nobel lecture slides

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How mammals sense infection: from endotoxin to the Toll-like

receptors Bruce Beutler

Center for Genetics of Host Defense UT Southwestern Medical Center

Dallas, TX

Ernest Beutler, M.D., 1928-2008

Galen 129 199, AD

Hippocrates 460 - 370, BCE

Moses ben-Maimon (Maimonides) 1135-1204, AD

Infections and their transmissible character were known in antiquity

Staphylococcus aureus

Neisseria meningitidis Herpes simplex

Chlamydia trachomatis Tinea faciei

Measles

Antonie van Leeuwenhoek 1632 - 1723

while microbes were only discovered in the 17th century

and the association between microbes and infection was only discovered in the 19th century.

Louis Pasteur 1822-1895

Robert Koch 1843-1910

What might be the nature of contact between microbe and host?

?

Soon after microbes were discovered, it was appreciated that mammals recognize them as foreign and mount an intense

inflammatory response

In 1891, Richard Pfeiffer, a student of Robert Koch, noted that heat-killed microbes caused a violent reaction in guinea pigs soon after they were injected into these animals. He coined the term endotoxin to describe the poisonous, heat-stable principle associated with bacteria, responsible for fever, inflammation, shock and sometimes death.

Gram negative bacteria Eschericia coli

Peptidoglycan Layer

Outer Membrane

Inner Membrane

Lipopolysaccharide (LPS)

O antigen

Outer core

Inner core

Lipid A

Biosynthesis of Lipid A

Christian R.H. Raetz 1946 - 2011

Lipid A Lipid IVa

Abraham Braude, 1917-1984

Wasting disease (cachexia) in a cow with African trypanosomiasis

Plasma triglyceride

LPL, AcCoAC, FAS

LPL suppression factor

Tumors

Other tissue

Endotoxin Trypanosome factors Tumor factors

Infection Malignancy

Adipocyte

?

?

LPL

Cachectin

RAW 264.7 macrophages

3T3-L1 pre-adipocytes

1983

Isolation of mouse cachectin Pressure dialysis of medium from ~500 10 cm plates

of LPS-activated RAW 264.7 cells (early harvest) ConA sepharose chromatography Isoelectric focusing in a glycerol gradient Preparative native gel electrophoresis Preparative SDS gel electrophoresis Yielded microgram quantities of an apparently pure

17.5 kD protein with approximately 2% yield of initial biological activity (prior to denaturing gel electrophoresis).

Cachectin comprised 1-2% of the protein secreted by RAW 264.7 cells during the first two hours following LPS activation.

Tumor necrosis and cytolysis

Triglyceride synthesis, LPL, FAS AcCoA carboxylase, glycerol release

FAT

TUMOR

MACROPHAGE LPS

This raised the question: might TNF mediate all

effects of LPS, including the lethal effect?

TNF

Purified TNF mimics LPS toxicity

Denatured TNF

Active TNF

1984

The lethal effect of LPS is attenuated by passive immunization against TNF

1985

N = 16 mice per point

Triglyceride synthesis, LPL, FAS, AcCoA carboxylase, glycerol release

FAT

Cytotoxicity, rearrangement, neutrophil adhesion, MHC expression, procoagulant, PLA2. Thrombomodulin

ENDOTHELIUM

MACROPHAGE

Degranulation, endothelial adhesion, lysozyme secretion, superoxide anion production, H2O2 release, phagocytosis

NEUTROPHIL

MUSCLE Transmembrane potential, glucose uptake

Proliferation of T cells

T CELLS

LPS

Tumor necrosis, cytolysis

TUMOR

TNF

The C3H/HeJ Mouse and the Lps Locus

Resistant to LPS (Heppner and Weiss, 1965)


Fail to make a cytokine response to LPS (for example, no TNF), suggesting a proximal defect.

