HOW WE SENSE MICROBES: GENETIC DISSECTION OF INNATE IMMUNITY IN INSECTS AND MAMM

November 28, 2007

HOW WE SENSE MICROBES: GENETIC DISSECTION OF INNATE IMMUNITY IN

INSECTS AND MAMMALS

Christoph Blase, Stiftungssekretariat

Internationale Balzan Stiftung " Fonds "

Informationsdienst Wissenschaft (Pressemitteilung) -

Bayreuth,NRW,Germany*

27.11.2007

http://idw-online.de/pages/de/news237418

Jules A. Hoffmann and Bruce Beutler

Balzan Prize 2007 for Innate Immunology

The fundamental challenges.

Infections have always been among the principal causes of death in

the human species. In the developed world, microbes may be perceived

as " manageable " because sanitation, immunization, and antibiotics

hold them in abeyance. But great plagues may lie before us, and our

practical success in the prevention and treatment of infection

belies the fact that we have much to learn about how immunity

operates.

The 20th century witnessed tremendous progress in our understanding

of adaptive immunity: the lymphocyte-based system of clonal

reactivity and expansion that leads to the production of antibodies

by B cells, and to the proliferation of cytotoxic T cells that hold

infection in check. Adaptive immunity is remarkable because of

its " anticipatory " quality (antibodies can be fashioned against

almost any molecular structure including those that do not exist in

nature), its exquisite specificity, and because of memory: the

ability to mount a faster and stronger response to foreign molecules

that we have encountered previously. But immunologists have known

that much of our resistance to microbes has nothing at all to do

with lymphocytes. Rather, resistance to most microbes is heritable

and multifaceted, depending upon neutrophils, macrophages, other

phagocytic cells, and upon widely expressed cell-autonomous immune

processes. Collectively, this heritable system of defense has been

termed innate immunity.

The molecular basis of innate immune recognition remained obscure

until recently, and was sought both in insects and in mammals. The

small number of molecules that form microbe detection systems, and

their clear importance in the initiation of powerful inflammatory

responses, have surprised many of us. Moreover, the new

understanding as to how infections are perceived by the host within

the first minutes following inoculation has wrought major changes in

the science of immunology.

Drosophila host defense: a paradigm for innate immunity

Since the beginning of the 20th century, it has been known that

insects are highly resistant to microbial infections. Furthermore,

it had been established at that time that septic injury leads to the

appearance in the cell-free hemolymph (blood) of a significant

antimicrobial activity. It took a half century until the first

molecules accounting for this inducible antimicrobial activity were

characterized in immune-challenged pupae of the Lepidopteran species

Hyalophora cecropia by H. Boman on Sweden. These molecules turned

out to be cationic, membrane-active peptides. Studies in other

insect species and, more generally, on a variety of metazoans

including mice and humans, have since shown that the synthesis of

antimicrobial peptides is a general phenomenon of the innate host

defense.

In the early nineties, the Hoffmann laboratory started the

investigation of the antimicrobial defense of Drosophila, with the

hope of using the remarkable possibilities offered by Drosophila

genetics to decipher the molecular mechanisms of this efficient

defense. Relying on a combination of physico-chemical methods and

molecular genetics, they first set out to identify the antimicrobial

molecules induced in response to septic injury in the fly. They were

able to identify seven distinct inducible antimicrobial peptides (or

peptide families). These molecules appear to be structurally diverse

with activity spectra directed against fungi or against Gram-

positive and/or Gram-negative bacteria. The predominant site of

biosynthesis of these peptides is the fat body, a functional

equivalent of the mammalian liver. Following their synthesis, the

peptides are secreted into the circulating hemolymph where their

total concentrations can reach values as high as 0,4 mM. It is

assumed that the combined activities of the seven distinct groups of

antimicrobial peptides largely contribute to the successful blocking

of the growth of the invading microorganisms.

When Hoffmann and colleagues analysed in the early nineties the

promoter regions of the gene encoding the antimicrobial peptide

diptericin, they noted the presence of nucleotide motifs similar to

mammalian binding sites for the inducible transactivator NF-kB,

reportedly one of the major regulators of immune gene expression in

mammals. Mutation of the Drosophila nucleotide sequences similar to

NF-kB response elements abolished the inducibility of the diptericin

gene. Subsequent studies established that the presence of functional

NF-kB response elements is a general phenomenon for the

antibacterial peptide genes and in several instances their mandatory

roles for challenge induced expression of these genes were confirmed.

