IFN-γ 对单层培养角质形成细胞和 3D 重建人表皮模型中犬尿氨酸/色氨酸比值的影响。

Effect of IFN-γ on the kynurenine/tryptophan ratio in monolayer-cultured keratinocytes and a 3D reconstructed human epidermis model.

机构信息

Biofilms - Research Center for Biointerfaces, Malmö University, Malmö, Sweden; Department of Biomedical Science, Faculty of Health and Society, Malmö University, Malmö, Sweden.

Department of Oral Biology, Faculty of Odontology, Malmö University, Malmö, Sweden.

出版信息

J Dermatol Sci. 2020 Sep;99(3):177-184. doi: 10.1016/j.jdermsci.2020.07.005. Epub 2020 Jul 17.

DOI:10.1016/j.jdermsci.2020.07.005

PMID:32782183

Abstract

BACKGROUND

Interferon-gamma (IFN-γ) represents a potent inducer for keratinocyte inflammatory and immune activation in vitro. Since tryptophan (trp) conversion to kynurenine (kyn) is involved in inflammation, the topical kyn/trp ratio may serve as a biomarker of skin inflammation. However, the trp metabolism in keratinocytes exposed to IFN-γ is not yet fully understood.

OBJECTIVE

The aim of this study was to establish a human epidermis model in order to quantify cytokine and kyn/trp secretion from IFN-γ stimulated cells and tissues. Moreover, to compare the cell response of 2D-cultured keratinocytes and the 3D epidermis model.

METHODS

Polycarbonate filters were used on which primary keratinocytes could attach and stratify in order to form the typical layers of reconstructed human epidermis (RHE). After IFN-γ treatment, secretion of kyn/trp was measured by high performance liquid chromatography. Gene and protein expression of indoleamine 2,3-dioxygenase 1 (IDO) was analyzed with real-time PCR and immunohistochemistry. The secretion of cytokines was quantified with ELISA.

RESULTS

Trp catabolism to kyn was significantly increased (P < 0.01) in the 2D culture in response to IFN-γ treatment. Before kyn secretion, IDO was strongly upregulated (P < 0.001). IFN-γ treatment also increased the secretion of IL-6 and IL-8 from the keratinocytes. In the RHE, IDO was upregulated by IFN-γ, and kyn secretion could be detected. Interestingly, IDO expression was only present in the basal cells of the RHE.

CONCLUSION

Our results suggest that IFN-γ acts as an inducer of trp degradation preferentially in undifferentiated keratinocytes, indicated by the IDO expression in the basal layer of the RHE.

摘要

背景

干扰素-γ（IFN-γ）在体外是角质细胞炎症和免疫激活的有效诱导剂。由于色氨酸（trp）向犬尿氨酸（kyn）的转化与炎症有关，因此局部 kyn/trp 比值可作为皮肤炎症的生物标志物。然而，暴露于 IFN-γ的角质细胞中的 trp 代谢尚未完全了解。

目的

本研究旨在建立人表皮模型，以定量测定 IFN-γ刺激细胞和组织中细胞因子和 kyn/trp 的分泌。此外，比较 2D 培养的角质细胞和 3D 表皮模型的细胞反应。

方法

使用聚碳酸酯过滤器，其上可附着和分层原代角质细胞，以形成重建的人表皮（RHE）的典型层。IFN-γ 处理后，通过高效液相色谱法测量 kyn/trp 的分泌。用实时 PCR 和免疫组织化学分析吲哚胺 2,3-双加氧酶 1（IDO）的基因和蛋白表达。用 ELISA 定量细胞因子的分泌。

结果

在 2D 培养中，IFN-γ 处理后 trp 向 kyn 的代谢明显增加（P <0.01）。在 kyn 分泌之前，IDO 强烈上调（P <0.001）。IFN-γ 处理还增加了角质细胞中 IL-6 和 IL-8 的分泌。在 RHE 中，IFN-γ 上调了 IDO，并且可以检测到 kyn 的分泌。有趣的是，IDO 表达仅存在于 RHE 的基底细胞中。