Transcript Toll-like Receptors and the Innate Immune Response to

TLR Variation & Susceptibility
to Pulmonary Pathogens
VIDI Symposium
January 22, 2009
Thomas R. Hawn, MD-PhD
University of Washington
Why do infections affect
individuals differently?
Pathogen
Virulence factors
Host
External Factors:
Exposure
Immunosuppression
Host
Genetics
{
Aims
1. Identify Variation
2. Pathogenesis
3. New Rx
Innate Immune Variation & Disease
? OTHER INNATE GENES ?
PATHOGEN
TLR VARIANTS
?
Cytokines &
Chemokines
INFLAMMATION
?
Mf or DC
?
T/B
MICROBIAL
INTERACTION
ADAPTIVE
? CLINICAL DISEASE ?
Outline
1. Mf Expression Profiles & TB Phenotypes
A. “Unbiased” Candidate Gene Selection
B. CCL1, EREG & Human Genetic Studies
2. TLR1 Deficiency & Mycobacteria
A. Cellular studies
B. Leprosy & TLR1 deficiency
3. TLRs & BCG Vaccine Response
Mycobacteria
M. tuberculosis
Infects 1/3 of the world
Leading cause of death
Drug resistance rising
BCG vaccine variable
efficacy
Host susceptibility factors
poorly understood
Animal models inadequate
M. leprae
Clinical phenotype
Th1 vs Th2
Immunologic Rxns
TB Pathogenesis
Droplet
Inhalation
No Infection
80%
Lung Apex
Pulmonary Dz
Lung 1° Infection
Alveolar Mf
Spread to LN
Bacillemia
Primary
Progressive Dz
Latent
90%
Seed Organs
“Rich Foci”
Meninges & Brain
R. Hewlett NEJM (2004)
Disseminated
Miliary
Genetics & Tuberculosis
I. Twin Studies
# Tb cases/100 co-twins
Monozyogotic
31.4
Dizyogotic
14.9
The Prophit Survey by Barbara Simonds. Comstock et al Am Rev Resp Dis 117: 621-624 (1978)
II. Genome-wide scans: TB susceptibility loci at:
15q & Xq, Gambia: Bellamy et al. PNAS 97: 8005-9 (2000)
2q35, Canada: Greenwood et al Am J Hum Gen 67: 405-16 (2000)
8q12-13, Morocco: Baghdadi et al JEM 203: 1679 (2006)
III. Primary immunodeficiency: Mendelian Disorders
IKKg (NEMO), IL-12p40, IL-12R, IFNgR1, IFNgR2, STAT1
Genetics & Tuberculosis
IV. Complex Inheritance: Candidate Gene Studies
Adaptive Immune Response
MHC Class II
Cytokines
IL-1Ra/IL-1b
MCP-1
IL-8
IFNg
Innate Immunity and Macrophage Function
NRAMP1 (SLC11a1)
VDR
P2X7
DC-SIGN
SP110 (IPR-1)
MBP
TLR2
TIRAP
Summary of TLRs and their Ligands
Bacteria--Gr+, Myco
Parasites, Yeast
PAM3
PAM2
LAM
19kd MTb
Gr-
Bacteria Viral
LPS Flagellin dsRNA
Viral
Bacteria &
Virus
ssRNA
CpG DNA
profilin
LPS
MALP2
PGN
?
2/1
2/6
2/X
4
5
3
7
8
9
10
11
Innate Immunity & Tb
Tb
TLR2
TLR1/6
TLR4
Extracellular
TLR9
Cytoplasm
MYD88
TIRAP
Tb
Endosome
Phagosome
NOD2
NF-kB
Nucleus
NF-kB
Activation of immune
response genes
TLR Pathway
Pathogen
CD14
TLR (TLR3 & 9 other TLRs
Extracellular
Adaptors: MyD88,TIRAP, TRIF, TRAM
IRAK1, IRAK2, IRAK4, IRAK-M
Cytoplasm
TLRs3,7,8,9
TRAF6
IKKa, IKKb, IKKg (NEMO)
I-kB
NF-kB
Endosome
Proteosomal
Degradation
Unc93b
Nucleus
NF-kB
Activate immune response:
TNF, IL6, IL12, etc
ER
IFNa,b
Mf array & SNP study:
Which Genes?
Jeremy Thuong Sarah
Farrar Nguyen Dunstan
Hypothesis
I. The gene expression profile of Tb-stimulated Mf differs
between individuals with meningeal, pulmonary, and
latent TB.
II. Polymorphisms in these genes are associated with
susceptibility to meningeal, pulmonary, and latent TB.
2 Problems:
1. Candidate Gene Bias
Known genes
Well-characterized genes
2. Animal Models Inadequate
No reliable animal model for some human phenotypes
(such as TB meningitis, latent TB)
Human vs. Mouse Models
Humans
Mice
Pathogen
Recent clinical sample Ancient lab strain
