Immunology and Host Defense in the Lungs.

Translational Research Training Program: Immunology and Host Defense in the Lungs Shawn Skerrett, MD Associate Professor of Medicine Pulmonary and Critical Care Medicine Components of the innate immune system: Afferent (Sensing) Efferent (Effector) Cytokines, defensins, cathelicidins, Lysozyme, BPI, complement, Lactoferrin, acute phase reactants Humoral LBP, CD14, collectins,C3b, properdin Cellular (Mac, PMN, NK, epith) TLRs, CD14, FLMPR NOD1, NOD2 Defensins, proteases, lipases, Adhesion molecules, ROI, RNI Perforin, granzymes TLR signaling is mediated by MyD88 Approaches to Study of TLRs in Innate Immunity to Bacterial Pneumonia • In vitro – Transfected cells » Gain of function: reporter assay for NFkB activation » Loss of function: dominant negatives, siRNA • In vivo – TLR-deficient mice • Ligand-deficient bacteria – Cells from TLR-deficient mice – Human cells: macrophages, epithelial cells, PMN Innate Immunity to Bacterial Pneumonia • Legionella pneumophila • Pseudomonas aeruginosa • Staphylococcus aureus • Francisella tularensis NK + Microbes IFN IL-1 GM-CSF TNF NO IFN IL-2 CD8 TH1 CD4 Lysis + Activate NK Alveolar Macrophage Suppress - IL-12 IL-18 + + TH0 IL-10 TGF IL-4 + - IL-10 IL-13 TH2 A Common Dominant TLR5 Stop Codon Polymorphism Abolishes Flagellin Signaling and Is Associated with Susceptibility to Legionnaires’ Disease Hawn TR, Verbon A, Lettinga KD, Zhao LP, Li SS, Laws RJ, Skerrett SK, Beutler B, Schroeder L, Nachman A, Ozinsky A, Smith KD, Aderem A J Exp Med 2003; 198:1563-1572 Identify TLR5 SNPs • Sequenced TLR5 coding region in 40 subjects • Found 4 SNPs (one stop) • Cloned into expr vector • Transfected into CHO and HEK cells for luciferase reporter assay Hawn J Exp Med 2003; 198:1563 Gain of Function Luciferase Reporter Assay Day 1: MD-2 CD14 Transient transfection of nonresponsive HEK293 cells: ELAM Firefly-Luc • HA-TLR expression construct(s) • Co-receptors MD-2, CD14 • ELAM-Firefly Luciferase • -actin-Renilla Luciferase • Vector DNA Day 2: -actin Renilla-Luc 4 hour stimulation Dual Luciferase Assay (Promega) Western blot for TLR expression HEK-293 One TLR5 polymorphism nonfunctional • Transfected into CHO • • • and HEK cells for luciferase reporter assay Stimulated with flagellin Measured light emission 3 SNPs functional, one not Hawn J Exp Med 2003; 198:1563 TLR5 mediates recognition of L. pneumophila • Lp activates NFkB via TLR5 • TLR4-deficient cells respond to Lp • MyD88-deficient cells do not Hawn J Exp Med 2003: 198:1563 Baseline Clinical Characteristics Cases n = 108 n (%) Male Smoking History of COPD DM Malignancy Rheum 8 (7.8) 10 (10.1) 4 (4.0) 2 (2.0) Mean ± SD 63.8 ± 10.8 39 (7.8) 14 (2.8) 12 (2.5) 13 (2.6) Mean ± SD 44.9 ± 13.6 1.01 (0.46, 2.23) 3.92 (1.69, 9.09) 1.64 0.78 0.98 <0.05 0.50a 1.00a P <0.05b 4 (4.5) 5 (5.6) 2 (2.3) 4 (4.5) Mean ± SD 54.6 ± 11.2 1.81 1.89 1.77 0.44 0.39a 0.29a 0.69a 0.43a P <0.05b 65 (60.2) 47 (49.0) n (%) 261 (52.4) 172 (40.6) All controls n = 508 OR (95% CI) 1.37 (0.90, 2.10) 1.41 (0.90, 2.19) P 0.14 0.13 n (%) 53 (60.2) 32 (42.7) Matched controls n = 89 OR (95% CI) 1.00 (0.56, 1.77) 1.29 (0.70, 2.37) P 1.00 0.41 Age TLR5 SNP Allele and Haplotype Frequencies in LD Cases and Controls SNP Cases n=108 freq freq All Controls N=508 OR (95%CI) P 0.05 0.03 0.98 freq Matched Controls N=89 OR (95% CI) P 0.18 0.04 0.70 0.02 0.09 1174T 0.083 0.050 1.75 (1.00, 3.05) 1775G 0.173 0.116 1.6 (1.04,2.45) 1846C 0.480 0.480 1.00 (0.72, 1.38) CG TA 0.045 