International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)

POSTER 122 - Phenotyping the innate immune response of inbred mouse strains using cDNA microarrays

Christine Wells
The Institute for Molecular Biosciences
The University of QLD
Brisbane 4072

Co-Authors: Ravasi T, Horton D, Wardrop S, Forrest A, Wainwright B, Grimmond S and Hume D.
Institutions: The Institute for Molecular Biosciences, The University of Queensland

Innate immunity describes the ability of an organism to mount a rapid defence against invading pathogens. In mammals this is mediated primarily through dendritic cells and macrophages that are resident throughout the body. Macrophages are large phagocytotic cells, which produce autocrine factors to co-ordinate activation of surrounding cells (cytokines), recruitment of granulocytes (chemokines) and prime the acquired (antibody producing) immune system through processing of pathogens and presentation of antigens. Macrophages are able to respond rapidly and with specificity to a diverse range of pathogens and their by-products, yet the receptor complexes underlying pathogen recognition are poorly understood. These complexes are triggered through the recognition of pathogen-associated molecular patterns (PAMPS) and lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, is the archetype PAMP. It is recruited to a large receptor complex through a serum binding protein, and is associated on the surface of the macrophage with a number of proteins CD14, MD-2, glycoproteins (HSP70) and the toll-like receptor Tlr-4. Tlr-4 associates with the adapter protein MyD88 to signal through a highly conserved cascade, culminating in the translocation of the transcriptional activator NF-kappa B to the nucleus. We have used cDNA microarrays to examine the transcriptional consequences of LPS activation of macrophages over a 24-hour time course. We chose two array strategies A/ the genomic approach, through the use of RIKEN 20K mouse cDNA arrays, and B/ Targeted arrays containing 2000 elements from a macrophage derived cDNA library. We have used inbred mouse strains to model the effects of genetic background on these responses. Four strains were initially chosen- C3H/HeJARC Lpsn, C3H/HeJ Lpsd, BALB/c and SJL. Lpsd mice have a point mutation on the signalling domain of Tlr4, preventing association of the receptor complex to the intracellular adapter protein MyD88, ablating transcriptional activation through NF-Kappa B. Comparison of Lpsd and Lpsn macrophages has revealed 4 classes of transcripts those that are dependant on the Tlr4-MyD88-NF Kappa B signalling cascade, such as TNF-alpha and IL-6; those that are activated regardless of the Tlr4 status, including MHC class II transcripts; those that are activated only in Tlr4 mutant mice; and those that are constitutively different between Lpsn and Lpsd mice. This data provides compelling evidence for the activation of macrophages by LPS through several pathways, and provides a mechanism underlying previously contradictory phenotypic observations in LPS hyporesponsive mice. BALB/c mice (which lack the iron transporter NRAMP-1 and thus show a delayed oxidative burst) showed an intermediate transcriptional response to LPS. This data provides insights into the NF-Kappa B associated activation of macrophages, and offers evidence of negative transcriptional regulation by these inflammatory mediators. In contrast, SJL mice have a response to LPS primed by over expression of interleukin12, leading to hyper-responsiveness and slower resolution of the inflammatory time course. Additionally, transcriptional differences identified between the strains provide evidence of novel genetic modifiers of the LPS signalling cascade.

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