MICR 201 Microbiology for Health Related Sciences

1. Lecture 9: Innate Immunity Edith Porter, M.D. MICR 201 Microbiology for Health Related Sciences

2. Lecture outline • Concept of immunity • Innate immunity • Adaptive immunity • Innate immunity: first line of defense • General aspects • Physical factors • Chemical factors • Normal microbiota • Cellular elements • Epithelial cells • Phagocytes • Effector molecule • Complement • Antimicrobial peptides • Interferons • Iron binding proteins • Acute inflammation

3. Concept of immunity Innate immunity Adaptive immunity Acquired, available within days High specificity Memory In higher vertebrates • Functional at birth • Rapid responses: preformed or available within hours after infection • Limited specificity: pattern recognition via toll like receptors • Widely present in nature including in plants, invertebrates and vertebrates

4. Overview of host defenses First Line of Defense Second Line of Defense • NK cells

5. Physical (mechanical) defense of skin • Outer body surface • Keratin barrier • Epithelial cell shedding • Dryness

6. Physical (mechanical) defense of mucosa • Inner body surfaces • Mucus • Viscous • Protects underlying cells • Contains antimicrobial factors • Lysozyme • Constant fluid flow • Tears • Saliva • Intestinal peristaltic • Urine production and urination • Vaginal secretions • Mucociliaryclearance • 1 – 3 cm/h • Epithelial cell shedding

7. Chemical defense • Sebum, unsaturated fatty acids • “antimicrobial lipids” • Low pH • Skin (pH 3-5) • Stomach (pH 1.5 – 3) • Vagina (pH 3 – 5) • Urine (pH 6) http://www.niams.nih.gov/Health_Info/Acne/images/normal.jpg

8. Normal microbiota as part of host defense • Competes with potential pathogens for nutrients • Directly inhibits potential pathogens • Lactobacilli: lactic acid, low pH • Bacteriocins Skin Tongue Esophagus

9. Cellular elements of host defense • Epithelial Cells • Leukocytes

10. Epithelial cells Keratinizing in skin Non-keratinizing elsewhere

11. Immune function of epithelial cells • Express toll-like receptors (TLR 1 –10) that recognize specific pathogen associated molecular patterns (PAMP) • LPS • PG • Produce antimicrobial peptides (AMP) • Kill microbes • Secrete pro-inflammatory cytokines • Alert the host

12. Epithelial cell defense Microbial Products (LPS, PG, etc) TLR Antimicrobial Peptides Cytokines

13. Leukocytes in peripheral blood Granulocytes Natural Killer Cells neutrophil eosinophil basophil Anti-parasitic Anti-allergic Anti-parasitic Anti-allergic Anti-bacterial Anti-fungal Anti-viral Monocytes Lymphocytes Anti-bacterial Anti-fungal B-Ly : antibody production T-Ly: orchestrate

14. Leukocytes in tissue Mast cell Macrophage in bone marrow Dendritic cell Anti-parasitic Allergic responses Clears bacteria and fungi in tissues Communicates with lymphocytes

15. Origin of leukocytes • All originate from bone marrow (red bone marrow) • Circulate in the body through vascular system and lymphatic system • Enter tissue as needed • Some differentiate here and remain in the tissue: macrophages, dendritic cells

17. Phagocytosis • Performed by phagocytes (professional eaters) • Neutrophils • Monocytes Macrophages • Refers primarily to the uptake of bacteria and fungi • Relatively inefficient without special opsonins • Opsonins are molecules that enhance phagocytosis • Make “food more edible” • Host derived molecules that cover the microbe and are recognized by phagocytes

18. Phagocytes in Action

19. Key steps of opsonophagocytosis (1)

20. Key steps of opsonophagocytosis (2) • Chemotaxis • Opsonization • Adherence (attachment) • Ingestion (engulfment) • Pseudopods • Phagosome • Phagolysosome • Killing and digestion

21. Chemotaxis • Chemical attraction of phagocytes to microorganisms and movement of phagocytes towards the source of infection • Induced by chemoattractants: • Microbial products (formyl-methionine-peptides) • Complement • Cytokines (“Chemokines”)

22. Opsonization • Phagocytes need their food (microorganisms) served on silver plates • Opsonines significantly enhance microbial uptake by phagocytes • Cover microbial surfaces and are recognized by specific receptors on phagocyte surfaces • Examples are: • Antibodies • Complement

23. Killing and digestion by phagocytes • Oxygen dependent • Oxidative Burst • Reactive oxygen and reactive nitrogen intermediates • Oxygen-independent • Antimicrobial peptides • Low pH • Enzymes (Hydrolases, proteases, phopholipases)

