1. Differential Expression Between Cufi cells and Nuli cells Breathe Project Yves Berthiaume Grégory Voisin Chantal Massé September 2006

2. Methodology Purpose: to note the differential expression between a CF cell and no- CF cell to understand inflammation in CF disease. Used cells: a new cellular model cell of epithelial pulmonary cells (immortalized lines) developped by J.Zabner. Nuli : non CF. Cufi : CF phenotype ( homozygote ∆F508) Technologie: Microarray analysis with Affymetrix Array U133.2.0 ( 54 000 transcripts), 3 technicals replicats Nuli and 3 technicals replicats Cufi. Statistic Analysis of microarray: bioconductor (Limma), based on a linear model (the expression probability ≥ to 50 % with a Pvalue minimum equals to 0,01). Analysis of Gene Ontology with Onto-Express (Sorin Draghici). Analysis of Pathway with Pathway-Express (Sorin Draghici).

3. Results Gene Ontology analysis 2335 modulated probesets = 1659 annoted genes (+202 NA annotation genes) 785 annotated Up-regulated genes + 32 not annotated Up-regulated genes 874 annotated Down-regulated genes + 170 not annotated Down-regulated genes Inflammatory response excessive Modification of metabolism: lipid, protein

4. Conclusion of GO analysis …about inflammatory response Up-regulation of GO = immune response, inflammatory response, chemotaxis, cell adhesion. Inflammatory actors : IL6, IL8, SPINK5, CXCL10, CXCL11, CXCL1, 2,3,5,6, IFIT1,3,IL1R2,TNFAIP6, S100A12.

5. Results of Metabolic Pathway analysis Two modulated pathways: Toll-like receptor Pathway (path: hsa04620): adjusted Pvalue : 8.231x10e-3 Jak/Stat signaling pathway (path: hsa04630): adjusted Pvalue : 3.285x10e-4

6. Expression Ratio Q-PCR analysis in Toll-Like Receptor Signaling Pathway CUFINULI

7. In summary, ratio expression in Cufi versus Nuli (Q-PCR) IL1beta: : expression X 5 IL6 : : expression X 4 IL8: : expression X 4 CXCL10 : expression X 5 CXCL11 : expression X 5

8. Other interesting observations GSTT1: x 0.1 (microarray ratio) CLCA4: : x 6 (microarray ratio)

9. Future experiments -1 Principal conclusion: Activation of TLR signaling pathway in Cufi cells. Experiments to be done to validate this conclusion: -NF-kB cascade. -activation (phosphorylation and translocation) of STAT 1,2. -activation of Myd 88. -measurement of protein expression (cytokines and signaling molecules).

10. Future experiments -2 1. To complete analysis of this gene expression response following oxidative stress. 2. Validation of information in human primary cells.