VL 3 - Innate PRR and paghocytosis

Prevention / combat of pathogen entry and infection
1. physical / chemical barrier (tight junctions)
2. anti-microbial peptides (defensines, S100 proteins)
3. neutralisation by secretory IgA
4. activation of coagulation cascade upon vessel damage
bradykinin è pain /
complement activation
4. PRR-mediated activation of epithelial / endothelial cells
prostaglandins / cytokines
è vasodilatation / permeabilisation
5. complement activation (alternative / MBL / classical)
opsonization / anaphylaxis / killing
6. mast cell activation
histamin / lipid mediators / cytokines / chemokines
è pain / vasodilat. / permeab. / immune cell activation + recruitment
7. local PRR-macrophage activation
8. recruitment of neutrophils and more macrophages
9. phagocytosis and killing of pathogens
Neutrophils only accumulate in tissues upon injury
Homeostasis
Staphylococcus infection
Prevention / combat of pathogen entry and infection
1. physical / chemical barrier (tight junctions)
2. anti-microbial peptides (defensines, S100 proteins)
3. neutralisation by secretory IgA
4. activation of coagulation cascade upon vessel damage
bradykinin è pain /
complement activation
4. PRR-mediated activation of epithelial / endothelial cells
prostaglandins / cytokines
è vasodilatation / permeabilisation
5. complement activation (alternative / MBL / classical)
opsonization / anaphylaxis / killing
6. mast cell activation
histamin / lipid mediators / cytokines / chemokines
è pain / vasodilat. / permeab. / immune cell activation + recruitment
7. local PRR-macrophage activation
8. recruitment of neutrophils and more macrophages
9. phagocytosis and killing of pathogens
What kind of PRRs do we know?
pathogen
Typical
Biochemical
structures=
patterns
Injury, mechanical stress, Trauma
PAMPs=
DAMPs=Alarmine
Pathogen
Associated
molecular
patterns
Damage
associated
molecular
patterns
Pattern recognition receptors = PRRs
(soluble and cell-associated)
pathogen
3) Sekretion of pro-inflammatory mediators
by immune cells (Makrophages, Dendritic cells)
bioactive lipids
(Thromboxanes,
Prostaglandines,
leukotriens)
Vasodilation
Higher blood circulation
rubor=redness
cytokines
Activation of
local endothelial
cells
chemokines
attract
„gorging“ phagocytic
cells
tumor=Swelling
Synthesis
of bioactive
Lipids
Pharmacological
Targets:
Phospholipase A2
Cyclooxygenase (COX)
Ibuprofen
Receptores for PAMPs =
Pattern recognition receptors (PRRs)?
LPS
LBP
CD14
??
NF-κB
Pro-inflammatorische
Zytokine (z.B. TNF-α)
Immune response of the fruit fly (Drosophila)
Nüsslein-Volhardt
discovery of Toll, Nobel prize 1995
Jules Hoffmann
innate immune response in
drosophila, Nobel prize 2011
together with Bruce Beutler and
Ralph Steinman
Drosophila
Human/Mice etc
• PAMPS-TLR-Specificities
• West AP, et al. 2006 in Annu. Rev. CellDev. + Biology TLRs can trigger immune response via 2 different signalling pathways
only
Myd88
Independent
(= TRIF dependent)
only
Myd88
dependent
TLR1/2
TLR2/6
TLR5
TLR7
TLR8
TLR9
TRIF
TLR3
TLRs can trigger immune response via 2 different signalling pathways
TLRs: Receptors as relays of
differences in dangers
Output: Patterned responses!
TLRs are located in different cell types and compartiments
mainly in macrophages and mDCs
mainly in
pDCs
NF-κB
TNFα etc.
IRF7
IFNβ
Nucleus
Also other (non-immune) cells (e.g. epithelial cells) can express TLRs
Other PRRs – NOD1 and NOD2 as intracellular PRRs
How can a virally infected non-immune cell,
which does not express TLR3, 7 or 9
sense the viral infection and induce IFNα expression?
Alternative (TLR independent) intracellular RNA recognition
MAVS signaling pathway.
RIG-I or MDA-5 contains 2
N-terminal CARDs and a
C-terminal RNA helicase
domain that interacts with
viral dsRNA (earlyreplication intermediates).
Interaction between RIG-I
or MDA-5 and MAVS
through CARD stimulates
nuclear factor (NF)-κB–
and interferon (INF)
regulatory factor (IRF)–
dependent pathways.
