The molecular mechanism of innate immunity.

The Shaw Prize Foundation (the Asia's Nobel Prize) announced that Bruce A. Beutler, chair of the Department of Genetics at The Scripps Research Institute, has been awarded a 2011 Shaw Prize in Life Science and Medicine, for “discovery of the molecular mechanism of innate immunity, the first line of defense against pathogens.” Beutler shares the $1 million prize with Jules A. Hoffmann of the University of Strasbourg (France), and Ruslan M. Medzhitov, the David W. Wallace Professor of Immunobiology at Yale University.

Beutler determined how innate immune system cells detect a potentially invasive microorganism present in the body. The same system that provides awareness of infection may sometimes drive inflammatory or autoimmune diseases such as systemic lupus erythematosus. Beutler has spearheaded the use of a technique called "forward genetics" to study genes used by the mammalian innate immune system to clear pathogens from the body.

The Genetics Department has assembled a highly interactive group of investigators with expertise in the theory and practice of forward genetics: the creation of phenovariance, its detection by phenotypic screening, and its solution by positional cloning or other methods.

While many biologists begin with hypotheses about how a particular biological phenomenon operates, geneticists begin instead with a phenotype: an altered form of the phenomenon in question. Our principal interest is mammalian immune function. If we wish to understand why the mouse immune system responds to a particular molecule, we find an exceptional mouse in which it doesn’t; if we wish to understand why most mice don’t have inflammatory disease, we find an exceptional mouse that does. When a phenotype is caused by a single gene mutation, it is generally possible to find the mutation. Then we have gained fundamental insight into the phenomenon itself.

Using a genetic approach, we established some years ago that the Toll-like receptors (TLRs) serve as the key sensors used by the mammals to perceive infection. This conclusion rested upon the positional cloning of a mutation (Lps^d) that prevented mice from sensing bacterial lipopolysaccharide [Poltorak et al., Science 282:2085-2088 (1998)]. Since that time, we have established many of the essential proteins active in TLR signal transduction, but many others remain to be found.

We use random chemical mutagenesis with N-ethyl-N-nitrosourea (ENU) to produce many thousands of mice with germline mutations that affect every aspect of normal biological function. We then screen the mice to detect phenotypic change in the innate immune response. For example, the ability of macrophages to sense molecules of microbial origin (for example, lipopolysaccharide, bacterial lipopeptides, double-stranded RNA, and unmethylated DNA) is measured in vitro. The ability to cope with specific pathogens (especially mouse cytomegalovirus) is measured in vivo. Mice with inflammatory colitis, or strong resistance to specific microbes, or an abnormal complement of immune cells are identified as well. When strong deviation from normal function is detected, transmissibility of the phenotype is examined. If the phenotype is transmissible (i.e., if a bona fide mutation exists), meiotic mapping is performed to confine the mutation to a particular genomic interval. The mutation is then sought among candidate genes within the interval. To date, 274 phenotypes of all kinds have been detected in our laboratory by screening random germline mutant mice, and 140 of these mutations have immunologic effects. 170 mutations have been mapped to chromosomes, and in 145 cases, the molecular identity of the defect has been established. We now know that hundreds of genes serve the innate immune responses to microbial infections, making a life-or-death difference to animals infected with a single defined pathogen. And we have made inroads into the principal pathways of innate immune response.

Along the way, we have found many mutations that have shed light on other biological phenomena: hearing, sight, iron absorption, and development. All of these are regarded with interest, and some have started entirely new lines of biological inquiry.


