Tumor Necrosis Factor TNF

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Tumor Necrosis Factor (TNF)

• From Pathogens to TNF
• Prof. Dr. J. Tavernier

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From Pathogens to TNF

• A historical perspective
• A very brief introduction to cytokines and
  cytokine receptors
• The innate and adaptive immunity
• The role of TNF in inflammation
• Actions of TNF at the molecular level
• Inhibition of TNF

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From Pathogens to TNF

• A historical perspective
• A very brief introduction to cytokines and
  cytokine receptors
• The innate and adaptive immunity
• The role of TNF in inflammation
• Actions of TNF at the molecular level
• Inhibition of TNF

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The beginning of the twentieth century

• Coleys toxin
• Coleys vaccin

Wiliam Coley
Spontaneous regression of tumors in patients
suffering from massive bacterial
infections Extract from Streptococcus pyogenes or
Serratia marcescens
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The seventies
Lloyd Old

• LPS induces the synthesis of a serum protein with
  strong antitumoral action both in vitro and in
  vivo
• In studying "hemorrhagic necrosis" of tumors
  produced by endotoxin, it was found that the
  serum of BCG-infected mice treated with endotoxin
  contains a substance (tumor necrosis factor TNF)
  which mimics the tumor necrotic action of
  endotoxin itself.
• Carswell, EA, Old LJ, et al. Proc Natl Acad Sci
  USA 1975 72 3666-3670

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The eighties
David Goeddel (Genentech)
Identification of a TNFa-producing macrophage-type
cell line
Purification of TNFa
Determination of the amino acid sequence
Isolation of the TNFa gene (1984) Its been
cloned, therefore it exists
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TNF as an anti-tumoral agent, anno 2007

• Problematic, given its strong toxic side-effects
  upon systemic delivery
• Cachexin
• Too much TNF induces shock, liver damage,
  intestinal necrosis, kidney and heart failure
• Local application isolated limb/organ perfusion
• Ongoing search to eliminate the systemic toxic
  side effects without affecting the antitumoral
  activity

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TNF has many functions

• SIRS systemic inflammatory response syndrome
• Very pleiotropic and pro-inflammatory cytokine
• ? provided a rationale for inhibiting TNF

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From Pathogens to TNF

• A historical perspective
• A very brief introduction to cytokines and
  cytokine receptors
• The innate and adaptive immunity
• The role of TNF in inflammation
• Actions of TNF at the molecular level
• Inhibition of TNF

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A very brief introduction to cytokines and
cytokine receptors

• A heterogeneous group of secreted proteins
  involved in intercellular communication. They are
  particularly important in coordinating immune and
  inflammatory responses.
• Examples
• Interferons
• Interleukins
• Colony-stimulating factors
• Chemokines
• Growth factors
• No clear-cut distinction with protein hormones

Endocrine
Paracrine
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Cytokines a structure-based classification
Growth hormone Erythropoietin Interleukin-2 Interf
eron
Tumor Necrosis Factor Interleukin-1
Interleukin-8 Chemokines
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Cytokines pleiotropy and redundancy
Pleiotropy
Redundancy
coordinated responses
buffered system clinical opportunity
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Cytokine receptors and signal transduction
Metabolic enzyme
Transcription factor
Cytoskeletal protein
Altered metabolism
Altered cell shape or movement
Altered gene expression
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Signals may result in variable effects
Cell division
Survival
Differentiation
Apoptosis
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Interference in signal transduction pathways
Negative interaction
Positive interaction
Effect
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From Pathogens to TNF

• A historical perspective
• A very brief introduction to cytokines and
  cytokine receptors
• The innate and adaptive immunity
• The role of TNF in inflammation
• Actions of TNF at the molecular level
• Inhibition of TNF

17
Two divisions of the mammalian immune system

• Innate immunity
• acts within minutes
• Containment of the infection
• Directed against both viruses and bacteria

