NOD-Like Receptor Signaling in Cholesteatoma

Abstract

Background. Cholesteatoma is a destructive process of the middle ear resulting in erosion of the surrounding bony structures with consequent hearing loss, vestibular dysfunction, facial paralysis, or intracranial complications. The etiopathogenesis of cholesteatoma is controversial but is associated with recurrent ear infections. The role of intracellular innate immune receptors, the NOD-like receptors, and their associated signaling networks was investigated in cholesteatoma, since mutations in NOD-like receptor-related genes have been implicated in other chronic inflammatory disorders. Results. The expression of NOD2 mRNA and protein was significantly induced in cholesteatoma compared to the external auditory canal skin, mainly located in the epithelial layer of cholesteatoma. Microarray analysis showed significant upregulation for NOD2, not for NOD1, TLR2, or TLR4 in cholesteatoma. Moreover, regulation of genes in an interaction network of the NOD-adaptor molecule RIPK2 was detected. In addition to NOD2, NLRC4, and PYCARD, the downstream molecules IRAK1 and antiapoptotic regulator CFLAR showed significant upregulation, whereas SMAD3, a proapoptotic inducer, was significantly downregulated. Finally, altered regulation of inflammatory target genes of NOD signaling was detected. Conclusions. These results indicate that the interaction of innate immune signaling mediated by NLRs and their downstream target molecules is involved in the etiopathogenesis and growth of cholesteatoma.

Figure 1

NOD1 and NOD2 mRNA expression in cholesteatoma. NOD1 (left bar) and NOD2 (right bar) mRNA expression in cholesteatoma compared to external auditory canal skin (EAS). Real time PCR reveals no significantly higher gene expression of NOD1 within the cholesteatoma, but a significantly higher expression of NOD2 compared to EAS. For normalization, the housekeeping gene GAPDH was used and compared to EAS. N = 10 samples; statistics was performed by GraphPad Prism with the use of an unpaired t-test, P < 0.05.

Figure 2

Localization of NOD2 and NOD1 in cholesteatoma and in external auditory canal skin (EAS). Protein expression in cholesteatoma demonstrates a higher expression of NOD2 within the cholesteatoma compared to EAS, whereas NOD1 displays no significant change compared to EAS. Upper column displays a positive immunofluorescence staining of NOD2 and NOD1 in EAS compared to cholesteatoma in the lower column imaging staining, using a confocal microscope. Red represents the target genes NOD1 and NOD2 and green represents nuclei.

Figure 3

Altered inflammatory gene regulation in cholesteatoma versus EAS via microarray analysis. Significant upregulation of the inflammatory-related genes IL1, cFlip, p65, and IRAK. Moreover, there was a significant upregulation of the pattern recognition receptor (PRR) NOD2, but no significant upregulation of NOD1, TLR2, or TLR4 and the adaptor protein RIPK2. Furthermore, the figure displays a downregulation of NGFR in cholesteatoma. Gene expression analysis was performed via microarray analysis.

Figure 4

Bioinformatical network analysis for NOD. Proteins are the nodes and the edges are protein activations/inactivations, such as phosphorylation and dephosphorylation across a set of proteins. The network reveals an upregulation of NOD2, NLRC4, and PYCARD and the downstream molecules IRAK1 and cFLAR (red), whereas SMAD3 seems to be downregulated (green). TLR2, TLR4, NOD1, and RIPK2 were not significantly altered (black). RIPK2 displayed a remarkable network with many genes involved in the inflammatory and apoptotic process within the cholesteatoma. N = 17 samples; means ± SEM; ^∗ P < 0.05.

Figure 5

TNFα and Il1β mRNA expression in cholesteatoma. TNFα and Il1β mRNA expression in cholesteatoma compared to external auditory canal skin (EAS). Real time PCR reveals a significantly higher expression of TNFα and Il1β within the cholesteatoma compared to EAS. For normalization, the housekeeping gene GAPDH was used and compared to EAS. N = 10 samples; statistics was performed by GraphPad Prism with the use of an unpaired t-test, P < 0.05.