X X CHIA1, D NIKOLIC-PATERSON1,2, G TESCH1,2
1Department of Nephrology, Monash Health, Clayton, Australia, 2Centre for Inflammatory Diseases, Monash University, Clayton, Australia
Aim: To investigate the role of spleen tyrosine kinase (SYK) in inflammatory cell signalling pathways in macrophages.
Background: SYK is expressed in B-lymphocytes and myeloid cells. Inhibition of SYK has been shown to prevent or reduce disease severity in animal models of inflammatory kidney disease, including LPS-induced acute kidney injury. This protection is associated with reduced expression of monocyte chemoattractant protein-1 (MCP-1), an important chemokine recruiting macrophages to sites of inflammation.
Methods: LPS stimulation assay was performed in cultured primary mouse bone marrow-derived macrophages (BMMac) and RAW264.7 murine macrophage cells. SYK inhibition was achieved using an SYK inhibitor (SYKi), GS-492429, and Sykf/f Csf1rCre mice with selective Syk gene knockout in myeloid cells. RNA expression of inflammatory markers was examined by RT-PCR and protein secretion over 24 hours into culture media was examined by ELISA.
Results: MCP-1 mRNA expression was significantly upregulated 6 hours after LPS stimulation, and this was reduced by 33% by SYKi in RAW264.7 cells (p<0.01). SYKi reduced LPS-induced MCP-1 secretion by 74% (p<0.01) in RAW264.7 cells over 24 hours. This finding was confirmed using BMMacs in which Syk knockout reduced LPS-induced MCP-1 secretion by 48% (p=0.01). Interestingly, SYKi treatment did not affect NFκB activation (IκBα degradation by Western blot) or up-regulation of TNF mRNA levels at 1 hr post-LPS. Finally, delaying SYKi addition until 1hr after LPS stimulation still inhibited MCP-1 secretion by 60% (p<0.01) in RAW264.7cells.
Conclusion: SYK inhibition reduces MCP-1 expression and release from macrophages after LPS stimulation. This appears to be independent of NFkB activation. These new finding highlight the potential role of SYK in inflammatory kidney disease.
Dr Xiu Xian Chia is a Nephrologist currently working towards a PhD at the Department of Nephrology, Monash Health. Dr Chia has a special interest in chronic kidney disease and is investigating the role of spleen tyrosine kinase signalling in models of acute kidney injury and chronic kidney disease.