These findings raised the tantalizing question whether an equivalent

of an NF-kB based transcriptional control is involved in the

antimicrobial response of Drosophila. At that time, it had been

established that Drosophila produces a member of the NF-kB family,

namely the protein Dorsal, which is involved in dorsoventral

patterning in the early embryo. Dorsal was also known to be retained

in the cytoplasm by binding to the NF-kB inhibitor Cactus.

Furthermore, dissociation of Dorsal from its inhibitor, allowing for

nuclear translocation of Dorsal and subsequent gene regulation, was

understood to be dependent on activation of the transmembrane

receptor Toll by a proteolytically cleaved form of the cysteine-knot

cytokine-like protein Spaetzle.

The striking similarities between the activation of Dorsal during

dorsoventral patterning in the Drosophila embryo and the cytokine-

induced activation of NF-kB during inflammation in mammals (e.g. IL-

1) induced the group to undertake an in-depth analysis of the

antimicrobial defenses in flies mutant for genes of the embryonic

regulatory cascade. A pivotal outcome of these studies, published in

1996, was the demonstration that the gene regulatory cascade

Spaetzle-Toll-Cactus controls the resistance to fungal infections.

It was later understood that this pertains also to the resistance to

Gram-positive bacterial infections. This result had an exciting

implication. The Toll receptor was indeed known to combine an

extracellular domain of leucine-rich repeats to an intracellular

Toll-Interleukin-Receptor (TIR) homology domain. LPS had been shown

to bind to a leucine-rich repeat domain in the membrane protein

CD14, and the TIR domain was known to signal to NF-kB in the case of

IL-1. CD14 being glycosylphosphoinositide-anchored, cannot signal by

itself to NF-kB. By demonstrating that the combination of an

extracellular leucine-rich repeat domain to an intracellular TIR

domain can mediate an NF-kB dependent immune response, the

Drosophila data provided a conceptual framework for the control of

innate immune defenses dependent on genome-encoded (versus

rearranged) receptors of microbial ligands.

It had meantime become apparent that mammals produce a family of

Toll-like receptors and a new field of study had opened in

immunology. Although their existence was reported as early as 1994,

such receptors had no known function in mammals. One year after the

publication of the role of Toll in the antifungal response of

Drosophila, C. Janeway and R. Medzhitov, with whom our group

collaborated in the framework of a Human Frontiers in Science

Program, reported the existence of a human homologue of Toll and

showed that the intracytoplasmic domain of this Toll-like receptor

could signal to NF-kB when transfected into a cultured cell line.

Significantly though, Janeway and Medzhitov had transfected a

contruct in which the extracytoplasmic domain was a CD4 ectodomain

consisting of immunoglobulin folds, and their studies did not reveal

the receptor's specificity or function.

In parallel to the development of the studies of mammalian TLRs, the

Hoffmann group pursued their interests in Drosophila Tolls which by

then had appeared to number nine distinct family members. They were

initially excited about to the idea that these individual Tolls

could bind distinct microbial ligands and signal to NF-kB to direct

the expression of various immune-response genes. However, it became

clear that -at least as far the control of antimicrobial peptide

genes in the systemic response is concerned- only Toll itself plays

a role. All Toll receptors, it should be stressed, have tightly

regulated, tissue -and stage- specific patterns of expression during

embryogenesis and in larval development and are primarily involved

in various facets of development. Apparently, only Toll amongst the

whole family, has been recruited to actively direct expression of

antimicrobial peptides destined to be released into the blood of the

fly (systemic immune response, as opposed to epithelial barrier

response). Furthermore, the data indicated that Toll activation

during the immune defense of Drosophila is mediated by the processed

form of the cytokine Spaetzle, but not by direct interaction with

microbial ligands. In the terminology coined by the late C. Janeway,

Toll does therefore not qualify as a bona fide " pattern recognition

receptor " , in contrast to the situation which prevails for mammalian

TLRs (see below).

The latter observation raised the question as to which protein

served the recognition function of microbes during infection. It was

only in 2001 that the Hoffmann group was able to generate a mutant

fly which failed to activate Toll in response to Gram-positive

bacterial infection. The unbiaised ethyl-methyl-sulfonate induced

mutation in this fly line appeared to affect a gene encoding a

member of a family of proteins initially discovered in the blood of

Lepidoptera through its binding to bacterial peptidoglycan, hence

the name of Peptidoglycan Recognition Proteins ( PGRP). The

Drosophila genome contains 13 genes coding for members of the PGRP

family and the newly generated mutation which affected recognition

of Gram-positive bacteria, had changed a conserved cysteine to a

tyrosine in the family member PGRP-SA. Interestingly, mammals

including humans, also express PGRPs. Mice in which genes encoding

PGRP members have been invalidated, show discrete immune phenotypes

which are however not of the magnitude with which knock-outs of some

of the Drosophila PGRP family members impound on the host defense.

