From Blood to Host Defense Innate Host Defense

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

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

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

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

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

Toll-like Receptor 4: The first human TLR discovered LPS Bruce Buetler MD 2 LBP TLR 4 CD 14 TLR 4: The long-sought signal transducer of LPS, cell wall component of Gram-negative bacteria LPS-nonresponsive mouse strains shown to harbor defective TLR 4 gene. Final product of cascade is the transcription factor NF-κB which leads to production of cytokines. Cell membrane My. D 88 IRAK TRAF 6 TAK 1 IKK Complex NF-k. B Ik. B p 65 p 50

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

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

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

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

The Innate Host Defense Response to Bacterial Infection of the Lung Bacterial Invasion Macrophages Alveolus of Lung TNF Epithelial cells Chemokines G-CSF Endothelial cells Neutrophils Bone marrow G-CSF Growth factor

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

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

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 5 0 ND NI Ab-TNF Ig. G 4 h BAL PMN 8 6 4 2 0 * NI Ab-TNF Ig. G 4 h Viable Bacteria 100 * % remaining 10 10 PMN number (x 106) U/ml x 103 15 2 h BAL TNF 50 0 NI Ab-TNF Ig. G

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

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 Mast cells, injured cells Many cells Macrophages and other immune and non-immune cells • Injured cells, neutrophils, macrophages

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.

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

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 – C 3 b – Antibody-Antigen complex – C-reactive protein

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.

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)

Local and Systemic Consequences of Microbe Contact with Phagocytes Microbial Contact with Phagocytes Phagocytosis Intracellellular Killing of microbes Secretion of Mediators Reg. of inflammatory process Extracellular Killing of microbes Reg. of overall body response to infection Activation of clotting & anticlotting pathways

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

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

Anti-inflammatory Mediators • Exogenous – – Synthetic glucocorticoids Non-steroidal anti-inflammatory (e. g. aspirin, ibuprofen) Catecholamines that are Beta 2 agonists Pro-inflammatory cytokine antagonists (Ab-TNF or s. TNF -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

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

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

Stress Suppresses the LPS-induced TNF Response: Prevented by Mixed β & β 2 -Adrenergic Antagonists TNF at 90 min post-LPS % of Control 100 50 * 0 Cntl - * b b 1 b 2 Antagonist Stress