Background The mechanistic target of rapamycin (mTOR) is known to couple energy and nutrient abundance to the execution of essential cellular processes, such as cellular growth and survival. As such, the unfolding biology of mTOR revealed a crucial role in metabolic diseases and ageing. Now, accumulating evidence suggests that mTOR may have an important role in the modulation of cellular responses to inflammatory cues, such as TNF. Whether or not this applies to rheumatoid fibroblast-like synoviocytes (FLS) remains to be determined.
Methods To assess mTOR activity in rheumatoid synovitis, immunohistochemistry (IHC) was performed using phosphospecific antibodies to mTOR. For functional in-vitro studies, the specific mTOR inhibitor Torin-1 was applied. The GeneChip® PrimeView array™ was used for gene expression profiling. Assessment of regulated genes was determined via SAM analysis. The expression of selected candidates was validated by Q-PCR. Finally, to further evaluate the significance of mTOR activity for the inflammatory mesencyhmal tissue response, a simplified 3-D model of the synovial tissue was used. IL-6 and IL-8 levels in the supernatants of 3-D cultures were measured by ELISA.
Results IHC revealed that mTOR is activated in RA synovitis, especially in fibroblast-like cells of the hyperplastic synovial lining layer. To determine the effect of mTOR activation for the TNF driven mesenchymal inflammatory response, isolated RA-FLS (n = 5) were exposed to TNF in the presence or the absence of Torin. TNF-stimulation resulted in the increased expression of 587 genes (fold induction>1,5; FDR<0,01). Surprisingly, 141 of those transcripts were further upregulated, when FLS were treated with TNF in the presence of Torin. Importantly, this group contained transcripts that are well known to be involved in RA pathogenesis, including IL-6, IL-8 and MMP1. Stimulation of the 3D synovial organ cultures with TNF resulted in hyperplasia of the lining layer at the surface of the spheres. Strikingly, Torin prevented TNF induced lining layer hyperplasia. In line with the gene expression studies, however, the combination of TNF with Torin resulted in increased production of IL-6 and IL-8, when compared to cultures that were solely exposed to TNF.
Conclusions These studies provide insight into the regulatory circuits that determine the synovial mesenchymal tissue response to inflammation and suggest a multifaceted regulatory role for the mTOR signalling circuit in rheumatoid synovitis.