Research presentation.
Among the largest issues in immunology is the question of self/non-self discrimination. How do we "know" when we have an infection? What are the receptors that alert us? For more than a century, and in fact, since microbes were recognized as the cause of infections, it has been clear that mammals are genetically programmed to recognize them.
Because the innate immune system must act promptly to contain an infection, mammals respond violently to purified molecules of microbial origin such as endotoxin (lipopolysaccharide; LPS). And it has long been known that sensing LPS is required for a mouse to overcome a Gram-negative infection (1;2). It has also been clear that cytokines, produced by mononuclear phagocytes in response to LPS, orchestrate the innate response and can be highly toxic when produced in large amounts (3-5). But the nature of the LPS receptor, which ignites the entire process, was long elusive.
It is now believed that each of the 12 mouse TLRs and 10 human TLRs dectect a limited number of the signature molecules that herald infection (LPS, lipopeptides, flagellin, unmethylated DNA, dsRNA, and ssRNA begin the best known examples). They may also detect molecular ligands of host origin under some circumstances, and may participate in sterile inflammation (observed in autoimmune diseases). The TLRs are the gatekeepers of the most powerful inflammatory responses known, and as such, are probably important in a wide range of diseases. And without TLR signaling, a state of severe immunocompromise exists (8).
The forward genetic approach entails the induction of thousands of random germline point mutations on a defined genetic background (C57BL/6) using N-ethyl-N-nitrosourea (ENU), the phenotypic screening of many thousands of mice for specific defects of immunity, and the positional cloning of those transmissible mutations that are detected. This classical genetic method does not depend upon hypotheses, nor upon assumptions about how innate immunity "should" work. Hence, it is unbiased, and errors of interpretation are extremely rare.

Over time, the effects of hundreds of millions of point mutations that change coding sense have been probed, and approximately 70% of all genes have so far been mutated to a state of detectable phenovariance. In terms of throughput, the ENU mutagenesis effort now underway in the Beutler laboratory is the largest in the world, and presently the only one primarily devoted to the decipherment of innate immunity.

In the Beutler lab, genetic screens are presently being applied to study four important topics in immunobiology. 1. Signaling pathways utilized by the TLRs and other innate immune sensors are kept under surveillance in screens designed to detect mutations that impair the detection of microbes. In the TLR signaling screen, signaling from seven TLRs is monitored by measuring tumor necrosis factor (TNF)-α production by peritoneal macrophages from ENU-mutagenized mice ex vivo. This screen has led to the decipherment of pathways for microbe sensing, identifying proteins that could not be "guessed" to participate in signaling (8-10). In addition, the study of several mutants identified in the screen has revealed subtleties in the nature of signaling from several TLRs (9;11;12). For example, the pococurante mutation of MyD88 demonstrated that signaling from TLR2 is inherently different from signaling through the other TLRs, requiring only one of two known sites of receptor-adapter interaction (Figure 1) (11). The Double-stranded DNA Macrophage Screen, to identify components involved in sensing cytoplasmic double-stranded DNA (dsDNA), and the NALP3 Inflammasome Screen, to identify components involved in sensing “danger signals,” are also being carried out in macrophages ex vivo. An in vivo screen for response to injected CpG oligodeoxynucleotides has recently been initiated.

2. By infecting mice with authentic pathogens using small inocula that are normally eliminated or contained by mice, mutations that impair host defense may be detected. Screens for susceptibility to mouse cytomegalovirus infection (MCMV Susceptibility and Resistance Screen), and for clearance of lymphocytic choriomeningitis virus (LCMV Clearance Screen) in vivo are currently underway. These screens rely on the highly reproducible behavior of mice challenged by infection, which assures that phenovariants may be easily discerned (Figure 2). Some of the identified mutations have also come as great surprises (13). For example, mayday mice die between 24 and 72 hours after infection with 5 x 10⁴ PFU of MCMV, and were found to carry a mutation in the gene encoding an inwardly-rectifying potassium (K+) channel subunit, Kir6.1 (Figure 2) (14). Screens for control of MCMV, adenovirus, influenza, and Rift Valley Fever Virus are being performed in macrophages ex vivo (Ex Vivo Macrophage Screen for Control of Viral Infection).

