using ipa to study immune cells csc workshop on pathway analysis espoo, november 12, 2007 helena...
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Using IPA to study immune cells
CSC workshop on Pathway AnalysisEspoo, November 12, 2007
Helena AhlforsTurku Centre for Biotechnology
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Lehtonen & Ahlfors et al. Journal of Leukocyte Biology, 2007
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Aim of the study
• Identification of cell-type specific differences in gene expression profiles of monocytes, macrophages (Mf) and dendritic cells (DCs)
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Experimental setting
Monocyte
MacrophageGM-CSF
GM-CSF+ IL-4
Dendriticcell
Buffy coat
Ficoll gradient
Percoll gradient
depletion ofT and B cells
Gene expression profiling with Affymetrix HG-U133A arrays
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CD14
CD1b
DC-SIGN=CD209
Monocyte 3d M 7d M3d DC 7d DC
Known Mf and DC markers
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Monocyte
0d0d 3d3d 7d7d
Dendriticcell
Macrophage
405376
824342
441265
733389
+ GM-CSF, IL-4
+ GM-CSF
Number of regulated genes
Filtering criteria:not NCnot AASLR ≥ 1reproducible differences
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Genes regulated both at 3-d and 7-d time point
354236
76120
342194
* Mf vs DC
*
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TGF
20
15
10
5
FZD2
6
4
2
100
80
60
40
20
WNT5A
10
8
6
4
2
SOCS1
16
12
8
4
FcR1A
FcR1A
30
20
10
1
0.1
0.01
C3 TCF7L21.0
0.6
0.2
Mo M DC M DC
3d 7d
Mo M DC M DC
3d 7d
Mo M DC M DC
3d 7d
Validation of the expression of a selected set of genes by quantitative
RT-PCR
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IPA / Canonical pathways
Mf
DC
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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.
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Kumar et al. Molecular Systems Biology, 2007, In press
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
1Immunology 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
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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.
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BCR signaling network
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Experimental setting
mock
CaMKII
Pyk2
PLCγ
PKCδ
Transfecting the cells with siRNA oligos
Stimulating the cells with anti-IgG for 30 min
Gene expression profiling with Illumina mouse-6 beadchips
B cell
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Alterations in phosphorylation profiles
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Array results
mock
PLCγ
CaMKII
Pyk2
PKCδ
210
2255
930
350
449
Filtering criteria:SLR ≥ 1reproducible differences
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IPA results
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PKCδ
PLCγ
Pyk2
IPA / network 1CaMKIImoc
k
Network 1:Cancer,Cell cycle,Skeletal and Muscular Disorders
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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.
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Acknowledgements
Turku Centre forBiotechnologyProf Riitta Lahesmaa
National Public Health
InstituteProf Ilkka Julkunen Anne Lehtonen Ville Veckman Minja Miettinen
International Centre forGenetic Engineering andBiotechnology (ICGEB),Delhi, IndiaProf Kanury V.S. Rao Dhiraj Kumar Ravichandran SrikanthFinnish Microarray
Centre Miina Miller Päivi Junni Tiia Heinonen
National GraduateSchool of Informationaland Structural Biology
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IPA results