Innate Immune Variation & Disease ? CLINICAL DISEASE ?
ADAPTIVE Cytokines & Chemokines INFLAMMATION T/B MICROBIAL
INTERACTION PATHOGEN M or DC ? ? ? TLR VARIANTS ? OTHER INNATE
GENES ?
Slide 4
Outline 1. M 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
Slide 5
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
Slide 6
TB Pathogenesis Droplet Inhalation No Infection R. Hewlett NEJM
(2004) Lung Apex Pulmonary Dz Seed Organs Disseminated Miliary Rich
Foci Meninges & Brain 80% Lung 1 Infection Alveolar M Spread to
LN Bacillemia 90% Primary Progressive Dz Latent
Slide 7
Genetics & Tuberculosis I. Twin Studies # Tb cases/100
co-twins Monozyogotic31.4 Dizyogotic14.9 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 IKK (NEMO), IL-12p40, IL-12R, IFN R1, IFN R2,
STAT1
Slide 8
Genetics & Tuberculosis IV. Complex Inheritance: Candidate
Gene Studies Adaptive Immune Response MHC Class II Cytokines
IL-1Ra/IL-1 IL-8 MCP-1IFN Innate Immunity and Macrophage Function
NRAMP1 (SLC11a1)VDR P2X7 DC-SIGN SP110 (IPR-1) MBP TLR2TIRAP
Slide 9
CpG DNAFlagellin PGN LPS PAM3 MALP2 PAM2LAMssRNA ? Summary of
TLRs and their Ligands 2/12/62/X45379108 Bacteria--Gr+, Myco
Parasites, Yeast dsRNA Viral Gr- BacteriaViralBacteria & Virus
19kd MTb 11 profilin
Slide 10
Innate Immunity & Tb Activation of immune response genes
NF- B Extracellular Nucleus Cytoplasm NF- B Tb TLR2 TLR1/6 TLR9
Endosome MYD88 TIRAP Phagosome Tb TLR4 NOD2
M array & SNP study: Which Genes? Hypothesis I. The gene
expression profile of Tb-stimulated M differs between individuals
with meningeal, pulmonary, and latent TB. II. Polymorphisms in
these genes are associated with susceptibility to meningeal,
pulmonary, and latent TB. Jeremy Thuong Sarah Farrar Nguyen
Dunstan
Slide 13
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)
Slide 14
Human vs. Mouse Models HumansMice Pathogen Recent clinical
sampleAncient lab strain Low, impure doseHigh, pure dose Natural
human tropismRare animal tropism Exposure Natural routeArtificial
route Many organismsOne by one in aseptic condition Host
OutbredInbred Frequent vaccinationsRare vaccinations Variable
agesStandardized age Long life spanShort life span From Casanova
& Abel (2004) Nat Rev Immunol 4: 55-66.
