1. Immune System

2. INNATE IMMUNITY Zuyue SUN, MD, PhD Department of Immunology Collage of Basic medicine, Room 320

3. INNATE IMMUNITY -SPRING 2015 Innate immunity predates development of adaptive immunity Does not produce protective immunity No memory response Prerequisite for developing adaptive immunity Non-antigen-specific immunity Found in plants, invertebrates and vertebrates

4. Immune Mechanisms INNATE –Present at birth –Self-discrimination –Limited diversity –Nonspecific defense –No memory –Examples: skin, cough, gastric pH, fever, inflammation (phagocytic cells) ACQUIRED –Appears after contact with antigen –Self-discrimination –Vast diversity –Specific defense –Memory responses –Examples: antibody, cytotoxic lymphocytes

5. INNATE IMMUNITY Present at birth Nonspecific Response does not target one specific immunogen Limited diversity Fixed, repeating, broad responses to a limited number of foreign substances No memory primary and secondary responses are identical

6. COMPONENTS OF INNATE IMMUNITY Physical/mechanical barriers Intact skin, epithelial layers, cough, fever Nonspecific chemical factors Antimicrobial peptides & fatty acids, gastric pH, lysozyme Inflammation Phagocytes (engulf and digest microbes) Proinflammatory factors (cytokines, complement proteins) Natural killer cells (nonspecific cytotoxic cells) Interferon (produced by virus-infected cells and induces antiviral state in neighboring uninfected cells)

8. IT ISN’T EASY BEING A PATHOGEN What a pathogen must do in order to cause disease: 1. Gain access to the body. 2. - Attach to, and/or enter cells of its host. - Receptors on pathogen must fit, lock-and- key, with receptor sites on host cell. 3.Reproduce while avoiding host’s immune system long enough to produce harmful changes. Images: Helicobacter pylori, Yutaka Tsutsumi, M.D; Deep gastric ulcer, Samir; Histopathology of H.pylori from a gastric biopsy, KGHHelicobacter pyloriDeep gastric ulceHistopathology of H.pylori Multiple flagella allow H. pylori to penetrate the coating of the stomach epithelium. H. pylori from a gastric biopsy

9. FIRST LINE OF DEFENSE _____________ Structures, chemicals, processes that work to prevent pathogens entering the body. Includes the skin and mucosal membrane of the respiratory, digestive, urinary, and reproductive systems. Images: : Castle, S. JervisCastle First Line of Defense Your skin

10. SKIN – _________ COMPONENTS OF DEFENSE Two major layers: 1. _____________ Outer layer composed of multiple layers of tightly packed cells Few pathogens can penetrate these layers Shedding of dead skin cells removes attached microorganisms Epidermal dendritic cells phagocytize pathogens. These cells extend out among other cells of the epidermis, forming a network to intercept invaders. 2. ___Demis__________ Contains protein fibers called collagenprotein Give skin strength and pliability to resist abrasions that could introduce microorganisms Image: “Skin” tattoo, Source unknown; Skin diagram, Daniel de Souza Telles Skin diagram First Line of Defense

11. SKIN – __________ COMPONENTS OF DEFENSE _______________ secreted by sweat glands Salt- inhibits growth of pathogen by drawing water from their cells Antimicrobial peptidespeptides Lysozyme- destroys cell wall of bacteria Sebum secreted by sebaceous (oil) glands Helps keep skin pliable and less likely to break or tear Lowers pH of skin to a level inhibitory to many bacteriapH First Line of Defense Images: Cartoon of castle being defended, Source unknown; Hair follicle, WikiHair follicle

12. MUCOUS MEMBRANES Line all body cavities open to the outside environment. Unlike surface epidermal cells, epithelial cells are living. Epithelial cells packed tightly to prevent entry of pathogens, but often only one cell layer thick, so pathogens sometimes breech the barrier. Continual shedding of cells carries attached microorganisms away Besides producing mucus, mucous membranes also produce lysozyme and other antimicrobial peptides.peptides OMG U R Nasty > Every day you swallow and digest about 1 liter of mucus. Images: Photo mucous membrane, Source unknown, Drawing of mucous membrane, Gray’s Anatomy Drawing of mucous membrane First Line of Defense