• Adaptive immunity
• develops slowly over days to weeks
• Eradication of the infection
• Fine-tuned, highly-specific counter-attack
• Memory

Key players
Macrophage
Complement factors
Lymphocyte
Plasma cell
Neutrophil
Antibodies
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Pathogen recognition in the adaptive immunity
depends upon highly specific receptors and
antibodies
Antigen presenting cell
B lymphocyte
Processed antigen
Pathogen
T lymphocyte
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The innate immune response causes inflammation

• Innate immune response is a well-orchestrated
  series of events (and may cause collateral
  damage to normal tissues)
• What is the molecular basis for pathogen
  recognition?
• Many different effects how are they coordinated?

Celse (25 BC - 50 AD) Virchow (1821-1902)
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Pattern recognition receptors
Charles Janeway Jr.

• Predicted in 1989 that the innate immune system
  uses pattern recognition of infection-associated
  molecules that are crucial for the pathogen...
• ... implying the existence of receptors with a
  dual function
• recognition and binding of fundamental
  components of pathogens
• initiation of an immune response
• 1997 cloning of the first human Toll-like
  Receptor (TLR)

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Toll-Like Receptors play a key role in the
innnate immunity

• In 1997 first human member (TLR4) cloned at
  present 10 human members described (TLR1-10)
• Essential components of innate immunity1998
  LPS-resistent mice have a defective TLR4 and are
  hypersensitive to infection
• Recognition of crucial bacterial and viral
  components e.g. LPS, dsRNA, flagellin (PAMPs)
• Very ancient defence system TLRs are present in
  virtually all multicellular organisms
• Innate immune response first described in
  starfish role of phagocytes (1883, Mechnikov
  phagocytosis, cell-based immunity)
• Plants (A. thalliana) have over 200 TLRs origin
  predates separation of animal and plant kingdoms

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TLRs sense different pathogens
Toll/IL-1 receptor (TIR) domain
? peptidoglycan
TLR2
leucin-rich repeat (LRR) domain
TLR7
TLR4
LBP
ss RNA

• Characterized by an extracellular Leucine Rich
  Repeat (LRR) domain
• and and intracellular Toll/Interleukin-1
  Receptor domain (TIR)

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LPS
Gram negative bacterium
LPS
Outher membrane
Inner membrane
Lipoprotein
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TLRs transduce an alarm-signal in the cell

• First step in the activation of both the innate
  and adaptive immune systems
• Signaling cascades initiated from the TIR
  domains, ultimately leading to the activation of
  several transcription factors (NF?B,)

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TLR signal transduction
TLR2
TLR3
Peptidoglycan
dsRNA
JNK
Kinase
NF?B
IRF-3
Other transcription factors
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TLR malfunction in man

• TLR4 underactive form associated with increased
  bacteremia
• TLR5 inactive variant often observed in
  patients who die from Legionella infection
• TLR4 over-activation involved in sepsis
• TLR9 recognition of self-DNA involved in lupus
• Mal functional variant associated with
  protection against malaria and tuberculosis
• A balancing act

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More PRRs

• TLRs
• Mannose receptor
• Present on the cell-surface of macrophages and
  dendritic cells
• Recognizes pathogen-specific carbohydrates
• Triggers phagocytosis
• RNA helicases
• In cytosol of most cells
• Recognize dsRNA (a replication intermediate of
  many viruses)
• Triggers interferon production
• NOD-like receptors (NODs and NALPs)
• In cytosol of most cells
• Many members, all have LRRs broad spectrum of
  pathogen recognition
• Trigger production of pro-inflammatory cytokines

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More PRRs
? peptidoglycan
TLR2
dsRNA ?
CARD
CARD
RNA helicase
RIG-I
TLR7
TLR4
LBP
ss RNA
RNA helicases
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From Pathogens to TNF

• A historical perspective
• A very brief introduction to cytokines and
  cytokine receptors
• The innate and adaptive immunity
• The role of TNF in inflammation
• Actions of TNF at the molecular level
• Inhibition of TNF

