There is growing interest in the tissue-protective effects of some cytokines, including
erythropoietin (EPO) and the IL-6 family cytokine leukaemia inhibitory factor (LIF);
both have receptors, and exert their effects, on cells other than their primary targets.
In the nervous system, these cytokines could aid in the treatment of demyelinating
diseases, such as multiple sclerosis, by protecting myelin from damage and
supporting remyelination after damage has occurred. Previous work has shown that
EPO increases myelination in oligodendrocytes, the cells responsible for myelin
deposition in the central nervous system. I aimed to determine if LIF shares the
promyelinating effects of EPO and understand more about the mechanisms
mediating tissue-protective cytokine-induced myelination.
A model of rat oligodendrocyte precursor cells was used and their myelinating
capacity was measured as represented by myelin oligodendrocyte glycoprotein
(Mog) expression. Initially I studied EPO and LIF’s effects on these cells before
defining the molecular mechanisms causing their effects using microarray gene
EPO increased myelination by eight-fold, a level that was sustained at concentrations
up to and including 400ng/ml. After treatment with LIF at 0.2ng/ml Mog expression
was increased by two-fold, but concentrations above 2ng/ml caused a reduced
expression of Mog. Interestingly, when LIF and EPO were added simultaneously
there was a significant reduction in EPO-induced Mog expression suggesting that
LIF induced an inhibitory feedback that was responsible for blocking not only its
own, but also EPO’s effect. The inhibitory feedback was replicated when LIF was
replaced by ciliary neurotrophic factor (CNTF) and oncostatin M (OSM),
glycoprotein 130 (GP130) cytokines that use the same receptor as LIF.
The signalling mechanisms that may have caused the inhibition of EPO-induced
Mog were then investigated. Socs3, a known inhibitory feedback of LIF and other
IL-6 cytokines, negatively correlated with Mog expression, as the higher
concentration of LIF and the simultaneous EPO and LIF treatment induced the
greatest Socs3 expression.
Gene expression microarray analysis was performed to elucidate further mechanisms
that may cause the inhibition of EPO-induced Mog. A variety of candidate genes
were identified and their expression validated by qPCR. The roles of Tlr2/Myd88
and lipocalin 2 were investigated further and Tlr2 activation showed a functional
effect on Mog expression.
The results showed that LIF and other GP130 cytokines inhibited EPO’s positive
effect on myelination and clarified some of the mechanisms that resulted in
inhibition. The implications of my work could be an increase in efficacy of EPO
treatment, as the work has elucidated mechanisms that could inhibit EPO’s promyelinating
effect. Increased efficacy of EPO would impact new therapies and
therapeutic approaches using tissue protective cytokines in regenerative medicine.
|Date of Award||Jul 2016|