Fig 2: Lack of evidence for bacterial or fungal organisms in Splunc1-/- middle ears. Middle ear sections from Splunc1-/- mice with otitis were examined for the presence of microorganisms. (A) A modified Gram stain was negative for bacteria. (B) Gomori methenamine silver (GMS) staining was performed to survey for fungal organisms. In this representative image, GMS staining produced typical black staining of both goblet cell mucus and luminal secreted mucus, but was negative for fungal organisms in luminal material. Scale bar: 57 µm (A); 38 µm (B).

Fig 3: The middle ears of Splunc1-/- mice harbor increased inflammatory cells and cellular debris relative to wild-type littermate controls. The top panel depicts a representative image of a middle ear from a Splunc1+/+ mouse, in which the middle ear epithelium is covered by a layer of fluid containing globular fluid material. In the Splunc1-/- middle ear image (bottom panel) this fluid contains multiple punctate eosinophilic ‘ghost’ cells (black arrowheads) along with a small number of solitary PMNs (black arrows). Scale bar: 43 µm.

Fig 4: Splunc1 protein is expressed in the mouse middle ear and Eustachian tube. Splunc1 (brown stain) is present in the ciliated and non-ciliated cells of the surface epithelium lining the Eustachian tube (A) and middle ear (B) in the mouse. Splunc1 is also abundant in the serous cells of the submucosal glands in these tissues (A,C). Splunc1 expression is completely absent in the middle ears and Eustachian tubes of Splunc1-/- mice (D-F). L, lumen of the Eustachian tube; SMG, submucosal glands; M, mucous tubules; S, serous acini. Scale bar: 200 µm.

Fig 5: Summary of histopathological analysis of Splunc1-/- and wild-type mouse middle ears. Middle ear sections from Splunc1-/- mice and age-matched wild-type littermate controls were examined for histological evidence of otitis or other significant changes in the middle ear, using scoring rubrics described in the Materials and Methods. Sections were scored for several parameters including the infiltration of PMNs and macrophages in the middle ear lumen (panels A and B, respectively), accumulated cell debris (C), and the degree of hyperplasia and remodeling exhibited by the epithelium (D). Scores are summarized as box and whiskers plots. In these plots, the median score is denoted by a thick line, the box indicates the 25th and 75th percentiles, and the minimum and maximum values are depicted by the outer bars. Mann–Whitney tests were used to test for statistically significant differences (n=18 Splunc1+/+ and n=26 Splunc1-/- mice).