Low, impure dose
High, pure dose
Natural human tropism Rare animal tropism
Exposure
Natural route
Many organisms
Artificial route
One by one in aseptic condition
Host
Outbred
Frequent vaccinations
Variable ages
Long life span
Inbred
Rare vaccinations
Standardized age
Short life span
From Casanova & Abel (2004) Nat Rev Immunol 4: 55-66.
Mf array study
Goals
I. Identify genes with distinct expression profiles with:
1. TB-stimulated Mf
2. TB-stimulated Mf in different TB clinical types
II. Identify SNPs in these genes associated with
susceptibility to MTb
III. Understand function of these genes & SNPs in Tb
pathogenesis
Nguyen et al PLoS Pathogens 2008
Tuberculosis &
Vietnam
Demographics
N
Mean Age (y):
HIV + (%)
Hemiparesis (%)
Death (%)
Meningitis
175
37.1
0
14.3
18.9
Ho Chi Minh City, Vietnam
The Hospital for Tropical Diseases
Pham Ngoc Thach Hospital for Tuberculosis
Oxford University Clinical Research Unit
Pulmonary
183
40.0
0
Collaborators:
S Dunstan
TT Nguyen
G Thwaites
C Simmons
J Farrar
NTN Lan
TTH Chau
HT Quy
TT Hien
NT Thuong
Array Study Design
Draw Blood
MTB, PTB, LTB
N=4 per group
Affymetrix HGU133 array
47,000 transcripts
30,000 genes
Isolate PBMC
Prepare MDMf by incubating
for 5 days ex vivo
Stimulate: PBS
MTb H37Rv
Lysate
Extract RNA
1. Identify Genes
2. Validate with RT-PCR
(n=34 samples)
3. Functional studies:
A. Human genetic studies
B. Molecular & cellular
Array Results
I. Identify Genes
II. Unsupervised Cluster
Analysis
>5 ratio
(# genes)
2<ratio<5
(# genes)
Select 1608 genes with > 2-fold
change
Up
185
1075
Down
14
334
All TB
Clinical Phenotypes
MTB/PTB
33
450
PTB/MTB
18
500
MTB/LTB
60
1763
LTB/MTB
37
1474
PTB/LTB
54
1688
LTB/PTB
59
1519
Analyzed with V. Thorsson & A. Aderem, ISB
TIGR MeV v 4.0
RT-PCR Validation
N= 86 genes
N=34 independent samples
Overall expression:
90% (52/58) validated
Clinical forms: Difficult to confirm at single gene level.
Genetics & Immunodeficiency
Inheritance
Complexity
Penetrance
Frequency
Age Onset
Features
Primary
“Common”
Mendelian
Monogenic
High
Rare
Early
Severe
Complex
Polygenic
Low for single allele
Common
Variable
Mild
Single Nucleotide Polymorphisms
Genomic
DNA
ATGGACTGGA ...
ATGGTCTGGA ...
SNP
Human Genome:
3 billion bases
30-40k genes
SNPs ~0.1%
Do SNPs:
A. regulate innate immune responses?
B. influence susceptibility to infection?
Genome-Wide Association Study
Genotype 250,000 SNPs with Affymetrix NspI Chip
TBM
n=114
PTB
n=159
Controls
n=188
Oxford University Clinical Research Unit
SJ Dunstan
JJ Farrar
TT Nguyen
Genome Institute of Singapore
Mark Seielstad
Martin Hibberd
Welcome Trust Centre for Human Genetics
University of Oxford
Yik Y. Teo
SNPs & TB Susceptibility
Examine SNP associations among 86 genes identified by
arrays
34 genes with SNPs associated with TB susceptibility
10 with 1 SNP
9 with 2 SNPs
9 with 3-5 SNPs
6 with >5 SNPs
EREG expression in Mtb stimulated MDM
EREG- Epiregulin
soluble-EREG
EGFR
membrane-EREG
Cell Growth & Homeostasis
Autocrine growth factor in human
keratinocytes
Ligand for EGFR
Inhibits growth of epithelial tumor cells.
Stimulates proliferation of cells.
High level expression in various
carcinomas.
Lung metastasis signature genes
Immune Response
Influences cytokine response to
bacterial products (PGN, LPS)
No studies on: TB & EREG
TLRs & EREG
EREG SNPs are associated with TB by GWA
6/12 SNPs are associated with PTB
5’UTR
3’UTR

