GASTROINTESTINAL SYSTEM CONTACT SURFACES Physical, chemical, biological borders Oral cavity Esophagus Stomach Alimentary tract GASTROINTESTINAL SYSTEM EYE Sinuses Trachea Lungs AIRWAY SYSTEM WALDEYER RING Tonsils, adenoids Palatinal, pharyngeal lingual and tubar tonsils SKIN Kidney BladderVagina UROGENITAL SYSTEM Demage Infection Mucus glycoproteins, proteoglycanes, enzymes

DEFENSE LINES OF NATURAL IMMUNITY ANATOMICAL BORDERS Skin Inhibits entry of pathogens, pH3 – 5 inhibits growth Mucosa Normal bacterial flora competes for binding sites and nutrients Mucus keeps away pathogens from the surface Cilia remove pathogens PHYSIOLOGICAL BORDERS Temperature Physiological body temperature and fever inhibits growth of certain pathogens Low pH Most pathogens are destroyed in the stomach Chemical Lysosyme degrades bacterial cell wall Type I interferons induce anti-viral resistance The complement system is able to lyse bacteria and promotes phagocytosis PHAGOCYTOSIS/ENDOCYTOSIS Many cells can take up microorganisms by receptor-mediated internalization Special professional phagocytes (monocyte, neutrophil, macrophage) are able to internalize, kill and degrade microorganisms INFLAMMATION Tissue demage and infection results in the leakage of anti-bacterial proteins and peptides to the affected tissue Phagocytic cells leave the blood stream and enter inflammed tissues

AS AN IMPORTANT FIRST LINE OF DEFENSE THE EPITHELIUM AS AN IMPORTANT FIRST LINE OF DEFENSE EPITHELIAL MONOLAYER

ORGANIZATION OF IMMUNE CELLS UNDER EPITHELIAL SURFACES Dendritic cell Epithelial cells Stroma cells Granulocyte Macrophage NK cell NKT cell DC TISSUE – SPECIFIC CELLULAR COMMUNICATION NETWORKS PERIFÉRIÁS SZÖVETEK PERIFÉRIÁS LIMFOID SZERVEK

MONOCYTE – MACROPHAGE – DENDRITIC CELL FAGOCYTE SYSTEM GRANULOCYTES MONOCYTE – MACROPHAGE – DENDRITIC CELL Defence against infectious diseases Elimination of tumor cells Transplantation Gatekeeper function Sensing commensals and pathogens Rapid activation of innate immunity Priming adaptive immune responses Maintenance of self tolerance

COLLECTION OF ENVIRONMENTAL INFORMATON, CONCENTRATION TISSUE MACROPHAGES AND DENDRITIc CELLS INTERNALIZE SOLUBLE MATERIAL AND PARTICLES ION CHANNELS ABC TRANSPORTERS CARBOHYDRATES PROTEINS C-TYPE LECTINS MMR/CD206 DC-SIGN/209 Langerin/CD207 OPSONIZING RECEPTORS FcR, CR INFECTED DEAD CELLS SCAVENGER RECEPTORS CD36, SR-A LIPID RECEPTORS LDL-R, CD91 LIPIDS UPTAKE Macropinocytosis Receptor-mediated endocytosis Fagocytosis COLLECTION OF ENVIRONMENTAL INFORMATON, CONCENTRATION

EXPRESSION OF THE RECEPTOR DETERMINES INTERACTION WITH LIGANDS Chemoattractant cytokines called chemokines direct migration of leukocytes to the appropriate anatomical sites EXPRESSION OF THE RECEPTOR DETERMINES INTERACTION WITH LIGANDS

Engagement of PRR triggers phagocytosis and cytokine production PHAGOCYTOSIS, RECOGNITION RECEPTORS, SIGNAL TRANSDUCTION, EFFECTOR MECHANISM Pattern recognition Receptors (PRR) Engagement of PRR triggers phagocytosis and cytokine production Lectins: receptors ( or plasma proteins) that bind to carbohydrates. On macrophages one finds mannose rec. And glucan receptors. Scavenger receptor binds various substrates generally negatively charges (sulfated polysaccharides, nucleic acids, lipoteic acid bound in the cell wall of Gram positive bacteria)