Slide 15
M array study Goals I. Identify genes with distinct expression
profiles with: 1. TB-stimulated M 2. TB-stimulated M in d ifferent
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
Slide 16
Collaborators: S DunstanNTN Lan TT NguyenTTH Chau G ThwaitesHT
Quy C SimmonsTT Hien J FarrarNT Thuong Ho Chi Minh City, Vietnam
The Hospital for Tropical Diseases Pham Ngoc Thach Hospital for
Tuberculosis Oxford University Clinical Research Unit
DemographicsMeningitisPulmonary N 175183 Mean Age (y): 37.140.0 HIV
+(%) 0 0 Hemiparesis (%)14.3 Death (%)18.9 Tuberculosis &
Vietnam
Slide 17
Array Study Design Draw Blood MTB, PTB, LTB N=4 per group
Isolate PBMC Prepare MDM by incubating for 5 days ex vivo
Stimulate: PBS MTb H37Rv Lysate Extract RNA Affymetrix HGU133 array
47,000 transcripts 30,000 genes 1. Identify Genes 2. Validate with
RT-PCR (n=34 samples) 3. Functional studies: A. Human genetic
studies B. Molecular & cellular
Slide 18
Array Results >5 ratio (# genes) 2
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
Slide 24
EREG expression in Mtb stimulated MDM
Slide 25
soluble-EREG membrane-EREG EGFR EREG- Epiregulin Immune
Response Influences cytokine response to bacterial products (PGN,
LPS) No studies on: TB & EREG TLRs & 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
Slide 26
EREG SNPs are associated with TB by GWA 6/12 SNPs are
associated with PTB rs1812194 rs12641042 3UTR 75,425K 75,445K
75,435K 75,455K 75,465K 75,475K EREG 5UTR 75,400K EPGN rs2125398
rs6846401 rs12646889 rs12646908
Slide 27
EREG SNPs are associated with TB by Sequenom 7/12 SNPs are
associated with TB rs968061 OR 0.7; P 0.03 rs4694190 OR 0.6; P 0.02
3UTR 75,425K 75,445K 75,435K 75,455K 75,465K 75,475K EREG 5UTR
75,400K EPGN rs2085594 OR 0.6; P 0.0006 rs12641042 OR 0.7; P 0.02
rs1563826 P 0.007 rs6446991 OR 0.7; P 0.03 rs6446992 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
Slide 28
EREG SNPs are associated with TB 12/24 SNPs are associated with
TB rs1812149 rs12646908 3UTR 75,425K 75,445K 75,435K 75,455K
75,465K 75,475K EREG 5UTR 75,400K EPGN rs2085594rs2125398 rs6846401
rs12646889 rs12641042 rs1563826 rs4694190 rs968061 rs6446991
rs6446992
Slide 29
EREG expression is modulated by the TLR pathway
Slide 30
Conclusions 1.EREG Induced by TB and TLR ligands 2. EREG SNPs
Associated with TB 3. EREG expression is TLR-mediated TB TLRs EGFR
EREG Cytokines IL6, TNFa, IL10 MM MM
Slide 31
CCL1 Expression Increased in PTB GeneArrayPPCRP PTB/LTB
CCL112.80.0041.90.020 Genotyping CCL1 Gene : 6/33 SNPs associated
with TB
Slide 32
29,550,00029,600,00029,650,00029,700,00029,750,000 CCL2 CCL7
CCL11 CCL8 CCL13 CCL1 rs210837 OR 0.6, P 0.014 rs159294 OR 0.5, P
0.001 rs159291 OR 1, P 0.023 rs159290 OR 0.7, P 0.019 rs159289 OR
0.7, P 0.015 rs159319 OR 0.7, P 0.023 rs3091324 rs10491110 OR 1.7,
P 0.012 OR P comparison between PTB and controls OR P comparison
between TBM and controls significantly associated in PTB vs TBM
comparison CCL SNPs & TB rs3138031 OR 1.7, P 0.024
Slide 33
CCL1 Induced by TLR Ligands PBMC HeLa & A549 cells: not
induced
Slide 34
CCL1 Expression TLR-mediated
Slide 35
CCL1 (I309) Antigen Presenting Cell CD4 Th2 cell B7 Monocyte
CD4 FcR Cytokines: IL-4, IL-5, IL-13 CCL1 IL-1 Or LPS IgG + CCR8
1989: Secreted by activated T cells (Miller JI) 1992: Monocyte
chemoattractant (not PMNs) (Miller, PNAS) 1997: Monocytes express
CCL1 1997: CCR8 identified as single CCL1 receptor (Tiffany JEM)
CCR8 expressed in Th2 & Treg cells CCL1-CCR8 implicated in
cutaneous T cell homing (Schaerli JEM) 2000+Involved in cell
aggregation
Slide 36
CCL1-CCR8 & Granulomas Antigen Presenting Cell T B7
CCL1-CCR8 axis regulates M aggregation (Hoshino JI 2007) CCL1 CCR8
CCL1 upregulated in M. bovis extract induced granulomas (Chiu et al
AJRCMB 2003) TTTTTTTTTTTTTTTTT MM
Slide 37
M 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
Slide 38
TLR Immunogenetics Immune Response membrane LIGAND RECOGNITION
SIGNALING Goals: 1. TLR SNP discovery 2. Human genetic studies 3.