3. ENU mutations can also render mice highly resistant to infection by specific pathogens, or result in autoimmune and inflammatory disease. The MCMV Susceptibility and Resistance Screen and Influenza Resistance Screen may identify mutations that ultimately point to targets for intervention during infection. Such mutations disclose the existence of a "latent innate immune system," in that not all mechanisms for host resistance have been exploited. Rather, the genome has much untapped potential, and innate immunity is a work in progress. The DSS-induced Colitis Screen is designed to discover mutations resulting in susceptibility to chemically-induced colitis, which is thought to arise from excessive and sustained inflammatory host immune responses against commensal intestinal microbes. The screen monitors weight loss, rather than mortality in the case of MCMV or influenza, as an indication of colitis (Figure 3), and for this reason, sensitizing mutations are easily retrieved. Mutations that inappropriately activate immune responses to normal intestinal flora may be revealed by looking for exceptions to the norm in DSS sensitivity. Because of their potential to activate both innate and adaptive immune systems, mutations identified in each of these screens may also reveal molecules that contribute to autoimmune disease.

Figure 3a. Screening of G3 mice for susceptibility to DSS-induced colitis. 1% DSS was administered in the drinking water and body weight was determined daily. C57BL/6 mice receiving 1% DSS (black, n=20) or 0% DSS (gray, n=10) served as controls. Putative mutants are circled.

Using genetics to understand the molecular mechanisms of intestinal bowel disease (IBD). Colitis can be induced by the chemical dextran sodium sulphate (DSS), which is toxic to intestinal epithelial cells (IECs) and compromises the mucosal barrier that usually protects the body from intestinal microflora. Detection of intestinal bacteria by Toll-like receptors (TLRs) present in IECs and immune cells in the lamina propria of the intestine results in activation of the immune system, production of inflammatory cytokines, and corrective responses. By screening ENU-induced mouse mutations (red text) with the DSS-induced Colitis Screen, we have identified a number of components that are important in protecting against IBD. These include TLR signaling, epithelial growth factor receptor (EGFR) signaling, aquaporin 3 (Aqp3), the response to unfolded proteins causing endoplasmic reticulum (ER) stress, and numerous factors involved in vesicle transport and secretion, which are required to release cytotoxic molecules necessary to kill invasive pathogens.

4. The nature of the innate:adaptive immune connection is being probed. Although the innate immune response clearly contributes to the development of an adaptive immune response, the mechanism by which this occurs remains unclear. Together with our colleagues in the Nemazee lab, we have recently shown that TLR signaling is not required for effective antibody production following immunization (15), nor for strong CTL responses (16). Focusing on CTL and NK responses (In Vivo NK Cell and CD8+ T Cell Cytotoxicity Screen), we have identified a number of mutations that impair either or both, consistent with the conclusion that a large number of genes have non-redundant function in supporting cytotoxic lymphoid immunity.

In addition, the functions of many genes are illuminated by the study of mice with visible phenotypes induced by random germline mutagenesis. In these mice, mutations may affect development, morphology, behavior, or even immune function, and are positionally cloned with interest. In this manner, the laboratory pursues a broad range of biological topics. Recently, mutations in TMPRSS6 and SHP1 were found to cause body iron deficiency due to impaired iron uptake (17), and autoimmune and inflammatory disease (18), respectively.

To date, 380 transmissible mutations that cause discernable phenotypes have been set aside for positional cloning in the Beutler laboratory; 238 mutations have been mapped to chromosomes, and in 217 instances, molecular identification of the causative mutation has been made. These mutations fall within 146 genes. 264 of the mutations studied affect immunity, and about half of the mutations affecting immunity that are cloned prove to be novel in the sense that no such phenotype had been predicted by knockout mutations, or knockouts had not been created. Only about 50% recessive saturation of the genome has been achieved to date in any given screen; therefore, it is expected that many key discoveries of function lie in waiting.

The long-range goal of the laboratory is to identify the key genes required for resistance to infection (the mammalian "resistome") and determine how they interact with one another. But as genetics is a form of exploration in which very surprising phenotypes can and do arise, many different lines of inquiry are pursued. In this way the lab has solved basic questions in many different fields. Please visit our Mutagenetix web site to view the expanding list of mutations that we have produced and solved.

