Innate immunity

Monika Raulf

Lecture 10.04.2019

Tasks of the immune system Body protection against damaging influences

Deployment of a strong defence

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Skin Mucosa Sweat Digestive fluids Lysozyme Lactoferrin Cilia Cough, Sneezing, Vomiting, Diarrhoea

The line of defence

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Innate immunity ⇒ natural/mechanistic barriers Skin, mucus moves by cilia (air- or fluid-flow along the epithelia; normal gut flora)

⇒ chemical barriers • proteolytic enzymes in body fluids • digestive enzymes (in stomach pH 1.9 – 2.6), lysozyme (enzyme able to cleave peptidoglycan of bacterial cell walls) sweat (pH 4.0 – 6.8) • low pH-value

⇒ two „Key player“-cell types Monocytes/macrophages + granulocytes → Endozytose ⇒ Complement system

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Problem:

How can the host differ between the large amount of different pathogens?

Immune defense

Answer:

Development of multiple receptors, which detect conserved motifs of pathogens, that do not occur on Eukaryotes.

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Pattern recognition receptors (PRR) 4 main groups (classified on the basis of their cellular localization and their function)

- free receptors in the serum (e.g. MBL) - membrane-bound phagocytic receptors (e.g. Dectin-1, Scavenger receptors)

- membrane-bound signaling receptors (e.g. TLR*1) - cytoplasmic signaling receptors (e.g. NODR*2)

*1 lead to an activation of the pro-inflammatory pathway *2 NOD = Nucleotide-binding Oligomerization Domain

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Comparison of the characteristics of recognition molecules of the innate and adaptive immune systems

Receptor characteristics Innate immunity

Adaptive immunity

Specificity inherited in the genome yes no

Expressed by all cells of a particular type (e.g. macrophages) yes no

Triggers immediate response yes no

Recognizes broad classes of pathogens yes no

Interacts with a range of molecular structures of a given type yes no

Encoded in multiple gene segments no yes

Requires gene rearrangement no yes

Clonal distribution no yes

Able to discriminate between even closely related molecular structures no yes

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Pathogen-associated molecular patterns (PAMPs)

Pathogen-associated molecular patterns

• Endotoxin (lipopolysaccharide) • Peptidoglycan • bacterial lipoprotein • 1→3β-D-glucan • Lipoteichoic acid • Flagellin • HSPs • dsRNA • Cpg • DNA

are recognized by pattern recognition receptors (PRRs) Monika Raulf 8

What is endotoxin?

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Structure of the lipopolysaccharides (LPS, endotoxins)

Hex

Hex Hex Hex

Hex Hep

Hep Hep Kdo

Kdo

Kdo

p

p

p

p

GlcN

GlcN

p

Variability

n

O-antigen Nucleus region

external internal

Lipid A (repeat 40 units)

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Interaction of the organism with endotoxin Route of exposure

• Contact with blood

• Oral

• Inhalative

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Interaction of LPS with cells

gram-negative bacteria

cell membrane

body barrier (e.g. respiratory tract)

LPS

LBP

LBP

sCD14

LBP = LPS-binding protein (Acute-Phase-Protein)

= Lipopolysaccharides, LPS

CD14

macrophages

NFkB

LBP

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Signal transduction way in detail

LPS

LBP

MyD88

IRAK

DNA nucleus

Transcription

IL-1β IL-6

IL-8 TNF-α

P

NFк B

Iк B

TRAF 6

Iк K

CD 14

Cytoplasma

RNA

Translation

MD 2

TLR 4

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Release of pro inflammatory cytokines

gram-negative bacteria

cell membrane

LPS

Body barrier (e.g. respiratory tract)

LBP

sCD14 macrophage

LBP

BPI

CD18

BPI

CD14

LBP CD14

blockade

LBP = LPS-binding protein (Acute-Phase-Protein)

= Lipopolysaccharides, LPS

BPI = Bactericidal-Permeability-Increasing-Protein

TNF-α

IL-1β IL-8

IL-6

NFkB

neutrophils

activation

O 2

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The receptor Toll was identified in Drosophila

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TLRs are members of the IL-1R superfamily and are membrane spanning molecules found on the cell surface and in endosomes

