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
Rationale
• COME is a chronic inflammatory disorder, but can resolve
Pro-inflammatory
reprogramming
IL-10, MAPK, NFκB, TGFβ, HIF
Disease Modifying
Anti-Rheumatic
Drugs
Conventional cytokines
IL-1β. IL-6, TNF
Pattern recognition receptors
TLRs
Glucocorticoids
Antibiotics
Bacterial
antigens
Chronic
OME
Acute
OME
Trigger
Hypoxia
Inducible
Factor
?
resolution
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
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
Cytology
Cell counts
70
60
50
40
Low cellularity
30
High cellularity
20
10
0
Serous
Intermediate
Mucoid
Cytology
bacterial phagocytosis
multinucleate macrophage
cholesterol crystal
Cytology preps by Chiara Piccinelli
Affymetrix whole genome array
• Affymetrix GeneChip® Human 2.0 ST Array on 12 samples
• >30,000 coding transcripts
• >11,000 non-coding transcripts
38_(HuGene−2_0−st).CEL
B5_(HuGene−2_0−st).CEL
44_(HuGene−2_0−st).CEL
24_(HuGene−2_0−st).CEL
od_(HuGene−2_0−st).CEL
R_(HuGene−2_0−st).CEL
8L_(HuGene−2_0−st).CEL
R_(HuGene−2_0−st).CEL
R_(HuGene−2_0−st).CEL
od_(HuGene−2_0−st).CEL
od_(HuGene−2_0−st).CEL
od_(HuGene−2_0−st).CEL
25_(HuGene−2_0−st).CEL
41_(HuGene−2_0−st).CEL
od_(HuGene−2_0−st).CEL
blood
R_(HuGene−2_0−st).CEL
R_(HuGene−2_0−st).CEL
8L_(HuGene−2_0−st).CEL
3L_(HuGene−2_0−st).CEL
R_(HuGene−2_0−st).CEL
R_(HuGene−2_0−st).CEL
4L_(HuGene−2_0−st).CEL
effusion
Transcriptionally upregulated genes
serous vs mucoid
The most highly upregulated
transcripts are shared in common
Enrichr web based pathway analysis
• Transcript data analysis using Enrichr
• Based on GO biological systems algorithm for pathways
– Inflammatory response
– Response to hypoxia
– Regulation of leucocyte activation
p<7.78E-08
p<6.61E-07
p<0.00002025
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)
Protein quantification
• Protein VEGF-A quantified
– MSD® MULTI-ARRAY® Human VEGF assay (n=37)
****
VEGF pg/ml
105
104
6,427 pg/ml in glue vs
69 pg/ml in blood
103
**** = p<0.0001
102
101
glue
plasma
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
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
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
Acknowledgments
• Roslin Institute
– 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