Beutlers Publications


Jules Hoffman and his publications, often free.

Discovery of insect-innate immune system and Toll receptor

Innate immunity is an essential host-defense system, which participates in the elimination of microbes from the body. The molecular mechanism of the innate immune system, especially the way of recognition of microbes, had been uncovered for a long time. Dr. Jules A. Hoffmann and his colleagues discovered that Drosophila Toll gene plays essential roles in innate immunity by using genetic approaches. Drosophila Toll functions as a sensor for microbes and activates intracellular signaling pathways, thereby inducing anti-microbial peptides. Their discovery is a breakthrough for the investigation of innate immune system of mammals, and leads discovery of mammalian Toll like receptor and role of their anti-microbial functions. Their findings are also contributes to the understanding of human immune systems and used for the development of adjuvant for vaccines and new anti-viral agents.

The evolutionary perspective. 1,

“The Antimicrobial Defence of Drosophila: a paradigm of innate immunity”

Today, immunologists consider the innate arm of immunity to be at least as equally important as the adaptive for the overall host defence. The innate immunity comprises a heritable, multifaceted and highly conserved defence system which its molecular basis only now has started to be elucidated. The fundamental questions on how the microbes interact with the host during the first minutes to hours following inoculation, what genes are induced and what molecular effectors are expressed are investigated extensively both in insects and in mammals.

Addressing these issues in the antimicrobial defence of Drosophila, a highly efficient innate defence system, has provided great insight and possibilities in immunology research. The results accumulated so far converge to a theatre where two major pathways act as the major actors of these mechanisms. The first is the Spatzle-Toll cascade, triggered by infection with fungi or gram-positive bacteria, while the second is the Imd (Immune deficiency) cascade, triggered by Gram-negative bacterial invasion. These pathways signal to NF-kB response elements, orchestrating the expression of several hundreds of immune-response genes. As to which protein family serves the infection discrimination function during the microbe invasion, several classes of the Peptidoglycan Recognition Proteins (PGRP) seem to be the possible culprit.

Although the knowledge about the innate immunity emerging from the Drosophila paradigm is still very elementary, several lines of investigation imply that the aforementioned complex signalling cascades are builded and act in a similar fashion in mammals also; every element of the Toll and Imd paths are represented in mammals by the TLR4 and TNF cascades respectively.

Ruslan M. Medzhitov, Yale bulletin
Medzhitov has made groundbreaking contributions to the understanding of innate immunity, which provides immediate defense against infection. His studies helped elucidate the critical role of toll-like receptors (TLRs) in sensing microbial infections, mechanisms of TLR signaling, and activation of the inflammatory and immune response.

Arming the Immune System talk. "We don't know how to make vaccines yet".
Toll like receptor and IL-1 receptor signalling, also capsases.
"Toll like receptors and innate immunity". R. Medzhitov. Nature Reviews
Immunology 1, 135, 2001.
Minireview 1997: Innate Immunity: The Virtues of a Nonclonal System of Recognition. Ancient Host Defense Pathway etc. with Il-1,6,8. Toll/NFkB pathway is conserved between insects and mammals and activates nonspecific defense mechanisms in both cases, while in mammals Toll also induces signals required for the activation of the adaptive immune response.
INNATE IMMUNE RECOGNITION, 2002

This man was a bit more interesting.

