1. Microbiology 532: Immunology Dennis E. Lopatin, Ph.D. Dept. of Biologic and Materials Sciences 4209 Dental Building Office Hours by Appointment Phone: 647-3912 Electronic mail: lopatin@umich.edu

2. Helpful Hints Readings in text are beneficial Readings in text are beneficial I expect you to read the relevant chapters in Nester. I expect you to read the relevant chapters in Nester. Understanding the concepts is not optional Understanding the concepts is not optional Think, rather than memorize Think, rather than memorize Test questions are based on concepts Test questions are based on concepts Ask questions Ask questions Don’t wait until the last minute to study Don’t wait until the last minute to study Check the website Check the website

3. Lecture 1 Immunology Introduction & Innate immunity

4. Why Does a Dentist Need to Understand Immunology? Many of the oral diseases have an immune component Many of the oral diseases have an immune component Periodontal disease Periodontal disease Caries Caries Sjögren’s Syndrome Sjögren’s Syndrome Current and future therapeutics affect the immune system and oral health Current and future therapeutics affect the immune system and oral health Systemic and Oral diseases are interrelated Systemic and Oral diseases are interrelated Cooperation with other health care professional requires a common language Cooperation with other health care professional requires a common language

5. What is immunity? “Protection” from infection, tumors, etc. “Protection” from infection, tumors, etc. Innate immunity is always available Innate immunity is always available Adaptive immunity distinguishes “self” from “non-self” and involves immune system “education” Adaptive immunity distinguishes “self” from “non-self” and involves immune system “education” Responses that may result in host tissue damage Responses that may result in host tissue damage

6. Two types of immunity Innate immunity (not antigen-specific) Innate immunity (not antigen-specific) Anatomical barriers Anatomical barriers Mechanical Mechanical Biochemical Biochemical Non-specific (eg. Low pH in stomach) Non-specific (eg. Low pH in stomach) Receptor-driven (eg. PAMP-recognition) Receptor-driven (eg. PAMP-recognition) Adaptive immunity (antigen-specific) Adaptive immunity (antigen-specific) Receptor-driven Receptor-driven Pre-existing clones programmed to make a specific immune response (humoral/cellular) Pre-existing clones programmed to make a specific immune response (humoral/cellular)

7. Antigen A substance (antigen) that is capable of reacting with the products of a specific immune response, e.g., antibody or specific sensitized T-lymphocytes. A substance (antigen) that is capable of reacting with the products of a specific immune response, e.g., antibody or specific sensitized T-lymphocytes. A “self” component may be considered an antigen even though one does not generally make immune responses against those components. A “self” component may be considered an antigen even though one does not generally make immune responses against those components.

8. Characteristics of Adaptive Immunity Immune response is highly specific for the antigen that triggered it. Immune response is highly specific for the antigen that triggered it. Receptors on surface of immune cells have same specificity as the antibody/effector activity that will be generated Receptors on surface of immune cells have same specificity as the antibody/effector activity that will be generated Exposure to antigen creates an immunologic “memory.” Exposure to antigen creates an immunologic “memory.” Due to clonal expansion and creation of a large pool of cells committed to that antigen Due to clonal expansion and creation of a large pool of cells committed to that antigen Subsequent exposure to the same antigen results in a rapid and vigorous response Subsequent exposure to the same antigen results in a rapid and vigorous response

9. Components of the immune system

10. Cells involved in immunity platelets megakaryocyte eosinophil neutrophil basophil mast cell common myeloid progenitor monocyte macrophage Natural Killer cell plasma cell B Lymphocyte T Lymphocyte common lymphoid progenitor Pluripotenthematopoietic stem cell

11. BloodBlood Serum or Plasma Leukocytes, Platelets and RBC Serum Proteins Mononuclear Cells Polymorphonuclear leukocytes (or Granulocytes) ImmunoglobulinsImmunoglobulins ComplementComplement Clotting factorsClotting factors Many othersMany others ImmunoglobulinsImmunoglobulins ComplementComplement Clotting factorsClotting factors Many othersMany others NeutrophilsNeutrophils EosinophilsEosinophils BasophilsBasophils NeutrophilsNeutrophils EosinophilsEosinophils BasophilsBasophils Lymphocytes (T cells, B cells & NK cells)Lymphocytes (T cells, B cells & NK cells) MonocytesMonocytes Lymphocytes (T cells, B cells & NK cells)Lymphocytes (T cells, B cells & NK cells) MonocytesMonocytes Where is that stuff?