Cytokines Pathogen-associated molecules Endogenous damage signals

Cell-membrane

TIR IL-1-family TLR 1-11

IL-1RII

TIR

Signal transduction

Cytokines

IL-1R, IL-18R, T1/ST2, IL-RAcP, IL-18RAcP

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LPS (Lipid A)

TLR4

TIR

NF-κB IκB IL-8

nucleus NF-κB

Lipid protein Peptidoglycan

TLR2

Toll-like-Receptors

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Microbe

LPS

CD14

Toll-like receptor Seven α-helical

transmembrane-receptors

Mannose receptor

N-formylmethionyl Peptide

Chemokine

Lipid Mediators

Detection of the microbes, mediators

Cell response

Appropriate results

increased integrin activity; cytosceletal changes

Cytokine production, reactive oxygen intermediate (ROIs)

Phagocytes of microbe into phagosome

Migration into tissues

Killing of microbes

Killing of microbes

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DAMPs = Damage-associated molecular patterns (molecules that are responsible for endogenous adjuvant activity) PAMPs = Pathogen-associated molecular patterns; exogenous molecules which share a number of different recognizable biochemical features

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from Yang D. et al. : Alarmins link neutrophils and dendritic cells. Trends Immunol 2009; 30: 531-537

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Rock KL et al. Innate and adaptive immune responses to cell death. Immunol Rev 2011; 243: 191-205

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Innate immunity – relevant cells

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1. First Cellular Frontline

2. Cellular Defence line Phagocytes

Macrophages (in tissue (mononuclear))

• long-lasting • at activation cytokines* and other mediators are released, which allure among others neutrophile granulocytes

Neutrophile granulocytes = PMN=polymorphonuclear neutrophil leucocytes (just in blood, not in tissue)

*important for local inflammatory response and mediation of the induced, not adapted reaction

• short-living

(development)

Monocytes (in blood)

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Cellular response

Polymorph neutrophils

1) Phagocytic cells: • Neutrophile granulocytes (60-70%) → bacteria

2) NK-cells (natural killer cells)

• Monocytes (→ macrophages) (5%) • Eosinophilic granulocytes (1.5%) → parasites

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Phagocytosis, activation of anti bacterial mechanisms

Killing of parasites (by antibodies coated)

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Like mast cells, to find in blood

Release of granule, which contain e.g. histamine and other active substances

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Phagocytosis and activation of anti bacterial mechanisms Antigen presentation

Antigen admission in the periphery Antigen presentation in the lymph node

Found in tissue and blood; characteristic markers: CD1a, CD11c, MHC II

DC take up proteins by pinocytosis and present fragments of these to T-cells

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Phagocytosis

Microbe coated with antibodies which

bind to Fc receptors on the sell surface

The macrophage wall encased the microbe

Fusion of the membranes and generation of a

vesicle = phagosom

Lysomes fused with the phagosom: generation of a phagolysosom

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Opsonisation I „Seasoning the victim“

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A bacterial infection triggers an inflammatory response

from: „Immunologie“, Janeway et al.

Microbes activate macrophages releasing

cytokines and chemokines

Vascular dilatation and enhanced permeability of the vascular wall caused redness,

swelling and heating

Inflammatory cells invaded the tissue and released inflammatory mediators,

which provoked pain

Fluid

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Inflammatory response

• Reinforcement of the first line of defence (macrophages) by further effector-molecules and -cells • Construction of a physical barrier, which prevents the distribution • Encouragement of the cure of the damaged tissue

⇒ Marker of the inflammation: pain, redness, heat, swelling

⇒ The release are the cytokines

Induced by detection of pathogens and at tissue damages

Three important functions of the inflammation in the fight against infection:

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Cytokines MW ~ 25 kDa • released of different cells • bind on specific receptors

Special class Chemokine = chemo attractors, e.g. IL-8

Three structural main families • haematopoietic family, e.g. IL-6 • TNF family • Chemokine family

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Effect • autocrine • paracrine • endocrine

Effect of the cytokines

bloodstream

target cell

hormone molecules

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From the cytokine to the biological function

Cytokine

Receptor

Intracellular signal kascade

Cell nucleus

Gene activation

Biological function

Redundant function TNF

IL-1

IL-6

fever

Pleiotropic effect

IL-1

fever

cytokine production

proliferation

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Pro-inflammatory cytokines have a broad spectrum of functions

from: „Immunologie“, Janeway et al.

