Scientific Report 2005

Immunology

The Genetic Basis of Resistance to Infection

B. Anchonda, K. Benson, B. Croker, K. Crozat, X. Du, P. Georgel, C. Gil-Lamaignere, K. Hoebe, Z. Jiang, N. Mann, S. Mudd, S. Rutschmann, L. Shamel, S. Sovath, K. Tabeta, H. Uy, Z. Zhou, B. Beutler

No “universal pathogens” exist, and even the most virulent human pathogens (e.g., HIV and smallpox virus) are innocuous in most mammalian species. Conversely, no human has ever died of mouse cytomegalovirus (MCMV) infection. In general, interspecies differences in susceptibility to infectious diseases cannot be explained on the basis of differences in adaptive immunity. The combinatorial system for generating diversity in T- and B-cell receptors is similarly effective in all mammals. Rather, innate immunity is the characteristic that differs remarkably among species. And even among members of a single species, differences in innate immunity may foretell life or death in the event of an infection. Evidently, a large number of genes confer resistance to infection. For this reason, in humans, susceptibility to death caused by infection has greater heritability than susceptibility to death from any other cause.

Our broad goal is to identify all the genes required for a strong innate immune response. Most genes that serve this function in mice are also required for innate immunity in humans, and, indeed, conservation of core innate immune processes is such that many of the same genes are even used for innate defense by fruit flies. To find nonredundant components of the innate immune system, we create inherited innate immunodeficiency states through the use of a randomly acting germ-line mutagen, N-ethyl-N-nitrosourea. Those exceptional animals that have compromised immunity are detected by phenotypic screening. The mutations are brought to homozygosity and mapped by using classical genetic methods. DNA sequencing is then applied to pinpoint the culpable mutation and hence find the gene (and protein) with an indispensable role in disease resistance. We use 2 general types of phenotypic screens.

Components of the Toll-Like Receptor Signal Transduction Pathways

We earlier determined that the mammalian Toll-like receptors (TLRs) are responsible for perceiving diverse infections, ranging from those caused by bacteria and fungi to those caused by protozoa and viruses. TLRs alert the host to the presence of conserved molecules that are synthesized by these microbes, such as lipopolysaccharide, DNA with unmethylated cytosine-guanine dinucleotides, double-stranded RNA, and various diacylglycerides and triacylglycerides. We have implemented a focused screen for defects in signaling from TLRs to the level of TNF production.

Using this screen, we identified 11 mutations, which affect 10 genes. Of the 10 genes, 5 encode “known” components of the TLR signaling apparatus, and 5 encode components that were not previously known. Among the new elements are an adapter protein for TLR signaling (TRIF), a coreceptor for sensing microbial diacylglycerides (CD36), a protein kinase (feckless) that links the sensing of double-stranded RNA to the activation of NF-κB (a key inflammatory transcription factor), and a component of the endoplasmic reticulum (3d) that is required for signaling via 4 of the TLRs. On the basis of the number and types of mutations found, we calculated that about 50 proteins are required for signaling from the TLRs to the level of TNF production.

The protein 3d is remarkable because it is required not only for TLR signaling but also for the processing of antigens for activation of adaptive immune responses. Thus, 3d links the innate and adaptive immune systems. Further investigation of the mechanism of action of 3d may reveal much about how autoimmune diseases are initiated and sustained.

Susceptibility to MCMV

C57BL/6 mice normally are strongly resistant to infection by MCMV. However, we found that a substantial fraction of the mouse genome is devoted to the creation of resistance. Mutations in approximately 300 genes can cause profound susceptibility to MCMV. Approximately 10% of these genes have now been altered by mutation, and several of the mutations have been brought to homozygosity, a necessary precondition for positional cloning.

The MCMV “resistome,” those genes that serve nonredundant functions in resistance to this pathogen, will gradually be defined through a forward genetic approach. Many of the mutations so far that diminish resistance to MCMV have broad effects on resistance to infection overall. We therefore think that the MCMV resistome is not much smaller than the “universal resistome,” the complement of genes required for resistance to all microbes.

Novel Pathways for the Activation of Adaptive Immune Responses

The TLRs mediate most infection-related phenomena, including “bad” effects such as fever and shock and “good” effects such as activation of the adaptive immune response. This last effect has been of particular interest to immunologists, because it is an important factor in the development of vaccines. However, we discovered that TLR-independent pathways exist for initiation of an adaptive immune response. One pathway impels a highly efficient CD8 T-cell response to foreign proteins expressed by cells undergoing apoptosis (programmed death). This “death-driven” adjuvant pathway is the most efficient means of CD8 activation ever described. It presumably evolved to permit the detection of pathogens that trigger an apoptotic response. Our assumption is that the host has retained pathogen-specific pathways for cell death and uses them to encourage an adaptive immune response. Aberrant activation of these pathways may be important in the development of dysfunctional adaptive immune responses to host proteins.

