Toll-like receptors Bridging the innate and adaptive immune responses June 1, 2005 MIMG 261 Stephan Krutzik, Ph.D.

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

HUMAN TOLL-LIKE RECEPTORS (TLRS) 19% 25% LRR Cys- Rich dTollTLR 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/TLR1 TLR2/TLR6 lipoproteins TLR7 ssRNA immiquimod 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 LRR in the ECD of TLRs Involved in ligand recognition A 2 v 700 A 2 for Ab TLR extracellular domain

TLR intracellular domain TIR domain Death domain MyD88 IL-1R TIR domain TLR NF  B TIR=Toll/IL-1R domain

MyD88 is required for TLR activation Kawai et al Immunity Volume 11, Issue 1, 1999, Pages Volume 11, Issue 1

CONSERVED PATHWAYS IN INNATE IMMUNITY Hoffmann JA, et al. Science May 21;284(5418):1313-8

Kawai et al Immunity Volume 11, Issue 1, 1999, Pages Volume 11, Issue 1 Cells from MyD88 -/- did not produce cytokines but did have delayed activation of NF  B 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 -/- ---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 TLR4 TLR2/TLR1 TLR2/TLR6 TLR5 TLR7 TLR9 IRAK TRAF6 NF  B IRF-3 TRIF IFN-  TLR3 TRIF TRAM TRAF6 NF  B TBK-1

InnateAdaptive 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 GFP Inert 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 30min2hr TLR signaling not required for phago-lysosomal fusion of apoptotic cells Blander et al Science 2004

TLRs and antimicrobial pathways RIP TLR 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, 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 TRIF TIRAP/MAL MyD88 IFN-  TLR3TLR4 TLR2/TLR1 TLR2/TLR6 TLR5 TLR7 TLR9 TLR signaling pathways TRIF IRAK TRAF6 NF  B TRAM TRAF6 NF  B

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

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 InnateAdaptive

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

Th1Th2

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 OVA Measure 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 TLRL 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 OVA Measure 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