tlr variation & susceptibility to pulmonary pathogens vidi symposium january 22, 2009 thomas r....

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 ? CLINICAL DISEASE ? ADAPTIVE Cytokines & Chemokines INFLAMMATION T/B MICROBIAL INTERACTION PATHOGEN M or DC ? ? ? TLR VARIANTS ? OTHER INNATE GENES ?

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

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 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

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

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

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

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

TLR Pathway CD14 TLR (TLR3 & 9 other TLRs Pathogen Adaptors: MyD88,TIRAP, TRIF, TRAM IRAK1, IRAK2, IRAK4, IRAK-M Activate immune response: TNF, IL6, IL12, etc NF-kB Extracellular Nucleus Cytoplasm TRAF6 IKK , IKK , IKK (NEMO) NF- B I- B Proteosomal Degradation TLRs3,7,8,9 IFN , Endosome Unc93b ER

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

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 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.

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

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

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

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

EREG expression in Mtb stimulated MDM

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

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

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

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

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 TLRs EGFR EREG Cytokines IL6, TNFa, IL10 MM MM

CCL1 Expression Increased in PTB GeneArrayPPCRP PTB/LTB CCL112.80.0041.90.020 Genotyping CCL1 Gene : 6/33 SNPs associated with TB

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

CCL1 Induced by TLR Ligands PBMC HeLa & A549 cells: not induced

CCL1 Expression TLR-mediated

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

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

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

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

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

Common Variation in Cytokine Response to Bacterial Lipopeptide Whole Blood Cytokine Assay PAM3PAM2 Di-acylated TLR2/1TLR2/6 Tri-acylated

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

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

1805G (602S) NF- B Signaling Impaired HEK293 cells NF-kB luciferase MTB M. leprae

Impaired IL6 response to M. leprae in 1805GG Primary Cells PBMCs

TLR SNPs Regulate DC Cytokines T1T5+ T1T5-

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

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

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

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

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

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 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

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: 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

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

TLR1_T1805G varies by ethnicity TTTGGG Vietnam European-American1913 African American1482 Vietnam 36870 Cape Town121312

In vivo: TLR1_T1805G & BCG p=0.3

TLR SNPs & Lipopeptides TLR1 Regulates PAM3 IL6 PAM3 & TB TB TLR1 T1805G TLR2 C597T TLR2 meningeal TB (OR 1.5, p=0.007) meningeal > pulmonary Cytokine response unknown TIRAP C558T TIRAP meningeal TB (OR 3.0, p pulmonary IL6 Lipopeptide [ng/ml] * *

TLR2_C597T & TLR Ligands Media PAM2, P=0.02, CC vs TT PAM3, P=0.057 CC vs TT LPS

Adaptive: TLR2_C597T & BCG P=0.8 P=0.67

Adaptive: TIRAP_C558T & BCG p=0.69p=0.09

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

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

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 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

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

tlr variation & susceptibility to pulmonary pathogens vidi symposium january 22, 2009 thomas r....

Documents

profilin slide

stat1 slide

latent tb

tb susceptibility loci

mbp tlr2tirap slide

innate genes

gene expression profile

endosome unc93b er slide