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Title: Investigations into the role of endogenous Annexin-A1 in dendritic cell biology
Author: Huggins, Anthony
Awarding Body: Queen Mary, University of London
Current Institution: Queen Mary, University of London
Date of Award: 2012
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A school of literature has shown that Annexin-A1 (Anx-A1) is an endogenous anti-inflammatory protein that exerts a regulatory control over the innate immune system in order to restore homeostasis after an inflammatory reaction. Surprisingly, recent published works have highlighted that Anx-A1 has an alternate role in the adaptive immune system by positively modulating the strength of TCR signalling and biasing helper-subset differentiation. Dendritic cells are a class of innate leukocytes, poised at the environmental interface, that are the essential immune cells responsible in the initiation of T-cell driven responses. These findings provided the foundation for this PhD project, the principal aim of which is to provide a link between the disparate effects of Annexin-A1 in innate and adaptive immunity by investigating the role of endogenous Annexin-A1 in dendritic cell biology and its effector function as an antigen-presenting cell towards T cell activation and differentiation. To address this hypothesis, I cultured bone marrowderived dendritic cells from AnxA1-deficient mice or control littermates and stimulated with LPS (100ng/ml) then compared phenotypic and functional characteristics. My results demonstrate that Anx-A1-/- bone marrow derived dendritic cells show an increased number of CD11c+ cells expressing high levels of some maturation markers such as CD40, CD54 and CD80 and a decreased capacity to take up antigen compared to control Anx-A1+/+ cells. However, analysis of LPS-treated dendritic cells from Anx-A1-/- mice demonstrated a diminished up-regulation of maturation markers, a decreased migratory activity in vivo and an attenuated production of the inflammatory cytokines Interleukin (IL)- 1β, Tumour Necrosis Factor (TNF)-α and IL-12. This defect was resultant of an impaired Nuclear Factor (NF)-κB/DNAbinding activity due to lack of Anx-A1 signalling as demonstrated by the reduced activation of Extracellular-signal Regulated Kinase (ERK) 1/2 and protein kinase B (PKB)/Akt compared to cells from control littermates. As a consequence of these defects, I assessed the antigenpresenting/ T-cell activating capabilities of these DC. Anx-A1-/- DC showed an impaired capacity to stimulate T cell proliferation and differentiation in allogeneic mixed leukocyte reaction. To dissect this biologically relevant phenomenon further, I employed an antigenspecific, T-cell restricted model; a co-culture system of chicken ovalbumin peptide-pulsed, LPS-matured bone marrow-derived DC incubated with transgenic TCR T cells from OT-I/RAG-1-/- (OT-I, OTI/ CD8+) or OT-II/ RAG-1-/- (OT-II, OT-II/CD4+) mice. Peptide-pulsed, LPS-matured AnxA1-/- DC failed to initiate an appropriate T cell activation in both OT-I and OT-II T cells indicated by reduced cell proliferation when compared to T cells co-cultured with peptide6 pulsed, LPS-matured AnxA1+/+ DC. Additionally, comparison of peptide-pulsed, LPS-matured AnxA1-/- DC with AnxA1+/+ DC counterparts detected severely diminished levels of IL-2 from cocultures with OT-I T cells and ablated IFN-γ production from cocultures with both OT-I and OT-II T cells. In conclusion, AnxA1 seems to act as a positive modulator of immunogenic activation of DC, whereby the AnxA1 signal pathway has a probable synergism with the TLR4 signalling cascade. DCderived AxnA1 appears to contribute in promoting T cell activation with a larger influence on OT-I/CD8+ T cells than OT-II/CD4+ T cells. Altogether these findings suggest that inhibition of Anx-A1 expression or function in dendritic cells might represent a useful way to modulate the adaptive immune response and pathogen-induced T cell-driven immune diseases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Medicine ; Biochemical Pharmacology