AbstractMacrophages are an important part of the innate immune response. They are capable of sensing pathogens via pattern recognition receptors (PRRs) and then initiating an appropriate inflammatory response. Dysregulation of the inflammatory response can result in chronic inflammation and tissue damage. This thesis aimed to investigate signaling pathways regulating production of cytokines and chemokines in response to PRR agonists.
Interleukin (IL)-10 is a potent anti-inflammatory cytokine which suppresses the production of pro-inflammatory cytokines such as TNFa, IL-6 and IL-12. In this thesis, a requirement for an interferon (IFN)b-mediated feedback loop was identified for sustained transcription of IL-10 in response to lipopolysaccharide (LPS), a Toll-like receptor (TLR)4 agonist. Ruxolitinib treatment of macrophages led to an increase in pro-inflammatory cytokine production in response to LPS. Another JAK inhibitor, Tofacitinib, also blocked the sustained transcription of IL-10 and led to elevated secretion of pro-inflammatory cytokines in response to LPS.
The IFNb-mediated feedback loop also regulated the transcription of another mediator in the inflammatory response, monocyte chemotactic protein-1 (MCP-1). Blocking a type I IFN-mediated feedback loop prevented maximal transcription and secretion of MCP-1 in response to LPS or poly(I:C). IFNb directly activated MCP-1 transcription and was shown to promote binding of STAT1 to a STAT binding site in the MCP-1 promoter.
Numerous transcription factors such as CREB, Sp1 and NFkB play a role in regulating cytokine production in response to LPS. Other transcription factors may be important in mediating TLR signaling. MEF2D was identified as being phosphorylated at Ser121 in response to LPS. Interestingly, phosphorylation of STAT3 was increased in MEF2D- deficient macrophages compared to wildtype macrophages. MEF2D KO macrophages have elevated levels of IL-10 mRNA and protein. Elevated secretion of IL-10 resulted in decreased levels of pro-inflammatory cytokines TNFa, IL-6 and IL-12. IL-10 mRNA levels were increased in response to a range of TLR agonists. Expression of the regulatory macrophage markers (Arg1, LIGHT and SphK1) was enhanced in MEF2D-deficient cells.
Regulatory macrophages are characterised by high expression of IL-10 amongst other markers. Interestingly, Zymosan stimulation of macrophages leads to a regulatory-like phenotype. Expression of SphK1 mRNA is strongly induced by Zymosan but not by LPS. Three SphK inhibitors were used to investigate the role of SphK1 in the Zymosan-stimulated macrophages. Interestingly, SphK inhibitors blocked the transcription of IL-10 in response to Zymosan. Treatment of Zymosan-stimulated macrophages with SphK inhibitors also reduced phosphorylation of Akt and STAT3. LPS-stimulated macrophages had lower IL-10 secretion and reduced activation of Akt and STAT3 when treated with a SphK inhibitor. Addition of exogenous DHS1P or sphingosine 1-phosphate (S1P) did not restore phosphorylation of STAT3 in the presence of a SphK inhibitor. This suggests the SphK inhibitors had off-target effects that reduced IL-10 transcription.
The work presented in this thesis demonstrates novel pathways that regulate the transcription of the important mediators, IL-10 and MCP-1.
|Date of Award||2014|
|Supervisor||Simon Arthur (Supervisor)|