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Effect of cytokines I

• IL-1 = Fever, T-cell- and macrophage-activation

• IL-2 = Increase of T-cells

• IL-3 = Haematopoiesis (Basophile)

• IL-4 = Th2-cell activation, B-cell activation, IgE-switch

• IL-5 = Growth and differentiation of eosinophils

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Effect of cytokines II

• IL-6 = Acute phase proteins, increase of T- and B-cells

• IL-9 = Mast cell activation

• IL-10 = Inhibition of macrophages and T-cells

• IL-12 = Differentiation of Th1, Activation of NK-cells

• IL-13 = B-cell activation, Inhibition of macrophages

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Effect of cytokines III • TNF-α = Local inflammation

• IFN-γ = Macrophage-activation, MHC-expression high, antiviral

• IFN-α/β = antiviral, MHC I high, Th1

• IL-8 = Chemotaxis of neutrophils and T-cells

• MCP = Chemotaxis of monocytes

• MIP-1α = Chemotaxis of monocytes, T-cells and eosinophils

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Chemokine = small chemoattract proteins that stimulate the migration and activation of cells

They induce a directed chemotaxis at adequate reactive cells

Abbreviation: CCL and CXCL

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Chemotaxis: „Come to the rescue“

Steps of an inflammatory reaction: • Chemotactic substances (e.g. C5a, LTB4, IL-8) are released or activated and diffuse from the site of infection to the surrounding tissue and the capillaries • Pavementation: Adhesion of the phagocytes to the endothelia (epithelia) of the blood vessels • Diapedesis: Resolving of the basement membrane by the phagocytes and pass through the vascular wall • Chemotaxis: Inflow/immigration against the concentration gradient

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Chemokine are a family of proteins with similar structures, binding on chemokine receptors, which belong on the other hand to the big family of the G-protein-coupled receptors

• all chemokine own similar amino acid sequences and their receptors are all integral membrane proteins with seven membrane traverses Helices. Classification in two large groups: C-C – Chemokine CCR (1-9) e.g. MCP-1 (spec. monocytes) C-X-C – Chemokine CXCR (1-5) e.g. IL-8 (spec. neutrophils)

IL-8 and MCP-1 possess similar, but complementary functions • IL-8 leads PMN to leave the blood circulation and to immigrate in the surrounding tissue • MCP-1 however, affects monocytes and influences their wandering of the vessels and therewith their development to tissue macrophages • also fMLP (bacterial peptide) = Chemokine for neutrophils

binds on

bind on

(C=Cystein)

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Unspecific (innate) immune response

• genetically fixed (innate)

• not directed toward specific pathogens but against pathogen associated molecular patterns PAMPs

• reduces invasion of pathogens into the tissue

• reacts immediately, slows down distribution of pathogen until specific immunity has evolved

• e.g. : humoral: protease, complement, Interferons… cellular: macrophages, granulocytes…

humoral = solubilized in blood (lat. umor = Liquid)

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Important immune responses

B

B

B B

T

T

T T

Penetration of infective agents (antigens) Skin, mucosal membrane

Macrophage

Release of cytokines and chemotactic factors as IFN-α, IL-1 and IL-6, TNF, etc.; Neopterin

T- and B-lymphocytes helper cell

Lymphocyte activation

Lymphocyte- proliferation

T-cell-mediated immunity Production and release of antibodies (Immunglobulines M, G, A and E)

Release of cytokines as IFN-γ and IL-2

Receptor-detection of antigens in association with MHC II

Antigen-presentation in association with MHC II

Antigen-processing

Phagocytosis

innate

adaptive

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Co-stimulatory molecules

Cytokines

Pathogens

Intake

Detection

naive T-cell

Innate immune system

Th1

Adaptive Immune system

IFNg

Th2

IL-4

Antigen presentation

IL-4

TLRs

APC

IL-4, IL-5, IL-10, IL-13

Effector-cytokines

IL-2, IFNγ

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Which cells are involved in the antigen presentation?

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