EPGN
75,400K


75,425K
Marker
Description
rs12646908
rs12641042
rs12646889
rs6846401
rs1812149
rs2125398
up 17306 bp
up 17522 bp
up 17584 bp
up 17693 bp
up 17792 bp
up 22341bp

75,445K
75,435K
Control
1
192 (0.54)
205 (0.55)
209 (0.56)
209 (0.56)
205 (0.54)
183 (0.52)
2
162 (0.46)
167 (0.45)
167 (0.44)
167 (0.44)
167 (0.46)
169 (0.48)

EREG
75,455K
PTB
1
238 (0.65)
248 (0.65)
248 (0.65)
248 (0.65)
231 (0.65)
232 (0.63)
75,465K
75,475K
CvP allelic comparison
P value
2
128 (0.35) 0.003
136 (0.35) 0.008
136 (0.35) 0.011
136 (0.35) 0.011
127 (0.35) 0.009
136 (0.37) 0.003
OR (95%CI)
0.64 (0.47-0.86)
0.67 (0.50-0.80)
0.69 (0.51-0.92)
0.69 (0.51-0.92)
0.68 (0.50-0.92)
0.67 (0.50-0.91)
EREG SNPs are associated with TB by Sequenom
7/12 SNPs are associated with TB
5’UTR
3’UTR

EPGN
75,400K
75,425K
75,435K
75,445K

75,455K
OR P comparison between PTB and controls
OR P comparison between PTB and controls
OR P comparison between TBM and controls
†significantly associated in PTB vs TBM comparison
 not associated with TB
  EREG
75,465K

