alone. Moreover, this proinflammatory response was inhibited by pretreatment with dexamethasone, showing that MDM-OME are also amenable to drug treatment. Dual-labeled immunofluorescence confocal microscopy revealed that MDM were the sole source of TNF-α production within MDM-OME. These data show functional activity of MDM-OME and illustrate their usefulness for investigations aimed at monitoring the immune response of the oral mucosa to pathogens, biomaterials, and for tissue toxicity and anti-inflammatory drug delivery studies.

中文翻译：

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含有巨噬细胞的免疫反应性组织工程口腔粘膜等效物

巨噬细胞在协调宿主对口腔黏膜中入侵生物或非自身分子的免疫反应方面发挥着关键作用。包含口腔成纤维细胞和角质形成细胞的三维 (3D) 口腔黏膜等效物 (OME) 被广泛用于模拟人类口腔黏膜，它们已被用于检查对细菌和真菌病原体以及生物材料的先天免疫反应。尽管免疫细胞的存在对于产生免疫反应至关重要，但很少有研究将白细胞纳入 OME，迄今为止，还没有研究包含原代人类巨噬细胞。在这项研究中，我们报告了具有免疫能力的 OME 的产生，以研究对细菌挑战的免疫反应。与培养相比，原代人单核细胞衍生的巨噬细胞 (MDM) 在 3D 水凝胶中培养时，在促炎细胞因子（IL-6、CXCL8 和 TNF-α）基因表达和蛋白质分泌方面对细菌脂多糖 (LPS) 攻击具有响应性作为二维单层。MDM 与口腔成纤维细胞和根尖表面一起被掺入 1 型胶原水凝胶，口腔角质形成细胞接种以产生含有 MDM 的 OME。全层 MDM-OME 显示分层的鳞状上皮细胞和含有 CD68 阳性 MDM 的成纤维细胞填充结缔组织，可以很容易地将其分离到单细胞群中，以便通过胶原酶处理和流式细胞术进行进一步分析。当用 LPS 刺激时，MDM-OME 会增加促炎细胞因子的分泌，最显着的是 TNF-α 与单独的 OME 相比增加了 12 倍。此外，地塞米松预处理抑制了这种促炎反应，表明 MDM-OME 也适合药物治疗。双标记免疫荧光共聚焦显微镜显示 MDM 是 MDM-OME 中 TNF-α 产生的唯一来源。这些数据显示了 MDM-OME 的功能活性，并说明了它们对旨在监测口腔粘膜对病原体、生物材料的免疫反应的研究以及组织毒性和抗炎药物递送研究的有用性。双标记免疫荧光共聚焦显微镜显示 MDM 是 MDM-OME 中 TNF-α 产生的唯一来源。这些数据显示了 MDM-OME 的功能活性，并说明了它们对旨在监测口腔粘膜对病原体、生物材料的免疫反应的研究以及组织毒性和抗炎药物递送研究的有用性。双标记免疫荧光共聚焦显微镜显示 MDM 是 MDM-OME 中 TNF-α 产生的唯一来源。这些数据显示了 MDM-OME 的功能活性，并说明了它们对旨在监测口腔粘膜对病原体、生物材料的免疫反应的研究以及组织毒性和抗炎药物递送研究的有用性。