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TNF in inflammation and innate immunity
Activation of endothelium Secundary cytokines ?
Adhesion molecules ? Permeablility
IL-6
IL-1
INF?
Elimination of infection and tissue repair
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TNF is a pleiotropic cytokine
Production of pro-inflammatory cytokines Productio
n of NO, prostaglandins, leukotriens,
... Vasodilatation and increased
permeability ICAM-1 induction Induction of
Tissue Factor initiation of coagulation
Production of pro-inflammatory cytokines
Activation
Raised adherence to activated endothelium Raised
oxydative burst
Induction of fever by activation of hypothalamic
neuron circuits Effect on synaptic
connectivity Production by microglia cells toxic
for oligodendrocytes (MS?)
Enhanced cell migration
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From Pathogens to TNF

• A historical perspective
• A very brief introduction to cytokines and
  cytokine receptors
• The innate and adaptive immunity
• The role of TNF in inflammation
• Actions of TNF at the molecular level
• Inhibition of TNF

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Two forms of TNF
soluble TNF? (17kDa)
Membrane-bound TNF? (26 kDa)
TNF?-Converting Enzyme
TACE
Nucleus
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The TNF receptor is a trimer
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Two TNF receptors
Death domain
TNFR1
TNFR2

• Similar structure of the extracellular domain
• Different intracellular domains
• Activation of different signal transduction
  pathways

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The TNF and TNF receptor superfamilies
Adapted form Locksley RM et al. Cell. 2001104487
RECEPTORS
LIGANDS
LT?3
TNFR2
TNF
TNFR1
N29
N19
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Function of other TNF superfamily members

• Host defence
• CD40 activation of B cells
• BAFF activation and survival of B cells
• Apoptosis
• Fas ligand cytoxicity T suppressor cells
• Organogenesis
• LT?3 en LT?1?2??lymphoid organs)
• RANKL (bone metabolism and breast gland)

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TNF receptor 1
Death domain

• The 55-60 KDa TNFR1 is expressed constitutively
  in nearly all tissues
• Induces programmed cell death (apoptosos) via a
  death domain in the intracellular part of the
  receptor
• Signalisation via TRADD, a death-domain-containi
  ng adaptor protein

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TNF receptor 2

• The 75-80 kDa TNFR2 contains no death domain
  and cannot induce apoptosis
• Signalisation via TRAF2, leading ultimately to
  the production of NF?B, cytokines and inhibitors
  of apoptosis

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TNF signal transduction
TNFR1
TNFR1
TNFR2
TRAF2
TRAFs
TRADD
RIP
Adapter proteins
Caspase activation
Protein kinase cascade
anti-apoptotic gene expression
NFKB
Inflammatory gene expression
I-KB
Apoptosis
NFKB
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The central role of NF?B
Cytokines (TNF, IL1)
Bacteria and viruses (via PPRs)
Physical and oxidative stress (UV, cigarette
smoke,)
NF?B
Anti-apoptotic proteins Cell cycle
proteins Growth factors Proteases
Cytokines Chemokines Enzymes Adhesion proteins
Immunity and inflammation
Cancer
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From Pathogens to TNF

• A historical perspective
• A very brief introduction to cytokines and
  cytokine receptors
• The innate and adaptive immunity
• The role of TNF in inflammation
• Actions of TNF at the molecular level
• Inhibition of TNF

43
Three classes of TNF inhibitors

• 1. Soluble receptor molecule

Etanercept (Enbrel)
Receptor
Fc
Human recombinant receptor/Fc fusion protein
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Three classes of TNF inhibitors

• 2. Monoclonal antibodies

Infliximab (Remicade)
Adalimumab (Humira)
Fab
Fc
mouse human
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Three classes of TNF inhibitors
3. Pegylated humanized Fab' fragment
Certolizumab pegol (Cimzia)
PEG
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Future

• Small molecules