Molecular analysis of IRF7-driven inflammatory gene

network (IDIN). Defining the role of the T1D gene networks

in pancreatic b-cell destruction.

Izortze Santin Gomez

Ciberdem – Diatrain Postdoctoral Fellowship Program

27/11/12

Background – I. Santin

Immunogenetics Research Laboratory – Cruces Hospital, Bilbao (2004-2009):

“Searching for novel susceptibility genes in autoimmune diseases: type 1

diabetes and celiac disease”

Background – I. Santin

Immunogenetics Research Laboratory – Cruces Hospital, Bilbao (2004-2009):

“Searching for novel susceptibility genes in autoimmune diseases: type 1

diabetes and celiac disease”

- Candidate gene studies - Innate immune system genes:

- Toll-like receptors (TLR) – SNPs in TLR2 and TLR4 (Santin et al, Ann N Y Acad

Sci, 2006; Santin et al, Tissue Antigens 2007)

- Killer cell Ig-like receptors (KIR) – Presence/absence (Santin et al, Human

Immunology, 2006; Santin et al, Genes and Immunity 2007)

- High throughput genotyping - Major Histocompatibility Complex:

- Conserved extended haplotypes (CEH) (DR3-DQ2) – Search for additional

risk variants using Illumina technology (Santin et al, Genes and Immunity, 2009)

Genetic association studies

Laboratory of Experimental Medicine – ULB, Brussels (2009-ongoing):

“Role of Type 1 diabetes genes in beta cell destruction”

Background – I. Santin

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*

*

* * * * * * * * * * * * * * * * * * * * * * *

Eizirik et al. PLoS genetics, 2012

Laboratory of Experimental Medicine – ULB, Brussels (2009-ongoing):

“Role of Type 1 diabetes genes in beta cell destruction”

Background – I. Santin

Background – I. Santin

PTPN2, a candidate gene for type 1 diabetes, modulates pancreatic b-cell apoptosis via

regulation of the BH3-only protein Bim

Santin et al. Diabetes 2011

Viral infection

Viral dsRNA

Receptors

(RIG-I, MDA5, TLR3…)

AP-1 NF-kB IRF3-7 STAT1

Pro-inflammatory cytokines and type I IFNs

INSULITIS

IFNAR IFN2R

Immune cells

IFNg

APOPTOSIS

PTPN2 ?

Santin et al. Diabetes 2011

PTPN2, a candidate gene for type 1 diabetes, modulates pancreatic b-cell apoptosis via

regulation of the BH3-only protein Bim

Viral infection

Viral dsRNA

Receptors

(RIG-I, MDA5, TLR3…)

AP-1 NF-kB IRF3-7 STAT1

Pro-inflammatory cytokines and type I IFNs

INSULITIS

IFNAR IFN2R

Immune cells

IFNg

APOPTOSIS

JNK1 JNK1

PTPN2 pBim

Santin et al. Diabetes 2011

PTPN2, a candidate gene for type 1 diabetes, modulates pancreatic b-cell apoptosis via

regulation of the BH3-only protein Bim

Viral infection

Viral dsRNA

Receptors

(RIG-I, MDA5, TLR3…)

AP-1 NF-kB IRF3-7 STAT1

Pro-inflammatory cytokines and type I IFNs

INSULITIS

IFNAR IFN2R

Immune cells

IFNg

APOPTOSIS

JNK1 JNK1

pBim PTPN2

Santin et al. Diabetes 2011

PTPN2, a candidate gene for type 1 diabetes, modulates pancreatic b-cell apoptosis via

regulation of the BH3-only protein Bim

Molecular analysis of IRF7-driven inflammatory gene

network (IDIN). Defining the role of the T1D gene networks

in pancreatic b-cell destruction.

Personal Research Proposal

IRF7-driven inflammatory

gene network – IDIN

Personal Research Proposal

IRF7-driven inflammatory

gene network – IDIN

IDIN genes contribute to T1D risk and implicate innate viral response

pathway in the etiology of T1D.