C3H/HeJ mice: resistant to LPS (and only LPS)

Viruses

Responsive Unresponsive

Gram negative bacteria

Responsive

Bacteria

Nucleic Acids LPS Flagellin Lipoproteins

Responsive

Bacteria

C3H/HeJ bone marrow transfer to C3H/HeN and vice versa: susceptibility to LPS-induced lethality is determined by the donor

C3H/HeJ Marrow

C3H/HeJ C3H/HeN

Alive Alive Dead Dead

C3H/HeJ Marrow C3H/HeN Marrow C3H/HeJ Marrow C3H/HeN Marrow

C3H/HeN Marrow

LPS h h


Hypersusceptible to authentic G(-) infections (Obrien, et al., 1980; Svanborg-Eden, et al., 1983)


LPS does not work as an adjuvant in C3H/HeJ mice (B.J. Skidmore et al, 1976)


Single locus (Lps); allelic to a mutation in the LPS-refractory C57BL/10ScCr strain (Coutinho and Meo,1978).


Lps mapped to Chr. 4 between Mup1 and Polysyndactyly loci by Watson et al. in 1978.

LPS

CD14

? IB

p50 p60

TNF

NF-B

R. Ulevitch and colleagues, 1990

Conventional searches for a difference between C3H/HeJ and C3H/HeN

3. Comparisons at the protein level.

C3H/HeJ C3H/HeN

1. Cross immunization of C3H/HeJ and C3H/HeN mice.

Macrophage

C3H/HeJ C3H/HeN

2. Transfect cDNA from C3H/HeN to

C3H/HeJ cells.

cDNA

C3H/HeN cells

mRNA

C3H/HeJ cells

AAAAA

AAAA

TNF?

AAA

LPS

Betsy Layton

Alexander Poltorak

Irina Smirnova

Christophe Van Huffel

Positional cloning entails

Genetic mapping (in our case, on 2093 meioses)

Physical mapping (in our case, entire interval cloned in 66 BACs and 2 YACs)

Exploration for genes (in our case, 1 authentic genes and 7 pseudogenes)

Mutation identification (find the one and only genetic change responsible for the phenotype.

Comparison of Mouse and Human LPS Gene Locus

24

23 22

21

12 13

11

13

12

21.1 21.2

22.3

22.1 22.2

32

31

34.1

21.3

33

11

34.2 34.3

Human 9 Mouse 4

TYRP1

ORM TAL2

PAPPA

IFNA

Pappa

lfna b

Tal2

Hxb Orm1

p

q

Mup1

Ps

1

Lps

Lps

Methods for finding genes within BACs

Exon trapping Hybridization selection Computational prediction

(GRAIL) Shotgun sequencing and EST

database searching

Creation of BACs

Fragmentation of BACs

Remote EST database

Cloning of Fragments BLASTing

Vector

Mammalian DNA

Lps

Lps

LPS LPS IL-1

TLR4 CD14 IL-1R

?

C3H/HeN

C3H/HeJ

Screen shot of a mutation in TLR4 distinguishing C3H/HeJ from C3H/HeN mice

1998

On Northern blot analysis, C57BL/10ScCr mice appear not to express Tlr4

1998

RT-PCR also shows non-expression of Tlr4 in C57BL/10ScSr mice

1998

Tlr4lps-n

Tlr4lps-d

C3H/HeN

C3H/HeJ

F I P G V

F I H G V 1998

712

Deletion of 74K in the C57BL/10ScCr mouse

1998

But the question remained:

Was there direct contact between TLR and LPS?

Lipid A: Agonist for both

mouse and human

Lipid IVa: Agonist for mouse;

antagonist for human

Mouse Mouse Human Human

C3H/HeJ

Responsive

C3H/HeJ C3H/HeJ C3H/HeJ

Lipid A Lipid IVa Lipid A Lipid IVa

Responsive Responsive Unresponsive

TNF TNF TNF

plasma membrane

TIR

TLR4

MD-2

LPS

BS Park et al. Nature 458, 7242 (2009) R Shimazu et al., J Exp Med 189, 11 (1999

Image created using PyMol Y. Xu et al. Nature 408, 6808 (2000)

TLR4 LPS

MD-2

> > > > > > > >

> > > > > > > >

CD14

> > > > > > > >

> > > > > > > >

MD-2

TLR4 Lps TLRs 2,1 TLR3 TLR7 TLR5 TLRs 2,6 TLR9

LPS LP

The role of TLR4 as a sensor of LPS suggested that other microbial ligands were sensed by other TLRs, and the

specificity of most TLRs was revealed by knocking the genes out one at a time,

largely in the Akira lab.

