Toll-like receptors

Bridging the innate and adaptive immune responses

June 1, 2005

MIMG 261

Stephan Krutzik, Ph.D.

skrutzik@mednet.ucla.edu

• Rapid Response• Dendritic cells, monocytes, NK cells • Pattern recognition receptors-

germ-line encoded– TLRs, mannose and scavenger

• Direct Response for host defense– Phagocytosis– Antimicrobial activity

Cytokines, co-stimulatory molecules

• Slow response• T and B cells• Recognition - initially low affinity

receptors Gene rearrangement

Clonal expansion

• Response - T and B cells with high affinity, very specific receptors and antibodies

• Immunological Memory

Innate Response Adaptive Response

The instructive role of the innate response on adaptive immunity

Lemaitre, et al. (1996) Cell 86, 973

DROSOPHILA HOST DEFENSE

• Regulated by Toll receptor family• Evidence for specificity in regulation• Different pathogens, different

response• Toll - Antifungal

18-wheeler - Antibacterial• Secretion of antimicrobial

polypeptides by the fat body• antifungal - drosomycin,

metchnikowan • antibacterial - cecropin, drosocin,

defensin, diptericin, attacin, metchnikowan

Toll 18- Wheeler

Fungus Bacteria

Antimicrobial Genes

HUMAN TOLL-LIKE RECEPTORS (TLRS)

19%

25%

LRR

Cys-Rich

dToll TLR250 1000

TLR2

TLR6

TLR7

TLR1

TLR5

TLR8

TLR4

TLR3

dToll

TLR9~IL-1R

TIR

TIR=Toll/IL-1R

In 1997, Charles Janeway cloned and sequenced the human homologue to Drosophila Toll

Immunomodulatory Genes

Cell signaling

TLR2/TLR1TLR2/TLR6

lipoproteins

TLR7

ssRNAimmiquimod

dsRNA

TLR3

LPS

TLR4

flagellin

TLR5

TLR9

CpG DNA

Toll-like receptors (TLRs) and their ligands

TLR8

ssRNA

TLR10-orphan (human)

TLR11-uropathogenic bacteria and protozoa(mouse)PAMPs (Pathogen associated molecular patterns)

Bell Trends Immunol

Leucine Rich Repeats

• 24-residue repeated sequence with characteristically spaced hydrophobic residues

• 19-25 LRR in the ECD of TLRs

• Involved in ligand recognition

• 6500-8000 A2 v 700 A2 for Ab

TLR extracellular domain

TLR intracellular domain

TIR domain

Death domain

MyD88

IL-1R

TIR domain

TIR domain

TLR

NFB

TIR=Toll/IL-1R domain

MyD88 is required for TLR activation

Kawai et al Immunity Volume 11, Issue 1 , 1999, Pages 115-122

CONSERVED PATHWAYS IN INNATE IMMUNITY

Hoffmann JA, et al. Science. 1999 May 21;284(5418):1313-8

Kawai et al Immunity Volume 11, Issue 1 , 1999, Pages 115-122

Cells from MyD88 -/- did not produce cytokines but did have delayed activation of NFB

These data suggest that an alternative, MyD88-independent signaling pathway is triggered by TLR4

Using subtractive hybridization in MyD88-/- macrophages, identified MyD88-independent specific genes (Kawai, Akira)

IP-10, GARG16, IRG1---- have ISRE (IFN-stimulated response element) and NFkB binding sites in promoter

IP-10

GARG16

IRG1

MyD88-/- MyD88-/-

--- Lipid A

TLR4 signaling leads to the activation of the transcription factor IRF-3 in a MyD88-independent manner

Identified an MyD88-independent pathway that triggers IRF-3

MyD88-dependent and –independent signaling

Immunomodulatory Genes

MyD88

TIRAP/MAL

MyD88 MyD88

TLR4TLR2/TLR1

TLR2/TLR6

TLR5 TLR7 TLR9

IRAKTRAF6

NFB

IRF-3

TRIF

IFN-

TLR3

TRIF

TRAM

TRAF6

NFB

TBK-1

Innate Adaptive

TLRs influence both innate and adaptive immune responses

Tissue injury

In 1884, Metchnikoff published studies on the water-flea Daphnia and its interaction with a yeast-like fungus. He demonstrated the ability of cells of the water-flea, which he termed phagocytes, to engulf the foreign spores. “The spores which reached the body cavity are attacked by blood cells, and- probably through some sort of secretion- are killed and destroyed”.

