1. Bulla fluid composition influences NTHi infection of the Junbo mouse middle ear.
Pratik P. Vikhe
Genetics and Pathobiology of Deafness
Mammalian Genetics Unit, MRC Harwell Institute,
United Kingdom

2. Junbo otitis media mutant mouse
Hearing deficient mouse line
mutation in Evi1 gene influences regulation of NFκB expression
inflammation confined to middle ear; altered tympanic membrane and middle ear fluid present from 4 weeks of age
+/+
Jbo/+
single intranasal inoculation with the human otopathogen non-typeable Haemophilus influenzae (NTHi) results in efficient middle ear infection
novel infection model
-natural route of infection
-robust long lasting middle ear infection
Hood et al. (2016) DMM 9

3. Middle ear viscosity/opacity vs. NTHi infection
Grade3
Grade 5
Post 7 day inoculation with NTHi 162sr the infection rate in Junbo is 78% (Hood et al. (2016) DMM 9).
Variation in the viscosity/opacity of the middle ear fluids of Junbo mouse.
Grade 4
Viscosity of 2 represents the most serous fluid and 5 the most viscous/ opaque.
Direct relationship between NTHi infection and viscosity of middle ear fluid.
NTHi Infected

4. Total cell count in non-inoculated Junbo middle ear fluid
Grade 2
Grade 3
Grade 4
Grade 5
Mention non inoculated
*P<0.05 vs. Grade 2 fluid

5. Flow-cytometry analysis of middle ear fluid
Gating strategy – Apoptotic & Necrotic cell content
Ear fluid viscosity Grade 2 Grade Grade Grade 5
Soon after initiating apoptosis, cells translocate the membrane phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface that can be detected by staining with a fluorescent conjugate of Annexin V, a protein that has a high affinity for PS.
Propidium Iodide (PI) binds to double-stranded DNA. PI cannot cross intact plasma membrane and therefore will only be present in DNA of cells where the plasma membrane has been compromised/ permeabilized thus can be used as a counterstain to discriminate necrotic/ dead cells from apoptotic cells.
n=11,*P<0.05, **P<0.01 vs. fluid viscosity of 2

6. Live vs. necrotic cell count in non inoculated Junbo middle ear fluid
*P<0.05 vs. Total noninoculated

7. Total cell count in Junbo middle ear fluid
Percentage of Ears +ve for NTHi
100 %
96.15 %
79.21 %
32.09% 0%
*P<0.05 vs. non inoculated

8. Relative proportion of immune cells in Junbo mouse middle ear fluid (Gating Strategy)
CD11b+Ly6G+ Neutrophils
CD11b+Ly6G-Ly6C-F4/80+ Macrophages
CD11b+Ly6G-Ly6C+F4/80+/- Monocytes
CD11b-Ly6G-CD5+ Lymphocytes
Mention the markers once and go to names of the cells.

9. Relative proportion of immune cells in Junbo mouse middle ear fluid
Relative percent
Relative percent
Relative percent
Relative percent
Relative percent
Neutrophils Macrophages Monocytes (F4/80+) Monocytes Lymphocytes
*P<0.05 vs. Grade 2 fluid

10. Percentage of Ears +ve for NTHi
Relative proportion of immune cells in Junbo mouse middle ear fluid – NTHi inoculated vs non inoculated.
Percentage of Ears +ve for NTHi
100 %
92.15 %
86.1 %
29.24%
0 %
Relative percent
Relative percent
Relative percent
Relative percent
Relative percent
Neutrophils Macrophages Monocytes (F4/80+) Monocytes Lymphocytes
*P<0.05 vs. non inoculated

11. Variation in cellular composition
Summary ?
Middle ear fluids of Junbo mouse vary in their viscosity/opacity.
Jbo/+
Molecular mechanism underlying variability in the middle ear fluid. (eg. Expression of epithelial cell receptors (CD34), Cytokines and signalling)
NTHi survive in high viscous fluid but not in serous.
Total cell content is high in viscous fluid compared to serous.
Grade 2
Grade 5
Live cell content is low whereas necrotic cell content is high in viscous fluid compared to serous.
Variability of other immune components in different grades of fluid (eg. immunoglobulins, CRP, PLUNC and MBL).
NTHi inoculation significantly increases the total and live cell proportion in high viscosity/ opacity fluids.
Variation in the middle ear fluids
NTHi colonisation
Proportion of immune cells varies on comparison between serous vs viscous fluids.
What does everything mean
Platform for understanding the proteom.
Mechanism behind ability of NTHi to survive and host factors responsible for inhibiting the growth of NTHi.
Variation in cellular composition
NTHi inoculation significantly increases the proportion of monocytes (Ly6C Hi) in Junbo middle ear fluids.

12. Disease Model Discovery
Acknowledgement
Deafness Group
Derek Hood
Tom Purnell
Jiewu Yang
Hilda Tateossian
Mike Bowl
Steve Brown
Clinical Chemistry
Paras Pathak
Helen Netukunda
Emily Page
Tertius Hough
MLC Ward 4
Lisa Ireson
Chris Brett
Tamzin Osborne
Lucie Vizor
Protein Core
Helen Hilton
Chris Esapa
Quarantine
Chris Jones
Julie Roberts
Disease Model Discovery
Paul Potter
Bioimaging
Jeremy Sanderson
Sara Wells
Histology
Caroline Barker
Adele Austin