Immunogenetics & TB Treatment Strategies: Hope or Hype?
Thomas Hawn, MD-PhD University of Washington Division of Allergy & Infectious Diseases
Advancing HDT for TB NIH Workshop April 15, 2014 Outline
1. The Challenge 2. Mendelian Lessons 3. Complex Lessons 4. Lessons from other infections 5. Future: Therapeutic Implications 1. Massive Genetic Variation: Random or Regulatory?
Genomic DNA ATGGACTGGA ... ATGGTCTGGA ...
Single Nucleotide Polymorphism
• Human Genome: 3 billion bases • SNPs ~0.1%
Which SNPs: alter cell function? affect susceptibility to TB? 2. Redundancy: Signaling Nightmares
47 Chemokines 23 Type I Cytokines 17 Type II Cytokines 9 TNF Family
Aderem & Smith 2004 Why do TB and BCG affect individuals differently?
Pathogen Virulence factors
Aims Host Host 1. Identify Variation External Factors: Genetics 2. Pathogenesis Exposure Immunosuppression 3. New Rx Complex Inheritance: TB
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) Genetics & Immunodeficiency
Alcais et al JCI 2009 Casanova Ann Rev Gen 2013
Primary Major Gene “Common” Inheritance Mendelian Complex Complexity Monogenic Polygenic Penetrance High Moderate Low for single allele Frequency Rare Common Age Onset Early Variable Features Severe Mild Type Mutations Polymorphisms Genetics & Mycobacteria Rare Mendelian Inheritance Mostly BCG, NTM infections & Salmonella Tb TLR Mor DC IL-12 Mutations IKK IKK T cell IL-12p40 IL-12R IL-12R IFNR1 IFNR2 IFNR IFN STAT1 Abs
STAT1 JL Casanova & L Abel Annu Rev Immunol 20: MICROBIAL 581-620 (2002) KILLING Specificity & TLR Mendelian Diseases Extracellular Pathogen CD14 TLRs 1/2/4/5/6 MyD88,TIRAP, TRIF, TRAM IRAK1, IRAK2, IRAK4, IRAK-M TLR3
Unc93b TRIF IKK, IKK, IKK (NEMO) TRAF3, TBK1 I-B Susceptible to: NF-B IRF3 & 7 Nucleus Extracellualar HSV Bacteria
NF-kB IRF3& 7 TNF, IL12, IL6 IFN, Mendelian Resistance HIV & CCR532 & Maraviroc
Malaria & Duffy Factor (DARC) Plasmodia
RBC
Duffy Factor Receptor required for P. vivax entry Sickle Cell Anemia pts protected against malaria Complex Inheritance: TB IIA. Genome-wide linkage scans
TB Disease 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)
TB Infection 11p14, 5p15 South Africa: Cobat et al JEM 2009 2q21, 5p13 Uganda: Stein, Boom et al Plos1 2008
IIB. GWAS 18q11.2 Thye et al Nat Gen 2010; 11p3 Thye Nat Gen 2012 BCG Pathogenesis Primary Droplet Lung 1° Infection Progressive Dz Inhalation Alveolar M Spread to LN Latent 90% Bacillemia No Infection 10%
80% Reactivate
Lung Apex “Rich Foci” Disseminated Pulmonary Dz Meninges & Brain Miliary “Reactivation” Lymph Node Bone (Pott’s Disease) R. Hewlett NEJM (2004) Genitourinary Why Failure?
Paucity of phenotypic discrimination TB infection vs TB Disease Site of TB Disease Pulmonary vs meningeal vs other Primary vs reactivation Children vs adults Treatment response Zhang et al NEJM 2009
N=3254 cases 5955 controls TLR1 T1805G & Leprosy
A. Misch & Hawn PLoS NTD 2008 1805G & leprosy RR B. Johnson et al J Immunol 2007 1805G & leprosy: OR 0.48 (0.29-0.80)
C. Wong et al PlosPathogens 2010
Genotyped 50,000 SNPs in 2,092 genes
2 major associations: 1. HLA-DRB1/DQA1 2. TLR1 T1805G (I602S) & leprosy
Combined analysis of 3 case-control studies 1805G: OR 0.37 P=1.7 x 10-9 Human TLR1/6 Deficiency epidemiology
Signal High frequency ↓ ↓ lipopeptide defect TLR2 ethnic variation signaling TLR1/6 SNPs
Cellular phenotype Clinical PAM3:TT/GT PAM3:GG phenotypes 3000 2500 2000 1500 1000
[IL-6 pg/ml] 500 0 0 100 200 300 400 [PAM3 ng/ml] ?leprosy RR, sepsis, malaria HCV & Innate Immunity
Extracellular HCV GWAS IL28B (IFNL3) SNPs HCV PAMPs TLR3 & HCV self cure PKR, RIG-I, MAVS TRIF HCV Rx TRAF3, TBK1 response IRF3 & 7 IRF3 & 7
IRF3& 7 IFN,, Kills HCV Complex Inheritance: TB
IV. TB Candidate Gene Association Studies Adaptive Immune Response MHC Class II
Cytokines/Chemokines/Inflammatory Pathways IL-1Ra/IL-1 IL-8 LTA4H MCP-1 IFN-
Innate Immunity and Macrophage Function NRAMP1 (SLC11a1) VDR P2X7 DC-SIGN SP110 (IPR-1) MBP TLR1, 2,8 TIRAP NOD2 TOLLIP LTA4H, TNF, & TB Meningitis
LTA4H SNP rs17525495 associated with higher promoter activity, higher mRNA levels, higher CSF WBC, and improved survival after dexamethasone Rx
Tobin et al Cell 2010, Tobin et al Cell 2012 TOLLIP rs5743899 & susceptibility to TB?