Toshitaka Yajima, and Kirk U. Knowlton Circulation.
2009;119:2615-2624
Intracellular DNA and RNA sensors
Typ-­‐I-­‐Interferone Bildung • Induk@on von IFN-­‐β durch dsRNA (TLR3 oder PKR) in allen Zellen • Verstärkung autokrin oder parakrin über IFN-­‐R , Bildung von IFN-­‐α (wirkt dann unabhängig von NFkB) • Interferon-­‐producing cells (IPC) (1 % der Leukozyten, sehen wie Plasmazellen aus), können 1000x mehr IFN produzieren als andere Zellen) und besitzen verschiedene TLR und differenzieren zu plasmacytoid dendri@c cells (pDCs) Folgen • Bildung von Oligoadenylat-­‐Synthetase (Synthese von 2´-­‐5´-­‐oligoA, ak@viert Endonuklease, baut virale RNA ab) • verstärkt Prolifera@on und Cytotoxizität von NK-­‐Zellen und bewirkt Expression von Liganden für NK-­‐Zellen (MIC-­‐A, MIC-­‐B) • Induk@on von PKR als PRR Inhibition of virus replication by IFNα/β
IFNα/β
2-5OA
RNAseL
Degradation of dsRNA
PKR
eIF2-p
PKR = RNA-dependent protein kinase
2-5OA = 2´-5´ oligoadenylate synthetase
translation
• Other PRRs – C-­‐type lecDns C-­‐Typ-­‐LekDn-­‐Rezeptoren typisch ist eine Ca2+-­‐abhängige CRD-­‐Domäne (carbohydrate recogni@on domain) • Mφ mannose receptor • DC-­‐SIGN • Dec@n-­‐1 •  Mφ mannose receptor is a constantly recycling endocy@c receptor, recogni@on of terminal mannose/
fucose > NAc-­‐Glc (CRD) and sulphated-­‐NAc-­‐Gal •  DC-­‐SIGN (DC-­‐specific ICAM3 grabbing non-­‐integrin) is largely restricted to DC, recognises highmannose oligosaccharides •  DecDn-­‐1 exhibits specificity for β-­‐(1,3/1,6)-­‐glucans in fungal cell walls expressed on monocytes, MØ, neutrophils and subsets of DC Mφ Mannose
Receptor
DC-SIGN
Dectin-1
http://users.path.ox.ac.uk/~cholt/Lecture1%20handout.pdf
Scavenger receptors: PPRs for both infecDous nonself and modified self • 
• 
• 
• 
Gruppe von Rezeptoren, die modifizierte „low density“ Lipoproteine (mLDL) und anionische Substanzen (z.B. LPS, Lipoteichonsäuren, einige Polynukleo@de) erkennen Aufnahme von acetyliertem und oxidiertem LDL und haben eine Bedeutung bei der Entstehung der Arteriosklerose (CD36 und SR-­‐A) Aufnahme von Listerien, Staphylokokken, Neisserien und apopto@schen Zellen (SR-­‐A) Aufnahme von Streptokokken (MARCO, MAcrophage Receptor with COllagenous structure) one receptor – many ligands LDL receptor ist spezifischer splice-­‐Varianten 2 TM N-­‐term. extrazellulär http://miami.uni-muenster.de/servlets/DerivateServlet/Derivate-131/d_einleitung.pdf
Alarmins = DAMPs
(damage/danger associated molecular patterns)
Necrotic dying cells release danger signals
ECM
infection
MSU
Cell death
Inflammasome
activation
ATP
Trauma
Injury
burn
DC
HMGB1
HSPs
inflammation
Plasma enzymes
HMGB = high mobility group B
HSPs = heat shock proteins
MSU = monosodium urate; uric acid crystals
ECM = extra cellular matrix
ECM
Degradation
(e.g. Hyalunoric acid)
TLR
Stimulation
(also other
receptors)
cytokines
NOD-like receptors –
regulators of inflammation
NOD1 and
NOD2 as
intracelluar
PAMP
sensors
NLRPs =
inflammasome
as DAMP sensor
ASC = apoptosis-associated speck-like protein
containing a carboxy-terminal CARD
Mustererkennungrezeptoren (PRRs) • 
membrangebundene PRRs scavenger Rezeptoren Toll-­‐like Rezeptoren C-­‐Typ-­‐Lek@n-­‐Rezeptoren Erkennung nach Opsonisierung: mit An@körpern: Fc-­‐Rezeptoren mit C3b: Komplement-­‐Rezeptoren CR3 • 
cytosolische PRRs NOD-­‐like Rezeptoren (NOD1, NOD2) RIG-­‐I-­‐like RNA-­‐Helikase • 
sezernierte Proteine Collek@ne: (gehören auch zu C-­‐Typ-­‐Lek@nen) Mannose-­‐bindendes Lec@n (MBL) und surfactant protein A, D (SP-­‐A, SP-­‐D) Ficoline Pentraxine: Serum Amyloid, C-­‐reak@ves Protein, • 
endozy@erende Rezeptoren • 
signalgebende Rezeptoren: → adap@ve Immunantwort (Expression von Cytokinen , kos@mulatorischen Proteinen • 
Komplement-­‐Ak@vierung Summary PRRs Summary PRR signaling
Other cytokines (IL-1, TNF-α) stimulate similar pathways
IL-17
IL-1R
Such cytokines activate (amplify) inflammation – they act as danger signals !
