Steven O'Reilly

Tumor necrosis factor–costimulated T lymphocytes from patients with systemic sclerosis trigger collagen production in fibroblasts

OBJECTIVE The role of tumor necrosis factor (TNF) in systemic sclerosis (SSc) remains controversial. The present study was undertaken to investigate the influence of TNF receptor (TNFR)-costimulated lymphocytes on collagen expression in fibroblasts. METHODS TNFR expression on mononuclear cells from the dermis and blood of SSc patients was assessed by flow cytometry. Peripheral blood CD3+ lymphocytes were activated with CD3/CD28 beads and costimulated with TNFR-selective variants. Expression of interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), IL-10, and IL-13 was detected by enzyme-linked immunosorbent assay or quantitative reverse transcription-polymerase chain reaction. Healthy fibroblasts were incubated with conditioned media from TNFR-costimulated T lymphocytes, and type I collagen expression was quantified. RESULTS TNFRI and TNFRII were up-regulated on dermal T lymphocytes from patients with diffuse cutaneous SSc. TNFRII expression correlated with skin thickening. After CD3/CD28 activation, peripheral blood lymphocytes from SSc patients produced more IL-6, sIL-6R, and IL-13 compared to healthy lymphocytes. Costimulation with TNFRI-selective ligands and soluble TNF further increased IL-6 expression, whereas costimulation with TNFRII led to greater release of sIL-6R. IL-10 expression, which normally occurs after TNFRII costimulation, was impaired in SSc T cells. Supernatants of TNF-costimulated SSc lymphocytes induced higher type I collagen expression in fibroblasts, which was partially reversible by dual inhibition of IL-6 and IL-13. Expression of TNFR and IL-6 in the dermis was reversible in a patient who received lymphoablative therapy prior to autologous hematopoietic stem cell transplantation. CONCLUSION TNF-costimulated T lymphocytes from SSc patients have a propensity to secrete profibrotic cytokines, while the ability to produce IL-10 is weakened. These results suggest that T lymphocytes in SSc support fibrosis, but might lack the capacity to resolve inflammation.

Serum amyloid A induces interleukin-6 in dermal fibroblasts via Toll-like receptor 2, interleukin-1 receptor-associated kinase 4 and nuclear factor-κB

Systemic sclerosis is an autoimmune idiopathic connective tissue disease, characterized by vasculopathy, inflammation and fibrosis. There appears to be a link between inflammation and fibrosis, although the exact nature of the relationship is unknown. Serum amyloid A (SAA) is an acute‐phase protein that is elevated up to 1000‐fold in times of infection or inflammation. This acute‐phase reactant, as well as being a marker of inflammation, may initiate signals in a cytokine‐like manner, possibly through toll‐like receptors (TLRs) promoting inflammation. This study addressed the role of SAA in initiating interleukin‐6 (IL‐6) production in dermal fibroblasts and the role of TLR2 in this system. We show that SAA induces IL‐6 secretion in healthy dermal fibroblasts and that blockade of TLR2 with a neutralizing antibody to TLR2 or specific small interfering RNA attenuated the SAA‐induced IL‐6 secretion and that this was also mediated through the TLR adaptor protein IL‐1 receptor‐associated kinase 4. The effect is nuclear factor‐κB‐mediated because blockade of nuclear factor‐κB reduced the induction. We also demonstrate that dermal fibroblasts express TLR2; this is functional and over‐expressed in the fibroblasts of patients with systemic sclerosis. Taken together these data suggest that SAA is a danger signal that initiates IL‐6 signalling in systemic sclerosis via enhanced TLR2 signalling.

Role of toll-like receptors in systemic sclerosis.

Accumulative evidence demonstrates the crucial role of evolutionary conserved Toll-like receptors (TLRs) in identifying microbial or viral compounds. TLRs are also able to recognise endogenous molecules which are released upon cell damage or stress and have been shown to play a key role in numerous autoimmune diseases including systemic sclerosis (SSc). A classic feature of SSc, is vascular injury manifested as Raynauds phenomenon and ischaemia of the skin, resulting in the release of endogenous TLR ligands during inflammation and local tissue damage. These locally released TLR ligands bind TLRs possibly complexed to autoantibodies, and initiate intracellular signalling pathways and may be one of the mechanisms that initiate and drive autoimmunity and subsequent fibrosis. Activation of the immune system results in interferon (IFN) sensitive gene transcription. There is also an IFN gene signature in SSc peripheral blood. TLRs may represent the link between immune activation, common in SSc, and tissue fibrosis. Therefore, a better understanding of the mechanisms of TLR-mediated pathogenesis and therapies targeting individual TLRs, may provide a more specific approach of treating multi-systemic autoimmune diseases. This review aims to integrate the current knowledge of TLR function in the autoimmune disorders with particular emphasis on SSc. We suggest the TLR system as a new therapeutic target.

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

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.

Interleukin-6: a new therapeutic target in systemic sclerosis?

