The Jeff Mouse Mutant Model for Chronic Otitis Media Manifests Gain-of-Function as Well as Loss-of-Function Effects.

Chronic otitis media (OM) is the most common cause of hearing loss worldwide, yet the underlying genetics and molecular pathology are poorly understood. The mouse mutant Jeff is a single gene mouse model for OM identified from a deafness screen as part of an ENU mutagenesis program at MRC Harwell. Jeff carries a missense mutation in the Fbxo11 gene. Jeff heterozygotes (Fbxo11 Jf/+ ) develop chronic OM at weaning and have reduced hearing. Homozygotes (Fbxo11 Jf/Jf ) display perinatal lethality due to developmental epithelial abnormalities. In order to investigate the role of FBXO11 and the type of mutation responsible for the phenotype of the Jeff mice, a knock-out mouse model was created and compared to Jeff. Surprisingly, the heterozygote knock-outs (Fbxo11 tm2b/+ ) show a much milder phenotype: they do not display any auditory deficit and only some of them have thickened middle ear epithelial lining with no fluid in the ear. In addition, the knock-out homozygote embryos (Fbxo11 tm2b/tm2b ), as well as the compound heterozygotes (Fbxo11 tm2b/Jf ) show only mild abnormalities compared to Jeff homozygotes (Fbxo11 Jf/Jf ). Interestingly, 3 days after intranasal inoculation of the Fbxo11 tm2b/+ mice with non-typeable Haemophilus influenzae (NTHi) a proportion of them have inflamed middle ear mucosa and fluid accumulation in the ear suggesting that the Fbxo11 knock-out mice are predisposed to NTHi induced middle ear inflammation. In conclusion, the finding that the phenotype of the Jeff mutant is much more severe than the knock-out indicates that the mutation in Jeff manifests gain-of-function as well as loss-of-function effects at both embryonic and adult stages.

Mutation in Fbxo11 Leads to Altered Immune Cell Content in Jeff Mouse Model of Otitis Media.

The Jeff mouse mutant carries a mutation in the F-box only 11 gene (Fbxo11) and heterozygous animals display conductive deafness due to the development of otitis media (OM). The Fbxo11 locus is also associated with chronic otitis media with effusion (COME) and recurrent OM in humans. The Jeff mutation affects the ability of FBXO11 to stabilize p53 that leads to perturbation in the TGF-beta/Smad2 signaling pathway important in immunity and inflammation. In the current study, we evaluated the effect of the Jeff mutation on the immune cell content using multicolor flow cytometry. In blood of Jeff heterozygotes, we observed a significant increase in the number of NK, dendritic (CD11b+), neutrophils, and natural killer T (NKT) cells and a significant decrease in effector T-helper and B-lymphocytes compared to wild-type controls. The percentage of NK cells significantly decreased in the lungs of Jeff heterozygotes, with a concomitant reduction in B-lymphocytes and T-cytotoxic cells. In the spleen, Jeff heterozygotes displayed a significant decrease in mature B-lymphocytes, effector T-helper, and naïve T-cytotoxic cells. Neutrophils, dendritic, and NKT cells dominated bulla fluid in Jeff heterozygote mice. Similar analysis carried out on Fbxo11tm2b/+ heterozygotes, which carry a null allele, showed no difference when compared to wild-type. Cytokine/chemokine analysis revealed a significant increase in the G-CSF, GM-CSF, sTNFRI, TPO, and IL-7 levels in Jeff heterozygote serum compared to wild-type. This analysis increases our understanding of the role played by Fbxo11, a gene associated with human OM, in the systemic and localized cellular immune response associated with increased susceptibility to OM.

Cellular Immune Response against Nontypeable Haemophilus influenzae Infecting the Preinflamed Middle Ear of the Junbo Mouse.

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing acute otitis media (AOM). The pathology of AOM increases during long-term infection in the middle ear (ME), but the host cellular immune response to bacterial infection in this inflamed environment is poorly understood. Using the Junbo mouse, a characterized NTHi infection model, we analyzed the cellular response to NTHi infection in the Junbo mouse middle ear fluid (MEF). NTHi infection increased the total cell number and significantly decreased the proportion of live cells in the MEF at day 1, and this further decreased gradually on each day up to day 7. Flow cytometry analysis showed that neutrophils were the dominant immune cell population in the MEF and that NTHi infection significantly increased their proportion whereas it decreased the monocyte, macrophage, and dendritic cell proportions. Neutrophil and macrophage numbers increased in blood and spleen after NTHi infection. The T-cell population was dominated by T-helper (Th) cells in noninoculated MEF, and the effector Th (CD44+) cell population increased at day 2 of NTHi infection with an increase in IL-12p40 levels. Sustained NTHi infection up to 3 days increased the transforming growth factor ß levels, decreasing the effector cell population and increasing the T-regulatory (T-reg) cell population. In the preinflamed ME environment of the Junbo mouse, neutrophils are the first responder to NTHi infection followed by T-reg immune suppressive cells. These data indicate that sustained NTHi infection in the ME induces the immune suppressive response by inducing the T-reg cell population and reducing immune cell infiltration, thus promoting longer-term infection.

Cellular content plays a crucial role in Non-typeable Haemophilus influenzae infection of preinflamed Junbo mouse middle ear.

Non-typeable Haemophilus influenzae (NTHi) is a major pathogen causing acute otitis media (AOM). The relationship between the cellular content of the middle ear fluid (MEF) during AOM and infection of NTHi is poorly understood. Using the Junbo mouse, a characterised NTHi infection model, we analysed the cellular content of MEF and correlated the data with NTHi titres. The MEF of the Junbo mouse was heterogeneous between ears and was graded from 1 to 5; 1 being highly serous/clear and 5 being heavily viscous/opaque. At seven-day post-intranasal inoculation, NTHi was not found in grade-1 or 2 fluids, and the proportion of MEF that supported NTHi increased with the grade. Analyses by flow cytometry indicated that the cellular content was highest in grade-4 and 5 fluids, with a greater proportion of necrotic cells and a low-live cell count. NTHi infection of the middle ear increased the cell count and led to infiltration of immune cells and changes in the cytokine and chemokine levels. Following NTHi inoculation, high-grade infected MEFs had greater neutrophil infiltration whereas monocyte infiltration was significantly higher in serous noninfected low-grade fluids. These data underline a role for immune cells, specifically monocytes and neutrophils, and cell necrosis in NTHi infection of the Junbo mouse middle ear.