Department of Otolaryngology, Head and Neck Surgery, Medical School OWL Campus Klinikum Bielefeld, Bielefeld University, Teutoburger Str. 50, 33604, Bielefeld, Germany.
Department of Cell Biology, Bielefeld University , 33619, Bielefeld, Germany.
Cell Commun Signal. 2021 Feb 24;19(1):25. doi: 10.1186/s12964-020-00690-y.
Cholesteatoma disease is an expanding lesion in the middle ear. Hearing loss and facial paralysis alongside with other intracranial complications are found. No pharmaceutical treatment is available today and recurrence after surgical extraction occurs. We investigated possible TLR4-based mechanisms promoting recurrence and explore possible treatments strategies.
We isolated fibroblasts and epidermal stem cells from cholesteatoma tissue and healthy auditory canal skin. Subsequently, their expression under standard culture conditions and after stimulation with LPS was investigated by RT-qPCR. Cell metabolism and proliferation were analysed upon LPS treatment, with and without TLR4 antagonist. An indirect co-culture of fibroblasts and epidermal stem cells isolated from cholesteatoma tissue was utilized to monitor epidermal differentiation upon LPS treatment by RT-qPCR and immunocytochemistry.
Under standard culture conditions, we detected a tissue-independent higher expression of IL-1β and IL-8 in stem cells, an upregulation of KGF and IGF-2 in both cell types derived from cholesteatoma and higher expression of TLR4 in stem cells derived from cholesteatoma tissue. Upon LPS challenge, we could detect a significantly higher expression of IL-1α, IL-1β, IL-6 and IL-8 in stem cells and of TNF-a, GM-CSF and CXCL-5 in stem cells and fibroblasts derived from cholesteatoma. The expression of the growth factors KGF, EGF, EREG, IGF-2 and HGF was significantly higher in fibroblasts, particularly when derived from cholesteatoma. Upon treatment with LPS the metabolism was elevated in stem cells and fibroblasts, proliferation was only enhanced in fibroblasts derived from cholesteatoma. This could be reversed by the treatment with a TLR4 antagonist. The cholesteatoma fibroblasts could be triggered by LPS to promote the epidermal differentiation of the stem cells, while no LPS treatment or LPS treatment without the presence of fibroblasts did not result in such a differentiation.
We propose that cholesteatoma recurrence is based on TLR4 signalling imprinted in the cholesteatoma cells. It induces excessive inflammation of stem cells and fibroblasts, proliferation of perimatrix fibroblasts and the generation of epidermal cells from stem cells thru paracrine signalling by fibroblasts. Treatment of the operation site with a TLR4 antagonist might reduce the chance of cholesteatoma recurrence. Video Abstract.
胆脂瘤病是中耳的一种扩张性病变。可发现听力损失、面瘫以及其他颅内并发症。目前尚无药物治疗方法，且手术后复发。我们研究了可能促进复发的 TLR4 相关机制，并探索了可能的治疗策略。
我们从胆脂瘤组织和健康耳道皮肤中分离出成纤维细胞和表皮干细胞。然后，通过 RT-qPCR 研究它们在标准培养条件下和 LPS 刺激后的表达情况。在 LPS 处理时，分析了成纤维细胞和表皮干细胞的代谢和增殖情况，同时使用 TLR4 拮抗剂。利用间接共培养从胆脂瘤组织中分离出的成纤维细胞和表皮干细胞，通过 RT-qPCR 和免疫细胞化学监测 LPS 处理后表皮的分化情况。
在标准培养条件下，我们在干细胞中检测到一种组织独立性的更高的 IL-1β 和 IL-8 表达，在胆脂瘤来源的两种细胞类型中均检测到 KGF 和 IGF-2 的上调，以及在干细胞中检测到更高的 TLR4 表达。在 LPS 刺激下，我们可以在干细胞中检测到更高的 IL-1α、IL-1β、IL-6 和 IL-8 表达，以及在胆脂瘤来源的成纤维细胞和干细胞中检测到更高的 TNF-a、GM-CSF 和 CXCL-5 表达。在成纤维细胞中，生长因子 KGF、EGF、EREG、IGF-2 和 HGF 的表达显著升高，尤其是来源于胆脂瘤的成纤维细胞。用 LPS 处理后，干细胞和成纤维细胞的代谢增加，只有来源于胆脂瘤的成纤维细胞的增殖增强。这可以通过 TLR4 拮抗剂的治疗来逆转。LPS 可以刺激胆脂瘤成纤维细胞促进干细胞的表皮分化，而没有 LPS 处理或没有成纤维细胞存在的 LPS 处理不会导致这种分化。
我们提出，胆脂瘤复发是基于 TLR4 信号在胆脂瘤细胞中的印迹。它诱导干细胞和成纤维细胞过度炎症，基质周围成纤维细胞增殖，并通过成纤维细胞的旁分泌信号从干细胞产生表皮细胞。在手术部位用 TLR4 拮抗剂治疗可能会降低胆脂瘤复发的机会。
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