Metchnikoff’s study of Daphnia

Metchnikoff’s innate immune system

Thus Metchnikoff had described the key functions of cells of the innate immune system

• rapid detection of microbes• phagocytosis• antimicrobial activity

Through the studies of TLRs, we have a better understanding of how the innate immune system can mediate these events

Tolls and Phagocytosis

TLR activation triggers direct host defense against invading pathogens

1. Enhances phagocytosis

Measure GFPInert

microspheres

Blander et al Science 2004

TLR activation triggers direct host defense against invading pathogens

2. Induces phago-lysosomal fusion

Blander et al Science 2004

Green- E. coli GFP

Red- LysoTracker

Green- (CFSE) apoptotic cell

Red- LysoTracker

30min 2hr

TLR signaling not required for phago-lysosomal fusion of apoptotic cells

Blander et al Science 2004

TLRs and antimicrobial pathways

RIPTLR activation

TLR2 activation induces anti-mycobacteria activity

1. Monocytes infected with M. tb

2. Activated with TLR2/1 ligand +/-

TLR2

3. Measured M. tb CFU

+/- TLR2 L

Human monocyte

CFU

Thoma-Uszynski et al Science 2001

Doyle et al Immunity, 2002

1. macrophages infected with MHV68

2. Activated with TLR ligands

3. Measured early replication proteins

(CM=conditioned media from TLR stimlulated macrophages)

TLR L

Western blot

MyD88-independent pathways trigger anti-viral activity

TLR3 and TLR4 induce antiviral responses via IFN

Immunomodulatory Genes

IRF-3

MyD88

TRIFTIRAP/MAL

MyD88 MyD88

IFN-

TLR3TLR4TLR2/TLR1

TLR2/TLR6

TLR5 TLR7 TLR9

TLR signaling pathways

TRIF

IRAKTRAF6

NFB

TRAM

TRAF6

NFB

Thus TLRs can mediate key functions of the innate immune system described by Metchnikoff

• rapid detection of microbes• phagocytosis• antimicrobial activity

TLRs bridge the innate and adaptive immune responses

TLRs bridge the innate and adaptive immune responses

Adaptive immune response is dependent on:

1. Density of peptides (Signal 1)

2. Types and levels of co-stimulatory molecules on APC (Signal 2)

3. Types of cytokines secreted by APC (Th skewing)

4. Being kept in check-- Peripheral T cell tolerance controlled by CD80/CD86 levels and Treg cells

TLR activation can influence all 4 factors

TLR

Innate Adaptive

Signals required for T cell activation

Treg

TLR

Mature Dendritic cell

immature Dendritic cell

1. Density of peptides (Signal 1)

2. Types and levels of co-stimulatory molecules on APC (Signal 2)

Hertz et al J Immunol 2001

Increase in levels of MHCII and co-stimulatory molecules enhances antigen presenting capacity

Signal 3 (Th skewing) can come directly from APC or from surrounding cells/tissue

Th1 Th2

TLR activation is important for triggering Th1 immune response

MyD88-/- mice have a deficient Th1 immune response…

Wt

MyD88-/-

Caspase-1 -/-

Mice were immunized with OVA and CFA

Harvest lymph nodes

OVAMeasure proliferation and cytokine production

8 days

Schnare Nat Immunol 2001

CFA=killed mycobacteria in oil and water

But intact Th2 immune response (antigen specific Th2 immunoglobulin intact)

Mice were immunized with OVA and CFA

Measure serum immunoglobulin levels

Wt

Myd88-/-

Caspase-1 -/-

MyD88-/- DC do not increase co-stimulatory molecules and do not secrete IL-12 upon TLR activation

Mature Dendritic cell

immature Dendritic cell

IL-12

mycobacteria

Wt

Myd88-/-

Caspase-1 -/-

T reg cells

CD4+CD25+

Foxp3 expression

Secrete TGF-, IL-6, IL-10

Block T cell proliferation

Thought to be involved in maintaining peripheral tolerance

But may also block pathogen-specific T cell activation

So, there needs to be a way to turn them OFF during infection

TLRs control Treg function

DC activation via TLRs inhibits Treg cells and allows T cell activation (“Signal 4”)

T cell prolif

T cell prolifTLRL

Pasare et al Science 2003

DC activation releases Treg block via IL-6

T cell prolif

IL-6 required for in vivo T cell activation

Mice were immunized with OVA and LPS

Harvest lymph nodes

OVAMeasure T cell activation

Thus, TLR activation is able to bridge the innate and adaptive immune responses by:

1. Detecting invading microbes

2. Enhancing APC function by increasing levels of MHCII and co-stimulatory molecules

3. Triggering the release of cytokines that skew adaptive immune response

4. Controlling T reg function