Publications

Anfossi, N., Robbins, S.H., Ugolini, S., Georgel, P., Hoebe, K., Bouneaud, C., Ronet, C., Kaser, A., DiCioccio, C.B., Tomasello, E., Blumberg, R.S., Beutler, B., Reiner, S.L., Alexopoulou, L., Lantz, O., Raulet, D.H., Brossay, L., Vivier, E. Expansion and function of CD8+ T cells expressing Ly49 inhibitory receptors specific for MHC class I molecules. J. Immunol. 173:3773, 2004.

Beutler, B. SHIP, TGF-β, and endotoxin tolerance. Immunity 21:134, 2004.

Beutler, B. The Toll-like receptors. In: Genetic Susceptibility to Infection. Kaslow, R.L., McNicholl, J., Hill, A.V.S. (Eds.). Oxford University Press, New York, in press.

Beutler, B., Crozat, K., Koziol, J.A., Georgel, P. Genetic dissection of innate immunity to infection: the mouse cytomegalovirus model. Curr. Opin. Immunol. 17:36, 2005.

Beutler, B., Hoebe, K., Georgel, P., Du, X. Forward genetic analysis of TLR pathways: a shared system for the detection of endotoxin and viral infection. In: Toll and Toll-Like Receptors: An Immunologic Perspective. Rich, T. (Ed.). Kluwer Academic/Plenum, New York, 2005, p. 168. Molecular Biology Intelligence Unit.

Beutler, B., Hoebe, K., Georgel, P., Tabeta, K., Du, X. Genetic analysis of innate immunity: TIR adapter proteins in innate and adaptive immune responses. Microbes Infect. 6:1374, 2004.

Georgel, P., Crozat, K., Lauth, X., Makrantonaki, E., Seltmann, H., Sovath, S., Hoebe, K., Du, X., Rutschmann, S., Jiang, Z., Bigby, T., Nizet, V., Zouboulis, C.C., Beutler, B. A Toll-like receptor 2-responsive lipid effector pathway protects mammals against skin infections with gram-positive bacteria. Infect. Immun. 73:4512, 2005.

Hawn, T.R., Verbon, A., Janer, M., Zhao, L.P., Beutler, B., Aderem, A. Toll-like receptor 4 polymorphisms are associated with resistance to Legionnaires’ disease. Proc. Natl. Acad. Sci. U. S. A. 102:2487, 2005.

Hoebe, K., Beutler, B. LPS, dsRNA and the interferon bridge to adaptive immune responses: Trif, Tram, and other TIR adaptor proteins. J. Endotoxin Res. 10:130, 2004.

Hoebe, K., Beutler, B. Unraveling innate immunity using large scale N-ethyl-N-nitrosourea mutagenesis. Tissue Antigens 65:395, 2005.

Hoebe, K., Georgel, P., Rutschmann, S., Du, X., Mudd, S., Crozat, K., Sovath, S., Shamel, L., Hartung, T., Zähringer, U., Beutler, B. CD36 is a sensor of diacylglycerides. Nature 433:523, 2005.

Hoebe, K., Janssen, E., Beutler, B. The interface between innate and adaptive Immunity. Nat. Immunol. 5:971, 2004.

Jiang, Z., Georgel, P., Du, X., Shamel, L., Sovath, S., Mudd, S., Huber, M., Kalis, C., Keck, S., Galanos, C., Freudenberg, M., Beutler, B. CD14 is required for MyD88-independent LPS signaling. Nat. Immunol. 6:565, 2005.

Mattner, J., DeBord, K.L., Ismail, N., Goff, R.D., Cantu, C. III, Zhou, D., Saint-Mezard, P., Wang, V., Gao, Y., Yin, N., Hoebe, K., Schneewind, O., Walker, D., Beutler, B., Teyton, L., Savage, P.B., Bendelac, A. Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature 434:525, 2005.

Rietschel, E.T., Rietschel, M., Beutler, B. How the mighty have fallen: fatal infectious diseases of divine composers. Infect. Dis. Clin. North Am. 18:311, 2004.

Smythe, I., Du, X., Taylor, M.S., Justice, M.J., Beutler, B., Jackson, I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis. Proc. Natl. Acad. Sci. U. S. A. 101:13560, 2004.

Theofilopoulos, A.N., Baccala, R., Beutler, B., Kono, D.H. Type I interferons (αβ) in immunity and autoimmunity. Annu. Rev Immunol. 23:307, 2005.

Zhou, Z., Hoebe, K., Du, X., Jiang, A., Shamel, L., Beutler, B. Antagonism between MyD88- and TRIF-dependent signals in B7RP-1 up-regulation. Eur. J. Immunol. 35:1918, 2005.