THE TWO „ARMS” OF THE IMMUNE SYSTEM

1. THE TWO „ARMS” OF THE IMMUNE SYSTEM • INNATE/NATURAL IMMUNITY • ACQUIRED IMMUNITY

2. WHY IS THE IMMUNE SYSTEM SO IMPORTANT? Virus Viruses 18 - 30 years 3 hours 3 hours VARIABILITY Rapid evolution Adaptation Selection Biomass: 90% microbes Animal mass < 5 – 25x microbes PATHOGENS Bacteria Monocellularparazites Multicellular parazites (helminths)

3. CHARACTERISTICS OF INNATE IMMUNITY • NATURAL/INNATE • Rapid, prompt response (hours) • No variable receptors • No improvement during the response • No memory • Not transferable • Can be exhausted, saturated COMMON EFFECTOR MECHANISMS FOR THE ELIMINATION OF PATHOGENS • ADAPTIVE/ACQUIRED • Time consuming (several days) • Variable antigen receptors • Efficacy is improving during the response • Memory • Can be transferred • Regulated, limited

4. TWO LINES OF IMMUNE DEFENSE INNATE/NATURAL IMMUNITY ACQUIRED/ADAPTIVE IMMUNITY Phagocytes (monocyte/macrophage, neutrophil, dendritic cell) Killer cells (NK cell, δ T cell) B1 lymphocytes (CD5+) B lymphocytes (B2) T lymphocytes helper T cell cytotoxic T cell CELLS HUMORAL FACTORS Enzymes (lysozyme, pepsin, trypsin) Antibacterial peptides Complement system Cytokines, chemokines Antibodies

5. Cells Receptors SENSING RECOGNITION SENSING RECOGNITION Signaling pathways Cell-Cell collaboration SIGNALING SIGNALING Effector functions RESPONSE RESPONSE DEFENSE SYSTEMS ADAPTIVE IMMUNITY INNATE IMMUNITY

6. PHYSICAL BARRIERS PROTECTING OUR BODY FROM THE ENVIRONMENT BRONCHIAL TRACT EYES Sinuses Trachea Lungs Kidney Bladder Vagina SKIN Damage UROGENITAL SYSTEM Infection GASTROINTESTINAL SYSTEM Oral cavity esophagus Stomach Intestines WALDEYER RING Tonsils, adenoids Palatinal, pharyngeal lingual and tubar tonsils

7. EPITELIAL SURFACES ARE IMPORTANT IN THE FIRST LINE OF DEFENSE

8. α2-macroglobulin inhibits potentially damaging proteases About 10% of serum proteins are protease inhibitors.

9. Human defensins are variable antimicrobial peptides Peptides of 30-40 amino acids, amphipathic character They penetrate microbial membranes Ongoing race between pathogens and the immune system of the host

10. Normal flora Cells of human body: 90% microbes, 10% human Symbiotic, non-pathogenic microbes – mucosal membrane, skin Bacteria, Fungi, Protozoa Gut – colonalization after birth 1012 bakteria/g (1.5 kg) intestinal content 1000 species 100-times more bacterial genes then eukaryotic „peaceful” commensalisms vitamins (i.e. K1 vitamin) real ecosystem, survival of the fittest, competition with pathogenic organism the few who brake in through the gut epithelium induce local immune response Important role in: -development of mucosal and systemic immunity -normal development of peripheral lymphoid organs - maintenance of basic level of immunity

11. RECOGNITION BY THE INNATE IMMUNE SYSTEM

12. INNATE/NATURAL IMMUNITY RECOGNITION Richard Pfeiffer, a student of Robert Koch – ENDOTOXIN There must be a receptor that recognizes endotoxin Lipopolysaccharide (LPS) receptor remained elusive The Dorsoventral Regulatory Gene Cassette Spätzle/Toll/Cactus controls the potent antifungal response in Drosophila adults Bruno Lemaitre, A Hoffmann et al, Cell, 1996 Spätzle: Toll ligand Toll: Receptor Cactus: I-kB Dorsal: NF-kB Drosomycin

13. TOLL RECEPTORS ACTIVATE PHYLOGENETICALLY CONSERVED SIGNAL TRANSDUCTION PATHWAYS Fungus Protease Spätzel Toll Tube Relish Pelle Cactus NFkB Peptid Bacterium LPB LPS TLR4 CD14 MyD88 IRAK IL-1R associated Kinase Inflammation Acute phase response Danger signal IL-6 Drosophila Macrophage

14. 9-13various Toll-receptors TLR family Several millions antigen receptors Acquired immunity Innate immunity 450 million years Ancient WHAT IS RECOGNIZED BY INNATE AND ACQUIRED IMMUNITY? RECEPTORS Common pattern of groups of pathogens Pathogen Associated Molecular Pattern PAMP Recognition by receptors Pattern Recognition Receptor PRR Unique structural elements Antigenic determinant Recognition by highly specific antigen receptors B cell receptor BCR (sIg) T cell receptor TCR

