THU0088 Monocyte downregulation of mitochondrial translocator protein may be a contributory mechanism to inflammation in ra
Authors: N. Narayan, H. Mandhair, T. Sekine, A. Sabokbar, P.C. Taylor
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> The translocator protein is an 18 kDa mitochondrial transporter, increasingly thought to play a critical role in cholesterol efflux in macrophages. Recent work demonstrates that macrophages engineered to over-express TSPO, exhibit increased cholesterol efflux, and reduced ability to form a pro-inflammatory ('M1') phenotype, with significant reduction in the ability to produce TNF-α. Additionally, there is growing data to demonstrate a difference in TSPO expression in monocytes in those with inflammatory disease compared with healthy, as exemplified by studies of multiple sclerosis, suggesting a role for TSPO in the generation of inflammation. <h3>Objectives</h3> In this study, we investigate the expression of TSPO in healthy and RA peripheral blood monocytes, and in monocyte derived macrophages (MDM), differentiated to an M1 (pro-inflammatory), and M2 (reparative) phenotype. <h3>Methods</h3> Using positive magnetic-activated cell sorting, we use peripheral blood mononuclear cells from 24 RA patients with active disease (as determined by clinical examination, and DAS28 CRP score), and 24 healthy controls, to isolate peripheral blood monocyte mRNA and protein, to ascertain any differences in TSPO expression at monocyte level. MDM were generated <i>in vitro</i> through differentiation of monocytes with 100 ng/ml M-CSF for 7 days, followed by activation to an M1 phenotype using LPS and IFN-γ, and a reparative 'M2' phenotype using IL-4, TGF-β or glucocorticoid, followed by quantification of TSPO mRNA utilising real-time PCR, and TSPO protein, utilising western blotting and radioligand binding. <h3>Results</h3> Our data establishes that both healthy and RA peripheral blood monocyte derived macrophages (MDM) exhibit a statistically significant downregulation of TSPO at mRNA and protein level, when activated to a pro-inflammatory 'M1' macrophage phenotype, with no change in TSPO expression in MDM activated to a reparative 'M2' phenotype. Our mRNA data also suggests that M1 macrophages from both healthy and RA donors, exhibit a significant reduction in expression of key cell components promoting cholesterol efflux in macrophages, including CYP27A1, and ABCA1. Our data additionally demonstrates a significant reduction in expression of TSPO between healthy and RA monocytes, at both mRNA and protein level (mean fold change <i>TSPO</i> mRNA of 1.00 for healthy monocytes, and 0.47±0.24, p&lt;0.001 for RA monocytes and mean TSPO optical densitometry of 1.01±0.10 for healthy monocytes and 0.85±0.02 p&lt;0.05 for RA monocytes relative to β-actin). <h3>Conclusions</h3> Our findings indicate that pro-inflammatory activation of both healthy and RA monocyte-derived macrophages downregulates TSPO, and is also associated with reduction in key components of the cholesterol efflux pathway, in line with pre-existing studies of TSPO silencing and over-expression in human macrophages. Furthermore, we demonstrate that RA peripheral blood monocytes themselves may have a predisposition to a pro-inflammatory phenotype through downregulation of TSPO expression, which could indicate an as yet uninvestigated cellular mechanism contributing to synovial inflammation in RA. <h3>References</h3> [1] Taylor, et al. Targeting mitochondrial 18 kDa translocator protein (TSPO) regulates macrophage cholesterol efflux and lipid phenotype. Clin Sci2014. [2] Harberts, et al. Translocator protein 18 kDa (TSPO) expression in multiple sclerosis patients. J Neuroimmune Pharmacol2013. <h3>Disclosure of Interest</h3> None declared