24. Effector molecules of host defense • Complement system • Kills and helps in phagocytosis • Antimicrobial Peptides: • Kill • Interferons • Strengthen basic host cell defenses • Iron binding proteins • Expressed by both host and microbe • Competition for iron

25. Complement system • System of over 30 serum proteins • Active components (C-) and inhibitors • Widely distributed in body • Many cells can synthesis complement factors • Major producers: • Hepatocytes (liver cells) • Monocytes/macrophages • Fibroblasts

26. The Complement Cascade • Early events: proteolytic cascade generates bioactive cleavage fragments • C1 C4 C2 C3 C5  C6  C7 C8C9n • Late events: Protein polymerization generate a pore on target cell

27. Activation of complement system • Classical pathway • Antibodies bound to microbes change conformation and open up binding sites for C1 • Lectin pathway • Sugar-binding molecule with similar structure to C1 binds to the microbe and activate complement C2 and C4 • Alternative pathway • C3 binds directly to the microbial surface aided by factors B, D, and P and activates C5

28. Outcomes of Complement Activation • Always the same • Pore formation on microbe and direct killing (C5b- C9n) • Opsonization and improved phagocytosis (C3b) • Inflammation and recruitment of phagocytes (C5a, C3a, C4a)

29. Complement mediated enhancement of phagocytosis Figure 16.9 (3 of 5)

30. Pro-inflammatory action of complement • Action on blood vessels and Mast cells • Dilation  reddening and heat • Leakage of blood components  edema • Make endothelial cells and leukocytes sticky • Transmigration of leukocytes  pus • Chemoattractant for leukocytes

31. Microbial killing by complement C5b-C9n

32. Practice questions 1) Innate immunity is A) The body's ability to ward off diseases. B) The body's defenses against any kind of pathogen. C) The body's defense against a particular pathogen. D) The lack of resistance. E) Increased susceptibility to disease. 3) Which of the following does NOT increase blood vessel permeability? A) Kinins B) Prostaglandins C) Lysozymes D) Histamine E) Leukotrienes 2) The complement protein cascade is the same for the classical pathway, alternative pathway, and lectin pathway beginning with the activation of A) C1. B) C2. C) C3. D) C5. E) C6. 4) Which of the following does NOT provide protection from phagocytic digestion? A) Preventing formation of phagolysosomes B) Killing white blood cells C) Causing formation of phagolysosomes D) Ability to grow at a low pH E) Biofilms

33. + + + + + + Antimicrobial peptides • Found in phagocytes and epithelial cells • Small (< 100 amino acids) • Cationic • positive net charge at physiological pH • Arginine and/or lysine rich • Amphiphilic: also hydrophobic domains • Microbial killing through membrane permeabilization and other mechanisms • Example: defensins + + +

34. Interferons (IFNaand IFNb): act on host cells to increase production of antiviral proteins

35. Iron binding proteins • Host derived proteins • Transferrin: blood and tissue fluid • Lactoferrin: milk, saliva, mucus • Bind iron which is essential to microbe • Microbes counteract with siderophores or iron binding protein receptors

36. Acute inflammation • Relatively uniform response to a variety of causes • Infection • Physical agents like heat, radiation etc • Chemical agents like acids, bases etc. • Key signs are rubor (redness), dolor (pain), calor (heat), and tumor (swelling) • Local response includes vasodilation and increase of permeability, phagocyte migration and phagocytosis, tissue repair • Systemic response mediated by TNFa and acute phase proteins like C-reactive protein up to 1000x fold increased) can lead to fever, shock, disseminated coagulation

37. Process of acuteinflammation

38. Important to remember • Innate immunity is widely conserved, functional upon birth, operates via pattern recognition and TLR, has no memory • Key cells in innate immunity are epithelial cells and phagocytes • Phagocytosis is enhanced by opsonins • Phagocytes kill via oxygen radicals, antimicrobial peptides, low pH, and enzymes. • The effector molecules of innate immunity are complement (killing, inflammation, enhanced phagodytosis), antimicrobial peptides (killing), interferons (activation of host antiviral defenses), and iron binding proteins (deprive microbes of iron).

39. W2011 MICR 450 Innate Immunity (4) First line defense from concept to molecules Emphasis on primary research and hands on training in current methods in innate immunology including flow cytometry Lec: MW 9:50 - 10:40 am * Lab: M 10:50 – 2:20 pm * Rec: W 10:50 – 11:40 am Prerequisites: One of the following; MICR 201+MICR 202, MICR 300, BIOL 380, or instructor consent. Questions? email/call Dr. Edith Porter at eporter@calstatela.edu , (323) 343 6353 or drop by at ASCL 355