From Howard Hughes Medical Institute:
Medzhitov’s interest in immunology was ignited in the early 1990s - a bleak time for science in Russia. Medzhitov witnessed this disintegration first-hand. Scientific resources drained away, until just a single battered copy of the weekly journals made the rounds at Moscow University. As a graduate student there, Medzhitov yearned to keep up with the latest advances, and his
weekly hour with Science and Nature wasn't enough. So he headed to the Academy of Natural Sciences, which was then engaged in its own detente with the university. For various bureaucratic reasons, university students weren't allowed access to the library. “So I had to go and flirt with the librarians—there were several of them—and eventually they all knew me and
let me in secretly and told me not to tell anyone,” says Medzhitov.
There, in the stacks, the young biology student stumbled on a copy of Cold Spring Harbor Symposia. In it was the paper that launched his career. Written by the late Yale immunologist Charles Janeway (an HHMI investigator), the article sketched a new theory for how the immune system recognizes and responds to pathogens. Little was known then about the so-called innate immune system and how it identifies and reacts to invaders. Janeway’s ideas
ignited Medzhitov, sending him to his university’s sole e-mail terminal. “I was able to send messages once a week,” says Medzhitov. “And my first message was to Charlie.” Medzhitov asked the professor for more details about his ideas. To Medzhitov’s delight, Janeway responded, and the pair exchanged several more messages.
“Charlie's paper was the only paper that made sense of a lot of things,” says Medzhitov. “That was the point I first thought about being a researcher in immunology. As an undergraduate student, I never had a course on immunology.”
With a career path now in mind, Medzhitov landed a fellowship at the University of California, San Diego. There, working with protein evolution pioneer Russell Doolittle, Medzhitov contacted local immunologist Richard Dutton, who knew Janeway and recommended Medzhitov for a postdoctoral position in Janeway’s lab. Janeway said yes. “I felt very lucky,” says
Medzhitov.
When he arrived at Yale—after a detour to Moscow to defend his thesis and sweat out a government coup and six months of uncertainty—Medzhitov felt overwhelmed. “Janeway’s lab was very famous, and I imagine competition to get in was very high. And I was coming from just a few e-mail exchanges and a recommendation. My challenge was, not only did I not speak English well, I also had never done any experiments. In Russia, there was no money to do anything. All I could do was sit in the library. So I arrived without any experience, basically zero. I had to learn as quickly as I could.”
It turns out that lack of experience helped Medzhitov in another way. Janeway’s theory of how innate immunity acted, by recognizing bits of invading organisms, was “extremely speculative.” And that meant it was risky to work on. But, being “oblivious to concerns about career,” Medzhitov jumped in on the project. “I was just happy to be in a place where I could do science,” he says.
In 1996, after just a few years working together, Janeway and Medzhitov made a breakthrough. They discovered receptors that alerted the second arm of the immune system, the more familiar T cells and B cells that attack pathogens. Studying these proteins, dubbed Toll-like receptors, quickly became one of the hottest areas in biology. “That was an extremely exciting time,” says Medzhitov. “We didn't realize how much would come out of it eventually, that it would become such a huge area of research.”
In the years since then, Medzhitov has piled one discovery after another upon the first, dramatically expanding our understanding of the key roles Toll-like receptors play in infection control, chronic inflammation, and even the growth of tumors. At the same time, he's branched off in a dozen directions:
One example of many, Medzhitov is learning how commensal bacteria—which live in our guts and help us digest food—also help protect our intestines from injury.
Medzhitov now thinks that Toll-like receptors and related proteins may trigger the chronic inflammation that leads to coronary artery disease, Alzheimer’s, and diabetes—some of our biggest killers. “I like a lot of areas of biology and it's hard for me to focus on only one,” he says. Now, with plenty of journals to read and experiments to conduct, he doesn't have to.

Research Summary

Research in this laboratory focuses on many aspects of innate immunity and includes the following areas:

• Molecular mechanisms of innate immune recognition: Identification and analysis of receptors involved in innate immune recognition (Pattern Recognition Receptors) and signaling pathways activated by these receptors. Of particular interest is the recently identifiedfamily of Toll-like receptors, which plays an essential role in innate immune recognition in both mammals and insects.

• Control of adaptive immune responses by innate immune recognition. Signals induced upon innate immune recognition (co-stimulatory molecules, cytokines and chemokines) are necessary both for the initiation of adaptive immune responses and the control of effector functions. We are interested in molecular mechanisms that translate the signals recognized by Pattern Recognition Receptors into signals that control the activation of naive lymphocytes and their differentiation into effector cells.

