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

2. Vessel Bone marrow Stem cell PU-1 Szerv/szövetMakrofág populáció BoneOsteoclast Central nervous system Microglia Connective tissue Histiocytes PlacentaHofbauer cells KidneyMesengial cells LiverKupffer cells PeritoneumPeritoneal macrophages LungAlveolar macrophages SkinEpidermal and dermal macrophages Macrophages can act as stromal cells to help the differentiation of other cells. Monocyte Macrophage Tissues organs DEVELOPMENT OF MACROPHAGES

3. RECEPTORS AND OTHER MOLEKULES OF MACROPHAGES LPS receptor (CD14) + TLR4 MHCI MHCII TLR – pathogen pattern CR1 (CD35) CR3 (CD11b/CD18) LFA1 (CD11a/CD18) Fc  RIII (CD16) Fc  RII (CD32) Fc  RI (CD64) Ag + IgG complex Mannose receptor Scavanger receptor peroxidáz hidroláz

4. Copyright ©2008 American Society of Hematology. Copyright restrictions may apply. Dale, D. C. et al. Blood 2008;112:935-945 Activation of macrophages

5. RECEPTORLIGANDFUNCTION FcRIgG, IgEOpsonized phagocytosis, ADCC, release of inflammatory mediators CR3iC3B, ICAM-1Opsonized phagocytosis Macrophage Mannose Receptor Lectin,Endocytosis, phagocytosis, antigen capture and transport SR-ALPS, polianions, lipoteikolic acid Endocytosis, phagocytosis, adhesion CD14LPSTransduces LPS aktivation, TNFa release CCR1MIP1a, MCP-3Recruitment, migration of monocytes CCR3EotaxinHaematopoiesis, HIV-1 coreceptor CCR5MIP1Haematopoiesis, HIV-1 coreceptor CXCR4SDF-1aHaematopoiesis, HIV-1 coreceptor Receptors and molecules of macrophages

6. Activation of macrophages

7. IFN  IL-12 IL-18 Th 1 cell NK cell Inflammatory cytokines Antimicrobial substances Alternative activation: Mannose receptor – endocytosis Th2 chemokines NOS inhibition Tissue regeneration IL-4 IL-13 Th 2 cell Microorganism TNF IL-6IL-12 IL-10 T cell APC Inactivation Activation of macrophages Inflammatory cytokines

8. Functions of activated macrophages in anti-bacterial immunity Macrophage Response ** Role in Cell-mediated Immunity Production of reactive oxygen intermediates,Killing of microbes in phagolysomes nitric oxide; increased lysosomal enzymes(effector function of macrophages) Secretion of CytokinesTNF- , IL-1: leukocyte recruitment (TNF- , IL-1, IL-12)(inflammation) IL-12: T H -1 differentiation, IFN-  production (induction of response) Increased expression of:Increased T cell activation CD80, CD86(amplification) Class I, Class II MHC ** These macrophage responses are induced by CD40 ligation to CD154 (CD40L) and T cell- derived IFN-  in cell-mediated immunity; similar responses are induced by microbial products, particularly LPS, and NK cell-derived IFN-  in innate immunity.

9. Phagocytosis

10. Intracellular Bacterial Killing

11. Intracellular bacterial killing Reactive oxygen species Intracellular bacteria in phagolysosomes are susceptible to reactive oxygen species, which damage cell wall components and fragment genomic DNA. O 2 + NADPH NADPH Oxidase NADP + O 2 - + H + O 2 - + H + SOD O 2 + H 2 O 2 Reactive Oxygen Intermediate (ROI) production is initiated by membrane- bound NADPH oxidase, which is activated by IFN- . O 2 - is further metabolized by superoxide dismutase (SOD).

12. Intracellular bacterial killing Reactive oxygen species In the presence of appropriate iron catalysts, the Haber-Weiss reaction takes place: O 2 - + Fe 3+ O 2 + Fe 2+ H 2 O 2 + Fe 2+ OH + OH - + Fe 3+ O 2 - + H 2 O 2 OH +OH - + O 2 O 2 - is transformed into 1 O 2. 1 O 2 and OH are short-lived, powerful oxidants with high antibacterial activity, causing damage to DNA, membrane lipids, and proteins. Nomenclature O 2 - - superoxide anion OH – hydroxyl radical containing a free electron 1 O 2 – singlet oxygen, a highly reactive form of O 2

13. Intracellular bacterial killing Reactive oxygen species myeloperoxidase-dependent killing The lysosomes of granulocytes and monocytes/macrophages contain the enzyme myeloperoxidase (MPO). This enzyme catalyzes the following reaction: H 2 O 2 + Cl - OCl - + H 2 O MPO Hypochlorous acid and chloramines are formed – both agents further increase the bactericidal power of the ROI system by destroying biologically important proteins through chlorination (Halogenation).