1.93 (0.74, 5.03) 0.101 1.83 (1.02, 3.28) 0.460 1.09 (0.71, 1.68) 0.173 0.115 1.68 (1.10, 2.59) 0.02 0.094 2.11 (1.16, 3.85) 0.083 0.049 1.90 (1.07, 3.35) 0.049 0.038 2.53 (0.86, 7.49) Hawn J Exp Med 2003; 198:1563 TLR5 SNPs Associated with LD in Nonsmokers SNP 1174T 1775G CG TA Smokers OR (95% CI) 0.72 (0.18, 2.86) 1.33 (0.54, 3.26) 1.33 (0.55, 3.25) 0.76 (0.19, 3.05) P 0.64 0.54 0.53 0.70 Nonsmokers OR (95% CI) P 2.16 (0.89, 5.23) 1.84 (0.97, 3.48) 1.99 (1.04, 3.80) 2.43 (1.00, 5.89) 0.09 0.06 0.04 0.05 Hawn J Exp Med 2003; 198:1563 Respiratory epithelial cells respond to flagellin but not LPS • A,B: A549 cells • C,D: Calu-3 cells • E: Whole blood • Stimulated with: • IL-8 release Hawn J Exp Med 2003; 198: 1563 o Salmonella fliC LPS  HK WT Legionella X HK flaA- Legionella • PBMC from subjects heterozygous for TLR5 stop codon respond poorly to flagellin • PBMC from subjects • homozygous for TLR5 wild type (RR) or heterozygous for TLR5 stop codon (R*) Stimulated with: – F: Salmonella fliC – G: Salmonella LPS • IL-6 release Hawn J Exp Med 2003; 198: 1563 Conclusions • Respiratory epithelial cells recognize L. pneumophila via TLR5/flagellin • TLR5 stop codon polymorphism has a dominant effect on PBMC responses • TLR5 polymorphisms are associated with susceptibility to Legionnaires’ disease among nonsmokers Recognition of L. pneumophila in the lungs is partially dependent on flagellin 700 Corby FlaA 6,000,000 5,000,000 Corby FLA-A Lung TNF [pg/ml] 600 500 400 300 200 100 0 PMN/lung 4,000,000 3,000,000 2,000,000 1,000,000 0 4 24 48 72 144 4 24 48 72 144 Hours after infection Hours after infection Clearance of L. pneumophila from the lungs does not require flagellin-mediated signaling 10,000,000 1,000,000 100,000 CFU/Lung Corby FLA-A 10,000 1,000 100 10 1 0 24 48 72 96 120 144 Hours after infection TLR2 Mediates Recognition of L. pneumophila Augmented by TLR1, TLR6 0.100 0.090 0.080 0.070 0.060 0.050 0.040 0.030 0.020 0.010 0.000 Relative Luciferase Units Unstim PG or LPS IL-1B Lp Vector Control mTLR1 mCD14 mMD2 mTLR1 mTLR2 mCD14 mMD2 mTLR2 mCD14 mMD2 Transfection mTLR2 mTLR6 mCD14 mMD2 mTLR6 mCD14 mMD2 mTLR4 mCD14 mMD2 Effect of Anti-TLR2, -TLR4, and CD14 on TNF Release by HAM 3500 3000 IgG1 2500 aTLR2 aTLR4 aCD14 1500 1000 500 0 1 Live Lp 10 Live Lp 100 Live Lp 0.1 011:B4 1 011:B4 10 011:B4 TNF [pg/ml] 2000 Resistance to L. pneumophila is impaired in TLR2-deficient mice 4,500,000 4,000,000 3,500,000 3,000,000 2,500,000 2,000,000 1,500,000 1,000,000 500,000 0 4 24 1000000 WT TLR2 KO 100000 PMN/Lung CFU/Lung 10000 1000 100 10 1 WT TLR2 KO 0 24 48 72 96 120 144 Hours after infection 72 120 Hours after infection TLR signaling is mediated by MyD88 Macrophage Recognition of Legionella Requires MyD88 MyD88+/+ No Lp +Lp MyD88-/No Lp +Lp Protected probes IL-10 IL-1 IL-1RA MIF IL-6 L32 GAPDH MyD88 is required for recognition of L. pneumophila in the lungs 700 600 500 400 300 200 100 0 4hr Lung TNF [pg/ml] PMN/Lung MyD88 WT MyD88 KO 4,500,000 4,000,000 3,500,000 3,000,000 2,500,000 2,000,000 1,500,000 1,000,000 500,000 0 WT MyD88-/- 24hr 4 24 72 Hours after infection MyD88 is required for clearance of L. pneumophila from the lungs 10,000,000 1,000,000 100,000 10,000 1,000 100 10 1 0 24 48 72 96 120 144 CFU/Lung WT MyD88-/- Hours after infection Translational Research in Pulmonary Host Defense • In vitro studies • Animal models • Clinical investigation – Cell lines – Cells from normal and genetically modified animals – Human cells – Genetically modified animals