A2.20 The Role of Serum Factors and Toll-Like Receptor Signaling in the Induction of Profibrotic TIMP-1 by Monocytes in Systemic Sclerosis

Background and ObjectivesSystemic sclerosis (SSc) is an autoimmune disease characterised by fibrosis, vascular dysfunction and abnormal activation of immune cells including monocytes. Monocytes along with fibroblasts play an important role in the production of profibrotic factors such as IL-6 and TIMP-1 (tissue-inhibitor of metalloproteinase-1). TIMPs are specific inhibitors of matrix metalloproteinases (MMPs) regulating extracellular matrix (ECM) turnover. Importantly, the balance between TIMPs and MMPs is altered in most pathological stages including SSc and is associated with abnormal ECM formation. However, the exact factors which drive both profibrotic TIMP-1 secretion are not fully defined. We aim to test whether circulating monocytes from SSc patients produce TIMP-1 in response to TLR activation and serum factors, which contributes to excessive matrix deposition and consequently disease progression.Materials and Methods25 patients with SSc, one IRAK4 deficient patient and 20 HC (healthy control) were included in this study. Peripheral blood monocytes were further separated by CD14+ microbeads. Production of TIMP-1, IL-6 by monocytes was determined by ELISA, qRT-PCR or functional assay in response to either panel of conventional TLR agonists or HC and SSc sera. Skin section from SSc patient was stained with CD14+ and TIMP-1 antibodies and further analysed by confocal microscopy.ResultsTIMP-1 production by monocytes was observed in the SSc skin section and was upregulated in SSc patients compared to HC. Incubation of HC monocytes with SSc sera resulted in functionally active TIMP-1 production. However, pre-treatment with MyD88 inhibitor, but not control peptide, decreased TIMP-1 secretion. Furthermore, SSc-mediated TIMP-1 induction by monocytes was attenuated when FcγR was blocked and also when SSc sera were treated with DNA/RNA endonuclease prior to stimulation. This indicates that SSc sera contain RNA/DNA agonists inducing TIMP-1 production. Indeed, direct treatment of HC and SSc monocytes with a panel of TLR ligands demonstrated strong TIMP-1 and IL-6 production following triggering with TLR8 agonists (ssRNA). TLR8-mediated TIMP-1 production was reduced in monocytes from a patient with a genetic TLR signalling defect or HC monocytes cultured with MyD88 inhibitory peptide. Furthermore, matrix assay of TLR8 stimulated monocytes also confirmed functional TIMP-1 secretion, as matrix metalloproteinase-1 activity was significantly inhibited.ConclusionsThis study indicates a potential link between SSc serum factors and TLR signalling resulting in excessive TIMP-1 secretion by circulating monocytes from SSc patients.