1. BY THE INNATE IMMUNE SYSTEM
RECOGNITION
BY THE INNATE IMMUNE SYSTEM

2. FUNCTIONAL ATTRIBUTES OF INNATE AND ADAPTIVE IMMUNITY

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

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

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

6. 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

7. 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

8. SIGNALING
IN INNATE IMMUNITY

9. 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.

10. 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.
TIR domain: Toll/Interleukin-1R (TIR) domain.
MYD88: Myeloid differentiation primary response gene 88, an adapter that signals for all TLRs except TLR3
IRAK: interleukin-1 receptor-associated kinase
TRIF: TIR-domain-containing adapter-inducing interferon-β
IRF3: Interferon regulatory factor 3
TRAM: TRIF-related adaptor molecule

11. 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.

12. Activation of macrophages induces secretion of multiple
pro-inflammatory cytokines

13. Systemic release of TNFa initiates septic shock
Local production of
A TNFα (and IL1) is beneficial,
Protective, BUT systemic release
May cause death
Drop in blood volume and hence
blood pressure
Disseminated intrvascular
coagulation
The panels on the left describe the causes and consequences of the release of TNF-α within a local area of infection. In contrast, the panels on the right describe the causes and consequences of the release of TNF-α throughout the body. The initial effects of TNF-α are on the endothelium of blood vessels, especially venules. It causes increased blood flow, vascular permeability, and endothelial adhesiveness for white blood cells and platelets. These events cause the blood in the venules to clot, preventing the spread of infection and directing extracellular fluid to the lymphatics and lymph nodes, where the adaptive immune response is activated. When an infection develops in the blood, the systemic release of TNF-α and the effect it has on the venules in all tissues simultaneously induce a state of shock that can lead to organ failure and death. H, heart; K, kidney; L, liver; S, spleen.

14. from blood to infected tissues (extravasatio)
Pro-inflammatory cytokines activate endothel which recruits immunocytes
from blood to infected tissues (extravasatio)
Inflammatory mediators and cytokines produced as the result of infection induce the expression of selectin on vascular endothelium, which enables it to bind leukocytes. The top panel shows the rolling interaction of a neutrophil with vascular endothelium as a result of transient interactions between selectin on the endothelium and sialyl-Lewisx (s-Lex) on the leukocyte. The bottom panel shows the conversion of rolling adhesion into tight binding and subsequent migration of the leukocyte into the infected tissue. The four stages of extravasation are shown. Rolling adhesion is converted into tight binding by interactions between integrins on the leukocyte (LFA-1 is shown here) and adhesion moleules on the endothelium (ICAM-1). Expression of these adhesion molecules is also induced by cytokines. A strong interaction is induced by the presence of chemoattractant cytokines (the chemokine CXCL8 is shown here) that have their source at the site of infection. They are held on proteoglycans of the extracellular matrix and cell surface to form a gradient along which the leukocyte can travel. Under the guidance of these chemokines, the neutrophil squeezes between the endothelial cells and penetrates the connective tissue (diapedesis). It then migrates to the center of infection along the CXCL8 gradient. The electron micrograph shows a neutrophil that has just started to migrate between adjacent endothelial cells but has yet to break through the basement membrane, which is at the bottom of the photograph. The blue arrow points to the pseudopod that the neutrophil is inserting between the endothelial cells. The dark mass in the bottom right-hand corner is an erythrocyte that has become trapped under the neutrophil. Photograph (x 5500) courtesy of I. Bird and J. Spragg.

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

16. Killing of bacteria by neutrophils: azurophilic and specific granules
After phagocytosis (first panel), the bacterium is held in a phagosome inside the neutrophil. The neutrophil's azurophilic granules and specific granules fuse with the phagosome, releasing their contents of antimicrobial proteins and peptides (second panel). NAPDH oxidase components contributed by the specific granules enable the respiratory burst to occur, which raises the pH of the phagosome. Antimicrobial proteins and peptides are activated and the bacterium is damaged and killed. A subsequent decrease in pH and the fusion of the phagosome with lysosomes containing acid hydrolases results in complete degradation of the bacterium. The neutrophil dies and is phagocytosed by a macrophage.
azurofil ic specific granuls
Lyzozyme NADPH oxidase
Defensins Lyzozyme
Mieloperoxidase
Cathepsin G
elastase

17. help destroy pathogens
Phsgocyte oxidase (Phox) produces reactive oxidative species (ROS) that
help destroy pathogens
In the absence of infection the antimicrobial proteins and peptides in neutrophil granules are kept inactive at low pH. After the granules fuse with the phagosome the pH within the phagosome is raised through the first two reactions, involving the enzymes NADPH oxidase and superoxide dismutase. Each round of these reactions eliminates a hydrogen ion, thereby reducing the acidity of the phagosome. A product of the two reactions is hydrogen peroxide, which has the potential to damage human cells. (In hair salons and the manufacture of paper it is used as a powerful bleach.) The third reaction, involving catalase, the most efficient of all enzymes, promptly gets rid of the hydrogen peroxide produced during the neutrophil's respiratory burst, raising the pH of the phagosome and enabling activation of the antimicrobial peptides and proteins.