13. COMPONENTS OF THE SECOND LINE OF DEFENSE Leukocytes Phagocytosis Extracellular killing by leukocytes Nonspecific Chemical Defenses Inflammation Nonspecific response to tissue damage. Damages cells release histamines, which increase vasodilation. - Heat, swelling pain. Fever Second Line of Defense Images: Inflamed ingrown toenail, MenetekelInflamed ingrown toenail From the Virtual Microbiology Classroom on ScienceProfOnline.comVirtual Microbiology ClassroomScienceProfOnline.com

14. COMPONENTS OF THE SECOND LINE OF DEFENSE Second Line of Defense Images: The sick girl, Michael AncherThe sick girl Body temp above normal range of 36.5–37.5 °C (98–100 °F). Results when chemicals called ________trigger the hypothalamus to increase body’s core temperature. Various types of pyrogens  Bacterial toxins  Cytoplasm of bacteria released by lysis  Antibody-antigen complexes  Interleukin-I (IL-1 a cytokine) Benefits  Speed of immune system reaction increased  Inhibits growth of some temp sensitive microorganisms

15. FEVER TRIGGERED BY GRAM- BACTERIA 1.When infected with a Gram- bacteria…Gram- bacteria 2. 2 nd line of defense responds with phagocytes. 3. Macrophages engulf invader in a vesicle called a phagosome. 4.The phagosome fuses with a lysosome. Q: What happens to the bacteria when the phagosome and lysosome fuse? 5.When the macrophage is exposed to Lipid-A (part of the LPS membrane that is a pyrogen) the macrophage secretes interleukin (a type of cytokine that is a pyrogen). 6.Interleukin is picked up by the blood and transported to the brain. 7.In the brain, interleukin stimulates the hypothalamus to secrete prostaglandin. 8.Prostaglandin attaches to receptors in the hypothalamus and cause it to reset the thermostat  fever. 9.Ibuprophin and Acetominaphen are antiprastoglandins (They temporarily remove the prostoglandin, interrupting the fever-generating process). Interleukin Prostaglandin LPS Macrophage Phagocytizing G- Bacteria

16. INNATE IMMUNITY 1. Provides a barrier to prevent the spread of infection Mechanical (tight junctions, movement) Chemical (fatty acids, enzymes, pH, antimicrobial peptides) Microbiological (normal flora) Mucosal surfaces Nasopharyngeal, Oral, Respiratory, Intestinal tract Urogenital tract Skin (epithelial cells) Wounds, burns, insect bites 2. Identifies and eliminates pathogens Non-adaptive recognition systems Activates molecules that target the microbe and aid in it’s identification. These factors may be expressed at the surface or within cells, released from immune cells or are secreted and present within circulatory system

17. INNATE IMMUNITY 3. Initiates an inflammatory response Reaction to injury or infection Trauma to tissues or cells Presence of foreign matter (self vs. non-self) Infectious agents (viruses, bacteria, fungi) Delivers effector molecules & immune cells to the site of infection Components Leukocytes & secreted factors Blood vessels Plasma proteins 4. Provides signals to activate and regulate the type of adaptive immune response generated Stimulation of co-stimulatory molecules B7 family (CD80/86, PD-L, ICOSL) TNFR family (OX40L) Induction of a cytokine/chemokine response Cytokines: IL-12, IL-23, IL-4 Chemokines: CXCR1, CXCR2, CCL20 a variety and depends on stimulus

18. THE PHASES OF IMMUNITY

19. IDENTIFICATION OF MICROBES Recognition Receptors – Pattern Recognition Receptors (PRRs) Fixed in the genome, ie gene rearrangement is not needed Distribution Non-clonal, ie all cells of a class are identical Differentiation Pathogen vs. Commensal

20. IDENTIFICATION OF MICROBES PRR Recognize conserved molecular patterns on microbes called microbe associated molecular patterns (MAMPs) which are not present on the host Not limited to pathogens Identify a class of microbes LPS, LTA, peptidoglycan, lipoarabinomannan, dsRNA, mannans, b-glycans MAMPs are often essential for microbe survival Action Time Immediate activation of effectors Delays need for adaptive immunity

