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Macrophage Biology and Innate Immunity Andreas Lengeling.

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Presentation on theme: "Macrophage Biology and Innate Immunity Andreas Lengeling."— Presentation transcript:

1 Macrophage Biology and Innate Immunity Andreas Lengeling

2 Consortium members will be drawn from the following organizations. The Roslin Institute (www.roslin.ed.ac.uk; Hume, Lengeling, Mabbott, Manson, Freeman),www.roslin.ed.ac.uk The Royal (Dick) School of Veterinary Studies Pathology Unit (Philbey, Harrison) Wellcome Trust Centre for Immunity, Infection and Evolution (http://ciie.bio.ed.ac.uk/; Allen)http://ciie.bio.ed.ac.uk/ Centre for Molecular Medicine (www.mmc.med.ed.ac.uk; Rheumatic Diseases Unit (Ralston), Gastrointestinal Diseases Unit (Satsangi)www.mmc.med.ed.ac.uk Edinburgh Cancer Research UK Centre (www.ecrc.ed.ac.uk; Frame)www.ecrc.ed.ac.uk MRC Centre for Inflammation Research (www.cir.med.ed.ac.uk; Dransfield, Rossi, Iredale, Forbes, Gregory)www.cir.med.ed.ac.uk MRC Human Genetics Unit (www.hgu.mrc.ac.uk, Dorin)www.hgu.mrc.ac.uk MRC Centre for Regenerative Medicine (www.crm.ed.ac.uk; ffrench-Constant)www.crm.ed.ac.uk Edinburgh Division of Pathway Medicine (www.ed.ac.uk/schools- departments/pathway-medicine; Ghazal)www.ed.ac.uk/schools- departments/pathway-medicine

3 Macrophage Activation Innate immune defence Wound repair Homeostasis Chronic inflammation Multiple organ failure Tumour growth Fibrosis/Atherosclerosis Obesity Why study macrophages!

4 We aim to establish an in vitro screening panel to examine the impact of knockouts on myeloid differentiation, innate immunity and antigen presentation.

5 Gene Prioritisation More than 70% of the genes in the genome are expressed in macrophages in some state of activation Knockouts of macrophage-specific genes, or conditional knockouts of essential genes in macrophages, are unlikely to be lethal. Condition Knockouts can use lysM-cre, CD11b/c- cre, Csf1r-cre

6 Biogps.org

7 Genomics Aug;88(2): Transcriptional network dynamics in macrophage activation. Nilsson R, Bajic VB, Suzuki H, di Bernardo D, Björkegren J, Katayama S, Reid JF, Sweet MJ, Gariboldi M, Carninci P, Hayashizaki Y, Hume DA, Tegner J, Ravasi T. Transcriptional regulatory networks govern cell differentiation and the cellular response to external stimuli. However, mammalian model systems have not yet been accessible for network analysis. Here, we present a genome-wide network analysis of the transcriptional regulation underlying the mouse macrophage response to bacterial lipopolysaccharide (LPS). Key to uncovering the network structure is our combination of time-series cap analysis of gene expression with in silico prediction of transcription factor binding sites. By integrating microarray and qPCR time-series expression data with a promoter analysis, we find dynamic subnetworks that describe how signaling pathways change dynamically during the progress of the macrophage LPS response, thus defining regulatory modules characteristic of the inflammatory response. In particular, our integrative analysis enabled us to suggest novel roles for the transcription factors ATF-3 and NRF-2 during the inflammatory response. We believe that our system approach presented here is applicable to understanding cellular differentiation in higher eukaryotes

8 Clustering of the BioGPS data using Biolayout

9 The phagocyte cluster

10 Genes in the phagocyte cluster

11

12 Bone marrow cells can be frozen, recovered, and cultured in appropriate colony-stimulating factors to generate all of the major myeloid lineages. We propose that IMPC routinely freeze marrow from all viable lines. Foetal liver is an alternative for late gestational or postnatal lethals.  Transcriptomic profiling of culture-derived macrophages  Measurements of differentiation (expression of markers), proliferation and apoptosis  Assay of the outcome of interactions of macrophages with a range of pathogens including mycobacteria, salmonella, listeria, group A streptococcus, candida, leishmania, influenza, adenovirus and cytomegalovirus.  Assay of classical (interferon gamma) and alternative (IL-4) activation based on induction of standard marker genes  Assay of antigen-presentation based upon activation of T cells from the OT-II transgenic mouse line in response to ovalbumin. Research Plan

13 The principal of phenotyping using frozen marrow has been confirmed by ourselves and others. We are developing to the pipeline to test the phenotype of an ENU mutant in the Mpeg1 macrophage-specific gene. We proposed to develop a programme grant application to the MRC that would include provision for funding of a dedicated technician to collect and freeze bone marrow at Harwell. Although we have prioritised a set of macrophage-expressed genes of specific interest to our own research, we would also be interesting in participating in other proposals. A priori, we can provide information on whether any gene is expressed in an informative manner in macrophages generated from bone marrow. We also propose to establish a major Centre of excellence in comparative pathology at DickVet/Roslin that would be please to be named and involved in any phenotyping activity. Funding/Research Strategy


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