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

19. RECOGNITION BY SOLUBLE MOLECULES MANNOSE BINDING LECTIN

20. GLYCOSYLATION OF PROTEINS IS DIFFERENT IN VARIOUS SPECIES
Eukariotic cells
Prokariotic cells
Mannose
Galactose
Glucoseamin
Mannose
Neuraminidase

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

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

23. RECOGNITION
CYTOPLASMIC SENSORS

24. CONSERVED RECEPTORS SENSING DANGER SIGNALS
NLR nod-like receptors
Leucin rich repeats
Nucleotide binding domain
TLR N
NBD C
PYR
NLRP1 – ASC
NLRP3 – ASC – CARDINAL
CARD
NOD1/2, IPAF/NLRC4
NBD
BIR
IPAF
NBD
NOD proteins: NOD1 : Muramyl dtipeptide in Gram negatives, NOD2: muramyl-dipeptide
Caspase recruitment domains, or Caspase activation and recruitment domains (CARDs)
MEMBRAN
TLR3
Fibroblast
Epithelial cell DC
CYTOPLASM
CARD-CARD-helicase
RLH

25. NOD-like receptors Group of abot 20 proteins named after NOD1 and NOD2
NOD family: --- intracellular bacteria
Activate NF-kB and induce chemokine secretion
NALP family regulation of cytokine release mainly IL1, 18, 33,
produced in an inactive form. (via regulation of caspases)
With other adaptor proteins they form „Inflammosomes”
Activation: by various bacterial pore forming toxins
endogenous compounds, mono sodium urate
(MSU), Ca pyrophosphate dihydrate (CPPD), ATP

26. INTERFERON RESPONSE

27. EFFECTS OF TYPE I INTERFERONS NATURAL INTERFERON PRODUCING CELLS – IPC
vírus
Plasmacytoid dendritic cells produce 1000x more type I interferon than other cells
NATURAL INTERFERON PRODUCING CELLS – IPC
After viral infection they are accumulated at the T cell zone of the lymph nodes

28. VIRUS INDUCED TYPE I INTERFERON PRODUCTION
Type I IFN receptor
IFN response
Virus
IFN-
IRF-3
NFB
AP-1
IRF-3
IFN-
paracrine
IFN-
IRF-7
autocrine
TLR3 binds dsRNA, IRF3 gets phosphorylated, dimerizes and translocate into the nucleus. IFN-beta expression is induced. Autokrine and paracrine effects.
Infected cell
IFN response
IFN-
subtypes
IRF: interferon regulatory factor

29. GAS – promoter elements
Type I. IFN receptor
Type III. IFN receptor (IFNλ)
Type II. IFN receptor
IFNAR1/2
IFNLR1
IL-10R2
IFNG1/2
TYK2
JAK1
TYK2
JAK1
JAK2
JAK1
Plasma membrane
Cytoplasm
Signal Transducers and
Activators of Transcription
STAT1
STAT1
STAT2
ISGF-3
STAT1
STAT2 P
STAT1 P
PKR. Ser/thr protein kinase activated by ds RNA. Autophosphorylactivated by dsRNAation and also eIF2. Translation shut off.
OAS: activated by ds RNA. 2’5’ oligo A syntehsized, RNAse L is dimerized and activated. ssRNA is cleaved.
GAS: Gamma activating sequence
IRF9
Interferon-stimulated genes
Nucleus
STAT1 P
STAT1
STAT2 P
ISG15, Mx,
OAS and
PKR
GAS: Gamma
Activating sequence
ISRE
GAS – promoter elements
Antiviral immunity
Interferon-stimulated
Regulatory elements
Antimycobacterial immunity

30. INTERFERON EFFECTOR PATHWAYS induction of the „antiviral state
1. Mx GTPase pathway
Trap viral particles in Endoplasmic Reticulum
2. 2',5'-oligoadenylate-synthetase (OAS)-directed Ribonuclease L pathway
degrade viral RNA
3. Protein kinase R (PKR) pathway (Ser/Thr kinase, dsRNA-dependent)
inhibit translation
4. ISG15 ubiquitin-like pathway
modify protein function
CONTROL ALL STEPS OF VIRAL REPLICATION

31. Mechanism of action of MxA, OAS1 and PKR cleaved RNA Oligomer
accumulation
in cytoplasmic
membranes
(e.g. ER)
(Nucleus)
(Cytoplasm)
ISRE
MxA
MxA monomer
MxA oligomer
Trapped viral
components
Mechanism of action of
MxA, OAS1 and PKR
(Nucleus)
(Cytoplasm)
ISRE
PKR
Inactive PKR monomer
Active PKR dimer
Induction by
viral RNAs
EIF2a P
Inhibition of
translation
(Nucleus)
(Cytoplasm)
ISRE
OAS1
Inactive OAS1 monomer
Induction by
viral dsRNA
Active OAS1 tetramer
synthetized
pppA(2’p5’A)n
inactive
RNaseL
monomer
active
dimer
cleaved RNA