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

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

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

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

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

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

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

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

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 http://carl1anderson.files.wordpress.com/2012/01/clinical_trials2.jpg

“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 http://writerdood.wordpress.com/2009/11/14/genetically-engineered-talking-mice/ Sacrifice and analysis PBMC (i.p.) Air pouch induction Inflammatory stimulus d0 d21 d27 d28

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

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

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

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

Questions?