12. Lymphoid Organs Primary or central lymphoid organs Primary or central lymphoid organs bone marrow and thymus bone marrow and thymus where lymphocytes are generated where lymphocytes are generated Secondary or peripheral lymphoid organs Secondary or peripheral lymphoid organs where adaptive immune responses are initiated where adaptive immune responses are initiated

13. Distribution of Lymphoid Tissues

14. Response to Initial Infection

15. Stages of Response to Infection

16. Course of Typical Acute Infection

17. Innate Host Defense Mechanisms Anatomic Factors Anatomic Factors Mechanical Factors Mechanical Factors Biochemical Factors Biochemical Factors

18. Skin Stratified and cornified epithelium provides a mechanical barrier Stratified and cornified epithelium provides a mechanical barrier Indigenous microbiota competes with pathogens Indigenous microbiota competes with pathogens Acid pH inhibits growth of disease producing bacteria Acid pH inhibits growth of disease producing bacteria Bactericidal long chain fatty acids in sebaceous gland secretions Bactericidal long chain fatty acids in sebaceous gland secretions

19. Respiratory Tract Upper Respiratory Tract Upper Respiratory Tract Nasal hairs induce turbulence Nasal hairs induce turbulence Mucous secretions trap particles Mucous secretions trap particles Mucous stream to the base of tongue where material is swallowed Mucous stream to the base of tongue where material is swallowed Nasal secretions contain antimicrobial substances Nasal secretions contain antimicrobial substances Upper respiratory tract contains large resident flora Upper respiratory tract contains large resident flora Lower Respiratory Tract Lower Respiratory Tract Particles trapped on mucous membranes of bronchi and bronchioles Particles trapped on mucous membranes of bronchi and bronchioles Beating action of cilia causes mucociliary stream to flow up into the pharynx where it is swallowed Beating action of cilia causes mucociliary stream to flow up into the pharynx where it is swallowed 90% of particles removed this way. Only smallest particles (<10µ in diameter) reach alveoli 90% of particles removed this way. Only smallest particles (<10µ in diameter) reach alveoli Alveoli Alveoli Alveolar macrophage rapidly phagocytize small particles Alveolar macrophage rapidly phagocytize small particles

20. Alimentary Tract General defense mechanisms General defense mechanisms Mucous secretions Mucous secretions Integrity of of mucosal epithelium Integrity of of mucosal epithelium Peristaltic motions of the gut propel contents downward Peristaltic motions of the gut propel contents downward Secretory antibody and phagocytic cells Secretory antibody and phagocytic cells Stomach Stomach Generally sterile due to low pH Generally sterile due to low pH Small Intestine Small Intestine Upper portion contains few bacteria Upper portion contains few bacteria As distal end of ilieum is reached flora increases As distal end of ilieum is reached flora increases Colon Colon Enormous numbers of microorganisms Enormous numbers of microorganisms 50-60% of fecal dry weight is bacteria 50-60% of fecal dry weight is bacteria

21. Genitourinary Tract Male Male No bacteria above urethrovesicular junction No bacteria above urethrovesicular junction Frequent flushing action of urine Frequent flushing action of urine Bactericidal substances from prostatic fluid Bactericidal substances from prostatic fluid pH of urine pH of urine Bladder mucosal cells may be phagocytic Bladder mucosal cells may be phagocytic Urinary sIgA Urinary sIgA Female (Vagina) Female (Vagina) Large microbial population (lactobacilli) Large microbial population (lactobacilli) Microorganisms produce low pH due to breakdown of glycogen produced by mucosal cells Microorganisms produce low pH due to breakdown of glycogen produced by mucosal cells