Extracellular IL-1R MTB Autophagy TLRs
MyD88 MyD88 IRAK1 IRAK1
LC3 Phagosome p62 Auto- NF-B TOLLIP phagosome Nucleus Auto-phagolysosome with MTB Killing NF-kB TNF, IL12, IL6 NO, ROI, anti-microbial peptides Is TOLLIIP-deficiency Associated with Adult TB?
N Cord Blood Controls (N) 762 Adults with Pulmonary TB (N) 385 Adults with Meningeal TB (N) 307 HIV-infected (%) 0 Age (Mean) 38 Jeremy Farrar Sarah Dunstan Sex (% female) 37 Ho Chi Minh City, Vietnam The Hospital for Tropical Diseases Pham Ngoc Thach Hospital for Tuberculosis Oxford University Clinical Research Unit TOLLIP rs5743899 Associated with risk of both pulmonary & meningeal TB Case Control & TOLLIP
Control Case (frequency) (frequency) Geno Dom 00 01 11 00 01 11 P P OR TOLLIP rs5743854 0.416 0.495 0.089 0.535 0.351 0.114 0.001 0.002 0.619 rs5743890 0.939 0.061 0.000 0.974 0.026 0.000 0.037 0.037 0.405 rs5743899 0.316 0.504 0.180 0.394 0.459 0.147 0.092 0.033 0.711 rs5743915 0.340 0.470 0.190 0.250 0.518 0.232 0.039 0.013 1.545 rs7481967 0.308 0.527 0.166 0.403 0.437 0.160 0.026 0.009 0.659
Cape Town Collaborators: W. Hanekom, M. Hatherill, T. Scriba, M. Shey, M. Musvosi, & SATVI Team Human TOLLIP Deficiency Rs5743899 mimics knockdown
C 20 * 15 10 6 4
TOLLIP mRNA 2 0 Javeed Shah A/A A/G G/G rs5743899
Associated with susceptibility to adult TB in Vietnam Genotypic analysis P<1x 10-7 TOLLIP: Negative Regulator of TLR1/2/4/6-mediated IL-6
A Experiment 20000 none * 1. shRNA Knockdown 15000 control in monocytes 10000 TOLLIP 2. Stimulate with TLR 5000 * * ligands or MTB x 3000 24h [IL-6 pg/ml] 2000 1000 3. ELISA 0 Med PAM2PAM3 LPS MTb TOLLIP: Negative Regulator of TLR1/2/4/6-mediated IL-6
ExperimentA D 20000 1. Isolate PBMC,none n=64 * 10000 ** 15000 control 8000 healthy10000 volunteersTOLLIP 2. Stimulate5000 with TLR 6000 * 4000 3000 * ligands or MTB x pg/ml] [IL-6 [IL-6 pg/ml] [IL-6 2000 2000 24h1000 0 0 A/A A/G G/G A/A A/G G/G A/A A/G G/G 3. ELISAMed PAM2PAM3 LPS MTb LPS PAM3 MTB rs5743899 N= 35 21 7 shRNA knockdown of TOLLIP in human IL-6 secretion in human monocytes monocytes Stratified by rs5743899 TOLLIP inhibits rapamycin- induced autophagy Tollip KD 1. siRNA Knockdown in THP1s Neg7 Cont 2. Rapamycin induced autophagy x 4h (55%) 6 3. P62 or LC3 western blot 5 Media Rapamycin Rapa Media Rapa 4 Media Rapamycin P62 3
2 Fold p62, by Actin Fold Change
LC3 B-Actin 1 p62 0 Control TOLLIP Auto-phagosome formation ↑LC3II P62:Actin 0.81 0.28 0.55 1.81 ↓p62 Data: Javeed Shah, Stephen Hawn TOLLIP Deficiency Summary
TLR2 TLR2 TLR4 TOLLIP SNP TLR1 TLR6 1. ↓ mRNA 2. Altered Monocyte TOLLIP Cytokines Regulates TLR1/2/4/6 3. TB Susceptibility Adult TB Vietnam Pediatric TB SA IL-6 IL-10 TNF TOLLIP Knockdown 4. ↑MTB Replication 5. ↓ autophagy Genetics & TB HDTs
Genetic Finding Progress: Pathogeneis or HDT 1. Mendelian Mycobacteria: IL12/IFN Others: HSV, Pyogenic, Candida HIV: CCR5 & Maraviroc
2. GWAS TB: Positive findings, but no genes yet Leprosy: NOD2/RIPK2, HLA, TLR1 HCV & IL28B
3. Candidate Gene Abacavir hypersensitivity & HLAB5701
TB examples: LTA4H & dexamethasone Rx TOLLIP & autophagy Genetics & TB HDTs What is next?
1. Target IL-12/IFN- axis? 2. Fine map GWAS and genome- wide linkage findings 3. Study more discrete phenotypes 4. Examine Genetics of TB Rx Response Acknowledgements: Collaborators
SATVI Vietnam CWRU W. Hanekom S Dunstan H Boom T. Scriba J Farrar C Stein M. Hatherill NTT Thuong M. Musvosvi NTB Yen M. Shey G Thwaites Uganda G. Hussey H Mayanja-Kizza B. Abel Seattle BioMed M Joloba A. Keyser A. Aderem B. Kagina D. Sherman L. Lerumo NJ, UMDNJ M. De Kock G. Kaplan A. Hawkridge B. Pleixoto J. Hughes Acknowledgements: UW Lab
Rick Wells Chetan Seshadri Tom Dave Horne Javeed Shah Bill Berrington Matt Arentz Ann Misch Jay Vary Randi Simmons, Kelsey Nebeck, Monica Campo, Glenna Peterson
Funding: NIH, Burroughs-Welcome Fund, Dana Fnd