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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
1
2
3
4
5
6
7
HL
A
INS
PT
PN
22
ER
BB
3
IL2
RA
FU
T2
SH
2B
3
PT
PN
2
CT
RB
1/2
RN
LS
CO
BL
CY
P2
7B
1
RA
SG
RP
1
PR
KC
Q
CL
EC
16
A
CE
NP
W
IL1
0
GA
B3
CC
R5
GP
R18
3
IFIH
1
CT
LA
4
CT
SH
IL2
7
TY
K2
IL2
BA
CH
2
OR
MD
L3
UB
AS
H3
IKZ
F1
SK
AP
2
GL
IS3
C1Q
TN
F6
AF
F3
RG
S1
STA
T4
DL
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TN
FA
IP3
CD
69
TA
GA
P
SM
AR
CE
1
T1D candidate genes
Od
ds
Ra
tio
> 60% of T1D Candidate genes are expressed
in human islets
*
*
* * * * * * * * * * * * * * * * * * * * * * *
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