BY THE INNATE IMMUNE SYSTEM RECOGNITION BY THE INNATE IMMUNE SYSTEM

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 Macrophage, dendritic cell – ACT AS TISSUE SENSORS (GATE KEEPERS) Neutrophil granulocytes – MIGRATE FROM THE BLOOD TO THE SITE OF INFLAMMATION

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 is not synthesized

complement INNATE (NATURAL) IMMUNITY Toll-receptors C. elegans RECOGNIZING RECEPTORS PROTECTIVE MECHANISMS Enzyme systems Multicellular (Metazoa) Sea urchin 600 million years Toll-receptors C. elegans Drosophila 700 million years complement IN PLANTS

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

DANGER SIGNALS ARE TRANSLATED TO CYTOKINE SECRETION THROUGH VARIOUS MOLECULAR SENSORS IN DC SUBTYPES 4 6 6 2 1 1 5 10 3 7 9 7 8 NLR RLH RLH NLR=NOD/NALP (IL-1β) RLH=RIG-1/MDA5 (IFN) Plasmacytoid DC IFNαβ NK/DC IL-1β IL-12/23 IL-10 Th1/Th17/Th2 Conventional DC 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 – mdified viral nucleotides

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

SIGNALING IN INNATE IMMUNITY

TOLL RECEPTORS ACTIVATE PHYLOGENETICALLY CONSERVED SIGNAL TRANSDUCTION PATHWAYS Fungus Toll Cactus Tube Spätzel Peptid Protease Pelle Relish Bacterium LPS LPB CD14 TLR4 TRIF IRF3 STAT1 IFN TLR3 TLR4 CD14 MyD88 NFkB IRAK IL-1R associated Kinase TIR-domain-containing adapter-inducing interferon-β Inflammation Acute phase response Danger signal IL-6 Drosophila Macrophage

IL- 6 THE ACUTE PHASE RESPONSE Mannose binding lectin/protein MBL/MBP COMPLEMENT C-reactive protein COMPLEMENT Liver Serum Amyloid Protein (SAP) Mannose/galactose binding Chromatin, DNA, Influenza Fibrinogen IL-6 induces the production of acute phase protiens

Sensing of LPS by TLR4 leads to activation of the Transcription factor NFkB and the synthesis of inflammatory cytokines. First panel: LPS is detected by the complex of TLR4, CD14, and MD2 on the macrophage surface. Second panel: the activated receptor binds the adaptor protein MyD88, which binds the protein kinase IRAK4. IRAK4 binds and phosphorylates the adaptor TRAF6, which leads via a kinase cascade to the activation of IKK. Third panel: in the absence of a signal, the transcription factor NFkB is bound by its inhibitor, IκB, which prevents it from entering the nucleus. In the presence of a signal, activated IKK phosphorylates IκB, which induces the release of NFκB from the complex; IκB is degraded. NFκB then enters the nucleus where it activates genes encoding inflammatory cytokines. Fourth panel: cytokines are synthesized from cytokine mRNA in the cytoplasm and secreted via the endoplasmic reticulum (ER). This MyD88-NFκB pathway is also stimulated by the receptors for cytokines IL-1 and IL-18.

TLR4 activation can lead to the production of either inflammatory cytokines or antiviral type I interferons. TLR4 can stimulate two different intracellular signaling pathways, depending on whether the adaptor protein MyD88 or TRIF is recruited to the activated receptor. TLR4 signaling through TRIF leads to activation of the transcription factor interferon response factor 3 (IRF3) and the production of type I interferons. Signaling through MyD88 leads to activation of the transcription factor NFκB and the production of inflammatory cytokines such as IL-6 and TNF-?. TLR3 also uses the TRIF pathway.

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.