Functional studies in vitro signaling in vivo murine models TIR
LRR
Slide 39
TLR5 392STOP is Associated with Legionnaires Disease Base
Pair:C1174T Amino Acid:R392STOP [Flagellin 30 ng ml -1 ] WT HET J
Exp Med 2003 TLR5 Stop Genotype freq OR P95% CI Legionnaires Dis
0.167 Total Controls0.095 1.90 0.031.06-3.42
Slide 40
Common Variation in Cytokine Response to Bacterial Lipopeptide
Whole Blood Cytokine Assay PAM3PAM2 Di-acylated TLR2/1TLR2/6
Tri-acylated
Slide 41
Innate Immunity & Tb Activation of immune response genes
NF- B Extracellular Nucleus Cytoplasm NF- B Tb TLR2 TLR1/6 TLR9
Endosome MYD88 TIRAP Phagosome Tb TLR4 NOD2
Slide 42
TM T1805G (I602S) Leucine Rich Repeat TIR domain
intracellularextracellular 602I 1805T 602S 1805G TLR2 TLR1 TLR2
TLR1 TLR1 SNP T1805G is Associated with Impaired Signalling
Signalling (defective) Ann Misch
Slide 43
1805G (602S) NF- B Signaling Impaired HEK293 cells NF-kB
luciferase MTB M. leprae
Slide 44
Impaired IL6 response to M. leprae in 1805GG Primary Cells
PBMCs
Slide 45
TLR SNPs Regulate DC Cytokines T1T5+ T1T5-
Slide 46
Anandaban Leprosy Hospital, Nepal Collaborators Murdo McDonald
Chaman Ranjit Bishwa Sapkota Ruby Siddiqui Gilla Kaplan Case
Control Study n Leprosy930 LL,BL,BB582 TT, BT348 RR237 ENL107
Slide 47
TLR1 SNP T1805G is Associated w/ Protection from Reversal
Reaction in Leprosy TLR1 SNP T1805G Genotype Frequency TTTGGGOR
(95%CI) TT v. TG/GG P 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 SNP T1805G
Allele Frequency TGOR (95%CI)P No RR 1115(0.926)89(0.074) Yes RR
392(0.961)16(0.039)0.51 (0.30-0.88)0.01
Slide 48
TLR1 T1805G Association Studies TLR2TLR1 T1805G I602S 1.G
Leprosy Reversal Rxn Hawn EJI 2007 Misch & Hawn PLoS NTD 2008
2. G Leprosy Johnson JI 2007 3. TT TB Ma Plos1 2007 4. TT sepsis
mortality Wurfel AJRCCM 2008 5. G malaria Leoratti CID 2008
Slide 49
Leprosy M or DC TLR Myco IL-12 T cell IL-12R IL4/IL13 TH2
LEPROMATOUS NO GRANULOMA ABUNDANT BACILLI IFN IFN R TH1 TUBERCULOID
REVERSAL RXN GRANULOMA RARE BACILLI IL2 Isolated lesions Diffuse
lesions
Slide 50
BCG Efficacy All AgesRR of TbDeathMeningitis Rand trials0.49
0.290.36 (0.34-0.70)(0.16-0.53)(0.18-0.70) Case control0.50
(0.39-0.64) BUT, Efficacy varies from 0 to 80% Why: 1. BCG Strain
Differences 2. Environmental Effect 3. Host Genetics Colditz et al
JAMA 1994
Slide 51
Hypothesis: Innate immune gene polymorphisms regulate in vivo
immune response to BCG & vaccine efficacy Cape Town
