Abstract
Otitis media (OM), the most common childhood illness, can be caused by bacterial and/or viral infection. Hyperplasia of the middle ear (ME) mucosa is an important component of OM that contributes to its deleterious sequelae. Our previous research revealed that ME mucosal hyperplasia in bacterially induced OM was associated with expression of the heparin-binding epidermal growth factor (HB-EGF) gene, and that HB-EGF induced the proliferation of ME mucosal explants in culture. We used single-cell RNA-Seq to identify ME cells that express Hbegf and related genes involved in mediating responses to this factor. To determine the degree to which a viral infection might induce mucosal hyperplasia, and to assess the role of HB-EGF in hyperplasia in vivo, we used, Poly(I:C) to simulate a ME viral infection, Western blotting to confirm ME protein expression, and a specific inhibitor to block the effects of HB-EGF during OM. Genes for HB-EGF and its receptor were expressed in the ME primarily by epithelial, stromal and endothelial cells. Poly(I:C) induced prominent ME mucosal hyperplasia, peaking two days after ME injection. Immunostaining revealed that cleavage of proHB-EGF into its soluble form (sHB-EGF) was strongly induced in response to Poly(I:C). Inhibition of the sHB-EGF receptor dramatically reduced the hyperplastic response of the mucosa. The results demonstrate that a synthetic analog of viral double-stranded RNA interaction can induce OM including a strong proliferative response of the ME mucosa, independent of bacteria. They also indicate that HB-EGF is the dominant growth factor responsible for ME mucosal hyperplasia in vivo.
Highlights
Otitis media (OM), one of the most common childhood infections (Casselbrant et al, 1993), can be a serious disease
Single-cell RNA-Seq was employed to determine whether genes related to heparin-binding epidermal growth factor (HB-EGF) signaling are expressed in the middle ear (ME), and to identify the cells involved
The fact that Poly(I:C) induced the major pathophysiologic elements of OM in the rat ME is perhaps not surprising, since double-stranded RNA (dsRNA) interaction with Toll-like receptor 3 (TLR3) and other nucleic acid receptors including MDA5 and RIG-1 induces interferons, and many of the same inflammatory mediators produced by bacterial infection (Jensen and Thomsen, 2012)
Summary
Otitis media (OM), one of the most common childhood infections (Casselbrant et al, 1993), can be a serious disease. In the US, OM causes more pediatrician visits, antibiotic prescriptions, and surgeries than any other condition for children under 5 years of age (Rovers, 2008). Clinical and experimental studies have shown that infection of the ME induces inflammatory responses, a major feature of which is ME mucosal hyperplasia. The mucosa of the resting ME consists primarily of a simple squamous epithelial monolayer and a minimal stroma, with a thickness of approximately 15-30 mm (Lim and Hussl, 1969; Lim, 1976). The thickness of the mucosa can increase by tens to hundreds of mm in 23 days, reducing effective ME volume (Lim, 1979; Hernandez et al, 2008; Leichtle et al, 2010). Mucus and inflammatory mediators are secreted into the lumen by ME cells (Ryan et al, 2020). Hyperplasia of the ME mucosa is a major driver of inflammatory pathophysiology in OM
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