Immunomodulatory Response of the Middle Ear Epithelial Cells in Otitis Media

Abstract

Hypothesis: The middle ear (ME) epithelium transforms because of changed immunomodulation during infection.

Introduction: The epithelial cells of the tympanic cavity represent the first line of defense in the context of otitis media. They can convert from a typical mucosal site into a respiratory epithelium and vice versa. Our goal is to depict the specific immune response of epithelial cells after infection at the molecular level.

Methods: The investigations were carried out on healthy and inflamed ME tissue, removed during surgical interventions in mouse and human models, and in a human in-vitro cell model in human ME epithelial cell line. We determined the epithelial localization of the protein expression of Toll- and NOD-like immune receptors and their associated signaling molecules using immunohistochemistry. In addition, we examined growth behavior and gene expression due to direct stimulation and inhibition.

Results: We found clinically and immunobiologically confirmed transformation of the inflamed ME epithelium depending on their origin, as well as differences in the distribution of Toll-like receptors and nucleotide-binding oligomerization domain-like receptors in the epithelial cell lining. Dysregulated gene and protein expression of the inflammatory and apoptotic genes could be modulated by stimulation and inhibition in the epithelial cells.

Conclusions: The local ME mucosal tissue is believed to modulate downstream immune activity after pathogen invasion via intrinsic cellular mechanism. Using translation approaches to target these molecular pathways may offer more reliable clinical resolution of otitis media in the future.

Figure 1.

Representation of the signal cascades of the innate immune system of bacterial middle ear infections. Extracellular pathogen-associated molecular patterns (PAMP) via Toll-like receptors (TLRs) and cytoplasmic PAMP via NOD-like receptors (NLRs) lead to the activation of the nuclear factor of kappa-B (NF-κB), a key player in production inflammatory cytokines. Additionally, this production is stimulated by growth factors such as TNFα, which act via the mitogen-activated protein kinase (MAPK) pathway. NOD-2 also can induce PAMP autophagy. As a result of the inflammasome activation via NLRP3, the pro-forms of the cytokines are cleaved into their active form by caspase-1 and then released. In addition to these individual reactions to PAMP, there are also interactions between the various signaling pathways, so it is assumed that the immune response is modulated. [Figure created with BioRender]

Figure 2.

Histology of a middle ear of a wild-type mouse (C57/BL6) and pathological changes by NTHi-induced otitis media. Brackets represent the epithelial thickness. (A) HE staining on non-infected middle ear mucosa (MM) of the middle ear cavity (MEC) shows the tympanic membrane (TM), Eustachian tube (ET), and external auditory canal skin (EAS) (4×). (B) Morphological assessment by HE stain of pathophysiological changes in the middle ear on the 3rd day after NTHi inoculation (4×). (C) The region of interest (ROI) from (A) displays a thin, superficial epithelial layer of the middle ear cavity (40×) (D) Pseudostratified ciliated epithelium and goblet cells on 3rd day after NTHi inoculation in ROI from (B) (40×). (E) Gram staining of the thin, simple epithelial layer of the middle ear from (C) on the 3^rd day after NTHi(100×). (F) Gram-stained pseudostratified ciliated epithelium and goblet cells on the 3rd day after NTHi inoculation in ROI from (D) (100×). Arrow points to the kinocilium.

Figure 3.

Histology of the human middle ear tissue depending on its localization and subsequent immunohistochemical display of the expression of pattern recognition receptors in otitis media. (A) Epitympanum with simple epithelium and mesotympanum with prismatic epithelium with cilia and goblet cells, magnification 20× and 40×, HE section. (B) Immunohistochemical representation of the epithelial expression of innate immune receptors (NOD-1, NOD-2, and TLR-2) in epi- and mesotympanum. Low specific staining for NOD-1 in both localizations. NOD-2 and TLR-2 are found to be stable expressed in the mesotympanic region. Arrows point to the stained target molecules

Figure 4.

The real-time proliferation of HMEEC after triggering with NTHi. The proliferation of HMEEC in real-time is compared with HT29-WT (human colorectal adenocarcinoma cell line) and its variants overexpressing RIP2 (HT29-RIP2), NOD-1 (HT29-NOD-1), and NOD-2 (HT29-NOD-2) initially seeded at 30 000 (A) and 60 000 (B) cells/mL. While the proliferation rate of HT29 start to decrease after around 25 hours, HMEEC constantly proliferates in the time window of 50–60 hours and flattens down until 70 hours (nearly 3 days).

Figure 5.

Immunomodulation in the Human Middle Ear Epithelial Cell Line (HMEEC). (A) Gene expression of IL-1ß, IL-6, IL-8, and TNFα after 6 and 24 hours of consecutive stimulation by NTHi, Tri-DAP, MDP, and SB203580 or double stimulation by qPCR. Expression increase in IL-1ß, IL-6, IL-8, and TNFα both in the sole stimulation and occasionally in the presence of NTHi (B) Gene expression of apoptotic genes (caspase 3, caspase 7, BID) after 6 hours and 24 hours of consecutive stimulation with TNFα, Tri-DAP, MDP, SB203580, CHX and in double stimulation with additional NTHi using RT-PCR. CHX served here as a positive control. No significant increase in gene expression, even decreases after 24 hours. (C) Protein expression of IL-6 and IL-8 after 6 hours and 24 hours consecutive stimulation with NTHi, TNFα, Tri-DAP, MDP, and SB203580 and in double stimulation using the Cytometric Bead Array Flex Set System. Significant increase by NOD-1 and NOD-2 pathways.