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.

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 Fbxo11 tm2b/+ 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.

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).

Otitis Media (OM) is the inflammation of the middle ear (ME). Non-typeable Haemophilus influenzae (NTHi) is one of the leading otopathogens in causing OM. Phosphocholine (PCho) on the NTHi lipopolysaccharide influences host-pathogen interaction. C-Reactive Protein (CRP), an acute phase protein recognizes PCho, and can mediate bacterial killing. However, some strains of NTHi survive even in the presence of CRP. We aim to study the interaction of CRP with NTHi to understand its role in bacterial survival and OM. NTHi can efficiently infect the Junbo mouse, a characterised model of chronic and acute OM. CRP levels were highest 1 day post-intranasal inoculation in the ME fluid (MEF) and nasal passage (NP) washes. We show CRP is a localized response to NTHi as serum CRP levels were unaffected in NTHi inoculated and non-inoculated mice at 1, 3 and 7-day post intranasal inoculation. Further, we confirm the presence of NTHi influences CRP levels in the MEF and NP washes. We show CRP binding is influenced by the position and expression of PCho on the NTHi surface. Serum bactericidal assays indicate that the expression and position of PCho affects NTHi survival. The removal of CRP from the serum restores NTHi survival. The expression of PCho also influences opsonophagocytosis activity in macrophages, thereby confirming the importance of PCho in NTHi survival. The CRP-NTHi interaction is currently under investigation to advance our understanding of its role in the complex biological processes that influence bacterial killing and the onset, progression and resolution of OM caused by NTHi.

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 knockout mouse model was created and compared to Jeff. Surprisingly, the heterozygote knockouts (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 knockout 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 knockout 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 knockout indicates that the mutation in Jeff manifests gain-of-function as well as loss-of-function effects at both embryonic and adult stages.

Here we describe a mutation in the mitochondrial complex I assembly factor (Evolutionarily conserved signalling intermediate in Toll pathway) ECSIT which reveals tissue specific requirements for this factor in complex I assembly. Mitochondrial complex I assembly is a multi-step process dependant on assembly factors that organise and arrange the individual subunits, allowing for their incorporation into the complete enzyme complex. We have identified an ENU induced mutation in ECSIT (N209I) that exhibits a profound effect on complex I assembly only in heart tissue resulting in hypertrophic cardiomyopathy in the absence of other phenotypes. Mitochondrial function was reduced by 98% in mitochondria isolated from cardiac tissue but mitochondria from other tissues such as skeletal muscle, brain, liver, and kidney were unaffected. This data suggests the mechanisms underlying complex I assembly are tissue specific and has implications in understanding the pathogenesis of cardiomyopathy.

Aims Mitochondrial complex I assembly is a multi-step process which necessitates the involvement of a variety of assembly factors and chaperones to ensure the final active enzyme is correctly assembled. The role of the assembly factor ECSIT was studied across various murine tissues to determine its role in this process and how this varied between tissues of varying energetic demands. We hypothesised that many of the known functions of ECSIT were unhindered by the introduction of an ENU induced mutation, whilst it’s role in complex I assembly was affected on a tissue specific basis. Methods and Results Here we describe a mutation in the mitochondrial complex I assembly factor ECSIT which reveals tissue specific requirements for ECSIT in complex I assembly. Mitochondrial complex I assembly is a multi-step process dependent on assembly factors that organise and arrange the individual subunits, allowing for their incorporation into the complete enzyme complex. We have identified an ENU induced mutation in ECSIT (N209I) that exhibits a profound effect on complex I component expression and assembly in heart tissue, resulting in hypertrophic cardiomyopathy in the absence of other phenotypes. The dysfunction of complex I appears to be cardiac specific, leading to a loss of mitochondrial output as measured by Seahorse extracellular flux and various biochemical assays in heart tissue, whilst mitochondria from other tissues were unaffected. Conclusions These data suggest that the mechanisms underlying complex I assembly and activity may have tissue specific elements tailored to the specific demands of cells and tissues. Our data suggest that tissues with high energy demands, such as the heart, may utilise assembly factors in different ways to low energy tissues in order to improve mitochondrial output. This data have implications for the diagnosis and treatment of various disorders of mitochondrial function as well as cardiac hypertrophy with no identifiable underlying genetic cause. Translational Perspective Mitochondrial diseases often present as multi system disorders with far reaching implications to the health and well being of patients. Diagnoses are often undertaken by characterisation of mitochondrial function from skin or muscle biopsy, with the expectation that any affect on mitochondrial function will be recognisable in all cell types. However, this study demonstrates that mitochondrial function may differ between cell types with the involvement of tissue specific proteins or isoforms, as such, current diagnostic techniques may miss diagnoses of a more specific mitochondrial dysfunction.