As early as the mid-nineties the studies of the Hoffmann laboratory

had further revealed that the defenses to Gram-negative infection in

Drosophila rely on a pathway independent from Toll which the authors

named Imd (for Immune deficiency) pathway. The end effect of a

complex intracellular signaling cascade, initiated by binding of

Gram-negative bacterial peptidoglycan to a transmembrane receptor

(PGRP-LC), is the phosphorylation and cleavage of the NF-kB family

member Relish: this transactivator carries an inhibitory Cactus

(IkB)-like domain in its C-terminal region and is evocative of the

mammalian NF-kB family member p105. This pathway comprises gene

products homologous (or similar) to mammalian RIP, FADD, the MAP3

kinase TAK1, IKKb and IKKg/NEMO. This contrasts with the Drosophila

Toll signaling cascade, which lacks all the partners listed above.

Overall, the Imd pathway is evocative of the mammalian TNF-µ

receptor pathway and strikingly, overexpression of genes encoding

upstream members of the Imd pathway can led to apoptosis of immune-

responsive cells.

The sum of the data accumulated so far leads to a picture in which

two distinct pathways regulate the expression of immune-responsive

genes: the Toll pathway, which is triggered primarily by fungal and

Gram-positive bacterial infection, and the Imd pathway which serves

predominantly in the defense against Gram-negative bacterial

aggressions. It should be stressed that each pathway controls the

expression of several hundreds of genes. This list includes, but is

by no means restricted to, the genes encoding the identified

antimicrobial peptides. More than half of the induced genes are of

unknown function, underlining that our understanding of the host

defense is still very fragmentary.

The prevalent impression which has emerged is that the Drosophila

and the mammalian immune pathways have evolved from a reduced number

of common ancestral building blocks to their present configurations.

Whether the parallels result from convergent evolution or reflect a

common ancestry is difficult to decide at present.

Perception of microbes in mammals.

From the mammalian perspective, a different line of investigation

was followed, but one with clear parallels to the insect studies

described above, and one that converged with the insect research in

a remarkable way. Mammals had been viewed as a model for the study

of human infectious diseases for more than a century, and in the

1890s, Pfeiffer and Koch began to decipher the inherent toxicity of

microbes and molecules derived from them. They identified a heat-

stable toxin derived from Gram-negative bacteria, capable of causing

shock and death in guinea pigs, mimicking the effects of an

authentic infection. Termed " endotoxin, " this substance was

ultimately found to be equivalent to lipopolysaccharide (LPS), the

major glycolipid constituent of the outer leaflet of the outer

membrane of Gram-negative bacteria. Herein lay the seeds of much

future discovery, because a microbial molecule of defined structure

could clearly trigger powerful inflammatory responses through

receptor(s) yet unknown.

LPS is neither toxic to insects, nor to most vertebrates, but is

toxic to most mammals, including mice and humans. The effects of LPS

are mediated by macrophages, and specifically, by macrophage-derived

cytokines, which collectively orchestrate the inflammatory response.

In 1985, Beutler and colleagues isolated an LPS-induced cytokine now

known as tumor necrosis factor, or TNF, and demonstrated its

contribution to LPS-induced shock in mice. Subsequently TNF gene

regulation was shown to depend upon NF-?B responsive motifs in the

TNF promoter region. The TNF mRNA was also responsive to LPS

induction, and translational activation of nearly 200-fold was

reported, mediated by the 3'-untranslated region of the TNF mRNA and

in particular, by the UA rich element common to the TNF mRNA and

other mRNA molecules encoding inflammatory cytokines.

These observations provided molecular endpoints to be used in

finding the LPS receptor, ultimately responsible for activating the

macrophage. In 1990, Ulevitch and his colleagues established

that CD14, a glycosylphosphoinositide-anchored cell membrane protein

expressed predominantly by macrophages, was important for LPS

signaling as well. Because CD14 has no cytoplasmic domain, it was

not immediately clear how the LPS signal might be transduced across

the cell membrane. Ulevitch and colleagues noted that CD14 was a

molecule composed of leucine-rich repeats, commenting on its

similarity Toll, among other molecules. The significance of this

observation was unclear at the time, however, and played no part in

the search for a transmembrane element within an LPS receptor

complex that could carry the LPS signal into the cytoplasm.

Conventional biochemical methods were pursued in several

laboratories in the search for such a molecule, but were

unproductive.

The genetic option.

