From Blood to Host Defense Innate Host Defense

1. From Blood to Host Defense Innate Host Defense Gregory J. Bagby, Ph.D. gbagby@lsuhsc.edu Office: 310 (CSRB)

2. From Blood to Host Defense • Blood • Components and function • Hemostasis and clotting • The host defense system • General overview • Innate immune system • pathogen recognition • inflammatory response • Adaptive immune system • Humoral immune system and antibodies • Cell-mediated immune system

3. Innate Immune Defense Defenses at the body surfaces Response of host defense cells that have an innate ability to respond to foreign molecules or altered/injured self Innate defense cells recognize some general property of foreign substances or cells Such identity tags are often found in particular classes of carbohydrates or lipids in microbial cell walls – pattern recognition receptors The inflammatory response is part of innate host defense General sequence of inflammation Lung inflammatory response to eradicate infection Non-infectious inflammatory response Type I interferons Tissue repair Anti-inflammatory mediators

4. Body Surfaces Minimize Entry of Pathogens Skin Respiratory Tract Upper GI tract Mucus Cilia Hair Cough Sneeze Normal Flora Sweat Glands (lacrymal…tear) pH (acidity) Mucociliary escalator

5. Toll-like receptors of the Innate Immune System Toll-like Receptors are importantPPR (Pattern Recognition Receptors) Mycoplasmal lipoprotein Bacterial CpG DNA Bacterial lipoprotein LPS dsRNA Flagellin TLR-1 TLR-6 TLR-2 TLR-3 MD-2 TLR-4 TLR-5 TLR-9 Prototypical TLR agonists and corresponding TLRs

6. p50 p65 IkB MyD88 IRAK TRAF6 TAK1 IKK Complex Toll-like Receptor 4: The first human TLR discovered LPS Bruce Buetler MD2 TLR4 LBP CD14 Cell membrane TLR4: The long-sought signal transducer of LPS, cell wall component of Gram-negative bacteria LPS-nonresponsive mouse strains shown to harbor defective TLR4 gene. Final product of cascade is the transcription factor NF-κB which leads to production of cytokines. NF-kB

7. Inflammatory Response • Local (or systemic) response to infection or injury • The local manifestations of inflammation are redness, swelling, heat, and pain • Systemic manifestations are fever, ache all over feeling, mallaise. • Purpose it to initiate destruction or inactivation foreign invaders or damaged/abnormal cells, and to set the stage for tissue repair

8. Cell Types of Innate Immunity“players of the game” • Neutrophils - blood • Eosinophils – blood • Basophils – blood • Monocytes – blood • Mast Cells – tissues • Macrophages – tissues • Resident macs. mostly barrier tissues (lung skin, GI tract, liver) • Dendritic cells - tissues • Natural Killer Cells – blood and tissues

9. Sequence of Events in Inflammatory Response to Bacteria • Bacterial invasion and recognition by macrophages and other cells. • Production of pro-inflammatory cytokines • Phagocytosis of invading/damaged cells • Microvascular damage and changes • Entry and activation of plasma proteins that amplify inflammatory response • Vasodilation of microcirculation to increase blood flow (red & heat) • Local increased vascular permeability of capillaries and venules resulting in fluid leak including plasma proteins (edema) • Chemotaxis – movement of leukocytes into infected area Initiation of host response • Killing and phagocytosis by neutrophils • Set stage for tissue repair

10. 2) SIGNALS 4) CAPILLARY PERMEABILITY 6) PHAGOCYTOSIS Water Proteins Signals 5) CHEMOTAXIS Macrophage 1) BACTERIAL INVASION 7) KILLING/REPAIR Cytokines 3) VASODILATION Blood flow Capillary * Neutrophil

11. The Innate Host Defense Response to Bacterial Infection of the Lung BacterialInvasion TNF Chemokines G-CSF G-CSF Alveolus of Lung Macrophages Epithelial cells Endothelial cells Neutrophils Bonemarrow Growth factor

12. The Normal Response to Intra-pulmonary Bacterial Infection Intrapulmonary bacterial challenge Percent viable bacteria remaining in the lung Neutrophil recruitment into the Alveolar compartment Cytokine response (TNF, IL-8, G-CSF) 0 6 12 18 24

13. Cells Found in Bronchoalveolar Lavage Fluid 4 hours after intratracheal bacterial challenge 4 hours after intratracheal PBS Neutrophil recruitment is essential for bacterial clearance.

