Download Investigating the role of CD14 in apoptotic cell clearance in the lungs

Investigating the role of CD14 in apoptotic cell
clearance in the lungs
Dying (apoptotic) cells are common in vivo and, when they occur, are removed
efficiently to prevent undesired inflammatory consequences. Whilst so-called
professional phagocytes (e.g. macrophages) mediate much clearance, there is
increasing evidence that other cells (e.g. epithelial cells) can remove apoptotic cells.
This project aims to investigate apoptotic cell clearance by airways epithelial cells and
the recently discovered novel function for CD14 in these cells. Through this work we
aim to address the following questions:
How does CD14 function for apoptotic cell clearance in differentiated
epithelial cells in culture?
How do airways epithelial cells respond to apoptotic cells?
Do airways epithelial cells from diseased airways respond differently to
apoptotic cells? If so, how? In these latter two questions we will directly
address important inflammatory lung disorders.
The airways epithelial cells act as an impermeable barrier and are the first line of
defence against inhaled particles or pathogens. This puts the airways at the forefront
of innate immune responses that use pattern recognition receptors to identify
“threats”. CD14 is the prototypic pattern recognition receptor and has both pro- and
anti-inflammatory roles. It is well established that macrophage expression of CD14 is
vital for apoptotic cell clearance and that this apoptotic cell clearance is required to
resolve inflammation and to maintain homeostasis. CD14 expression was thought to
be limited to myeloid cells (macrophages, neutrophils, dendritic cells); however, our
recent work showed that airways epithelial cells also express CD14 [Thomas et al.,
PLoS One, 2013]. Importantly, we showed that epithelial CD14 is functional and
mediates apoptotic cell clearance by the epithelial cells.
Other research has shown the importance of apoptotic cell clearance from the airways
and the role that this plays in human health. For example, reduced apoptotic cell
clearance in the airways is observed in inflammatory lung conditions such as asthma
and the smoking-related lung condition- emphysema, and experiments using animal
models have shown that increasing the burden of dead cells in the airways can lead to
pathological changes typical of emphysema. These observations suggest that
alteration of CD14 expression by the airways epithelial cells will compromise dead
cell clearance and modify inflammatory responses.
This is an entirely novel role for CD14 in the airways and one that will be explored
further in this PhD. The project will use in vitro models of the airways developed in
our laboratories to examine the expression profile of CD14. Initially, we will use
confocal microscopy and flow cytometry to characterise CD14 expression by various
airways epithelial cell lines currently used in our lab. These cell lines are cultured at
the air-liquid interface in order to promote cellular differentiation to a physiological
barrier. We will profile the pro- and anti-inflammatory roles for CD14 in our airways
epithelial cell models by examining responses to pathogens and to apoptotic cells,
using our established methods. We will move on to examine CD14 expression and
function in inflammatory conditions of the airways, using primary cells, clinical
material and by developing our own smoke-exposed airways using the automatic
smoking machine that we have designed and developed.
Overall, this project will allow us to define the contribution of CD14 to lung
inflammation, to explore the function of CD14 in clearance of apoptotic cells in the
airways and ultimately will allow us to design therapeutic interventions for
inflammatory lung conditions.