CpG ssRNA Flagellin Poly I:C

Now, the mode of binding of several ligands to TLRs is understood

DMTOLL1

HUMTLR4

LU-U73916

Function of the TIR domain throughout the tree of life

Pamela Ronald, Discoverer of XA21

Images courtesy Dr. Pamela Ronald

Bacterial blight of rice

Xanthomonas oryzae (Xoo)

Monocots (Rice) Insects (Drosophila) Vertebrates (Mouse)

Making new phenotypes in mice

* *

* ** **

**

* *

* **

ENU

G1

G2

G3

ENU C57BL/6

As of 9/20/11, >151,000 G1 + G3 mice produced. ~60 coding/splicing changes per sperm.

Mask

Business Class WT

The process of finding mutations was greatly accelerated by the sequencing

and annotation of the mouse genome

Never again was it necessary to make a BAC contig or search for genes.

MGI genome browser (typical view)

Massively parallel short read sequencing has made targeted exon sequencing unnecessary in most cases.

The cost of sequencing a mouse genome to >90% coverage is now about $3,000. About 4 mouse genomes can be sequenced per week in our lab.

The price per base pair continues to drop by about 80% each year.

With minimal mapping, it is possible to find mutations extremely quickly.

TLR7 TLR9

From genetics to structures to mechanism

In plasmacytoid dendritic cells, specialized machinery is needed for TLR signaling

salt and pepper

Disbindin

Mutations we have found to cause MCMV susceptibility

Beutler Lab, December 15, 2009

Nancy Nelson

Betsy Layton

Cindy Boulton

Charles Ross Yu

Xia

Dan Popkin

Philippe Krebs

Carrie Arnold

Celine Eidenschenk

Christina Neppl

Kathi Brandl

Xiaohong Li

Pei Lin Xin

Du

Micha Berger

Nora Smart

Sunyong Won Eva

Moresco Christine Domingo

Liz Flores

Elaine Pirie

Sara Kalina

Bruce Beutler Diantha

La Vine

Stephen Lyon

Lei Sun

Hua Huang

Amanda Blasius

Owen Siggs Lara

Krieg

Wataru Tomisato

Others not shown in the last slide

Jiangfan Jiang Kasper Hoebe Ben Croker Koichi Tabeta Karine Crozat Sophie Rutschmann Philippe Georgel

For the cloning of the Lps locus

Alexander Poltorak Irina Smirnova Christophe Van Huffel Betsy Layton Xiaolong He Mu-Ya Liu Xin Du Dale Birdwell Erica Alejos

Chris Galanos and Marina Freudenberg (Max Planck Institute fur

Immunbiologie, Freiburg)

Paola Ricciardi-Castagnoli (University of Milan)

How mammals sense infection: from endotoxin to the Toll-like receptorsSlide Number 2Slide Number 3Slide Number 4while microbes were only discovered in the 17th centuryand the association between microbes and infection was only discovered in the 19th century.Slide Number 7Slide Number 8Slide Number 9LPSBiosynthesis of Lipid ASlide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23The C3H/HeJ Mouse andthe Lps LocusThe C3H/HeJ Mouse andthe Lps LocusSlide Number 26Slide Number 27The C3H/HeJ Mouse andthe Lps LocusThe C3H/HeJ Mouse andthe Lps LocusThe C3H/HeJ Mouse andthe Lps LocusThe C3H/HeJ Mouse andthe Lps LocusSlide Number 32Conventional searches for a difference between C3H/HeJ and C3H/HeNSlide Number 34Slide Number 35Slide Number 36Comparison of Mouse and Human LPS Gene Locus Slide Number 38Slide Number 39Slide Number 40Methods for finding genes within BACsSlide Number 42Slide Number 43Slide Number 44Slide Number 45Slide Number 46Slide Number 47Slide Number 48Slide Number 49Slide Number 50But the question remained:Slide Number 52Slide Number 53Slide Number 54Slide Number 55Slide Number 56Slide Number 57Slide Number 58Slide Number 59Slide Number 60Slide Number 61Slide Number 62The process of finding mutations was greatly accelerated by the sequencing and annotation of the mouse genomeMGI genome browser (typical view)Massively parallel short read sequencing has made targeted exon sequencing unnecessary in most cases.Slide Number 66Slide Number 67Slide Number 68Slide Number 69Slide Number 70Slide Number 71Slide Number 72Slide Number 73

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