The Regulation of Interleukin-10 and Interleukin-12 in Macrophages: Investigating the differential regulation of IL-10 and IL-12 in C57BL/6 and BALB/c mice
Ashleigh Frances Howes
June 2013
Division of Immunoregulation, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA
A thesis submitted to University College London for the Degree of Doctor of Philosophy
Declaration
I Ashleigh Frances Howes confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis.
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Abstract
Pattern recognition receptors (PRR) detect microbial products and induce cytokines which shape the immunological response. Interleukin-12 (IL-12) is a proinflammatory cytokine important for the differentiation of T helper 1 (Th1) cells which produce IFN-γ to activate macrophages and eradicate intracellular pathogens. In contrast, interleukin-
10 (IL-10) is an immunosuppressive cytokine that minimises immune-driven host pathology, but can also lead to defective pathogen clearance. The regulation of IL-10 and IL-12 is therefore of interest due to their central roles in the orchestration of an effective but regulated immune response. C57BL/6 and BALB/c mice differ significantly in their resistance to several pathogens. We observed that macrophages generated from these mice produce reciprocal levels of IL-10 and IL-12 in response to the bacterial ligands LPS and Pam3CSK4, which activate TLR4 and TLR2 respectively, and heat-killed Burkholderia pseudomallei, a Gram-negative bacterium which activates
TLR2 and TLR4. We have investigated this differential cytokine production in order to further dissect the molecular mechanisms underlying the regulation of IL-10 and IL-12.
Detailed analyses of protein production, signal transduction and transcriptional kinetics have identified a type I IFN dependent, but IL-27 independent mechanism for the differential production of IL-10 in LPS and heat-killed B.pseudomallei stimulated
C57BL/6 and BALB/c macrophages. Microarray analysis of LPS stimulated C57BL/6 and BALB/c macrophages further revealed potential regulatory networks that may differ between these mouse strains. These findings highlight key pathways responsible for the regulation of IL-10 and IL-12, and may provide valuable information on factors contributing to the ability of C57BL/6 and BALB/c mice to clear bacterial infections.
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Acknowledgement
I would like to begin by thanking my principal supervisor, Dr Anne O’Garra, for taking me on as a PhD student in her lab, for her guidance throughout the course of this project and for her support towards my future career. I would additionally like to thank the members of my thesis committee Dr Gregory Bancroft, Dr Steve Ley and Dr Jean
Langhorne, for their helpful suggestions and discussions at all stages of my PhD. I would like to thank Dr John Ewbank for the time he took to train me in the lab at the start of my PhD. I thank Dr Xuemei Wu and Dr Margarida Saraiva for their contributions to the initial findings which gave rise to this project, and their continued advice throughout this investigation. I am also very grateful to Dr Xuemei Wu and Dr
Damian Carragher for their organisation of mouse breeding and for doing their utmost to ensure I had the mice required to carry out my experiments. I thank Dr Christine
Graham and Dr Harsha Jani for their help with RNA preparation and microarray processing, and Evangelos Stavropoulos for his help with in vivo experiments and going out of his way to make sure I was always prepared for my experiments. I would like to thank Dr Simon Blankley and Dr Krzysztof Potempa for their helpful discussions on microarray analysis. My gratitude also goes to Dr Leo