14. Copyright ©2008 American Society of Hematology. Copyright restrictions may apply. Dale, D. C. et al. Blood 2008;112:935-945

15. Intracellular bacterial killing Reactive nitrogen species Phagocytes possess an additional pathway for generating reactive species that possess bactericidal activity. These species are the reactive nitrogen intermediates (RNI). The principal RNI is nitric oxide (NO), which is derived from the terminal guanidino-nitrogen atom of L-arginine. The reaction is catalyzed by the inducible form of nitric oxide synthase (iNOS; NOS2), leading to the formation of L-citrulline and NO.

16. Intracellular bacterial killing Reactive nitrogen species NO can act as an oxidizing agent alone, or it interacts with O 2 - to form unstable peroxynitrite (ONOO - ). This may be transformed to the more stable anions, NO 2 - and NO 3 -, or decomposed to NO. O 2 - + NOONOO - ONOO - + H + NO 2 - +. OH NO 3 - + H + ONOO - + H +. OH + NO. NO· and ONOO- are highly reactive antimicrobial agents. NO· may be transformed to nitrosothiols expressing the most potent antimicrobial activity. In contrast, NO 2 - and NO 3 - are without notable effects on microorganisms.

17. NO O2-O2- 0NO2- Destruction of mitochondria DNA destruction PARP activation CELL DEATH Producing ROI Decreased energy 1 NAD  ADP – ribose + NAM 4 ATP Protein destruction iNOS S-nitrosilation Effects of reactive species

18. NO : sera, (clinical) Gries Ilosvay assay (reduction of nitrit and nitrate to NO), Arginin –Citrulin reaction, detection of iNOS activity (IHC, Western blot), measure of released NO by DAF (FACS) Detection of mediators produced by macrophages Phagocytosis assay: yeast,uptake of fluorescent beads, Preopsonized FITC labeled E. coli (FACS) Citokines : TNFa, TGFb (ELISA, ELISPOT) ROI: NBT reduction assay Hydrogen peroxide assay Citochrome c reduction assay

19. Intracellular bacterial evasion of killing in phagocytes Intracellular bacteria have evolved strategies to evade killing by the mechanisms available to the phagocyte. DefensinsUnknown Phagosome acidificationPhagosome neutralization Phagosome–lysosome fusionInhibition of phagosome–lysosome fusion Lysosomal enzymesResistance against enzymes Intraphagolysosomal killingEvasion into cytosol Robust cell wall C3b receptor-mediated uptake, ROI ROI detoxifiers, ROI scavengers RNIUnknown (ROI detoxifiers probably interfere with RNI) Iron starvationMicrobial iron scavengers (e.g., siderophores) Tryptophan starvationUnknown Macrophage effector capacityMicrobial evasion mechanism

20. Diseases in which macrophages play a significant role Type Example Mechanism Lysosomal storage diseases Gaucher- syndrome Genetically coded, disfunction of glucocerebrozidase Niemann-Pick- syndrome Lack of sphyngomyelinase or disfunction of cholesterol estherization and –transport sphyngomyelin and cholesterol accumulation Tay –Sachs- syndrome Most prominent gangliosidosis, lack of hexose-aminidase-A, gangliosides accumulation in CNS

21. Diseases in which macrophages play a significant role Type Example Mechanism InfectionsAIDS Cellular immunodeficiency, lack of CD4+ T cells and macrophages Malaria Host mononuclear phagocyte system hyperplasia, massive splenomegaly Cerebrospinal diseases Alzheimer- dysease Senilis cerebralis amiloidosis, caused by improper elimination of amyloid-associated protein because of defects in macrophage enzymes

22. Diseases in which macrophages play a significant role Type Example Mechanism Chronic inflammation Silicosis Chrystal quartz powder phagocytosed by alveolar macrophages - progrediated nodular fibrotizing pneumoconiosis Asbestosis Asbestos filaments phagocytosed by alveolar macrophages - chromic desquamative alveolitis and interstitial inflammation become fibrosis Atherosclerosis Monocytes exit to the intima from the blood, become macrophages and store fat cytoplasmatically: foamy cells - inflammation

23. Granulomatosus inflammation - chronic inflammation - epitheloid cells in the infiltrate, these are modified macrophages whit pale cytoplasm and nucleus - cells with no intercellular substances (epithelial cell-like tight connections) - cells become multinucleated Langhans type giant cells ↓ Granulomatosus inflammation: granuloma formation with cell death

24. lymphocytes syncytium (multinucleated giant cells) Granulomas

25. Periapical granuloma = dental granuloma Modified granulation tissue containing elements of chronic inflammation, located adjacent to the root apex of a tooth with infected, necrotic pulp.