Blutgefäß Gewebe (Haut) Zytokine machen Entzündung Chemokine locken Leukozyten ins entzündete Gewebe Haut (Epithel) Chemokine Infektion Zytokine Gefäßwand (Endothel) transmigration Slow rolling rolling Blutfluss Leukozyt Entzündungsbotenstoffe weisen den Weg zur Infektionsstelle Gewebe (Haut) Haut (Epithel) Infektion PAMPs Komplement
aktivierung
MΦ
DCs
Mast
zelle
Histamin
Blutgefäß Zelltod DAMPs PGs
Öffnung
Vaso
Tight
dilatation junctions
Chemokine
Zytokine
(z.B. TNFα, IL-1) (z.B. IL-8 = CXCL8)
Influx
C +AK
Induktion
Influx
Adhäsions
Granulozyten
moleküle
PAMPs = pathogen associated molecular patterns
DAMPS = danger/damage associated molecular patterns
PRRs = pattern recognition receptors
Anlocken und ExtravasaDon von Neutrophilen Glykoprotein-­‐Selek@n CXCL8 (IL-­‐8) wirkt chemotak@sch auf Neutrophile Chemokin-­‐Chemokin-­‐Rezeptor bacterial products (fMLP), C5a, LT-­‐
B4 → Ca2+ → Cytoskelel Integrin-­‐Integrin-­‐Ligand PECAM Neutrophile wandern schneller zum Entzündungort (1 d) als Monozyten (2-­‐3 d) P-­‐selec@n glyco-­‐
protein ligand-­‐1 P(platelet)-­‐SelekDn aus intrazellulären Vesikeln (Weibel-­‐Palade-­‐bodies) Sekunden nach Signal, z. B. Histamin an die Zelloberfläche E(endothelial)-­‐SelekDn: Neusynthese induziert durch TNF-­‐α oder IL-­‐1 über NFκB TNF-­‐α, IL-­‐1 induzieren Bildung von Zell-­‐adhäsions-­‐
molekülen, erhöhte Gefäßpermeabilität (TNF-­‐α) IL-6
IL-12
Ak@vierung von NK-­‐Zellen Leber: Bildung von Akut-­‐
Phase-­‐Proteinen Fieber (IL-­‐1, TNF-­‐α, IL-­‐6) Clinical case development
•  Afternoon: ague (shivering fever) 38,5 oC. Local
inflammation signs enhanced.
•  Additonally signs of Lymphadenitis and swelling of
lymphnodes.
•  Medic prescribes Antibiotics (Ampicillin) and lokal
cooling Kühlung.
Start of acute phase response
„Warning“ remote response
cytokines
Tumor Necrosis
Factor alpha (TNF-α)
Interleukin-1 (IL-1)
Pyrogene cytokines
Interleukin-6
(IL-6)
Granulocytes/Monocytes
Colony Stimulating factor
GM-CSF
Hypothalamus
Temperature
Set point changes
Liver
Synthesis of
Acute-Phase-Proteins
Bone marrow
New synthesis of
Phagocytic cells
Fever
u.a. Opsonisation
„Linksverschiebung“
Akut Phase Proteine
Opsonisierung:
Markierung von Erregern
durch Bindung an Erregerstrukturen
für die erleichterte Phagozytose
Beispiele:
Collectins:
C reaktives Protein (CRP)
mannose binding lectin (MBL)
serum amyloid protein (SAP)
surfactant proteins (SP-A SP-D)
Komplement C3 (C3b)
Energy for the immune system
Adipocyted
Lipolyse
LipoproteinlipaseActivity
Lipid metabolism
Muscle cells
IL-1 and
TNF-alpha
Protein
metabolism
Appetite
cachexia and Wasting
Makrophage functions
1)  Secretion of pro-inflammatory mediators
a) cytokine
b) chemokines
c) bioactive lipids
2. Phase: attraction/accumulation
of more/other immune cells
2)  Phagocytosis
3)  Killing of phagocytosed pathogen
Phagocytosis receptors for opsonized pathogenes
? CRP-Rezeptor (CD32??)
