Abstract
BackgroundCholesteatoma 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.MethodsWe 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.ResultsUnder 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.ConclusionWe 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.184woRBw6stDeYDHN9byyNVideo
Highlights
Cholesteatoma disease is an expanding lesion in the middle ear
While investigating the expression of targets related to cholesteatoma disease under standard culture conditions (Fig. 1), we detected that the expression of inflammatory mediators IL-1β and IL-8 was massively enhanced in stem cells compared to fibroblasts, while IL-1α and TNF-α exhibited this effect in a less pronounced manner
Taken our experimental results together, the high recurrence upon infection of cholesteatoma [34] might be supported by an enhanced proliferation of Fibroblast from cholesteatoma tissue (ME-CF) and the increased epidermal differentiation of Stem cell isolated from cholesteatoma tissue (ME-CSC) upon paracrine stimulation of ME-CFs both caused upon Toll like receptor 4 (TLR4) stimulation
Summary
Cholesteatoma disease is an expanding lesion in the middle ear. Hearing loss and facial paralysis along‐ side with other intracranial complications are found. The cause of this hyperproliferation is not fully understood, but it is known that TLR4 agonistic pathogen-associated molecular patterns (PAMPs) [15] as well as damage associated molecular patterns (DAMPs) inside the cholesteatoma tissue will activate the expression of different cytokines and growth factors provoking this proliferation [16]. In accordance to this Jovanovic et al found that the most significantly differentially upregulated genes were linked to inflammation, epidermis development and keratinization [17]. In detail the expression of the cytokines, e.g. IL-1α [24] IL-1β and IL-6 [18], TNF-α [19], GM-CSF [20]
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