Investigations into the Role of Endogenous Annexin-A1 in Dendritic Cell Biology
Abstract
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.
Authors
Huggins, AnthonyCollections
- Theses [3822]