15. TOLL RECEPTORS RECOGNIZE VARIOUS MICROBIAL STRUCTURES Virus Bacteria ssRNS dsRNA CpG DNA Gram- Flagellin Peptidoglycane LPS Gram+ TLR9 TLR7 TLR8 TLR3 TLR2 TLR6 TLR5 TLR4 Interferon producing cell pDC IFN Macrophage/Dendritic cell ALL STRUCTURES ARE ESSENTIAL FOR THE SURVIVAL OR REPLICATION OF THE PATHOGEN

16. CONSERVED RECEPTORS/SENSORS THAT DETECT DANGER SIGNALS TLR TLR3 MEMBRANE Fibroblast Epithelial cell DC LRR CELL MEMBRANE Bacteria MEMBRANES OF INTRACELLULAR VESICLES virus TIR domain TIR: Toll-Interleukin Receptor signaling domain

17. PHAGOCYTES ARE ABLE TO RECOGNIZE PATHOGENS Toll receptor-mediated signaling FcR, CR Toll receptor PHAGOCYTES (macrophages, dendriticcells, neutrophil granulocytes) RECOGNIZE PATHOGENS BY PATTERN RECOGNITION RECEPTORS RECOGNITION IS ESSENTIAL

18. RECOGNITION CYTOPLASMIC SENSORS

19. VESZÉLYT ÉRZÉKELŐ KONZERVÁLT RECEPTOROK NLR: NOD-like receptor RLR: RIG-like receptor

20. CONSERVED RECEPTORS SENSING DANGER SIGNALS NLR nod-like receptors Nucleotide binding domain Leucin rich repeats TLR NOD1/2, IPAF/NLRC4 CARD IPAF BIR TLR3 RLH CARD-CARD-helicase MEMBRAN NBD N C PYR NLRP1 – ASC NLRP3 – ASC – CARDINAL NBD NBD Fibroblast Epithelial cell DC CYTOPLASM

21. DANGER SIGNALS ARE TRANSLATED TO CYTOKINE SECRETION THROUGH VARIOUS MOLECULAR SENSORS IN DC SUBTYPES 4 6 6 2 1 1 NLR RLH RLH IL-1β IL-12/23 IL-10 IFNαβ NK/DC Th1/Th17/Th2 5 10 7 3 9 7 8 NLR=NOD/NALP (IL-1β) RLH=RIG-1/MDA5 (IFN) Plasmacytoid DC Conventional DC TLR1 – bacteriallipoprotein (togetherwith TLR2) TLR2 – bacteriallipoprotein, peptidoglycane, lipoteicholicacid (heteromerwith TLR1 and TLR6) TLR3 – viraldsRNS, polyI:C TLR4 – bacterial LPS TLR5 – bacterialflagellin TLR6 – bacteriallipoprotein (with TLR2) TLR7 – viralssRNA TLR8 – GU richviralssRNS, imidazoquinolin (antiviraldrug) TLR9 – unmethylatedCpG DNA TLR10 – modifiedviralnucleotides

22. SIGNALING IN INNATE IMMUNITY

23. TOLL RECEPTORS ACTIVATE PHYLOGENETICALLY CONSERVED SIGNAL TRANSDUCTION PATHWAYS Fungus Protease Spätzel Toll Tube Relish Pelle Cactus NFkB Peptid Bacterium LPB LPS TLR4 CD14 MyD88 IRAK IL-1R associated Kinase Inflammation Acute phase response Danger signal IL-6 Drosophila Macrophage

24. TOLL RECEPTOR MEDIATED SIGNALLING NEW THERAPEUTIC TARGET Figure 3 The 'hourglass' shape of the innate immune response. Although microbial stimuli are chemically complex and although the innate immune response ultimately involves the activation of thousands of host genes, innate immune signals traverse a channel of low complexity. Ten Toll-like receptors (TLRs), four TIR (Toll/interleukin-1 receptor homologous region) adaptors and two protein kinases are required for most microbial perception. This circumstance lends itself to effective pharmacotherapeutic intervention. NF-B, nuclear factor-B; STAT1, signal transducer and activator of transcription 1.

