AbstractIn the healthy individual fungal infections are relatively benign, however in the rapidly increasing population of immunosuppressed patients fungal infections have become an increasing cause of morbidity and mortality. In the response to fungal pathogens, the innate immune system recognises a series of specific PAMPs via the Dectin-1 and TLR2 receptors, ultimately resulting in pro and anti-inflammatory cytokine production.
Following Dectin-1 activation, I show that blocking SYK activity with SYK inhibitor II
prevents MAPK and NF?B signalling, causing a reduction in both pro and antiinflammatory cytokine production. However, the clinically used inhibitor R406
(fostamatinib), which has been described as a SYK inhibitor, does not block these
signalling pathways downstream of Dectin-1 activation, but is able to abolish cytokine production. As R406 has these effects in response to not only Dectin-1 ligand stimulation, but also TLR2 and TLR4 stimulation, it is likely that these events are the result of an off target effect of R406, and not a result of SYK inhibition. In line with this, R406 was found to inhibit multiple kinases in an in vitro kinase screening panel.
To investigate signalling further downstream of Dectin-1, I attempted to elicit the kinase responsible for ERK1/2 activation. For most stimuli, Raf-1 activates MEK1/2 which activates ERK1/2, however in response to TLR signalling the kinase Tpl2 is required to activate MEK1/2. I show that the kinase responsible for ERK1/2 activation downstream of Dectin-1 is not Tpl2, but is an unidentified off target effect of the Tpl2 small molecule inhibitor SHN681.
MSK1 and 2 have previously been shown to be important in limiting inflammatory
cytokine production by macrophages in response to the TLR4 agonist LPS. This is in large part due to the ability of MSKs to regulate the production of the anti-inflammatory cytokine IL-10. In this thesis I show that MSKs are activated in macrophages by fungal ligands, including the Dectin-1 specific agonists curdlan and depleted zymosan, via the ERK1/2 and p38a MAPK pathways. Further, I show that although MSKs regulate Dectin-1 induced IL-10 transcription, this does not significantly affect pro-inflammatory cytokine production. This is in direct contrast to the inhibition of these pro-inflammatory cytokine that we see post LPS stimulation. Investigating further, I show that although IL-10 secreted in response to zymosan is unable to suppress pro-inflammatory cytokine production, it is still able to promote STAT3 phosphorylation. I suggest that Gfi1 is involved as I show that LPS can induce high levels of Gfi1 expression, whereas zymosan does not induce Gfi1. One possible explanation would be that without Gfi1 to inhibit
PIAS3, PIAS3 is binding the activated STAT3 and not allowing it to bind to DNA. This would result in STAT3 being unable to function, and hence no repression of proinflammatory cytokine expression would occur, regardless of the level of IL-10 present.
Finally, I show that activation of Dectin-1 in a SYK dependent fashion resulted in
macrophage switching to a regulatory macrophage phenotype. As regulatory
macrophages are thought of as essentially anti-inflammatory, this may help explain why many people suffer commensal fungal infections that can persist for long periods of time without developing the classical inflammatory signs of infection.
|Date of Award
|Simon Arthur (Supervisor)