IL-6 Amplifies TLR Mediated Cytokine and Chemokine Production: Implications for the Pathogenesis of Rheumatic Inflammatory Diseases

<div><p>The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.</p></div

Exposure to IL-6 enhances cytokine and chemokine production in SFMC.

<p>SFMC were left to adhere on plastic for 3 hours in DMEM supplemented with 10% fetal calf serum (FCS). SFMC were pre-exposed to IL-6/sIL-6R in combination with IgG1 or with TCZ for 1 hour. Cells were then stimulated for 18 hours with LPS (10 ng/ml) (<b>A</b>), and S100A8 (5 µg/ml) (<b>B</b>). IL-1β, CXCL8 and CCL2 levels were measured by ELISA. *p<0.05 for values from IL-6/sIL-6R -stimulated compared with NT cells.</p

Western blots showing the expression of phospho–STAT3 (Tyr⁷⁰⁵), phospho–NF-kB (Ser⁵³⁶) in total lysates of RA synoviocytes pretreated as in Figure 7B for 1 hour and then stimulated with IL-1β (1 ng/ml) or LPS (10 ng/ml) for 90 minutes.

<p>Expression of α-tubulin was used as a loading control. Results of densitometric analysis are shown above each blot. Data are representative of 3 independent experiments.</p

Increased p65 NF-κB activation in response to inflammatory stimuli in human PBMCs pre-exposed to IL-6/IL-6R.

<p>Western blots showing the expression of phospho–p65 NF-kB (Ser⁵³⁶) in total lysates from human PBMCs pre-exposed to IL-6 (10 ng/ml) in combination with sIL-6R (125 ng/ml) for 1 hour and stimulated with LPS, poly(I-C), MDP, CpG, IL-1β, PAM (used at the same concentrations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107886#pone-0107886-g002" target="_blank">Figure 2</a>) for 90 minutes. Expression of α-tubulin was used as a loading control. Results of densitometric analysis are shown above each blot. Data are representative of 3 independent experiments.</p

Exposure to IL-6 affects the production of inflammatory cytokines and chemokines in human PBMCs in response to TLR ligands.

<p>Human PBMCs were pre-exposed to IL-6/sIL-6R for 1 hour. Cells were then stimulated with poly(I-C) (20 µg/ml), CpG (5 µg/ml), PAM (200 ng/ml), MDP (10 µg/ml), LPS (10 ng/ml), S100A8 (5 µg/ml) for 18 hours. IL-1β (<b>A</b>) and CXCL8 (<b>B</b>) levels were measured by ELISA. *p<0.05 for values from IL-6/sIL-6R-stimulated compared with NT cells.</p

Exposure to IL-6 affects the production of inflammatory cytokines and chemokines in human PBMCs in response to IL-1β.

<p>Human PBMCs were pre-exposed to IL-6/sIL-6R for 1 hour. Cells were then stimulated with IL-1β (1 ng/ml) for 18 hours. CXCL8 (<b>A</b>), CCL2 (<b>B</b>), and IL-6 (<b>C</b>) levels were measured by ELISA. In (<b>C</b>), the levels of exogenous IL-6 were subtracted after the ELISA measurement. *p<0.05 for values from IL-6/sIL-6R-stimulated compared with NT cells.</p

TAK1 inhibition limits the expression of mesenchymal markers by MCs derived from peritoneal effluent of PD patients.

<p><b>A</b>, Confocal immunofluorescence (red) of MCs from a patient undergoing PD. Cells were treated with DMSO or NP-009245 (600 nM) for 72 h. Cells were fixed and stained with a monoclonal antibody against fibronectin. Nuclei were stained with Hoechst 33342 (blue). The immunofluorescence shown is representative of three independent experiments. <b>B</b>, Western blots showing the expression of fibronectin, E-cadherin and vimentin in total cell lysates of MCs treated with DMSO, NP-009245 (600 nM) or CI-1040 (2 µM) for 72 h. Expression of tubulin was used as a loading control. The results shown are representative of three independent experiments. <b>C</b>, Effect of TAK1 pharmacological inhibition on fibronectin and E-cadherin mRNA expression in MCs from patients undergoing PD. Quantitative RT-PCR was performed on total RNA from MCs treated as in A. Histone H3 mRNA levels were used for normalization. Bars represent means+s. e. m. of duplicate determinations in three independent experiments. <b>D</b>, Western blots showing the expression of E-cadherin, fibronectin and TAK1 in total cell lysates of MCs transfected with either control or specific TAK1-targeting siRNAs. Scr, cells transfected with control siRNA. Data are representative of three independent experiments. <b>E</b>, Effect of TAK1 siRNA silencing on E-cadherin mRNA expression in MCs. Cells were treated as in D. Quantitative RT-PCR was performed; bars represent means+s. e. m. of duplicate determinations in three independent experiments. * p<0.05 versus siScrambled treated cells.</p

TAK1 as a checkpoint of major signalling pathways controlling EMT and fibrosis.

<p>Rapidly induced by a wide array of pro-inflammatory and pro-fibrotic stimuli, TAK1 activation plays a central role in the induction of the EMT program. Besides controlling classical inflammatory pathways, such as NF-κB and MAPKs, TAK1 activation affects Smad3/Smad1–5 balance. TAK1 activation in MCs relies on cadherin switching, PAI-1 expression, increased ECM protein production, as well as the acquisition of invasive abilities. See text for details.</p

TAK1 inhibition limits the induction of EMT in HPMCs treated with TGF-β1 in combination with IL-1β.

<p>Effect of TAK1 pharmacological inhibition on E-cadherin (<b>A, C</b>), Snail (<b>B, D</b>), fibronectin (<b>E</b>) and type I collagen (<b>F</b>) mRNA expression in HPMCs. Quantitative RT-PCR was performed on total RNA from MCs pre-treated with DMSO, or NP-009245, (600 nM) for 1 h and then left untreated or stimulated with TGF-β1 0.5 ng/ml) in combination with IL-1β 2 ng/ml) (T/I) or TGF-β1 alone at the same concentration (T) for 24 h. Histone H3 mRNA levels were used for normalization. Bars represent means+s. e. m. of duplicate determinations in three independent experiments. * p<0.05 versus DMSO treated cells.</p

Exposure to IL-6 enhances cytokine and chemokine production in SFMC.

<p>SFMC were left to adhere on plastic for 3 hours in DMEM supplemented with 10% fetal calf serum (FCS). SFMC were pre-exposed to IL-6/sIL-6R in combination with IgG1 or with TCZ (50 µg/ml) for 1 hour. Cells were then left untreated (<b>A</b>) or stimulated with poly(I-C) and MDP (<b>B</b>), IL-1β (1ng/ml) (<b>C</b>). CXCL8, CCL2 and IL-1β levels were measured by ELISA. *p<0.05 for values from IL-6/sIL-6R-stimulated compared with NT cells.</p