Bruce Beutler Laboratory Research

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. Moreover, it has long been an obvious corollary that certain molecules of microbial origin must trigger a host response, and that specialized receptors of the host must mediate recognition of these molecules. This, after all, is how biological systems operate. But what were these receptors? A genetic approach was required to answer the question.

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). LPS has been investigated for many decades as a prototypic inducer of innate responses. 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.

The present activities of the Beutler laboratory stem from a longstanding interest in innate immune sensing and response, and involve the use of positional cloning as a method to decipher it. In 1998, the laboratory identified the mammalian LPS receptor as Toll-like receptor 4 (TLR4) by genetically mapping and then cloning a mutant allele known as Lpsd, which in homozygous form caused unresponsiveness to LPS in mice (6). This discovery-the first assignment of function to a TLR-led directly to the present concept that the mammalian TLRs serve as sensors of microbial infection (7). 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 Beutler laboratory systematically employs a forward genetic approach to identify genes that are essential for the mammalian innate immune response, and to determine their functions relative to one another. 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.

Four Major Areas of Focus

Important mutations do not always declare themselves without challenge. This is particularly true of mutations that affect immune function. Without an infection, immunocompromised mice may seem entirely normal. For example, the C3H/HeJ mouse is indistinguishable from other C3H substrains unless it is challenged with LPS or an authentic Gram-negative infection. It may be that many of our genes are conditionally important in the same way. In the Beutler lab, genetic screens are presently being applied to study four important topics in immunobiology.

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.
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).
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.
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. The phenotypes were first discovered in unchallenged mice because of visible defects: hair loss in Tmprss6<mask/mask> mice (Figure 4), and spontaneous foot inflammation in Ptpn6<spin/spin> mice (Figure 5). Of particular interest in the case of the SHP1 mutant spin, the autoimmune and autoinflammatory phenotypes were shown to depend upon a microbial trigger and upon IL-1 receptor signaling.

Because of the link between pigmentation and immune function, a wide variety of mutants with variations in coat color are also under investigation (Figure 6).

To date, 234 transmissible mutations that cause discernable phenotypes have been set aside for positional cloning in the Beutler laboratory; 135 mutations have been mapped to chromosomes, and in 120 instances, molecular identification of the causative mutation has been made. About 120 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 activities of the lab are sustained by dedicated staff highly trained in mutation mapping, DNA sequencing and mutation finding (much of which is performed robotically and computationally). A core devoted to germline cryopreservation, intracytoplasmic sperm injection (ICSI; required for rapid fixation of susceptibility alleles in a homozygous state), transgenesis, and gene targeting has also been established. These facilities make it possible to positionally clone 20-30 molecular targets each year. A skilled and dedicated postdoctoral fellow or graduate student may expect to identify 5 to 10 mutations over the course of his or her stay in the laboratory.

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.

All mutant stocks are deposited with MMRRC, JAX, or other repositories when published and are available to the scientific community through these sources.

Reference List

The Bruce Beutler Laboratory

The Major Questions

Four Major Areas of Focus