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. 2022 Jan 24:11:815627.
doi: 10.3389/fcimb.2021.815627. eCollection 2021.

Probing Immune-Mediated Clearance of Acute Middle Ear Infection in Mice

Kalyan K Dewan  1 , Colleen Sedney  1 , Amanda D Caulfield  1 , Yang Su  1 , Longhuan Ma  1 , Uriel Blas-Machado  2 , Eric T Harvill  1
Affiliations

Probing Immune-Mediated Clearance of Acute Middle Ear Infection in Mice

Kalyan K Dewan et al. Front Cell Infect Microbiol. .

Abstract

Acute otitis media (AOM) is commonly caused by bacterial pathobionts of the nasopharynx that ascend the Eustachian tube to cause disease in the middle ears. To model and study the various complexities of AOM, common human otopathogens are injected directly into the middle ear bullae of rodents or are delivered with viral co-infections which contribute to the access to the middle ears in complex and partially understood ways. Here, we present the novel observation that Bordetella bronchiseptica, a well-characterized respiratory commensal/pathogen of mice, also efficiently ascends their Eustachian tubes to colonize their middle ears, providing a flexible mouse model to study naturally occurring AOM. Mice lacking T and/or B cells failed to resolve infections, highlighting the cooperative role of both in clearing middle ear infection. Adoptively transferred antibodies provided complete protection to the lungs but only partially protected the middle ears, highlighting the differences between respiratory and otoimmunology. We present this as a novel experimental system that can capitalize on the strengths of the mouse model to dissect the molecular mechanisms involved in the generation and function of immunity within the middle ear.

Keywords: Bordetella bronchiseptica; adaptive immunity; natural infection; otitis media; protective immunity.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Colonization and growth of Bordetella bronchiseptica in the respiratory tract and middle ears of C57BL/6J mice. (A) Graphs represent the B. bronchiseptica colony-forming units (CFUs) recovered (log10/ml) from the nasal cavity, trachea, lungs, and middle ears (left to right) from individual C57BL6J mice (filled circles, n = 5 per group). For the middle ears, circles represent bacteria recovered from the left (filled circle) and right (open circle) middle ear bulla of an individual mouse. (B) Graphs represent the number of CFUs recovered over time from the nasal cavity, trachea, lungs, and middle ears of groups of C57BL/6J mice (n = 4 per group). Filled circles represent the mean value ± SEM. (C) Images represent developing inflammation in the middle ears from C57BL/6J mice. HE stains. Top panel: uninfected. In the left image, the small arrows point to the tympanic membrane, and in the lower right, to the Eustachian tube as it enters the middle ear. Scale bar = 500 μm. The right-side image represents higher magnification of the dashed boxed region of the middle ear. Scale bar = 200 μm. Middle panel: 3 dpi with B. bronchiseptica. The left image, the small arrows point to minimal accumulation of inflammatory exudate within the middle ear. Scale bar = 500 μm. The right-side image represents higher magnification of the dashed boxed region of the middle ear. Arrows point to minimal accumulation of neutrophils and macrophages arranged about or near the Eustachian tube (*) as it enters the middle ear. Scale bar = 200 μm. Lower panel: 7 dpi with B. bronchiseptica. In the left image, the arrowhead points to a moderate accumulation of inflammatory exudate within the middle ear. Scale bar = 500 μm. The right-side image represents higher magnification of the dashed boxed region of the middle ear. Arrows point to a densely cellular collection of neutrophils and macrophages arranged about or near the Eustachian tube (*) as it enters the middle ear, just below an ear polyp (Po; incidental finding). Scale bar = 200 μm. Inset (small dashed box): Bacteria attached to the cilia (arrowheads) on the epithelia lining the middle ear by the entrance of the Eustachian tube. Scale bar = 10 μm. EAM, external auditory meatus; Co, cochlea; ME, middle ear in bony tympanic bulla.
Figure 2
Figure 2
Growth of B. bronchiseptica in Rag-1−/− mice. Graphs represent the number of B. bronchiseptica RB50 CFU recovered from the nasal cavity, trachea, lungs, and middle ears of groups of Rag-1−/− mice over time following inoculation. Error bars represent the standard deviation (n = 4).
Figure 3
Figure 3
Growth of B. bronchiseptica in B- and T-cell-deficient mice. Graphs represent the number of CFU recovered from the nasal cavity, trachea, lungs, and middle ears of groups of B-cell-deficient (left, panel A) and T-cell-deficient (right, panel B) mice. Error bars represent the standard deviation (n = 4).
Figure 4
Figure 4
Adoptively transferred convalescent serum protects the lower respiratory tract but is less effective in the nasal cavity and middle ears. Graphs represent the number of CFUs recovered on 3 dpi from the nasal cavity (NC), trachea (TR), lungs (LNG), and middle ears (ME) of groups of C57Bl/6J mice. The adoptively transferred convalescent serum was collected from C57BL/6J mice (light gray columns) or control mice that received no serum (black columns). Dashed line represents the limit of detection. Error bars represent the standard deviation (n = 4). *p < 0.05, ** <0.001.
Figure 5
Figure 5
Effect of splenocyte transfer on the growth of B. bronchiseptica in Rag-1−/− mice. (A) Graphs represent the number of CFUs of B. bronchiseptica recovered at day 28 post-inoculation from the respiratory tract and middle ears of Rag-1−/− mice that either received no splenocytes (black) or mice that received splenocytes from naive mice (dark gray striped column) or splenocytes from convalescent mice (white column) 2 h before being inoculated with 500 CFUs of bacteria (n = 4 mice per group). Error bars represent the standard deviation. Dotted line represents the limit of detection. (B) Graphs represent the anti-B. bronchiseptica serum IgG titers (arbitrary units) from mice studied in (A) [control serum from d56 p.i. convalescent mice (striped column); no splenocytes (black); splenocytes from naive mice (dark gray), convalescent splenocytes (white)]. Error bars: ± SEM; *p < 0.05, *** <0.0001.
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