ABSTRACT

Purpose

Inflammation is associated with, and may be causal of, a variety of ophthalmic pathologies. These pathologies are currently difficult to model in vitro because they involve complex interactions between the innate immune system, stromal cells, and other cells that normally maintain ocular tissue homeostasis. Using transscleral drainage channel fibrosis after glaucoma surgery as an example of inflammation-associated ocular fibrosis, we have assessed a simple but novel 3D cell culture system designed to reveal the immunomodulatory impacts of ocular connective tissue cells on monocytes, a major cellular component of the circulating immune system.

Methods

Primary human Tenon’s capsule fibroblasts derived from five unrelated patients were activated into myofibroblasts in 3D collagen matrices under isometric tension, with and without exposure to an inflammatory cytokine-enhanced milieu, and co-cultured with an immortalized human monocyte cell line (THP-1 cells). Quantitative PCR analyses were performed on 8 candidate genes to assess the impacts of inflammatory cytokines on the myofibroblasts and the monocytes in mono-cultures and compared to cells in co-culture to clearly distinguish any co-culture-induced impacts on gene expression.

Results

Our data indicate that both Tenon’s capsule myofibroblasts in 3D mono-culture and THP-1 monocytes in suspension mono-culture were responsive to inflammatory cytokine stimuli. Co-culture with Tenon’s capsule myofibroblasts significantly modulated the gene expression responses of THP-1 monocytes to inflammatory cytokine stimulation, indicative of an immunomodulatory “feedback” system between these cell types.

Conclusion

Our findings provide proof of principle for the use of simple 3D co-culture systems as a means to enhance our understanding of ocular stromal cell interactions with cells of the innate immune system and to provide more informative in vitro models of inflammation-associated ophthalmic pathologies.

中文翻译：

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了解炎症相关眼科病理：单核细胞-肌成纤维细胞免疫调节的新型 3D 共培养模型

摘要

目的

炎症与多种眼科病症有关，并且可能是多种眼科病症的原因。这些病理目前难以在体外建模，因为它们涉及先天免疫系统、基质细胞和通常维持眼组织稳态的其他细胞之间复杂的相互作用。使用青光眼手术后的经巩膜引流通道纤维化作为炎症相关眼纤维化的一个例子，我们评估了一种简单但新颖的 3D 细胞培养系统，旨在揭示眼结缔组织细胞对单核细胞的免疫调节影响，单核细胞是循环系统的主要细胞成分免疫系统。

方法

来自五名无关患者的原代人 Tenon 囊成纤维细胞在等长张力下被激活为 3D 胶原基质中的肌成纤维细胞，有和没有暴露于炎性细胞因子增强的环境，并与永生化人单核细胞系（THP-1 细胞）共培养). 对 8 个候选基因进行定量 PCR 分析，以评估炎性细胞因子对单一培养物中肌成纤维细胞和单核细胞的影响，并与共培养物中的细胞进行比较，以清楚地区分任何共培养物诱导的对基因表达的影响。

结果

我们的数据表明，3D 单一培养物中的 Tenon 囊肌成纤维细胞和悬浮单一培养物中的 THP-1 单核细胞均对炎性细胞因子刺激有反应。与 Tenon 囊肌成纤维细胞共培养可显着调节 THP-1 单核细胞对炎性细胞因子刺激的基因表达反应，表明这些细胞类型之间存在免疫调节“反馈”系统。

结论

我们的研究结果为使用简单的 3D 共培养系统作为增强我们对眼基质细胞与先天免疫系统细胞相互作用的理解并提供更多信息的炎症相关眼科病理体外模型的手段提供了原理证明。