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

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

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

4. CELLS HUMORAL FACTORS 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 Enzymes (lysozyme, pepsin, trypsin) Antibacterial peptides Complement system Cytokines, chemokines Antibodies TWO LINES OF IMMUNE DEFENSE INNATE/NATURAL IMMUNITY ACQUIRED/ADAPTIVE IMMUNITY

5. SENSINGRECOGNITION SIGNALING RESPONSE INNATE IMMUNITY CellsReceptors Signaling pathways Cell-Cell collaboration Effector functions DEFENSE SYSTEMS ADAPTIVE IMMUNITY SENSINGRECOGNITION SIGNALING RESPONSE

6. Sinuses Trachea Lungs BRONCHIAL TRACT EYES Oral cavity esophagus Stomach Intestines GASTROINTESTINAL SYSTEM SKIN PHYSICAL BARRIERS PROTECTING OUR BODY FROM THE ENVIRONMENT Damage Infection Kidney Bladder Vagina UROGENITAL SYSTEM 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 10 12 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 ENDOTOXIN 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. Bacterium CD14 TLR4 LPSNFkBMyD88 IRAK LPB IL-6 Fungus Toll Cactus Tube Spätzel Peptid Protease Pelle Relish TOLL RECEPTORS ACTIVATE PHYLOGENETICALLY CONSERVED SIGNAL TRANSDUCTION PATHWAYS Inflammation Acute phase response Danger signal Macrophage Drosophila IL-1R associated Kinase

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

15. Macrophage/Dendritic cell TLR5 Flagellin Virus TLR3 dsRNA TOLL RECEPTORS RECOGNIZE VARIOUS MICROBIAL STRUCTURESTLR2 Peptidoglycane Gram+ TLR4 LPS TLR6 Gram- Interferon producing cell pDC IFN  Bacteria CpG DNA TLR9 TLR7TLR8ssRNS ALL STRUCTURES ARE ESSENTIAL FOR THE SURVIVAL OR REPLICATION OF THE PATHOGEN

16. TLR CONSERVED RECEPTORS/SENSORS THAT DETECT DANGER SIGNALS MEMBRANETLR3Fibroblast Epithelial cell DC CELL MEMBRANEBacteria MEMBRANES OF INTRACELLULAR VESICLES virus LRR TIR domain TIR: Toll-Interleukin Receptor signaling domain

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

18. RECOGNITION CYTOPLASMIC SENSORS

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

20. TLR CYTOPLASM CARD-CARD-helicase RLH CONSERVED RECEPTORS SENSING DANGER SIGNALS NLR nod-like receptors Leucin rich repeats Nucleotide binding domain NLRP1 – ASC NLRP3 – ASC – CARDINAL NBD NC PYR CARD NOD1/2, IPAF/NLRC4 MEMBRANTLR3 BIR IPAF Fibroblast Epithelial cell DC NBD NBD

21. Conventional DC Plasmacytoid DC 5 8 7 3 7 10 9 NLR=NOD/NALP (IL-1β) RLH=RIG-1/MDA5 (IFN) NLR IL-1β IL-12/23 IL-10 Th1/Th17/Th2 IFNαβ NK/DC 124616 RLH RLH DANGER SIGNALS ARE TRANSLATED TO CYTOKINE SECRETION THROUGH VARIOUS MOLECULAR SENSORS IN DC SUBTYPES TLR1 – bacterial lipoprotein (together with TLR2) TLR2 – bacterial lipoprotein, peptidoglycane, lipoteicholic acid (heteromer with TLR1 and TLR6) TLR3 –viral dsRNS, polyI:C TLR4 – bacterial LPS TLR5 – bacterial flagellin TLR6 – bacterial lipoprotein (with TLR2) TLR7 – viral ssRNA TLR8 – GU rich viral ssRNS, imidazoquinolin (antiviral drug) TLR9 – unmethylated CpG DNA TLR10 – modified viral nucleotides

22. SIGNALING IN INNATE IMMUNITY

23. Bacterium CD14 TLR4 LPSNFkBMyD88 IRAK LPB IL-6 Fungus Toll Cactus Tube Spätzel Peptid Protease Pelle Relish TOLL RECEPTORS ACTIVATE PHYLOGENETICALLY CONSERVED SIGNAL TRANSDUCTION PATHWAYS Inflammation Acute phase response Danger signal Macrophage Drosophila IL-1R associated Kinase

24. 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. TOLL RECEPTOR MEDIATED SIGNALLING NEW THERAPEUTIC TARGET

25. EFFECTOR MECHANISMS OF INNATE IMMUNITY

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

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

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. 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. PHAGOCYTOSIS

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 LyzozymeNADPH 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 dideasePROTECTION against bacteria and fungi is down regulated

34. NK-cell IL-12 macrophage IFN  cytokines neutrophil TNF-  INFLAMMATION – ACUTE PHASE RESPONSE hrs Plasma level LPS (endotoxin) (Gram(-) bacteria) TNF-  IL-1  IL-6 Kinetics of the release of pro- inflammatory citokines in bacterial infection TNF-  IL-1  IL-6 Few hours ACUTE PHASE RESPONSE Bacterium LPS DANGER SIGNAL ACTIVATION PRR 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. Liver C-reactive protein Phosphocolin binding (e.g.fungi) COMPLEMENT Serum Amyloid Protein (SAP) Mannose/galactose binding Fibrinogen Mannose binding lectin/protein MBL/MBP COMPLEMENT IL- 6 THE ACUTE PHASE RESPONSE IL-6 induces the production of acute phase protiens Phosphocoline binding Fungi, bacterial Cell wall.

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

43. RECOGNITION BY SOLUBLE MOLECULES MANNOSE BINDING LECTIN

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

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

46. NK cells Type I IFNs increase their cytotoxicity (100x) IL12, and TNFα are also able to activate them IFNγ production --- MF, DC activation - 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