21. PATTERN RECOGNITION RECEPTORS (PRRS) Three broad classes of PRRs based on expression profile, localization, function 1) PRRs that signal an infection Include the Toll Receptor Family Expressed external or internally Activation of “pro-inflammatory” signaling pathways NF  B and MAP kinase signaling pathways Antimicrobial peptides (Defensins) / lysozyme, Inflammatory cytokines (TNF , IL-8, IL-1) Regulate activation of adaptive immune response co-stimulatory molecules

22. PATTERN RECOGNITION RECEPTORS(PRRS) 2) Phagocytic (endocytic) PRRs Expressed on the surface of phagocytic cells (MQs, PMNs, DCs) Mediate uptake of microbe into phagocytes 3) Secreted PRRs Secreted by MQs, epithelial cells, liver Activate C’, opsonize microbial cells, function as accessory proteins for MAMP recognition

23. TOLL-LIKE RECEPTOR FAMILY Curr. Opin. Hematology 9:2-10, 2002 PRR receptor ●Found both on the surface and within cells ●First discovered in Drosophila ●Currently 13 receptors o1-9 mouse & human o10 human o11-13 mouse Toll-like Receptor family

24. PHAGOCYTIC (ENDOCYTIC) PRRS BIND CARBOHYDRATES 1. Macrophage Mannose Receptor (C-type lectin) Type 1 transmembrane receptor Recognizes patterns of mannose residues in a certain spatial orientation unique to microbes (CRD) Only found on macrophages (not monocytes or PMNs) 2. Glucan Receptor (Dectin-1) Type 2 transmembrane receptor Recognizes  -1,3 &  -1,6 linked glycans Present on all phagocytes

25. PHAGOCYTIC (ENDOCYTIC) PRRS: CONT. 3. Scavenger Receptors Recognize charged ligands Polyanionic ligands (ds-RNA, LPS, LTA) Acetylated low-density lipoproteins (LDL) Found on all phagocytes MARCO (macrophage receptor with collagenous struction) binds bacterial cell walls but not yeast Phagocytose apoptotic cells new factor MFG-E8 (released from activated macrophages and binds to apoptotic cells via phosphatidylserine)

26. SECRETED PRRS ACTIVATE THE COMPLEMENT (C’) SYSTEM Complement system is activated by innate immunity Recognition by Complement receptors (CR) CR1, CR2, CR3, CR4, C5a, C3a Comprised of plasma proteins that when activated forms a triggered enzyme cascade Zymogens – activated by the cleavage of other proteases Precursor enzymes Function Facilitates the uptake & destruction of pathogens by phagocytes Induces an inflammatory responses

27. b C4b + C2b C3b + Bb ACTIVATION OF C’ SYSTEM

28. SECRETED PATTERN RECOGNITION MOLECULES Acute Phase Proteins Activation of Complement Opsonization of microbial cells Primarily produced by the liver but can be produced by phagocytes

29. SECRETED PATTERN RECOGNITION MOLECULES 1. Collectins Recognizes microbial carbohydrates (CRD domain) Effector function mediated by collagenous domain Mannan-binding lectin (MBL) Recognizes patterns of mannose & fucose residues in a certain spatial orientation unique to microbes Initiates the lectin pathway of C’ cleaving C2 & C4 Can function as an opsonin Binds a receptor on phagocytes (C1qRp) Surfactant proteins (SP-A / SP-D) lung

30. COLLECTINS Structure is conserved and similar to other proteins with similar function: Some Complement proteins & Mannose Binding Protein Binds to bacteria, fungi & viruses Function by binding microbes and are important for mediating phagocytosis of alveolar macrophages Microbe C-type Lectin domain Collagen helix  -coiled helix

31. SECRETED PATTERN RECOGNITION MOLECULES – CONT. 2. Pentraxin Members include Serum amyloid protein (SAP) C-reactive protein (CRP) Recognize phosphorylcholines on microbes Functions as an opsonins Binds to C1q & activate classical C’ pathway

32. SECRETED PATTERN RECOGNITION MOLECULES – CONT. 3. Lipid Transferases LPS binding protein (LBP) Opsonin Bactericidal permeability increasing protein (BPI) Bactericidal protein 4. Peptidoglycan recognition proteins (PGRS) Recognizes peptidoglycans in evolutionarily distant organisms 4 human PGRS Function is unknown One has bactericidal effects Triggers a serine protease cascade in insects ? Complement cascade ?

