Immunomodulation as a Protective Strategy in Chronic Otitis Media

Abstract

IntroductionMajor features of the pathogenesis in otitis media, the most common disease in childhood, include hyperplasia of the middle ear mucosa and infiltration by leukocytes, both of which typically resolve upon bacterial clearance via apoptosis. Activation of innate immune receptors during the inflammatory process leads to the activation of intracellular transcription factors (such as NF-κB, AP-1), which regulate both the inflammatory response and tissue growth. We investigated these leading signaling pathways in otitis media using mouse models, human samples, and human middle ear epithelial cell (HMEEC) lines for therapeutic immunomodulation.MethodsA stable otitis media model in wild-type mice and immunodeficient KO-mice, as well as human tissue samples from chronic otitis media, skin from the external auditory canal and middle ear mucosa removed from patients undergoing ear surgery, were studied. Gene and protein expression of innate immune signaling molecules were evaluated using microarray, qPCR and IHC. In situ apoptosis detection determined the apoptotic rate. The influence of bacterial infection on immunomodulating molecules (TNFα, MDP, Tri-DAP, SB203580, Cycloheximide) in HMEEC was evaluated. HMEEC cells were examined after bacterial stimulation/inhibition for gene expression and cellular growth.ResultsPersistent mucosal hyperplasia of the middle ear mucosa in chronic otitis media resulted from gene and protein expression of inflammatory and apoptotic genes, including NODs, TNFα, Casp3 and cleaved Casp3. In clinical chronic middle ear samples, these molecules were modulated after a specific stimulation. They also induced a hyposensitive response after bacterial/NOD-/TLR-pathway double stimulation of HMEEC cells in vitro. Hence, they might be suitable targets for immunological therapeutic approaches.ConclusionUncontrolled middle ear mucosal hyperplasia is triggered by TLRs/NLRs immunoreceptor activation of downstream inflammatory and apoptotic molecules.

Essential Role of the Innate Immune Adaptor RIP2 in the Response to Otitis Media

“…The present study provides direct support for the hypothesis that RIP2 signaling, both NOD-dependent and independent, is critical for timely OM resolution and the prevention of COM. Of note our gene array date indicate that Tlr2 is strongly upregulated (Leichtle et al, 2022), which may have a potential role in RIP2 activation (Kobayashi et al, 2002). While RIP2-mediated responses to infection have been observed in leukocytes at other sites (McCully et al, 2008;Shimada et al, 2018;Honjo et al, 2021), in the ME Ripk2 and Nod gene expression was prominent only in epithelial and stromal cells (Figure 4).…”

“…The present study provides direct support for the hypothesis that RIP2 signaling, both NOD-dependent and independent, is critical for timely OM resolution and the prevention of COM. Of note our gene array date indicate that Tlr2 is strongly upregulated ( Leichtle et al, 2022 ), which may have a potential role in RIP2 activation ( Kobayashi et al, 2002 ).…”

Immunomodulatory Response of the Middle Ear Epithelial Cells in Otitis Media

“…It was carried out analogously to the protocol of previously published work of our group (28,31). Here, mRNA from HMEEC was obtained using RNeasy Mini Kits (Qiagen, Mississauga, ON, Canada).…”

“…3). Regarding our previous results in mice and human immune patterns in OM (31), we focused on specific immune expression in relation to the different epithelia of different origins. Expression of NOD-1, NOD-2, and TLR-2 was successfully detected with different distribution patterns.…”

“…Because of our previously published role of innate immune system in-vivo models (mouse and human tissue) in OM (31), investigations of additional effector molecules, apoptosis genes, and protein expression in the in-vitro model of HMEEC were carried out, with focus on epithelial behavior (Fig. 5).…”