Using IPA to study immune cells

CSC workshop on Pathway AnalysisEspoo, November 12, 2007

Helena AhlforsTurku 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

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

CD14

CD1b

DC-SIGN=CD209

Monocyte 3d M 7d M3d DC 7d DC

Known Mf and DC markers

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

Genes regulated both at 3-d and 7-d time point

354236

76120

342194

* Mf vs DC

*

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

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.

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

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

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

Alterations in phosphorylation profiles

Array results

mock

PLCγ

CaMKII

Pyk2

PKCδ

210

2255

930

350

449

Filtering criteria:SLR ≥ 1reproducible differences

IPA results

PKCδ

PLCγ

Pyk2

IPA / network 1CaMKIImoc

k

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 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

IPA results