A non-redundant pathway for LPS recognition was shown to exist in

the 1960s and 1970s when substrains of LPS-unresponsive mice

(C3H/HeJ mice and C57BL/10ScCr mice) were identified, and the

resistance phenotype was in both cases ascribed to spontaneous

mutations affecting a single locus, eventually known as the Lps

locus. All LPS responses were clearly dependent upon a single,

crucial molecule. This molecule was widely envisioned as the " LPS

receptor, " though over the years, no evidence of such a receptor

could be gleaned from binding studies, likely because of its low

abundance and the hydrophobic character of the ligand molecule.

Importantly, experiments with the C3H/HeJ mouse revealed that LPS

responses were beneficial in the context of a real infection. Mice

that could not sense LPS were quickly overwhelmed when injected with

small numbers of Gram-negative bacteria (which produce LPS), while

mice that could sense LPS readily contained the infection. Both LPS-

sensitive and LPS-resistance mice were competent to cope with Gram-

positive microbes (which don't produce LPS). Hence, sensing LPS and

responding to it during the first minutes following the inoculation

of Gram-negative microbes determined the ultimate outcome of

infection.

TNF production, measured by bioassay, was taken as the endpoint of

the mammalian response to LPS, and used to positionally clone the

Lps locus in the Beutler lab. On 2093 meioses, the locus was

confined to a genomic interval approximately 2.6 Mb in length. A BAC

and YAC contig was assembled to span the critical region. By shotgun

sequencing, Beutler and colleagues identified a gene encoding one of

several mammalian homologues of Toll: Toll-like receptor 4 (or

TLR4). The gene in question was homologous, in part, to the IL-1 and

IL-18 receptors. Further sequence analysis revealed that in C3H/HeJ

mice, the gene was modified by a point mutation that altered the

cytoplasmic domain of the protein. In C57BL/10ScCr mice, the gene

was deleted. Hence, TLR4 was found to be essential for LPS sensing.

Like CD14, TLR4 was a leucine-rich repeat protein. The common motif

structure suggested the possibility of a cooperative interaction,

with both subunits contributing to a common receptor. Moreover,

particularly given the immunological function of Toll in Drosophila,

discovered in the Hoffmann lab, the identification of TLR4 as the

signaling element of the LPS receptor suggested the likelihood that

each of the divergent mammalian TLR paralogues might recognize a

distinct subset of microbial ligands, activating a common set of

molecular events within the cell, and driving a relatively

stereotypic innate immune response. A total of five TLR paralogues

were known in mammals in 1998, numbered TLR1 though TLR5. Today we

know of 10 human TLRs, 12 mouse TLRs, and 13 TLRs in both species

combined. And to a remarkable degree, the hypothesis just stated has

been supported by experimental observations.

The subsequent knockout of the gene encoding TLR2 revealed a

function in sensing bacterial lipopeptides and lipoteichoic acid. In

turn, activating ligands for TLR9, TLR3, TLR7 and TLR8, and TLR5

were identified. TLRs 1 and 6 were shown to contribute to TLR2-

mediated sensing by forming heterodimeric complexes with TLR2. TLR1

was required for tri-acyl lipopeptide sensing, while TLR6 was

required for diacyl lipopeptide sensing. TLR9 detects DNA of either

microbial or host origin, TLR3 detects poly I:C (a mimetic of

dsRNA), and TLR7 and TLR8 detect ssRNA or nucleotide-based drugs

such as resiquimod (only TLR7, and not TLR8, is active in the mouse).

Accessory subunits contribute to the receptor complexes in some

cases. For example, TLR4 signaling depends upon MD-2 (a small

protein with a hydrophobic pocket for ligand binding), which binds

to the TLR4 ectodomain and may be required not only for initial

sensing of LPS but also to permit surface expression of TLR4. CD14

assists in TLR2 signaling and is entirely required for perception of

smooth (highly glycosylated) LPS via TLR4. CD36 assists in diacyl

lipopeptide sensing. Other cofactors for signaling may also exist.

TLRs are believed to operate as functional dimers, and the overall

subunit structure of TLRs is believed to be that of a curved

solenoid, or horseshoe, and to the present time, crystallographic

models of the ectodomains of TLR2/1, TLR2/6, TLR4/MD-2, and TLR3

have been published. In the case of the TLR2 and TLR4 complexes, a

rational explanation for ligand engagement has been offered. The

exact molecular events that transpire on the cytoplasmic side of the

membrane following activation have not been elucidated.

The range of microbes detected by TLRs

While TLRs 1, 2, 4, and 6 were initially found to recognize

components of bacteria and fungi, it later became apparent that the

TLR4 complex could recognize viral proteins as well (for example,

the env glycoprotein of MMTV, the F protein of RSV, and the G

protein of VSV). Later it became clear that TLR signaling leads to

the detection of viral nucleic acids as well. Particularly in the

case of herpesviruses (MCMV and HSV), immunocompromise results from

inadequacy of TLR signaling.