• Mechanisms of autoimmunity and allergy. Inflammation is a normal component of the host response to infection. However, excessive inflammation, or inflammation in the absence of infection, may lead to a variety of pathological states, including autoimmunity and allergy. We are studying the cellular and molecular basis of inflammatory disorders that are caused by the dysfunctions of the innate immune system.

Extensive Research Description

Innate immune recognition

The innate immune system relies on several distinct strategies of recognition, including pattern recognition and missing self recognition. We are interested in defining cellular and molecular mechanisms of innate immune sensing and signaling. There are several different classes of receptors involved in innate immune recognition. We are interested in the general design of the recognition and signaling modules of the innate immune system, their functional relationships, their roles in host defense and in control of adaptive immunity, and their contributions to immunopathology.

Host-Pathogen interactions

The disease state caused by microbial infection is a result of either microbial virulence or immunopathology (the host response to infection), or in some cases both. Thus immune sensing and responsiveness to infection are adjusted during evolution to achieve an optimal balance to maximize protection from infection, and to minimize the pathology caused by an overzealous immune response. This balance can presumably vary depending on infection. We are interested in studying the mechanisms (both hard-wired and adaptive) that allow for an optimal trade-off between these two conflicting goals. We are interested in understanding the role of virulence in host-pathogen interactions and the effect of microbial virulence on innate and adaptive immunity. We are also studying the affect of infection on the immune system and how the immune system handles co-infections.

Inflammation

Inflammation is a fundamental physiological process that underlies a multitude of normal and pathological conditions. We are studying both the basic biology of inflammation and the regulatory mechanisms that control initiation, quality and intensity of inflammatory responses. In particular, we are studying the links between inflammation and metabolism, inflammation and aging, and inflammation and cancer.

Control of adaptive immunity

Innate immune recognition plays a critical role in the control of adaptive immune responses. Multiple mechanisms underlie the connections between innate and adaptive immune systems, and most of them are poorly understood. We are studying basic mechanisms that couple innate immune recognition with activation and differentiation of adaptive immune responses. We are also studying the links between innate immune system and peripheral tolerance.

Cell biology of signal transduction

Most of what we know about cell signaling is based on biochemical and genetic studies. While these approaches provide essential information about the composition of signaling pathways, much less progress has been made in understanding the functional organization of signaling pathways, especially in the context of basic cell biological processes, such as protein sorting and vesicular trafficking. We are interested in basic principles that govern the cell biology of signaling transduction pathways.

Control of gene expression

Stimulation of macrophages through TLRs leads to changes in the expression (induction and suppression) of hundreds of genes. These changes are effected through a diversity of mechanisms. Gene regulation occurs at multiple levels (activation of trasnscription factors, chromatin remodeling and histone modifications) and has both signal-specific and gene-specific components. Different subsets of TLR-inducible genes are subject to differential regulatory influences, which are dependent on the function of the products they encode. We are interested in the basic principles of inducible gene expression, which are currently poorly characterized.

Cancer biology

We are studying the mechanisms whereby cancer cells can sense their 'oncogenic state' and communicate it to other cells of the host. We are also studying the role of inflammation and tissue repair in tumor progression.

Announcement at Science
All plants and animals have a built-in resistance to pathogens called innate immunity that is more basic and general than the better-known adaptive immunity that responds to specific infections or vaccines. Innate immunity is the first line of defense against pathogens in all plants and animals. Jules Hoffmann of the University of Strasbourg in France first identified a key molecule, called Toll, involved in the innate immune response in fruit flies. Ruslan Medzhitov of Yale University then found homologous molecules, Toll-like receptors, in humans. Bruce Beutler of the Scripps Research Institute in San Diego, California, completed the puzzle by showing how the Toll-like receptors activate the innate immune system.

There are also others involved, of course.