Differential Expression Between Cufi cells and Nuli cells

Breathe Project

Yves Berthiaume Grégory VoisinChantal Massé

September 2006

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).

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

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.

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

Expression Ratio Q-PCR analysisin Toll-Like Receptor Signaling Pathway

expression de STAT2Pvalue = 0.03 , ratio 1.8

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

Expression de IL1b par PCRp=0.0004 ratio = 3,6

0

0.5

1

1.5

2

2.5

3

3.5

4

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

Expression d'IL6p=0,01 , ratio= 5.4

0

1

2

3

4

5

6

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

Expression de IL8Pvalue= 0,002

0

0.5

1

1.5

2

2.5

3

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

EXPRESSION DE CXCL10p=0.028, ratio= 5,5

0.0000

1.0000

2.0000

3.0000

4.0000

5.0000

6.0000

7.0000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

expression de CXCL11p=0,01 , ratio = 4,9

0

1

2

3

4

5

6

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

expression de STAT1Pvalue = 0.006 , ratio= 2.1

0.0000

0.5000

1.0000

1.5000

2.0000

2.5000

3.0000

Nuli_CTL1 Nuli_CTL2 Nuli_CTL3 Cufi_CTL1 Cufi_CTL2 Cufi_CTL6

expression de C-FOSpvalue = 0.63, ratio = 0.9

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

expression de MYD88pvalue= 0,88 , ratio =1,1

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6 expression de MAPK8pvalue = 0.6 , ratio = 0.9

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

CUFINULI

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

Other interesting observationsexpression de GSTT1

(Q PCR)

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

expression de CLCA4 (Q PCR)pvalue = 0.0001 , ratio = 4,7

0.0000

0.5000

1.0000

1.5000

2.0000

2.50003.0000

3.5000

4.0000

4.5000

5.0000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

GSTT1: x 0.1 (microarray ratio)

CLCA4: : x 6 (microarray ratio)

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).

Future experiments -2

1. To complete analysis of this gene expression response following oxidative stress.

2. Validation of information in human primary cells.

Cufi - Physiological response - DMNQ 24h

Ctl

DMNQ 5

µM

DMNQ 1

0µM

DMNQ 1

5 µM

DMNQ 2

0 µM

0

25

50

75

100

0

100

200

300

400

500

600

700

800

traitement

Co

ura

nt

tota

l (µ

A)

Rés

ista

nce

(o

hm

s)