Toll-like receptor-mediated, enhanced production of profibrotic TIMP-1 in monocytes from patients with systemic sclerosis: role of serum factors

Abstract

Objectives: To investigate whether monocytes contribute to matrix deposition in systemic sclerosis (SSc) by production of tissue-inhibitor of metalloproteinase-1 (TIMP-1).

Methods: Matrix metalloproteinase-1 (MMP-1) and TIMP-1 expression and secretion were measured by qRT-PCR and ELISA in circulating monocytes from patients with SSc, patients with rheumatoid arthritis (RA) and healthy controls (HC) and in healthy monocytes cultured in the presence of SSc or HC serum samples. Production of TIMP-1 was determined in response to a panel of Toll-like receptor (TLR) agonists and MyD88 inhibitory peptide. The functional effect of conditioned media from SSc and HC serum samples or TLR8-stimulated monocytes was studied in an MMP-1 activity assay.

Results: TIMP-1 production by monocytes was upregulated in patients with SSc compared with patients with RA and HC. Incubation of HC monocytes with SSc serum samples resulted in functionally active TIMP-1 production. However, pretreatment with MyD88 inhibitor, but not control peptide, decreased TIMP-1 secretion. TIMP-1 production was significantly stronger when SSc and HC monocytes were stimulated with TLR8 (ssRNA) agonist, but the response was more pronounced in SSc monocytes. TIMP-1 production after TLR stimulation was also strongly reduced in the presence of MyD88 inhibitory peptide or in the monocytes isolated from a patient with a genetic TLR signalling defect. MMP-1 activity was significantly inhibited in media from serum samples or TLR8-stimulated monocytes indicative of functional TIMP activity.

Conclusions: This study demonstrates profibrotic properties of circulating monocytes from patients with SSc and a key role for TLR signalling, particularly TLR8, in TIMP-1 secretion and matrix remodelling.

Figure 1

Tissue inhibitor of metalloproteinase-1 (TIMP-1) in serum samples and circulating monocytes. (A) TIMP-1 protein concentration was measured in serum samples of healthy controls (HC) (n=14) or patients with systemic sclerosis (SSc) (n=19). (B) Relative TIMP-1 expression in freshly isolated circulating CD14 monocytes from HC (n=11) or patients with SSc (n=14) was obtained. Relative expression levels of TIMP-1 were normalised to the 18S housekeeping gene. Each value represents the mean; *p<0.05, **p<0.01. Paraffin-embedded SSc skin section. (C) The skin section was stained with CD14 and TIMP-1 primary and secondary antibodies (Abs; Alexa-Fluor488, Alexa-Fluor546, respectively) and further analysed by confocal microscopy (TCS SP2 UV LSM Leica Microsystems) using ×20 (top side of the image represents the dermis and bottom the epidermis) and ×40 magnification. White arrows indicate colocalisation between CD14 and TIMP-1.

Figure 2

The effect of serum samples on the tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-1 (MMP-1) induction. (A) Healthy control (HC) monocytes were treated for 4 h with serum samples from HC (n=11), patients with rheumatoid arthritis (RA) (n=11) or patients with systemic sclerosis (SSc) defined by autoantibody positivity—anticentromere antibodies (n=6), Scl-70 (n=7), antinuclear antibodies (n=9)—and TIMP-1 gene expression was measured. (B) MMP-1 gene expression in HC monocytes cultured in SSc or HC serum was also analysed. Expression levels of TIMP-1 and MMP-1 were normalised to the 18S housekeeping gene and each dot represents the mean. (C) HC monocytes were stimulated with HC, rheumatoid arthritis (RA) or SSc serum samples and TIMP-1 secretion was measured 24 h later. (D) HC monocytes were pretreated or not with MyD88 inhibitory peptide for 24 h and further stimulated over 4 h either with 10% HC or SSc serum samples. De novo TIMP-1 secretion by HC monocytes was measured 24 h later. Results are representative of mean±SEM of HC (n=11) or SSc (n=13) serum samples. (E) HC monocytes were pretreated with human IgG for 1 h before stimulation with HC or SSc serum samples (IgG block); HC or SSc serum samples were incubated with RNA/DNA endonuclease (benzonase) before sera stimulation; or IRAK4−/− monocytes were treated with HC and SSc serum samples and TIMP-1 secretion was measured. Statistical analysis showed that monocytes treated with SSc sera were comparable with monocytes of IgG blocked sera or monocytes stimulated with SSc sera treated with benzonase and *p<0.05, **p<0.01, ***p<0.001. Data shown in A–E represent separate experiments. IRAK, IL-1R-associated kinase.

Figure 3

Kinetic analysis of the effect of serum samples on tissue inhibitor of metalloproteinase-1 (TIMP-1) induction. (A, B) healthy control (HC) monocytes were pretreated with MyD88 inhibitory peptide (white columns), control peptide (grey columns) or media only (black columns) for 24 h and further stimulated over 4 h either with 10% HC or systemic sclerosis (SSc) serum samples. De novo TIMP-1 secretion by HC monocytes was measured directly after stimulation with sera (T0), 4 h (T1), 24 h (T2) or 48 h (T3) later. Results are representative of mean±SEM of two independent experiments for monocytes treated with HC or SSc serum samples.

Figure 4

Functional matrix assay of monocytes stimulated with serum samples. Preactivated matrix metalloproteinases-1 (MMP-1) was added to the conditioned media of healthy controls (triangle) or systemic sclerosis (SSc) (circle) sera-treated or unstimulated monocytes (square) and fluorescent FS-6 breakdown product was measured over the time (min)]. Data shown represent the mean from duplicate values of one of three independent experiments (*p<0.05 for comparison of the values of MMP-1 media only with MMP-1 SSc sera medium).

Figure 5

The effect of Toll-like receptor (TLR) agonists on tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression and protein secretion in monocytes. (A) Monocytes from healthy controls (HC) (n=10) (white bars) or patients with SSc (n=10) (black bars) and monocytes isolated from the mut-IRAK4 patient (grey bars) were incubated in the presence of different TLR agonists or media alone for 24 h, and TIMP-1 secretion was determined. Statistical analysis showed that HC monocytes compared with SSc monocytes after individual TLR stimulation. (B) Relative TIMP-1 gene expression was determined and normalised to 18S housekeeping gene of total RNA isolated from HC (n=3) or SSc (n=4) or mut-IRAK4 (grey bars) monocytes after 24 h stimulation with TLR8 agonist. (C) The effect of MyD88 inhibitor on TLR8-mediated TIMP-1 production. HC monocytes (n=4) were pretreated for 24 h with 10, 20, 50, 100 μM MyD88 inhibitory peptide (white bars) or control peptide (grey bars) or media alone (black bars) and further stimulated with TLR8 agonist (ssRNA). Twenty-four hours later TIMP-1 production was measured. Statistical analysis showed a comparison of TLR8-stimulated monocytes compared with MyD88 inhibitor or control peptide treated monocytes, respectively. Each bar represents the mean±SEM, *p<0.05, **p<0.01, ***p<0.001. IRAK, IL-1R-associated kinase.

Figure 6

Functional matrix assay of Toll-like receptor 8 (TLR8)-stimulated monocytes. (A, B) Preactivated matrix metalloproteinases-1 (MMP-1) was added to the conditioned media of untreated (black line) or TLR8-simulated (grey line) healthy control (HC) or systemic sclerosis (SSc) monocytes and fluorescent FS-6 breakdown product was measured over time (min). (C) Statistical analysis of three independent experiments showing MMP-1 inhibition by HC and SSc monocytes pretreated with TLR8 agonist.