1. Whole genome transcript analysis of COME reveals a hypoxic inflammatory environment
Mahmood Bhutta
TWJ & Colledge Family Memorial Otology Fellow
University of Western Australia
Royal Perth Hospital
James Ramsden
Steve Brown
Michael Cheeseman

2. ? Rationale COME is a chronic inflammatory disorder, but can resolve
Trigger
Acute
OME
Chronic
OME
Conventional cytokines
IL-1β. IL-6, TNF
Pattern recognition receptors
TLRs
Bacterial
antigens
Pro-inflammatory reprogramming
IL-10, MAPK, NFκB, TGFβ, HIF ?
Hypoxia Inducible
Factor
Antibiotics
Glucocorticoids
Disease Modifying
Anti-Rheumatic Drugs
resolution
Antibiotics no effect
Steroids no effect, and in other disorders they have not been shown to modify the course of the disease
Disease modifying ant-rheumatic drugs were first used in rheumatoid arthritis and slow disease progression, but are also now adapted for use in other inflammatory disorders e.g. SLE, Crohns, myaesthnia gravis, sarcoidosis. Use of a single disease modifying drug is often sufficient to effect clinically significant outcomes

3. Rationale
Undertake analysis of gene upregulation (RNA transcripts) in COME
Through this, identify pathways for molecular targeting
Methodology enabled by availability of large-scale transcript arrays, and by bioinformatic software
Global transcript analyses reported in animal models of OM, but never for human disease

4. Transcript analysis from COME
52 children aged <10 undergoing grommet insertion at Oxford University Hospital for COME
Effusion trapped and RNA chemically stabilised
Macroscopically classified as serous, mucoid, or intermediate
Cytology of specimens
Transcript analysis comparing effusion to serum

5. Cytology
Cell counts
Low cellularity
High cellularity

6. Cytology bacterial phagocytosis multinucleate macrophage
cholesterol crystal
Cytology preps by Chiara Piccinelli

7. Affymetrix whole genome array
Affymetrix GeneChip® Human 2.0 ST Array on 12 samples
>30,000 coding transcripts
>11,000 non-coding transcripts

8. Transcriptionally upregulated genes serous vs mucoid
effusion
blood
Transcriptionally upregulated genes
serous vs mucoid
The most highly upregulated transcripts are shared in common

9. Enrichr web based pathway analysis
Transcript data analysis using Enrichr
Based on GO biological systems algorithm for pathways
Inflammatory response p<7.78E-08
Response to hypoxia p<6.61E-07
Regulation of leucocyte activation p<

10. RT-PCR verification of hypoxia transcripts
Re-assessed hypoxia transcripts using quantitative Real-Time PCR on a custom Taqman array
32 samples
Control genes HPRT1, HRAS and NRAS
81% concordance (p<0.001)

11. Protein quantification
Protein VEGF-A quantified
MSD® MULTI-ARRAY® Human VEGF assay (n=37)
6,427 pg/ml in glue vs
69 pg/ml in blood
**** = p<0.0001

12. Hypoxia signaling
Hypoxia inducible factor (HIF) is upregulated in response to cellular hypoxia
Hypoxia is a common finding in inflamed microenvironments
Inflammation distances mucosa and leucocytes from vasculature
Inflammation consumes cellular oxygen
HIF changes cellular physiology to promote survival under hypoxic stress, but when persistently expressed reprograms cells to a pro-inflammatory state

13. Hypoxia as a therapeutic target
Synthetic disease modifying drugs have been developed to target hypoxia pathways
Hypoxia is found in genetic mouse models of chronic OM (Junbo, Jeff, TGIF1, Edison)
Oral administration of Anti-VEGF receptor molecules to the Junbo mouse moderates inflammation and hearing loss1
1 . Cheeseman et al, PLOS Genetics, 2011

14. Conclusions
An agnostic whole genome transcript analysis shows upregulation of hypoxia pathways in the effusion of children with using the affymetrix array
Quantitative PCR and VEGF protein analysis confirm upregulation of hypoxia pathways
Hypoxia pathways represent a potential therapeutic target

15. Acknowledgments Roslin Institute University of Oxford MRC Harwell
Prof Michael Cheeseman
Prof Elspeth Milne
Chiara Piccinelli
University of Oxford
James Ramsden
Lindsey Hobson
Jane Lambie
MRC Harwell
Prof Steve Brown
Debbie Williams
Hayley Tyrer
Ethical approval
Oxfordshire Research Ethics Committee