25. EFFECTOR MECHANISMS OF INNATE IMMUNITY

26. Phagocytosis Intracellular killing PHAGOCYTOSIS Phagocyte Bacterium NK-CELLS Lysis of infected cell NK-cell Virus-infected cell INFLAMMATION Cytokines TNF Neutrophil IL-12 Bacterium LPS NK-cell IFN Macrophage COMPLEMENT Complement proteins Lysis of bacteria Inflammation Bacterium Complement-dependent phagocytosis CELLULAR AND HUMORAL MECHANISMS OF INNATE IMMUNITY

27. Degradation PRR ACTIVATION Bacterium Intracellular killing Phagocyte Uptake Antigen + Antibody ACQUIRED IMMUNITY Antigen presentation T cell ACQUIRED IMMUNITY MECHANISMS OF INNATE IMMUNITY PHAGOCYTOSIS 0.5 - 1 hours The amount of internalized particles is limited

28. PHAGOCYTE SYSTEM NEUTROPHIL GRANULOCYTE MONOCYTE – MACROPHAGE – DENDRITIC CELL Defence against infectious diseases Elimination of tumor cells Gatekeeper function Sensing commensals and pathogens Rapid activation of innate immunity Priming adaptive immune responses Maintenance of self tolerance

29. PHAGOCYTOSIS Macrophages ingest and degrade particulate antigens through the use of long pseudopodia that bind and engulf bacteria. The engulfed bacteria are degraded when the phagosome fuses with a vesicle containing proteolytic enzymes (lysosome), forming the phagolysosome. Specialized compartments also exist in the macrophage to promote antigen processing for presentation to antigen-specific T cells.

30. Opsonization enhances the efficiency of phagocytosis of pathogens by phagocytes

31. Killing of bacteria by neutrophils: azurophilic and specific granules azurofil ic specific granuls Lyzozyme NADPH oxidase Defensins Lyzozyme Mieloperoxidase Cathepsin G elastase

32. Phagocyte oxidase (Phox) produces reactive oxidative species (ROS) that help destroy pathogens

33. Failure of phagocytes to produce reactive oxigen species in chronic granulomatous didease PROTECTION against bacteria and fungi is down regulated

34. INFLAMMATION – ACUTE PHASE RESPONSE PRR TNF- neutrophil LPS IL-12 NK-cell TNF- IL-1 IL-6 Bacterium IFN macrophage cytokines Few hours ACUTE PHASE RESPONSE LPS (endotoxin) (Gram(-) bacteria) DANGER SIGNAL ACTIVATION TNF- IL-1 IL-6 Kinetics of the release of pro-inflammatory citokines in bacterial infection Plasma level hrs MECHANISMS OF INNATE IMMUNITY

35. INFLAMMATORY RESPONSE

36. The classic symptoms of inflammation: redness (rubor) - vasodilation, swelling (tumor) - edema, heat (calor) – increased perfusion, pain (dolor) – factors stimulating nociceptors, loss of function (functio laesa)

37. CONSEQUENCES OF MACROPHAGE ACTIVATION SYNTHESIS OF CYTOKINES

38. Systemic effects of pro-inflammatory cytokines

39. Systemic release of TNFa initiates septic shock Septic shock Local production of TNFα (and IL1) is beneficial, and protective, BUT systemic release may cause death Drop in blood volume and hence blood pressure Disseminated intrvascular coagulation

40. Pro-inflammatory cytokines activate endothel which recruits immunocytes from blood to infected tissues (extravasatio)

41. THE ACUTE PHASE RESPONSE IL- 6 Mannose binding lectin/protein MBL/MBP COMPLEMENT C-reactive protein Phosphocolin binding (e.g.fungi) COMPLEMENT Fibrinogen Liver Phosphocoline binding Fungi, bacterial Cell wall. Serum Amyloid Protein (SAP) Mannose/galactose binding IL-6 induces the production of acute phase protiens

42. COMPLEMENT ACTIVATION COMPLEMENT Lysis of bacteria Complement-proteins Inflammation Chemotaxis Bacterium Lectin pathway Alternative pathway Complement-dependent phagocytosis MECHANISMS OF INNATE IMMUNITY Antigen + Antibody ACQUIRED IMMUNITY Few minutes – 1 hour Enzymes get fragmented, complement activity can be exhausted

43. RECOGNITION BY SOLUBLE MOLECULES MANNOSE BINDING LECTIN

44. Eukariotic cells Mannose GLYCOSYLATION OF PROTEINS IS DIFFERENT IN VARIOUS SPECIES Prokariotic cells Galactose Glucoseamin Neuraminicacid Mannose

45. PATTERN RECOGNITION BY MANNAN BINDING LECTIN Bacterium lysis Complement activation LECTIN PATHWAY CR3 Macrophage Phagocytosis Strong binding No binding

46. NK cells • 5-10% of lymphocytes in circulation • bigger than T or B lymphocytes • several granules in their cytoplasm • have no antigen binding receptors („null cells”) • participants of native immunity Type I IFNs increase their cytotoxicity (100x) IL12, and TNFα are also able to activate them IFNγ production --- MF, DC activation