|Screen||LPS Hypersensitivity Screen|
|Posted On||02/18/2010 12:24 PM|
|Author||Amanda L. Blasius, Sungyong Won|
|Science Writer||Nora G. Smart|
LPS or lipopolysaccharide is a glycolipid of the outer membrane of Gram-negative bacteria, and is a ligand for toll-like receptor 4 (TLR4) (1). LPS has long been known to cause fever, shock, and sometimes death by inducing cytokine production, particular tumor necrosis factor (TNF), by hematopoietic cells (2-4). However, recognition of LPS by cells of the innate immune system permits effective clearance of a Gram-negative infection (5). Intraperitoneal (i.p.) injection of mice with a high enough dose of LPS will cause death in wild type C57BL/6J animals, with 100% death at 300μg/20g bodyweight (Table 1) (6). Animals with mutations in the TLR4 signaling pathway or mutations that prevent the production of inflammatory cytokines are resistant to LPS-induced shock (1;7-9).
The low dose LPS screen was devised after the identification of an N-ethyl-N-nitrosourea (ENU)–induced mutation that caused mouse cytomegalovirus (MCMV) susceptibility (MCMV Susceptibility and Resistance Screen), termed mayday. These mice succumbed to normally survivable doses of MCMV infection. Moreover, they were generally more susceptible to spontaneous death, particularly when under added stresses such as viral infection (6). Mayday mice were found to have a mutation in Kcnj8, which encoded for a subunit of an ATP-sensitive potassium channel required for cardiac function (10). In addition to MCMV susceptibility and spontaneous death, homozygous mayday mice were also sensitive to injections of low dose LPS (Table 1), with 100% dying at a dose of 50μg/20g bodyweight. These mutants also succumb to i.p. doses of CpG containing oligonucleotides (127μg/20g bodyweight), which are recognized by TLR9, and double-stranded RNA or poly I:C (20μg/20g bodyweight), which are TLR3 ligands (6).
Table 1. Mortality in mayday homozygotes after LPS administration.
Based on the susceptibility of mayday homozygous mice to LPS, a screen for additional mutants that may sensitize mice to immunological or other stresses was devised. ENU-mutagenized G3 mice are injected with a low dose of LPS based on the doses reported in Table 1 (5-10μg/mouse). Alternatively, such mutations may also be identified by intravenous (i.v.) injected CpG-induced death (please see the In Vivo CpG Screen). The ability of the low dose LPS screen to pick up appropriate mutants was confirmed with the identification of a new allele of Kcnj8, termed sos.
An additional screen now in use uses ENU-mutagenized mayday mice to identify new mutations that can rescue the original hypersensitivity to LPS conferred by the mayday mutation. ENU-mutagenized homozygous G3 mayday mice are injected with a low dose of LPS (10μg/mouse; I.V. injection). Survivors of this initial low dose are then retested with 20μg/mouse of I.V. injected LPS.
|Reagents and Solutions|
LPS (Alexis, San Diego, CA; Catalogue No. 581-008-L002)
BD insulin syringe (Becton-Dickinson; Catalogue No. 329424)
Preparation of LPS
In vivo LPS screening
1. Poltorak, A., He, X., Smirnova, I., Liu, M.-Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M. A., Ricciardi-Castagnoli, P., Layton, B., and Beutler, B. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene, Science 282, 2085-2088.
2. Thomas, L. (1954) The physiological disturbances produced by endotoxins, Annu. Rev. Physiol 16, 467-490.
3. Beutler, B., Milsark, I. W., and Cerami, A. (1985) Passive immunization against cachectin/tumor necrosis factor (TNF) protects mice from the lethal effect of endotoxin, Science 229, 869-871.
4. Michalek, S. M., Moore, R. N., McGhee, J. R., Rosenstreich, D. L., and Mergenhagen, S. E. (1980) The primary role of lymphoreticular cells in the mediation of host responses to bacterial endotoxin, J. Infec. Dis. 141, 55-63.
5. Rosenstreich, D. L., Weinblatt, A. C., and O'Brien, A. D. (1982) Genetic Control of Resistance to Infection in Mice. CRC Crit. Rev. Immunol. 3, 263-330.
6. Croker, B., Crozat, K., Berger, M., Xia, Y., Sovath, S., Schaffer, L., Eleftherianos, I., Imler, J. L., and Beutler, B. (2007) ATP-sensitive potassium channels mediate survival during infection in mammals and insects, Nat. Genet. 39, 1453-1460.
7. Pasparakis, M., Alexopoulou, L., Episkopou, V., and Kollias, G. (1996) Immune and inflammatory responses in TNFa-deficient mice: A critical requirement for TNFa in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response, J. Exp. Med. 184, 1397-1411.
8. Pfeffer, K., Matsuyama, T., Kündig, T. M., Wakeham, A., Kishihara, K., Shahinian, A., Wiegmann, K., Ohashi, P. S., Krönke, M., and Mak, T. W. (1993) Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection, Cell 73, 457-467.
9. Dumitru, C. D., Ceci, J. D., Tsatsanis, C., Kontoyiannis, D., Stamatakis, K., Lin, J. H., Patriotis, C., Jenkins, N. A., Copeland, N. G., Kollias, G., and Tsichlis, P. N. (2000) TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway, Cell 103, 1071-1083.