Lps2: a new locus required for responses to lipopolysaccharide,revealed by germline mutagenesis and phenotypic screening

Abstract

Both forward and reverse genetic techniques have been used to define components of the mammalian lipopolysaccharide (LPS) receptor. TLR4, identified by a forward genetic approach as the product of the classical Lps locus, is the only known transmembrane component of the mammalian LPS receptor. Gene knockout work has also established that LPS signal transduction requires the integrity of CD14, MD-2, and, in part, MyD88, IRAK4, and TRAF-6. However, there is no reason to believe that these are the only proteins that make up the receptor/transducer apparatus. To examine the possibility that other proteins may be involved, we initiated a mutagenesis program, in which germline mutations are induced in mice using N-ethyl-N-nitrosourea (ENU), and macrophages from individual animals are screened for their competence to respond to LPS. We now report the existence of a new locus, Lps2, which is required for TNF production in response to LPS. The Lps2 mutation that we have identified is co-dominant, is similar in phenotypic effect to Lps^d , and does not represent a novel allele of any of the genes that are known to encode the `core' LPS signaling apparatus. The Lps2 mutation does not preclude signaling initiated by peptidoglycan or unmethylated DNA. Hence, genetic data suggest that there is at least one `missing' component of the LPS receptor complex that has yet to be found.

References

Brown SD , Peters J. Combining mutagenesis and genomics in the mouse - closing the phenotype gap . Trends Genet 1996; 12: 433-435.

Hrabe de Angelis M. , Balling R. Large scale ENU screens in the mouse: genetics meets genomics. Mutat Res 1998; 400: 25-32.

Justice MJ , Noveroske JK , Weber JS , Zheng B. , Bradley A. Mouse ENU mutagenesis. Hum Mol Genet 1999; 8: 1955-1963.

Brown SD , Balling R. Systematic approaches to mouse mutagenesis. Curr Opin Genet Dev 2001; 11: 268-273.

Poltorak A. , Smirnova I. , He XL et al. Genetic and physical mapping of the Lps locus - identification of the Toll-4 receptor as a candidate gene in the critical region. Blood Cells Mol Dis 1998; 24: 340-355.

Poltorak A. , He X. , Smirnova I. et al. Defective LPS signaling in C3H/HeJ and C57BL/l OScCr mice: mutations in Tlr4 gene. Science 1998; 282: 2085-2088.

Wright SD , Ramos RA , Tobias PS , Ulevitch RJ , Mathison JC CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 1990; 249: 1431-1433.

Haziot A. , Ferrero E. , Kontgen F. et al. Resistance to endotoxin shock and reduced dissemination of Gram-negative bacteria in CD14-deficient mice. Immunity 1996; 4: 407-414.

Shimazu R. , Akashi S. , Ogata H. et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med 1999; 189: 1777-1782.

10.

Nagai Y. , Akashi S. , Nagafuku M. et al. Essential role of MD-2 in LPS responsiveness and TLR4 distribution . Nat Immunol 2002; 3: 667-672.

11.

Kawai T. , Adachi O. , Ogawa T. , Takeda K. , Akira S. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 1999; 11: 115-122.

12.

Suzuki N. , Suzuki S. , Duncan GS et al. Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4. Nature 2002; 416: 750-756.

13.

Lomaga MA , Yeh WC , Sarosi I. et al. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev 1999; 13: 1015-1024.

14.

Moore RN , Goodrum KJ , Berry LJ Mediation of an endotoxic effect by macrophages. J Reticuloendothel Soc 1976; 19: 187-197.

15.

Michalek SM , Moore RN , McGhee JR , Rosenstreich DL , Mergenhagen SE The primary role of lymphoreticular cells in the mediation of host responses to bacterial endotoxin. J Infect Dis 1980; 141: 55-63.

16.

Freudenberg MA , Keppler D. , Galanos C. Requirement for lipopolysaccharide-resp onsive m acroph ages in galactosamineinduced sensitization to endotoxin. Infect Immun 1986; 51: 891-895.

17.

Beutler B. , Milsark IW , Cerami A. Passive immunization against cachectin/tumor necrosis factor (TNF) protects mice from the lethal effect of endotoxin. Science 1985; 229: 869-871.

18.

Chow JC , Young DW , Golenbock DT , Christ WJ , Gusovsky F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction . J Biol Chem 1999; 274: 10689-10692.

19.

Garrelds IM , Zijlstra FJ, Tak CJAM, Bonta IL, Beckmann I, Ben-Efraim S. A comparison between two methods for measuring tumor necrosis factor in biological fluids. Agents Actions 1993; 38 (Suppl. C): C89-C91.

20.

Heppner G. , Weiss DW High susceptibility of strain A mice to endotoxin and endotoxin-red blood cell mixtures. J Bacteriol 1965; 90: 696-703.

21.

Watson J. , Riblet R. Genetic control of responses to bacterial lipopolysaccharides in mice. I. Evidence for a single gene that influences mitogenic and immunogenic responses to lipopolysaccharides. J Exp Med 1974; 140: 1147-1161.

22.

Coutinho A. , Meo T. Genetic basis for unresponsiveness to lipopolysaccharide in C57BL/10Cr mice . Immunogenetics 1978; 7: 17-24.

23.

Fitzgerald KA , Palsson-McDermott EM , Bowie AG et al. Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction. Nature 2001; 413: 78-83.

24.

Horng T. , Barton GM , Medzhitov R. TIRAP: an adapter molecule in the Toll signaling pathway. Nat Immunol 2001; 2: 835-841.

25.

Adachi O. , Kawai T. , Takeda K. et al. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 1998 ; 9: 143-150.

26.

Hemmi H. , Takeuchi O. , Kawai T. et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408: 740-745.

27.

Hemmi H. , Kaisho T. , Takeuchi O. et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 2002; 3: 196-200.

28.

Lomaga MA , Henderson JT , Elia AJ et al. Tumor necrosis factor receptor-associated factor 6 (TRAF6) deficiency results in exencephaly and is required for apoptosis within the developing CNS. J Neurosci 2000; 20: 7384-7393.

29.

Xu Y. , Tao X. , Shen B. et al. Structural basis for signal transduction by the Toll/interleukin-1 receptor domains. Nature 2000; 408: 111-115.

30.

Rosenstreich D. , Vogel SN , Jacques AR , Wahl LM , Oppenheim JJ Macrophage sensitivity to endotoxin. Genetic control by a single codominant gene. J Immunol 1978; 121: 1664-1670.