? MBL-Rezeptor (CD14??)
CRP
MBL
Fibronektin
Komplement
(C3b)
Integrine
„Pathogene
Oberfläche“
C3b
Antikörper
Opsonisierungsprinzip
CR1 (CD35)
CR3 (CD11b/CD18)
CR4 (CD11c/CD18)
Fc-Rezeptoren
CD16 = FcγRIII
CD32 = FcγRII
CD64 = FcγRI
Makrophagen / Neutrophile
Phagozytosis receptors
for non-opsonized pathogenes
Scavenger-Rezeptoren:
SR-A I+II
MARCO
Lektin-type-Rezeptoren:
Mannose Rezeptor
β-Glukan-Rezeptor (Dectin-1)
LPS-Rezeptor
CD14
Phagozytose
1)  Aufnahme des Erregers
- Pseudopoiden
- Aufnahme in die Zelle
- Ablösung von Zellmembran
2) Verschmelzung mit Lysosom
- Phagolysosom
- saurer pH
3)  Abtöten des Erregers
- oxidative burst
(O2-Radikale; NO)
4) Abbau der Erregerbestandteil
- Enzyme
(Hydrolasen, Proteasen, etc...)
Phagozytose
1)  Aufnahme des Erregers
- Pseudopodien
- Aufnahme in die Zelle
- Ablösung von Zellmembran
2) Verschmelzung mit Lysosom
- Phagolysosom
- saurer pH
3)  Abtöten des Erregers
- oxidative burst
(O2-Radikale; NO)
4) Abbau der Erregerbestandteil
- Enzyme
(Hydrolasen, Proteasen ...)
Phagozytose effektiver bei 2 Signalen
1.  Fc-Rezeptor
2.  Komplementrezeptor
+ weitere Aktivierung über TLRs /
Zytokine / C5a
Principles of KILLING
ROS-formation
ROS = reactive oxygen species
„respiratory burst“
Stickstoffoxid-abhängiges Abtöten
Examples: Mycobacterium tuberclosis / leprae, Listeria monocytogenes, Salmonella enterica
Neutrophil ways of KILLING -­‐ NETosis Mechanism of Neutrophil Extracellular Traps
release. Neutrophils are stimulated by
contact with bacteria, protozoan, fungi (yeast
and hyphae forms) or their products (not
shown), leading to: (a) ultrastructural
alterations of nuclear shape with chromatin
decondensation, swollen and fragmentation
of the nuclear membrane, which allow the
association of granules and cytoplasmic
proteins with the chromatin, and (b) release
of extracellular structures consisting of a
DNA-backbone, decorated with histones,
neutrophil granular and cytoplasmatic
proteins, AMPs (NETs), which ensnare and
kill microorganisms.
Release of neutrophil mediators by degranula@on Mucosal Immunology (2012) 5, 354-366;
doi:10.1038/mi.2012.24
Phagozytose nicht möglich-Was nun?
Parasiten z.B. Würmer
„Frustrierte“ Phagozytose
Degranulation
C3a
C5a
Fcε
IgE
Mastzellen
Eosinophile
Basophile
Phagozytose nicht möglich-Was nun?
Hermetisch „Abriegeln“
Granulom
Gewebe (Haut) Haut (Epithel) Infektion PAMPs Komplement
aktivierung
MΦ
DCs
Mast
zelle
Histamin
Blutgefäß Zelltod DAMPs PGs
Öffnung
Vaso
Tight
dilatation junctions
Chemokine
Zytokine
(z.B. TNFα, IL-1) (z.B. IL-8 = CXCL8)
Influx
C +AK
Induktion
Influx
Adhäsions
Granulozyten
moleküle
PAMPs = pathogen associated molecular patterns
DAMPS = danger/damage associated molecular patterns
PRRs = pattern recognition receptors
Von der angeborenen zur erworbenen Immunität: Transport von Erregerbestandteilen zum Lymphknoten TLR Von der angeborenen zur erworbenen Immunität: Lymphknoten – Treffpunkt der Immunzellen tion infec