14. 4 h Viable Bacteria * 4 h BAL PMN 2 h BAL TNF 100 10 15 8 10 6 U/ml x 103 PMN number (x106) 50 % remaining 4 5 * 2 ND 0 0 0 Ab-TNF Ab-TNF Ab-TNF NI NI NI IgG IgG IgG Effect of Anti-TNF on Pulmonary Host Defenses against P. aeruginosa challenge Cytokine production/secretion and neutrophil recruitment are critical to effective killing of invading bacteria. Same adverse effect with anti-chemokine or anti-GCSF

15. SIGNALS 4) CAPILLARY PERMEABILITY 6) PHAGOCYTOSIS Water Proteins Signals 5) CHEMOTAXIS Injury 7) REPAIR 3) VASODILATION Blood flow Capillary * Neutrophil

16. Important Mediators of the Inflammatory Response • Kinins • Complement • Blood clotting • Histamine • Eicosanoids • Platelet-activating factor • Cytokines to include chemokines • Others • Lysosomal enzympes • Nitric oxide • Reactive oxygen intermediates • Plasma • Plasma • Plasma • Mast cells, injured cells • Many cells • Many cells • Macrophages and other immune and non-immune cells • Injured cells, neutrophils, macrophages

17. Vasodilation and Increased Permeability • Many mediators – kinins, histamine, completment • Two purposes • Increase delivery of plasma mediators and cellular participants in the inflammatory response • Increased diffusion of mediators and migration/diapedesis of leukocytes through the capillary or venule wall.

18. Functions of the Complement Proteins • > 30 identified plasma proteins • Activated by infection, damage (inflammation) or Ab-Ag complexes

19. Killing of Microbes by Phagocytosis • Phagocytosis is engulfing of microbes by cells such as macrophages and neutrophils • Phagosome + lysosome = Phagolysosome • Enhanced by host substances that bind to the microbe – opsonin (prepare for eating) • Complement – C3b • Antibody-Antigen complex • C-reactive protein

20. Killing of Microbes by Extracellular Killing • Phagocytes can also kill microbes by secreting antimicrobial substances • nitric oxide • reactive oxygen intermediates • Activated complement proteins • Membrane attack complex (MAC) – inserts into wall of microbe membrane to form a pore-like leaky channel to disrupt the intracellular ionic environment.

21. Role of the Macrophage in Host Defense • Play a critical role in recognizing invading bacteria via TLR. • Secrete antimicrobial chemicals (reactive oxygen intermediates) • Initiate inflammatory cascade by producing cytokines, chemokines and growth factors. • Able to process and present antigen to Helper T cells (but Dendritic cells are better). • Engulf (phagocytosis) and neutralize/kill pathogen. Accelerated via antibody-antigen complexes (opsonization)

22. Local and Systemic Consequences of Microbe Contact with Phagocytes Microbial Contact with Phagocytes Phagocytosis Secretion of Mediators Intracellellular Killing of microbes Reg. of inflammatoryprocess Extracellular Killing of microbes Reg. of overall body response toinfection Activation of clotting &anticlotting pathways

23. Role of Type I Interferons in Innate Host Defense • The innate system also participates in host defense against viruses • Virally infected cells secrete type I interferons • Type I interferon binds to uninfected cells to induce synthesis of antiviral proteins which inhibit viral replication

24. Tissue Repair • Final Stage of Inflammation • Cell Division (tissue dependent) • Collagen secretion by fibroblasts • Angiogenesis • Imperfect remodeling  scar

25. Anti-inflammatory Mediators • Exogenous • Synthetic glucocorticoids • Non-steroidal anti-inflammatory (e.g. aspirin, ibuprofen) • Catecholamines that are Beta2 agonists • Pro-inflammatory cytokine antagonists (Ab-TNF or sTNF-R) • Endogenous • Anti-inflammatory cytokines – IL-10 • Glucocorticoids (cortisol) • Rodent adrenalectomy – • Increase stimulation of LPS-induced TNF production by macrophage • Reversed by glucocorticoid administration • Catecholamines

26. Stress * * * The plasma TNF Response to the TLR4 Ligand LPS 12000 Bacterial LPS Control 8000 Plasma Units/ml 4000 0 0 1 2 3 4 Time (hours)

27. 9000 6000 TNF (Units/ml) 3000 * * * l ) m 0 60 90 120 Min. Post-LPS Administration Control Epinephrine Epinephrine Infusion and the Bacteria-induced Plasma TNF Response

28. Stress Suppresses the LPS-induced TNF Response: Prevented by Mixed β & β2-Adrenergic Antagonists TNF at 90 min post-LPS 100 % of Control 50 * * 0 - b b1 b2 Antagonist Cntl Stress