Transcript Using IPA to study immune cells

Using IPA to study
immune cells
CSC workshop on Pathway Analysis
Espoo, November 12, 2007
Helena Ahlfors
Turku Centre for Biotechnology
Lehtonen & Ahlfors et al.
Journal of Leukocyte Biology, 2007
Aim of the study
• Identification of cell-type specific
differences in gene expression profiles
of monocytes, macrophages (Mf) and
dendritic cells (DCs)
Experimental setting
Buffy coat
Gene expression
profiling with
Affymetrix
HG-U133A arrays
Ficoll gradient
Percoll gradient
depletion of
T and B cells
Monocyte
GM-CSF
Macrophage
GM-CSF
+ IL-4
Dendritic
cell
Known Mf and DC markers
Monocyte
CD14
CD1b
DC-SIGN
=CD209
3d Mf
3d DC
7d Mf
7d DC
Number of regulated genes
0d
3d
7d
Dendritic
cell
405
376
824
342
Monocyte
Filtering criteria:
not NC
not AA
SLR ≥ 1
reproducible differences
Macrophage
441
265
733
389
Genes regulated both at
3-d and 7-d time point
354
236
76 *
120
* Mf vs DC
342
194
Validation of the expression of a selected set
of genes by quantitative RT-PCR
C3
TCF7L2
1.0
1
0.1
30
20
0.6
0.01
10
0.2
TGFa
FceR1A
20
16
15
12
10
8
5
4
FcgR1A
10
SOCS1
8
6
4
2
Mo
FZD2
100
6
WNT5A
60
40
2
20
Mo
Mf
DC
3d
Mf
DC
7d
Mo
Mf
DC
3d
80
4
Mf
DC
3d
Mf
DC
7d
Mf
DC
7d
IPA / Canonical pathways
Mf
DC
Conclusions I
• GM-CSF and IL-4 regulate the expression of almost 900
genes during the differentiation of macrophages and DCs.
• Altogether 196 genes were differentially regulated in
macrophages and DCs throughout the 7-day differentiation.
• Most of these genes code for factors involved in the
signaling from cell surface to nucleus.
• Several novel genes with unknown molecular function were
identified. These genes may cooperate with the previously
known differentiation promoting factors and thus have an
essential role in macrophage and DC differentiation
process.
• Ingenuity Pathways Analysis revealed that canonical
pathways of particular interest are differentially regulated
in macrophages and DCs.
CAPTURING CELL-FATE DECISIONS FROM THE MOLECULAR
SIGNATURES OF A RECEPTOR-DEPENDENT SIGNALING RESPONSE
Dhiraj Kumar1, Ravichandran Srikanth1, Helena Ahlfors2, Riitta Lahesmaa2,
and Kanury V.S. Rao1,3
1
Immunology Group, International Centre for Genetic Engineering and Biotechnology
Aruna Asaf Ali Marg, New Delhi – 110067 INDIA
and 2Turku Centre for Biotechnology, Tykistokatu 6B, FIN-20521 Turku, FINLAND
Kumar et al.
Molecular Systems Biology, 2007, In press
Aim of the study
• Examine how the BCR-dependent
intracellular signaling network adapts to
targeted perturbations induced through
siRNA-mediated depletion of select
signaling intermediates.
BCR signaling network
Experimental setting
mock
PLCγ
B cell
Transfecting
the cells with
siRNA oligos
CaMKII
Pyk2
PKCδ
Stimulating the
cells with antiIgG for 30 min
Gene expression
profiling with
Illumina mouse-6
beadchips
Alterations in phosphorylation
profiles
Array results
mock
21
0
PLCγ
22
55
CaMKII
93
0
Pyk2
35
0
PKCδ
44
9
Filtering criteria:
SLR ≥ 1
reproducible differences
IPA results
IPA / network 1
mock
PLCγ
CaMKII
Pyk2
PKCδ
Network 1:
Cancer,
Cell cycle,
Skeletal and
Muscular
Disorders
Conclusions II
• The depletion of any given component from the BCR
signaling network resulted in significant alterations in
phophorylation profiles of the other intermediates.
• This effect was not localized but extended over to
intermediates that were not within the canonical signaling
pathways to which each of the depleted molecules
belonged.
• The depletion of individual nodes also reflected alterations
in signal processing with corresponding alterations in the
cellular phenotypic response as several tens of genes were
either strongly induced or downregulated.
• The most significant gene regulatory network identified by
IPA was a Myc-centric network with a proposed function in
cell cycle regulation.
Acknowledgements
Turku Centre for
Biotechnology
Prof Riitta Lahesmaa
National Public Health
Institute
Prof Ilkka Julkunen
Anne Lehtonen
Ville Veckman
Minja Miettinen
Finnish Microarray Centre
Miina Miller
Päivi Junni
Tiia Heinonen
International Centre for
Genetic Engineering and
Biotechnology (ICGEB),
Delhi, India
Prof Kanury V.S. Rao
Dhiraj Kumar
Ravichandran Srikanth
National Graduate
School of Informational
and Structural Biology
IPA results