75,475K
EREG SNPs are associated with TB
12/24 SNPs are associated with TB
5’UTR

3’UTR
EPGN
75,400K

75,425K


75,435K
75,445K


75,455K
  EREG
75,465K


75,475K
EREG expression is modulated by the TLR pathway
Conclusions
1. EREG Induced by TB and TLR ligands
2. EREG SNPs Associated with TB
3. EREG expression is TLR-mediated
TB
EGFR
TLRs
M
M
EREG
Cytokines
IL6, TNFa, IL10
CCL1 Expression Increased in PTB
Gene
Array
P
PCR
P
1.9
0.020
PTB/LTB
CCL1
12.8
0.004
Genotyping
CCL1 Gene : 6/33 SNPs associated with TB
CCL SNPs & TB
CCL2
29,550,000
29,600,000
CCL7
CCL11
CCL8
29,650,000
CCL13 CCL1
29,700,000
OR P comparison between PTB and controls
OR P comparison between TBM and controls
†significantly associated in PTB vs TBM comparison
29,750,000
CCL1 Induced by TLR Ligands
PBMC
HeLa & A549 cells: not induced
CCL1 Expression TLR-mediated
IgG
+
IL-1
Or
LPS
CCL1 (I309)
FcR
Monocyte
CD4
CCL1
CCR8
CD4
Cytokines:
Th2 cell
IL-4, IL-5,
IL-13
B7
1989:
1992:
1997:
1997:
Secreted by activated T cells (Miller JI)
Monocyte chemoattractant (not PMNs) (Miller, PNAS)
Monocytes express CCL1
CCR8 identified as single CCL1 receptor (Tiffany JEM)
CCR8 expressed in Th2 & Treg cells
CCL1-CCR8 implicated in cutaneous T cell homing
(Schaerli JEM)
Antigen Presenting
Cell
2000+ Involved in cell aggregation
CCL1-CCR8 & Granulomas
T
T
T
T
T
T
CCR8
T
T
CCL1
T
T
T
B7
T
Mf
T
T
T
T
CCL1-CCR8 axis regulates Mf
aggregation (Hoshino JI 2007)
Antigen Presenting Cell
T
T
CCL1 upregulated in M. bovis
extract induced granulomas
(Chiu et al AJRCMB 2003)
Mf Array Project: Summary
1. TB-stimulated genes highly replicated
2. Many novel candidates identified
3. TB clinical forms partially distinguished
4. Large number of immunologic genes
5. CCL1 & EREG expression upregulated
& SNPs associated with TB
TLR Immunogenetics
Goals:
1. TLR SNP discovery
LIGAND
RECOGNITION LRR
2. Human genetic studies
3. Functional studies
in vitro signaling
in vivo murine models
membrane
SIGNALING
TIR
Immune Response
392STOP
TLR5
is Associated with
Legionnaires’ Disease
Base Pair:
C1174T
Amino Acid: R392STOP
[IL-6 pg ml-1]
2000
1500
1000
500
0
392RR
392R*
[Flagellin 30 ng ml-1 ]
WT
HET
TLR5 Stop Genotype
freq
OR
P
Legionnaires’ Dis
0.167
Total Controls
0.095 1.90 0.03
95% CI
1.06-3.42
J Exp Med 2003
Common Variation in Cytokine Response
to Bacterial Lipopeptide
Tri-acylated
PAM3
3500
[IL-6 pg/ml]
3000
PAM2
PAM3
4
6
Di-acylated
PAM2
2500
2000
1500
1000
500
0
1
2
3
5
7
8
9 10
Whole Blood Cytokine Assay
TLR2/1
TLR2/6
Innate Immunity & Tb
Tb
TLR2
TLR1/6
TLR4
Extracellular
TLR9
Cytoplasm
MYD88
TIRAP
Tb
Endosome
Phagosome
NOD2
NF-kB
Nucleus
NF-kB
Activation of immune
response genes
TLR1 SNP T1805G is Associated with Impaired Signalling
extracellular
intracellular
TIR domain
Leucine Rich Repeat
TM
Ann Misch
T1805G (I602S)
TLR1
TLR2
TLR1
PAM3:TT/GT
Signalling
(defective)
PAM3:GG
3000
[IL-6 pg/ml]
TLR2
2500
2000
1500
1000
500
0
602I
1805T
602S
1805G
0
100
200
300
[PAM3 ng/ml]
400
RLU
1805G (602S) NF-kB Signaling Impaired