Collaborators: Willem Hanekom Gilla Kaplan Greg Hussey
Slide 52
BCG & T Cell Responses M or DC TLR BCG IL-12 T cell IL-12R
IFN IFN R TH1 ? PROTECTION MICROBIAL KILLING ? TLR SNP
IL4/IL5/IL10/IL13 TH2 ? SUSCEPTIBLE MICROBIAL GROWTH ? TLR SNP
IL2
Slide 53
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
Slide 54
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: GeneLigand TLR 2/1PAM3 TLR 2/6PAM2
TLR4LPS TLR9CpG DNA NOD2MDP/LPS co-stimulation MultipleMTb H37Rv
lysate Measure: 24h: TNF- , IL-6, IL-1b, & IL-12 48h:IFN- for
TLR9 stimulation April Randhawa
Slide 55
BCG & Adaptive Immune Response Birth: BCG vaccination 10
wks: Blood Draw Stimulate with BCG ex vivo x 7hrs Freeze
supernatants Measure: IFN- , IL-2, IL-10, IL-13
TLR2_C597T & TLR Ligands Media PAM2, P=0.02, CC vs TT PAM3,
P=0.057 CC vs TT LPS
Slide 60
Adaptive: TLR2_C597T & BCG P=0.8 P=0.67
Slide 61
Adaptive: TIRAP_C558T & BCG p=0.69p=0.09
Slide 62
Innate Immune Variation & Disease ADAPTIVE Cytokines &
Chemokines INFLAMMATION T/B EFFECTOR MECHANISMS PATHOGEN M or DC
TLR VARIANTS OTHER INNATE GENES: CCL1 EREG TLR1,2,4, 5 & TIRAP
CLINICAL DISEASE: Legionella, Tb, & Leprosy BCG Immunity
Slide 63
Acknowledgements: Collaborators Tb & Vietnam S DunstanNTN
Lan TT NguyenTTH Chau G ThwaitesHT Quy C SimmonsNT Thuong J
FarrarTT Hien Seattle: ISB & UW A Aderem V Thorsson P Bochud A
NachmanM Janer S RodriguesS Li S GagneuxP Small Other M Seielstad M
Hibberd YY Teo A Verbon NJ, PHRI G. Kaplan Nepal Murdo MacDonald
Deanne Hagge Bishwa Sapkota Chaman Ranjit Ruby Siddiqui South
Africa W. Hanekom G. Hussey M. Shey Seattle-HMC S Skerrett R Iyer M
Jansson- Hutson I Smith
Slide 64
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
Slide 65
Slide 66
TLR Genetics & Tb TIR LRR TLR2TLR1 TIRAP/MAL S180L A186A
TLR1 SNPs T1805G (I602S) G Leprosy Johnson JI 2007 G Leprosy RR
Misch PLoS NTD 2008 TB TT TB Ma Plos1 2007 R753Q N199N TLR2 SNPs 1.
C597T (N199N) Nguyen G&I 2007 2. Intron 2 GT VNTR Yim Genes Imm
2006 3. G2258A (R753Q) TB Lyme Ogus ERJ 2004 Schroeder JI 2005
TIRAP/MAL SNPs 1. C558T (A186A) Hawn JID 2006 2. C539T (S180L) TB
Malaria Pneumococcus Bacteremia Khor Nat Gen 2007 I602S
Slide 67
TLR SNPs & Function S180L A186A TLR1 T1805G (I602S) TLR2
G2258A R753Q) Ogus ERJ 2004 Schroeder JI 2005 TIRAP/MAL 1. C558T
(A186A) Hawn JID 2006 2. C539T (S180L) Khor Nat Gen 2007 TIR TLR4
A896G (D299G) Arbour Nat Gen 2000 LRR 4 5 2 1 MAL TLR5 C1174T
(R392*) Hawn JEM 2003 3 TLR3 C1236T (L412P) Yang NEJM 2008