Stem Cell-Induced Inflammation in Cholesteatoma is Inhibited by the TLR4 Antagonist LPS-RS.

Matthias Schürmann,Felix Oppel,Johannes F W Greiner,Holger Sudhoff,Barbara Kaltschmidt,Christian Kaltschmidt,Verena Volland-Thurn

doi:10.3390/cells9010199

Abstract

Cholesteatoma is a severe non-cancerous lesion of the middle ear characterized by massive inflammation, tissue destruction, and an abnormal growth of keratinized squamous epithelium. We recently demonstrated the presence of pathogenic stem cells within cholesteatoma tissue, unfortunately their potential roles in regulating disease-specific chronic inflammation remain poorly understood. In the presented study, we utilized our established human in vitro cholesteatoma stem cell model for treatments with lipopolysaccharides (LPS), tumor necrosis factor α (TNFα), and the TLR4-antagonist LPS from R. sphaeroides (LPS-RS) followed by qPCR, western blot, and immunocytochemistry. Middle ear cholesteatoma stem cells (ME-CSCs) showed a significantly increased expression of TLR4 accompanied by a significantly enhanced LPS-dependent pro-inflammatory gene expression pattern of TNFα, IL-1α, IL-1ß, IL-6, and IL-8 compared to non-pathogenic control cells. LPS-dependent pro-inflammatory gene expression in ME-CSCs was driven by an enhanced activity of NF-κB p65 leading to a TNFα-mediated feed-forward-loop of pro-inflammatory NF-κB target gene expression. Functional inactivation of TLR4 via the TLR4-antagonist LPS-RS blocked chronic inflammation in ME-CSCs, resulting in a nearly complete loss of IL-1ß, IL-6, and TNFα expression. In summary, we determined that ME-CSCs mediate the inflammatory environment of cholesteatoma via TLR4-mediated NF-κB-signaling, suggesting a distinct role of ME-CSCs as drivers of cholesteatoma progression and TLR4 on ME-CSCs as a therapeutic target.

Highlights

Cholesteatoma is a potentially life-threatening inflammatory lesion of abnormally growing keratinizing squamous epithelium in the middle ear (Figure 1A) resulting in clinical symptoms like hearing loss, ear discharge, and ear pain [1]
For isolation of stem cells, cholesteatoma tissue was obtained from the human posterior epitympanon, while auditory canal skin originated from the tympanomeatal flap (Figure 1A)
With tumor necrosis factor α (TNFα) being a major target gene of NF-κB and an important cytokine involved in cholesteatoma progression [8], we investigated a potential pro-inflammatory feed-forward-loop of NF-κB target gene expression in Middle ear cholesteatoma stem cells (ME-CSCs) mediated by TNFα

Summary

Introduction

Cholesteatoma is a potentially life-threatening inflammatory lesion of abnormally growing keratinizing squamous epithelium in the middle ear (Figure 1A) resulting in clinical symptoms like hearing loss, ear discharge, and ear pain [1]. The non-cancerous but intensely proliferating squamous epithelium can locally invade and destroy nearby structures like the temporal bone and the auditory ossicles [2]. The presence of inflammatory mediators in cholesteatoma tissue was studied and exhibits a higher presence of the cytokines TNFα, IL-6, IL-8, IL-1α, and IL-1ß in comparison to non-inflamed external auditory canal skin tissue [8,9]. Bone resorption and destruction was shown to be positively correlated to inflammation, the presence of bacterial lipopolysaccharides (LPS), and expression of inflammatory mediators like TNFα, IL-1α, and IL-6 in cholesteatoma [10,11,12]. An enhanced expression of the Toll-like receptor 4 (TLR4) was observed in cholesteatoma tissue compared to external auditory canal skin tissue [13]. The cellular mediators potentially linking NF-κB-signaling and the pro-inflammatory environment of cholesteatoma still remain unknown

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