22. Eye Flushing action of tears which drain through the lacrimal duct and deposit bacteria in nasopharynx Flushing action of tears which drain through the lacrimal duct and deposit bacteria in nasopharynx Tears contain a high concentration of lysozyme (effective against gram positive microorganisms Tears contain a high concentration of lysozyme (effective against gram positive microorganisms

23. Receptors Almost all of biology occurs because recognition Almost all of biology occurs because recognition Enzymatic action Enzymatic action Interactions between cells (cooperation/activation) Interactions between cells (cooperation/activation) Communication between cells Communication between cells Innate and adaptive immunity requires it Innate and adaptive immunity requires it

24. Innate Immune Recognition All multi-cellular organisms are able to recognize and eliminate pathogens All multi-cellular organisms are able to recognize and eliminate pathogens Despite their extreme heterogeneity, pathogens share highly conserved molecules, called “pathogen-associated molecular patterns” (PAMPs) Despite their extreme heterogeneity, pathogens share highly conserved molecules, called “pathogen-associated molecular patterns” (PAMPs) Host cells do not share PAMPs with pathogens Host cells do not share PAMPs with pathogens PAMPs are recognized by innate immune recognition receptors called pattern-recognition molecules/receptors (PRMs/PRRs) PAMPs are recognized by innate immune recognition receptors called pattern-recognition molecules/receptors (PRMs/PRRs)

25. Typical PAMPs Lipopolysaccharides Lipopolysaccharides Peptidoglycans Peptidoglycans Certain nucleotide sequences unique to bacteria Certain nucleotide sequences unique to bacteria Other bacterial components Other bacterial components

26. Endogenous Signals Induced by PAMPs Mediate inflammatory cytokines Mediate inflammatory cytokines Antigen-presenting cells recognize PAMPs Antigen-presenting cells recognize PAMPs Same APC processes pathogens into specific pathogen-derived antigens and presents them with MHC encoded receptors to T-cells Same APC processes pathogens into specific pathogen-derived antigens and presents them with MHC encoded receptors to T-cells T-cell responds only when presented with both signals T-cell responds only when presented with both signals Different Effector Cytokines in Response to Different Pathogens (Th1 vs. Th2) Different Effector Cytokines in Response to Different Pathogens (Th1 vs. Th2)

27. Antimicrobial Peptides/Defensins Four hundred peptides described to date Four hundred peptides described to date Defensins (3- 5-kD, four families in eukaryotes) Defensins (3- 5-kD, four families in eukaryotes)  -defensins (neutrophils and intestinal Paneth cells)  -defensins (neutrophils and intestinal Paneth cells)  -defensins (epithelial cells)  -defensins (epithelial cells) Insect defensins Insect defensins Plant defensins Plant defensins Defensins appear to act by binding to outer membrane of bacteria, resulting in increased membrane permeability. Defensins appear to act by binding to outer membrane of bacteria, resulting in increased membrane permeability. May also play a role in inflammation and wound repair May also play a role in inflammation and wound repair

28. Complement System Three pathways now known Three pathways now known Classical Classical Alternative Alternative Lectin or MBL pathway (binding to mannose- containing carbohydrates) Lectin or MBL pathway (binding to mannose- containing carbohydrates) Host cells have complement regulatory proteins on their surface that protect them from spontaneous activation of C3 molecules Host cells have complement regulatory proteins on their surface that protect them from spontaneous activation of C3 molecules

29. Inflammatory Mediators in Innate Immunity Cytokines secreted by phagocytes in response to infection include: Cytokines secreted by phagocytes in response to infection include: IL-1 IL-1 activates vascular endothelium and lymphocytes activates vascular endothelium and lymphocytes Increases adhesiveness of leukocytes Increases adhesiveness of leukocytes IL-6 IL-6 Induces B-cell terminal maturation into Ig-producing plasma cells Induces B-cell terminal maturation into Ig-producing plasma cells IL-8 IL-8 Induces expression of b2 integrin adhesion molecules on neutrophils, leading to neutrophil migration to infection site Induces expression of b2 integrin adhesion molecules on neutrophils, leading to neutrophil migration to infection site IL-12 IL-12 Activates NK cells and induces Th1-cell differentiation Activates NK cells and induces Th1-cell differentiation IL-18 IL-18 TNF-  TNF-  Activates vascular endothelium and increases vascular permeability, leading to accumulation of Ig and complement in infected tissues Activates vascular endothelium and increases vascular permeability, leading to accumulation of Ig and complement in infected tissues

