1. Air Pouch Inflammation model
Jonathan D Finn, PhD
Arthrogen BV, Academic Medical Center
Amsterdam, The Netherlands

2. Overview History of APM Examples Humanized mice Assays Pros vs. cons
Practical tips

3. Murine inflammation models
Collagen induced arthritis model (CIA)
Commonly used for RA investigation
Advantages:
Well characterized and widely used
Inflammation develops over weeks (chronic)
Disadvantages:
Long duration (~2 months)
Severe discomfort for animals
Often necessitating premature sacrifice
Can be technically challenging
Intra-articular injections (2-5 uL volume)
Unable to collect synovial fluid
Air pouch inflammation model
Alternative model for the investigation of synovial inflammation
Pietersz et al, Immunology and Cell Biology, 2009

4. Air Pouch Model: History
“The formation of a structure with the features of synovial lining by subcutaneous injection of air: an in vivo tissue culture system”
1981, Journal of Pathology, Edwards, Sedgwick, Willoughby
Subcutaneous injection of air on back of mouse (5 mL)
Air pouch lining forms quickly (5-6 days)
6-10 cells deep composed of fibroblast like (50-90%) and macrophage like (10-50%) cells
By d14, highly compact lining, resembling synovium from mouse knee
Kadl et al, Arthritis and Rheum 2009

5. Air-pouch model: role of TNF
“Carrageenan-induced acute inflammation in the mouse air pouch synovial model. Role of TNF”
Romano et al, Mediators of Inflammation, 1997
Protocol
Male CD1 mice
5 mL air on d0, re-inflated with 3 mL air d3
1% carrageenan (1mL) administered d6
Sacrifice and harvest tissue/fluid at various time points
Sacrifice and analysis
Sacrifice and analysis
Sacrifice and analysis
Air pouch induction
Inflammatory stimulus
Re-inflation d0 d3 d6 d7 d8
d13
Romano et al, Mediators of Inflammation, 1997

6. Results (1) Leukocyte recruitment increases over time
Cytokines (IL-6, TNF-a) rapidly increase and peak ~24 hrs
Romano et al, Mediators of Inflammation, 1997

7. Results (2) PGE2 increases over time
Treatment with glucocorticosteroids effective in reducing inflammation
PGE2 role in inflammation? Can resolve inflammation by upregulating lipid enzymes?
Anti-TNF also inhibits inflammation

8. Inflammatory stimulus
Carrageenan
linear sulfated polysaccharides from seaweed
Activates NFkB pathway via TLR4
1-2% in saline
Zymosan
Yeast cell wall extract consisting of protein-carbohydrate complexes
LPS
Gram negative bacteria cell wall component
Cytokines/chemokines
TNFa, IL-1, CXCL11, CXCL12, etc
RA SF

9. Humanized Air Pouch Model
Murine models of inflammation do not often predict human outcomes
~150 clinical trials testing new treatments for acute inflammation
100% failure rate (Seok et al, PNAS 2013)
Need for more relevant animal models
Humanized mouse air pouch model

10. “Chemokine receptor CXCR3 agonist prevents human T-cell migration in a humanized model of arthritic inflammation” O’Boyle et al, PNAS 2012
1) immune deficient mice
NSG (NOD-SCID-gamma)
(NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ)
2) human PBMC
1x107 cells by i.p. administration
3) induce air pouch 21 days post PBMC administration
4) Pouch filled with stimulus
CXCL11, CXCL12, CCL5, RASF
5) CD45+ human T cells quantified by FLOW
Sacrifice and analysis
PBMC (i.p.)
Air pouch induction
Inflammatory stimulus d0
d21
d27
d28

11. Repopulation of mouse spleen with human cells (50-80%)
Influx of human CD45+ cells into air pouch
CXCL11
RA SF
Treatment with human specific agonist effective in reducing cell infiltrates
O’Boyle et al, PNAS 2012

12. Assays Air pouch lavage Air pouch membrane
Cell quantification (Coulter counter)
ELISA for cytokines, chemokines, etc
FLOW to investigate cell type subsets
RNA analysis
Lavage cells
miRNA
exosomes
Air pouch membrane
Homogenize and examine gene expression profiles
Fix and stain
Immunohistochemistry
FLOW
Prepare tissue by enzyme digestion to release single cells

13. Summary Pros Cons Quick model (7-14 days start to finish)
Wide range of inflammatory stimulus available
Easy to perform
analyze air pouch fluid
Large amount of material for analysis
Air pouch lavage
Synovial-like membrane/lining
Investigate human inflammation
Humanized mice
Study human specific compounds
Less suffering for animals
Easier to get DEC approval
Cons
Acute inflammation
Not a good model for chronic inflammation
Not a true arthritis model
Does not involve cartilage or bone

14. Practical tips Important to keep air pouch inflated
Check every 2-3 days and re-inflate as necessary
May want to prepare inflammatory stimulus in 1-2% carboxymethylcellulose (CMC)
When using non-viscous inflammatory stimulus (e.g. LPS, cytokines)
Reference list available

15. Questions?