33. INFLAMMATORY RESPONSE

35. LEUKOCYTE ADHESION

36. NAÏVE AND MEMORY T CELLS TRAVEL IN DIFFERENT PATHS  Naïve (have not seen their antigen) T cells travel in the blood and lymphatics  Memory T cells (have been activated by their antigen) can also travel through tissues

37. Endothelial Cell 1. Tethering 3. Firm adhesion 2. Triggering 4. Diapedesis Lymphocyte Pathogens Macrophage Cytokines Chemokines Stromal cells Blood Vessel Steps in Lymphocyte Trafficking

38. PHAGOCYTOSIS Phagocytosis Definition: uptake of large particles (>0.5  m) Actin-dependent, clathrin-independent High rate & efficiency of internalization Professional phagocytic cells Macrophages Neutrophils These cells have phagocytic receptors External receptors FcR, CR3, Mannose receptor Internal receptors TLRs

39. Second Line of Defense Let’s look at phagocytosis in action! phagocytosis LEUKOCYTES: PHAGOCYTOSIS

40. MACROPHAGES (MQ) Blood - Called monocytes (1-6% WBC) Tissues - Called macrophages mature form of monocytes normally found in tissues such as gastrointestinal tract, lung, liver and spleen Functions: Phagocytose and kills after bactericidal mechanisms are activated (T cells) Produce cytokines/chemokines (initiates inflammation) Is an antigen presenting cell (co-stim. Molecules)

41. NEUTROPHILS (PMN) Present in blood (55-60% of WBC) Not normally present in tissues Short lifespan - 12 hours Functions: First at the site of infection/injury Ingest and kill microbes after bactericidal mechanisms are activated (binding to pathogen)

42. PHAGOCYTOSIS (MQ & PMN) Active process initiated by binding to pathogen Pathogen is surrounded and then internalized

43. SIGNALING INTERACTIONS DURING PHAGOCYTOSIS Ann. Rev. Immunol. 20:825-852, 2002

44. KILLING MECHANISMS Phagosome - membrane bounded vesicle that becomes acidified Lysozome - granules that contain products that damage or kill pathogens Enzymes Lysozyme - dissolves cell walls of some bacteria Acid hydrolases - digests bacteria Proteins Lactoferrin - binds Fe ++ needed for bacterial growth Vitamin B12-binding protein Peptides Defensins and cationic proteins - direct antimicrobials

45. KILLING MECHANISMS - CONT. Respiratory Burst Activated following phagocytosis Stimulated by PRR Requires increased oxygen consumption Produces substances that are directly toxic to the bacteria Oxygen-derived products O 2 -, H 2 O 2 & Myeloperoxidase Nitrogen-derived products NO (nitrogen oxide) Produced by inducible NO synthase (iNOS) enzyme Enzyme is induced by cytokines (LT, TNF  )

46. ENZYME REACTIONS OF RESPIRATORY BURST Respiratory Burst NADPH NADP+Superoxide + dismutase 2 O 2 2 O - H 2 O 2 Myeloperoxidase Enzyme which is stored in primary granules of PMN & MQ and uses the products of the respiratory burst. H 2 O 2 + C1 - Chloramines

47. PROFESSIONAL APC

48. REGULATION OF ADAPTIVE RESPONSE Veterinary Immunology & Immunopathology 91: 1, 2003

49. T CELLS RECIRCULATE TO “FIND” ANTIGEN-LOADED DENDRITIC CELLS Germinal Center Efferent lymphatics Afferent lymphatics Paracortical Area Follicular Area HEV