The TLRs are therefore responsible for restricting the growth of

many different kinds of microbes. Mice lacking all TLR signaling as

a result of mutations in the adapter proteins MyD88 and TRIF (see

below) often die of opportunistic infections before weaning, and are

difficult to maintain as a stock, although it is possible to do so

with effort. If they do manage to survive for several weeks, TLR

signaling ablated mice become increasingly resistant to spontaneous

infection, consistent with the interpretation that adaptive immune

function becomes more and more effective at protecting them

independent of innate function.

It is fair to conclude that the mammalian TLRs are a major arm of

innate immune perception, though not unique in this role. Some

microbes appear to be countered by the NOD/NALP proteins, and others

by the RIG-I-like helicases (two distinct classes of cytoplasmic

sensors). A full consideration of these pathways is beyond the scope

of discussion here, but it may be said that a remarkably small

number of protein sensors ignite the most powerful inflammatory

responses we know, and prevent small infections from growing out of

control prior to the initiation of an adaptive immune response.

Where TLR signaling is concerned, the same biochemical pathways that

assure survival following the introduction of small numbers of

microbes mediate shock and death when infection is out of control.

The role of TLRs in adaptive immunity.

Although numerous papers attest to a requirement for TLRs in the

activation of an adaptive immune response, and although there is no

doubt that TLRs mediate the adjuvant effect of LPS and a few other

molecules of microbial origin that have been known to be adjuvants

for many decades, there is no essential need for TLRs in the genesis

of an adaptive immune response. In mutant mice with no TLR signaling

potential, the production of class-switched immunoglobulins proceeds

to the extent that total Ig levels are normal. Antibody responses to

defined antigens are normal with all adjuvants tested (including

complete Freund's adjuvant, which contains whole mycobacteria

emulsified in oil ), and so too is the antibody response to live

pathogens; likewise the memory B cell response. Allograft rejection

is also unimpaired. On this basis, it may be said that while innate

immunity truly depends upon TLR activation, adaptive immunity does

not.

Forward Genetics in Mice and the Elucidation of TLR Signaling

Pathways

Beginning in the year 2000, a phenotype-driven genetic strategy has

been pursued to examine TLR signaling pathways, and to look broadly

at molecules required for host defense. The forward screening

approach has been carried to considerable depth, and has grown

increasingly productive as the speed of positional cloning has

accelerated due to technical improvements and its cost has declined.

TLRs were initially shown to signal in part via a cytoplasmic

adapter protein termed MyD88 and MAL (also known as Tirap). A

mutation produced in the Beutler laboratory, termed Lps2, disclosed

a new signaling adapter, now known as TRIF, that works independently

of MyD88 and Tirap to carry signals from TLR4 and TLR3. Additional

work disclosed a fourth adapter, now known as TRAM, which serves

only TLR4. When MyD88 and TRIF mutations were combined, it was found

that no TLR signaling could occur, and that mice were severely

immunodeficient although they retain adequate adaptive immune

responses.

Other mutations revealed that the TLR2/TLR6 complex requires CD36 in

order to detect some of its ligands; also that TLRs 3, 7, and 9 (the

nucleic acid sensing TLRs) all depend upon a multispanning membrane

protein called UNC-93B, which may support trafficking of the TLRs to

their proper location within the cell. Still another mutation

established that CD14, once known as a component of the TLR4

signaling apparatus, participates in TLR2 complex signaling as well.

In all, 18 mutations affecting TLR signal transduction have been

created in the Beutler laboratory, and most of them have been

positionally cloned. Other mutations, identified in screens for

susceptibility to mouse cytomegalovirus (MCMV) have allowed the

construction of a " map " of those processes that permit mice to

contain this pathogen.

Flies, mice and the future of innate immunity.

What conclusions can be drawn based on the genetic analysis of

innate immunity? First, that it is conserved to a remarkable degree.

Although pathways have been sundered by 800 million years of

divergence, almost every element of the Toll and Imd pathways is

represented in one form or another in mammals, and the great

majority of mammalian sensing and signaling proteins have their

counterparts in insects. Second, innate immunity has gained a status

equal to adaptive immunity with regard to its overall importance in

host defense. Third, the most powerful inflammatory responses we

know can be traced to a small number of receptors, which represent a

biological " bottleneck. " This has enormous implications for the

treatment of chronic inflammatory diseases, some of which do indeed

seem to be driven by TLR signaling.

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