Loci that perturb this gene network can be important for disease risk

Personal Research Proposal

Dota F et al. PNAS, 2007

Reactivity to VP-1 in

T1D pancreases

Personal Research Proposal

Enteroviral infections in pancreatic b-cells

T1D

C

TR

L

Dota F et al. PNAS, 2007

Reactivity to VP-1 in

T1D pancreases

Personal Research Proposal

Enteroviral infections in pancreatic b-cells

T1D

C

TR

L

Enteroviral infections

affects only b cells

b-c

ell

s

a-c

ells

Chehadeh W et al. Journal of Virology (2000)

Personal Research Proposal

Type I IFNs in T1D

IFNa expression in T1D pancreases

Dota F et al. PNAS (2007)

IFNa expression in T1D

patients blood

Chehadeh W et al. The Journal of Infectious Diseases (2000)

Personal Research Proposal

Type I IFNs in T1D

IPA analysis of T1D candidate genes expressed in b-cells

Santin I et al, unpublished

Personal Research Proposal

Personal Research Proposal

To analyze the role of the T1D gene network IDIN in pancreatic b-cell

inflammation and death, and to establish its interaction with potential

environmental factors

Personal Research Proposal

To analyze the role of the T1D gene network IDIN in pancreatic b-cell

inflammation and death, and to establish its interaction with potential

environmental factors

1. Culture of INS-1E cells and obtaining of primary rat b-cells.

2. Selection of relevant T1D candidate genes in the IDIN gene network.

3. Analysis of the role of IDIN candidate genes in viral dsRNA or type I

IFN-induced b-cell inflammation and death.

4. Analysis of the regulation of IDIN network in pancreatic b-cells.

5. Modulation of the IDIN network to protect pancreatic b-cells from

dsRNA- or type I IFN-induced destruction.

Specific aims

Personal Research Proposal

2. Selection of relevant T1D candidate genes in the IDIN gene network

IDIN gene list

Expression in pancreatic b-cells

(rodent and human)

Genetic association

Relevant function in b-cells

PR

IOR

ITY

T1D base

Beta cell gene Bank

Available GWAS

association data

Bibliographic search

3. Analysis of the role of IDIN candidate genes in viral dsRNA or type I IFN-

induced b-cell inflammation and death.

Personal Research Proposal

Viral infections:

1) Synthetic viral dsRNA (PIC)

2) Type I IFNs (IFNa)

Inflammation Apoptosis

- Chemokine expression by RT-PCR

- Chemokine release by ELISA

- Cell death by HO-PI staining

- Mechanisms:

- ER stress?

- NO production?

- Apoptotic pathways of cell death?

4. Analysis of the regulation of IDIN network in pancreatic b-cells.

Personal Research Proposal

Ebi2 (GPR183): Controls IRF7 in macrophages and represents a candidate for trans-

regulation of the human IDIN and for T1D risk

Ebi2 knockdown

Custom taqman array card of

selected IDIN genes

IDIN genes modulated by Ebi2 in

pancreatic b-cells. Potentially important

for b-cell inflammation and death (after

viral infection)

Cells treated/non-treated with

dsRNA or IFNa

5. Modulation of the IDIN network to protect pancreatic b-cells from dsRNA-

or type I IFN-induced destruction.

Personal Research Proposal

Viral infection

Chemokines and type I IFNs

INFLAMMATION

IFNAR

IDIN gene

network

Type I IFNs

APOPTOSIS

5. Modulation of the IDIN network to protect pancreatic b-cells from dsRNA-

or type I IFN-induced destruction.

Personal Research Proposal

Viral infection

Chemokines and type I IFNs

INFLAMMATION

IFNAR

IDIN gene

network

Type I IFNs

APOPTOSIS

EBI2 Overexpression / Inhibition