3000
2500
2000
1500
1000
500
0
pef 6
MTB
2/602I
2/602S
MED
1200
IRR 25
IRR 125
M. leprae
1000
CW 5
MEM 1
MEM 5
EV
T2+ 602I
T2+ 602S
800
RLU
CW 1
HEK293 cells
NF-kB luciferase
600
400
200
0
Media
P3 300
M2 100
ML 5
ML 50
ML 250
MLcw 1
MLcw10
Impaired IL6 response to M. leprae
in 1805GG Primary Cells
PBMCs
TLR SNPs Regulate DC Cytokines
IL6 [pg/ml]
T1T5+
T1T53500
1000
750
250
200
100
0
Media
LPS
PAM3 50
PAM3 250
FliC 50
Flic 250
Anandaban Leprosy
Hospital, Nepal
Collaborators
Murdo McDonald
Chaman Ranjit
Bishwa Sapkota
Ruby Siddiqui
Gilla Kaplan
Case Control Study
n
Leprosy
930
LL,BL,BB
582
TT, BT
348
RR
237
ENL
107
TLR1 SNP T1805G is Associated w/ Protection
from Reversal Reaction in Leprosy
TLR1 SNP T1805G Allele Frequency
T
No RR
Yes RR
G
1115(0.926)
OR (95%CI)
P
89(0.074)
392(0.961) 16(0.039)
0.51 (0.30-0.88)
0.01
TLR1 SNP T1805G Genotype Frequency
TT
TG
GG
OR (95%CI)
P
TT v. TG/GG
No RR
522(0.867)
71(0.118) 9(0.015)
Yes RR
188(0.922)
16(0.078) 0(0.000) 0.56 (0.32-0.97)
0.038
TLR1 T1805G Association Studies
TLR2
TLR1
1. G ↓Leprosy Reversal Rxn
Hawn EJI 2007
Misch & Hawn PLoS NTD 2008
2. G ↓Leprosy
T1805G
I602S
Johnson JI 2007
3. TT ↑TB
Ma Plos1 2007
4. TT ↑ sepsis mortality
Wurfel AJRCCM 2008
5. G ↑ malaria
Leoratti CID 2008
Leprosy
Myco
TLR
Isolated lesions
Diffuse lesions
Mf or DC
TH2
TH1
IL-12
IL-12R LEPROMATOUS
TUBERCULOID
REVERSAL RXN
T cell
IL2
IFNg
IL4/IL13
IFNgR
GRANULOMA
RARE BACILLI
NO GRANULOMA
ABUNDANT BACILLI
BCG Efficacy
All Ages
Rand trials
RR of Tb
0.49
(0.34-0.70)
Case control
0.50
(0.39-0.64)
Death
0.29
(0.16-0.53)
Meningitis
0.36
(0.18-0.70)
BUT, Efficacy varies from 0 to 80%
Why: 1. BCG Strain Differences
2. Environmental Effect
3. Host Genetics
Colditz et al JAMA 1994
Hypothesis:
Innate immune gene polymorphisms
regulate in vivo immune response to BCG
& vaccine efficacy
Cape Town Collaborators:
Willem Hanekom
Gilla Kaplan
Greg Hussey
BCG & T Cell Responses
? TLR
SNP
BCG
TLR
? TLR
SNP
Mf or DC
IL-12
IL-12R
TH1
? PROTECTION
TH2
? SUSCEPTIBLE
T cell
IL2
IFNg
IL4/IL5/IL10/IL13
IFNgR
MICROBIAL KILLING
MICROBIAL GROWTH
Immune Correlates Study Design
Birth: Vaccinate 11,680 infants w/ BCG
10 wks: Blood draw, cryopreserve
Immune assays
Innate
Adaptive
Genotype
Prospective Study over 5 years
A. Cases with TB
B. Exposed controls w/o TB
Study Design:
Innate Immune Assays
Birth: BCG vaccination, n=11,680
10 wks: Blood Draw, Cryopreserve PBMCs
4-6 yrs later: Thaw
Purify genomic DNA
Stimulate:
Gene
TLR 2/1
TLR 2/6
TLR4
TLR9
NOD2
Multiple
Ligand
PAM3
PAM2
LPS
CpG DNA
MDP/LPS co-stimulation
MTb H37Rv lysate
Measure:
24h: TNF-a, IL-6, IL-1b, & IL-12
48h: IFN-a for TLR9 stimulation
April
Randhawa
BCG & Adaptive Immune Response
Birth: BCG vaccination
10 wks: Blood Draw
Stimulate with BCG ex vivo x 7hrs
Freeze supernatants
Measure: IFN-g, IL-2, IL-10, IL-13
TLR1_T1805G varies by ethnicity
TT
TG
GG
Vietnam
European-American
African American
Vietnam
1
14
368
9
8
7
13
2
0
Cape Town
121 31
1805GG
2
1805GT
1805TT
[IL-6 pg/ml]
3000
2500
