The phenomenon of sepsis can be separated what might be called 'sensing' and 'post-sensing' phases. The sensing phase entails the innate immune perception of bacteria, and occurs largely via Toll-like receptors, which trigger a complex cytokine response. The post-sensing phase encompasses the reaction of tissues, organs, and organ systems to the cytokine response. The post-sensing phase is largely terra incognita. Much ofthe variability in clinical course and clinical outcome between individuals with comparable infections may be explained by differences manifested at this level. Some of the differences are agedependent. Others are clearly genetic. The purpose of the current proposal is to identify mutations that render mice highly susceptible to LPS through effects downstream of the initial innate immune response. We also hope to understand precisely how these mutations operate. A genetic approach will be used in our work. Random germline mutagenesis will be performed in the C57BL/6J strain using N-ethyl-N-nitrosourea (ENU). Mutations will be brought to homozygosity, and male mice will be screened for lethality or severe illness when injected with a normally harmless quantity of LPS (10-20 ug). When lethality or severe illness is encountered, sperm from the affected individual will be used to preserve the germline. We will then perform intracytoplasmic sperm injection (ICSI) using oocytes from the closely related C57BL/10J strain, for which we have established an informative mapping panel. Mapping will be accomplished using bulk segregation analysis: a technique we have also recently developed for the C57BL/6J x C57BL/10J strain combination. DNA from the index mouse will be sequenced to identify all candidate mutations. The mutation implicated in this process will be confirmed by BAC transgene rescue or gene trap complementation studies. At the same time, a suppression screen will be used to clarify the mode of action of one mutation that causes hypersensitivity to LPS, called MayDay. This mutation has been found to be suppressed by Bourgeoisie, an ENU mutation detected during the first cycle of funding. Bourgeoisie will be positionally cloned, and other mutations like it will be found, using a positional cloning strategy similar to the one just described. Our work will help to explain why some individuals with bacteremia are likely to develop sepsis while others are not. Moreover, by disclosing points of host vulnerability during infection, it may suggest pharmacotherapeutic interventions helpful to all patients with sepsis.
While many people experience bacteremia (bacteria in the blood) at some point during their lives, and exhibit a normal innate immune response, some develop overwhelming sepsis~a fall in blood pressure and severe damage to internal organs-while others do not. This research seeks to identify the genetic differences that explain variability in human responses to infection, particulariy infection with Gram-negative bacteria. This work may lead to an appreciation of who is at risk, and may also suggest therapies for sepsis in all people.
|Moresco, Eva Marie Y; Li, Xiaohong; Beutler, Bruce (2013) Going forward with genetics: recent technological advances and forward genetics in mice. Am J Pathol 182:1462-73|
|Kono, Dwight H; Haraldsson, M Katarina; Lawson, Brian R et al. (2009) Endosomal TLR signaling is required for anti-nucleic acid and rheumatoid factor autoantibodies in lupus. Proc Natl Acad Sci U S A 106:12061-6|
|Blasius, Amanda L; Brandl, Katharina; Crozat, Karine et al. (2009) Mice with mutations of Dock7 have generalized hypopigmentation and white-spotting but show normal neurological function. Proc Natl Acad Sci U S A 106:2706-11|
|Croker, Ben A; Lawson, Brian R; Rutschmann, Sophie et al. (2008) Inflammation and autoimmunity caused by a SHP1 mutation depend on IL-1, MyD88, and a microbial trigger. Proc Natl Acad Sci U S A 105:15028-33|
|Hoebe, K; Jiang, Z; Georgel, P et al. (2006) TLR signaling pathways: opportunities for activation and blockade in pursuit of therapy. Curr Pharm Des 12:4123-34|