Long-lasting activation of inflammation leads to chronic conditions and is particularly
common in autoimmune diseases. The causes of this self-activation in those
conditions are still unknown, but these diseases are often also associated with an
increase in oxidative stress. In fact, reactive oxygen species (ROS) are released
during the inflammatory response and can cause oxidative damage, which in turn can
lead to maintenance of inflammation. However, ROS are not only toxic species but
can also act as signalling molecules to regulate immune responses, for instance via
thiol modification. Thiols present in the cysteine residues of protein are among the
most sensitive targets of ROS. They can undergo many redox changes, including
glutathionylation or disulphide-linked dimerisation, all of which alter the protein and
thus its function, localisation and secretion. This “redox regulation” regulates many
cellular processes such as apoptosis, cell development and differentiation,
homoeostasis and the immune response.
In this project, we hypothesise that changes in thiol oxidation affect the inflammatory
response and two different approaches have been set up to track redox changes in
Firstly, the role of endogenous glutathione (GSH), the main thiol antioxidant, was
investigated. For this purpose, we used RAW cells, a mouse macrophage cell line.
Cells were depleted of endogenous GSH and then stimulated with a standard
inflammatory stimulus, bacterial lipopolysaccharide (LPS). A microarray analysis was
then performed to identify changes in the gene expression profile. Results indicated
that endogenous GSH does not decrease the inflammatory response but, on the
contrary, favours the host antiviral response as its depletion results in an impaired
LPS-induced increase in gene expression of genes in the interferon pathway,
including oas2, mx2 and irf9. The biological significance of these results was later
confirmed in cells infected with influenza A, showing that the antiviral response elicited
by LPS was inhibited by GSH depletion.
The second approach of this work was the use of a pegylated maleimide (MalPEG –
10kDa) to determine the redox state of three “redoxkines”, protein thiol/disulphide
oxidoreductases with inflammatory properties: Trx, Prx1 and Prx2. MalPEG covalently
binds to free thiols causing a mobility shift that can be detected by Western blot,
leading to differences in the migration of oxidised and reduced proteins. After LPS
stimulation, clear changes in the redox state were detected both intracellularly and in
secreted proteins. To identify potential membrane targets of redoxkines, we set up a
technique to identify proteins with redox-sensitive exofacial thiols on the cell surface.
The results of this work show that activation of inflammatory pathway in macrophages
brings about a number of redox changes in protein thiols, some of which may be
related to GSH signalling, which are important in the regulation of both inflammation
and host defence.
|Date of Award