2000
1500
1000
500
0
0
100
200
300
[PAM3 ng/ml]
400
In vivo: TLR1_T1805G & BCG
1400
1200
1200
1000
1000
IFN-gamma
1400
800
600
400
800
600
400
200
200
0
0
-200
-200
N=
2
31
121
G/G
T/G
T/T
p=0.3
N=
2
31
121
G/G
T/G
T/T
p=0.3
PAM3 & TB
TLR SNPs &
Lipopeptides
Lipopeptide
TB
TLR1
T1805G
TLR2
C597T
 IL6
  IL6
TLR1
Regulates PAM3
TIRAP
meningeal TB
(OR 3.0, p<0.001)
meningeal > pulmonary
TIRAP
C558T
1200.0
1000.0
[IL-6 pg/ml]
TLR2
meningeal TB
(OR 1.5, p=0.007)
meningeal > pulmonary
Cytokine response unknown
800.0
*
*
558CC
600.0
558TT
400.0
200.0
0.0
PAM2
80
PAM2 PAM3
320
80
PAM3
320
[ng/ml]
TLR2_C597T & TLR Ligands
700
200
TNF-alpha (pg/mL)
600
180
160
140
120
100
80
60
40
400
300
200
100
0
20
-100
0
N=
-20
N=
500
25
52
13
C/C
T/C
T/T
25
52
13
C/C
T/C
T/T
Media
LPS
700
600
600
TNF-alpha (pg/mL)
700
500
400
300
200
100
0
400
300
200
100
0
-100
N=
500
25
52
13
C/C
T/C
T/T
PAM2, P=0.02, CC vs TT
-100
N=
25
52
13
C/C
T/C
T/T
PAM3, P=0.057 CC vs TT
Adaptive: TLR2_C597T & BCG
1400
1200
1200
1000
1000
IFN-gamma
1400
800
600
400
800
600
400
200
200
0
0
-200
-200
N=
43
89
26
C/C
T/C
T/T
P=0.8
N=
43
89
26
C/C
T/C
T/T
P=0.67
Adaptive: TIRAP_C558T & BCG
2000
1400
1800
1200
IFN-gamma (pg/mL)
1600
1400
1200
1000
800
600
400
200
1000
800
600
400
200
0
0
-200
N=
-200
128
25
3
C/C
T/C
T/T
p=0.09
N=
128
25
3
C/C
T/C
T/T
p=0.69
Innate Immune Variation & Disease
PATHOGEN
TLR VARIANTS
TLR1,2,4, 5 & TIRAP
Mf or DC
Cytokines &
Chemokines
INFLAMMATION
BCG Immunity
T/B
OTHER INNATE GENES: CCL1
EREG
ADAPTIVE
CLINICAL DISEASE:
Legionella, Tb, & Leprosy
EFFECTOR
MECHANISMS
Acknowledgements: Collaborators
Seattle: ISB & UW
A Aderem
V Thorsson P Bochud
A Nachman M Janer
S Rodrigues S Li
S Gagneux P Small
Tb & Vietnam
S Dunstan NTN Lan
TT Nguyen TTH Chau
G Thwaites HT Quy
C Simmons NT Thuong
J Farrar
TT Hien
Seattle-HMC
S Skerrett
R Iyer
M JanssonHutson
I Smith
Other
M Seielstad
M Hibberd
YY Teo
A Verbon
South Africa
W. Hanekom
G. Hussey
M. Shey
NJ, PHRI
G. Kaplan
Nepal
Murdo MacDonald
Deanne Hagge
Bishwa Sapkota
Chaman Ranjit
Ruby Siddiqui
Acknowledgements
UW Lab
Matt Arentz
Bill Berrington
Carey Cassidy
E Ann Misch
April Randhawa
Rick Wells
Jay Vary
Funding: NIH, Dana Foundation, Heiser Program,
Puget Sound Partners for Global Health
TLR Genetics & Tb
TLR2
TLR2 SNPs
1. C597T (N199N)
Nguyen G&I 2007
N199N
LRR
2. Intron 2 GT VNTR
Yim Genes Imm 2006
3. G2258A (R753Q)
TB
Lyme
Ogus ERJ 2004
Schroeder JI 2005
TLR1
I602S
TLR1 SNPs
T1805G (I602S)
G ↓Leprosy
Johnson JI 2007
G ↓Leprosy RR
Misch PLoS NTD 2008
TB
TT ↑↑TB
Ma Plos1 2007
TIR
R753Q
A186A
S180L
TIRAP/MAL
TIRAP/MAL SNPs
1. C558T (A186A)
Hawn JID 2006
2. C539T (S180L)
TB
Malaria
Pneumococcus
Bacteremia
Khor Nat Gen 2007
TLR SNPs & Function
TLR4
A896G (D299G)
Arbour Nat Gen 2000
4
3
TLR5
C1174T (R392*)
Hawn JEM 2003
5
2
1
TLR1
T1805G (I602S)
LRR
TIR
TLR3
C1236T (L412P)
Yang NEJM 2008
MAL
A186A
TLR2
G2258A R753Q)
Ogus ERJ 2004
Schroeder JI 2005
S180L
TIRAP/MAL
1. C558T (A186A)
Hawn JID 2006
2. C539T (S180L)
Khor Nat Gen 2007