30. Other Mediators and Molecules Phagocytes Phagocytes Toxic oxygen radicals Toxic oxygen radicals Peroxides Peroxides Nitric oxide (NO) Nitric oxide (NO) Lipid mediators of inflammation Lipid mediators of inflammation Prostaglandins Prostaglandins LTB4 LTB4 Platelet activating factor Platelet activating factor Complement component C5a Complement component C5a Stimulates mast cells to release histamine, serotonin and LTB4 Stimulates mast cells to release histamine, serotonin and LTB4 IL-1, IL-6 and TNF-  IL-1, IL-6 and TNF-  Induce acute-phase response in liver Induce acute-phase response in liver Induce fever Induce fever IL-1 and IL-18 signaling pathways activate NF-  B, important in innate immunity IL-1 and IL-18 signaling pathways activate NF-  B, important in innate immunity

31. Immune Cells and Innate Immunity Phagocytes Phagocytes Neutrophils Neutrophils Moncyte/macrophage Moncyte/macrophage Eosinophils (to a lesser extent) Eosinophils (to a lesser extent) NK cells (large granular lymphocytes) NK cells (large granular lymphocytes) Antibody-dependent cell-mediated cytotoxicity (ADCC) Antibody-dependent cell-mediated cytotoxicity (ADCC) Have two major functions Have two major functions Lysis of target cells Lysis of target cells Production of cytokines (IFN-  and TNF-  ) Production of cytokines (IFN-  and TNF-  ) Act against intracellular pathogens Act against intracellular pathogens Herpesviruses Herpesviruses Leishmania Leishmania Listeria monocytogenes Listeria monocytogenes Act against protozoa Act against protozoa Toxoplasma Toxoplasma Trypanasoma Trypanasoma

32. Immune Cells and Innate Immunity (cont’d)  /  T cells  /  T cells Two types of T cell receptors Two types of T cell receptors One composed of  and  chains (basic T cell antigen receptor) One composed of  and  chains (basic T cell antigen receptor) One composed of  and  chains (minor population of T cells) One composed of  and  chains (minor population of T cells) Two groups of  /  T cells Two groups of  /  T cells One group found in lymphoid tissues One group found in lymphoid tissues One group located in paracellular space between epithelial cells One group located in paracellular space between epithelial cells Recognizes unprocessed target antigen in absence of APC help Recognizes unprocessed target antigen in absence of APC help B-1 cells (minor fraction of B cells, do not require T-cell help) B-1 cells (minor fraction of B cells, do not require T-cell help) Mast cells Mast cells Located in serosa, under epithelial surfaces and adjacent to blood vessels, nerves and glands Located in serosa, under epithelial surfaces and adjacent to blood vessels, nerves and glands Capable of phagocytosis Capable of phagocytosis Process and present antigen using MHC class I or II receptors Process and present antigen using MHC class I or II receptors LPS can directly induce release of mast cell mediators LPS can directly induce release of mast cell mediators Complement (C3a and C5a) induce mast cells to release mediators Complement (C3a and C5a) induce mast cells to release mediators Chemotaxis, complement activation, inflammation Chemotaxis, complement activation, inflammation TNF-  secreted by mast cells results in neutrophil influx into infected site TNF-  secreted by mast cells results in neutrophil influx into infected site

33. Summary of Innate Immunity External and mechanical barriers External and mechanical barriers Receptors for pathogen motifs Receptors for pathogen motifs Soluble antimicrobial proteins Soluble antimicrobial proteins Pattern of cytokines produced influences adaptive response Pattern of cytokines produced influences adaptive response