26. Phatogen: Mycobacterium tuberculosis Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti, and Mycobacterium microti can also cause tuberculosis, but these species do not usually infect healthy adults Tuberculosis most commonly attacks: the lungs (as pulmonary TB) central nervous system (meningitidis) lymphatic and circulatory system (miliary TB) genitourinary system, bones, joints skin From 2000 to 2004, 20% of TB cases being resistant to standard treatments and 2% resistant to second-line drugs. Tuberculosis (TBC) 2.000.000.000 infected worldwide

27. Mycobacterium infection DS 1.Infection of macrophages CD8+ T cell CD4+ T cell IL-12 2. Antigen presentation Macrophage perforin granulysin IFN-  TNF 3. T cell and macrophage activation Macrophage

28. Mycobacterium infection Healing (?) Acute tuberculosis - 10% (HIV infection) Symptom-free carriers 90% MTb. remains in granulomas Reinfection Dissemination Transmission Reactivation (10%) HIV infection: 800x more tuberculosis Other immune suppression DS CD8+ T cell CD4+ T cell IL-12 Macrophage perforin granulysin IFN-  TNF Macrophage

29. Morbus hungaricus Morbidity of TBC in Hungary: first decades of the later century: 340-380/100000 citizens 1955: 30/100000 citizens (lower than the European average!) reason: regular screening, vaccination, up-to-date therapy In last years: increasing numbers of TBC reason: optimistic attitude, ease of strict control The 90% of people infected with bacteria are symptome-free, living with latent TBC (LTBI), their opportunity is 10% to develop disease. Without treatment, 50% of TBC diseases are lethal. TBC is one of the three most dangerous infectious diseases worldwide, mortality is two times higher than to malaria. 2.000.000.000 infected persons Appearance and frequency of TBC

30. Skin and bone - tuberculosis

32. Primary infection

33. Miliaris tuberculosis

34. CNS tuberculosis

35. Lymphnode tuberculosis

36. Diagnostic testing of tuberculosis Tuberculin skin test(TST)  Most often applied tuberculin test: Mantoux’s test  PPD (purified protein derivative)  Size of induratio (after 48h) Disadvantages: Not specific for M. tuberculosis Positiv reaction: in case of atypical Mycobacterial diseases and BCG vaccination also

37. IFNγ release assay (IGRA) - ELISPOT  ESAT-6 (early secrete antigen target 6) and CFP-10 (culture filtrate protein) stimulatory antigens  Measuring: release of IFNγ by T cells  Results: SFU (Spot Forming Unit) Advantages : More specific than TST Can be repeated The testing protocol requires only one visit Disadvantages: Reversion: a previously positive IGRA results becomes negative upon revers testing, due to clearing of TB infection (spontaneous or due to treatment) biological variations among IGRA+ individuals the life cycle of M. tuberculosis, where the Mycobacterium enters a dormant state in which it may not be secrete ESAT-6 and CFP-10 antigens (but instead secrete other antigens which are not used in currently available IGRAs) Diagnostic testing of tuberculosis

38. Treatment First line tuberculosis drugs 3-letter1-letterDrug EMBEEthambutol INHHIsoniazid PZAZPyrazinamide RMPRRifampicin STMSstreptomycin Second line tuberculosis drugs CIP(none)Ciprofloxacin MXF(none)Moxifloxacin PASPp-aminosalicylic acid All first-line anti-tuberculous drug names have a standard three-letter and a single-letter abbreviation: Streptomycin is STM or S, isoniazid is INH or H, rifampicin is RMP or R, ethambutol is EMB or E, pyrazinamide is PZA or Z. The US commonly uses abbreviations and names that are not internationally recognised: rifampicin is called rifampin and abbreviated RIF; streptomycin is commonly abbreviated SM.

39. The standard "short" course treatment for tuberculosis (TB): 2 months: isoniazid, rifampicin, pyrazinamide, and ethambutol + 4 months: isoniazid and rifampicin alone For latent tuberculosis, the standard treatment is six to nine months of isoniazid alone