Investigations into the role of endogenous annexin-A1 in dendritic cell biology

A school of literature has shown that Annexin-Al (Anx-Al) 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-Al 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-Al in innate and adaptive immunity by investigating the role of endogenous Annexin-Al 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 marrow- derived dendritic cells from AnxAl-deficient mice or control littermates and stimulated with LPS (lOOng/ml) then compared phenotypic and functional characteristics. My results demonstrate that Anx-Al-/- bone marrow derived dendritic 4 cells show an increased number of CD11c+ cells expressing high levels of some maturation markers such as CD40, CD54 and CDSO 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)- 1~, Tumour Necrosis Factor (TNF)-a and IL-12. This defect was resultant of an impaired Nuclear Factor (NF)-KB/DNA- binding 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 antigen- presenting/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 antigen- specific, T-cell restricted model; a eo-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, OT- I/CDS+) or OT-P/ RAG-1-/- (OT-Il, OT-Il/CD4+) mice. Peptide-pulsed, LPS-matured AnxA1-/- DC failed to initiate an appropriate T cell activation in both OT - I and OT - Il T cells indicated by reduced cell proliferation when compared to T cells eo-cultured with peptide- 5 pulsed, LPS-matured AnxAl +/+ DC. Additionally, comparison of peptide-pulsed, LPS-matured AnxAl-/- DC with AnxAl +/+ DC counterparts detected severely diminished levels of IL-2 from eo- cultures with OT - I T cells and ablated IFN-y production from eo- cultures with both OT-I and OT-II T cells. In conclusion, AnxAl seems to act as a positive modulator of immunogenic activation of DC, whereby the AnxAl signal pathway has a probable synergism with the TLR4 signalling cascade. DC- derived AxnAl appears to contribute in promoting T cell activation with a larger influence on OT-I/CDS+ T cells than OT-IIjCD4+ T cells. Altogether these findings suggest that inhibition of Anx-Al 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.EThOS - Electronic Theses Online ServiceGBUnited Kingdo