Interleukin‐6 (IL‐6) is a classic pro‐inflammatory cytokine critical in mounting an effective immune response. It is secreted by a wide array of cell types; however, its effector cells are more restricted, owing to the fact that very few cells, except lymphocytes and hepatocytes, express the functional membrane IL‐6 receptor thus reducing the number of IL‐6‐responsive cells. Trans‐signalling, the shedding of the membrane‐bound form of the IL‐6 receptor into the local microenvironment, greatly increases the range of cells that can respond. IL‐6 has been demonstrated to have a pivotal role in the pathogenesis of rheumatoid arthritis, Castlemans disease and Crohns disease exemplified by the use of an anti‐IL‐6 biological therapy. However, IL‐6 is also associated with the autoimmune disease systemic sclerosis (SSc) and has been shown to be directly fibrotic. Elevated levels of IL‐6 are found in SSc patients and this correlates with skin thickness, suggesting a causal effect. This review focuses on the role of IL‐6 in SSc, a chronic autoimmune disease with fibrosis. In particular, we will examine the evidence base of the role of IL‐6 in fibrosis in this condition, especially the downstream effector pathways. We will then argue why molecular targeting of IL‐6 is a promising therapeutic target in this fibrosing disease.

Role of interleukin-13 in fibrosis, particularly systemic sclerosis.

Chronic inflammation can lead to altered extracellular matrix deposition and ultimately fibrosis. Interleukin‐13 (IL‐13) is a cytokine that was found to promote IgE class switching and inhibit proinflammatory cytokines. However, it is now recognized as an important mediator in allergy and most importantly fibrosis. Indeed, animal studies with genetically deleted mice have demonstrated its critical role in fibrosis and although it shares over lapping functions with IL‐4 it is the dominant cytokine in fibrosis. Systemic sclerosis is an autoimmune disease in which there is chronic inflammation and fibrosis. The disease is associated with a Th2 polarization and IL‐13 levels are elevated both in the blood and in the skin of patients. This review will examine the role of IL‐13 in driving fibrosis with a particular emphasis on systemic sclerosis as a prototypical fibrotic disease. It will highlight recent research into the role of IL‐13 and how this cytokine may be targeted in systemic sclerosis.

IL-13 mediates collagen deposition via STAT6 and microRNA-135b: a role for epigenetics

Systemic sclerosis is an autoimmune connective tissue disease in which T cells play a prominent role. We and others have previously demonstrated a role for T cell-derived IL-13 in mediating the induction of collagen in dermal fibroblasts and that blockade with IL-13 antibodies attenuates this increase. In this study we want to probe the signalling that underpins IL-13 mediated matrix deposition. Isolated dermal fibroblasts were incubated with recombinant IL-13 and gene expression by qRT-PCR was performed for collagen1A1 and TGF-β1. Small interfering RNA (siRNA) was used to knock down STAT6 and a small molecule inhibitor was also used to block this pathway. MiR-135b was transfected into fibroblasts plus and minus IL-13 to see if this miR plays a role. miR-135b was measured in systemic sclerosis fibroblasts isolated from patients and also in serum. Results showed that IL-13 increased collagen expression and that this is independent from TGF-β1. This is dependent on STAT6 as targeting this blocked induction. MiR-135b reduces collagen induction in fibroblasts and scleroderma fibroblasts have lower constitutive levels of the miR. We further demonstrate that miR135b is repressed by methylation and may include MeCP2. In conclusion we show that STAT6 and miR-135b regulate IL-13-mediated collagen production by fibroblasts.

Current frontiers in systemic sclerosis pathogenesis

Systemic sclerosis is an autoimmune disease characterised by vascular dysfunction, impaired angiogenesis, inflammation and fibrosis. There is no currently accepted disease‐modifying treatment with only autologous stem cell transplant showing clinically meaningful benefit. The lack of treatment options reflects our lack of understanding of the precise molecular mechanisms occurring in the disease. Recent investigations have begun to decipher the molecular pathways underpinning the different aspects of the disease and may provide a rational clinical target(s). Uncovering the molecular mechanisms of the disease is important in understanding systemic sclerosis treatment. The aim of this review was to examine the current thinking in SSc pathogenesis and will offer novel areas for research which may yield novel therapeutics.

Differentiation potential of CD14+ monocytes into myofibroblasts in patients with systemic sclerosis.

Background Circulating monocytes are a highly plastic and functionally heterogeneic cell type with an activated phenotype in patients with systemic sclerosis (SSc). CD14+ monocytes have the potential to differentiate into extra-cellular matrix (ECM) producing cells, possibly participating in fibrogenesis. Aim To study the effect of GM-CSF, IL-4 and endothelin -1 (ET-1) alone or in combination on monocyte differentiation into myofibroblasts. Methods CD14+ cells were isolated from peripheral blood from 14 SSc patients and healthy controls by positive selection and incubated with different combinations of GM-CSF, IL-4 and ET-1 for 14 days. Type-1 collagen and α-SMA were detected by Western blot, qPCR and confocal microscopy. HLA-DR, CD11c and CD14 expression was analysed by flow cytometry. A collagen gel contraction assay was performed for functional myofibroblast assessment. Results GM-CSF both induced collagen and α-SMA expression after 14 days. ET-1 further increased GM-CSF-induced collagen expression in a dose dependent manner up to 30-fold. IL-4/GM-CSF combination leads to a more DC-like phenotype of monocytes associated with reduced collagen and α-SMA expression compared to GM-CSF alone. Collagen and α-SMA expression was higher in monocytes from SSc patients and monocytes were more prone to obtain a spindle form. In contrast to controls, ET-1 and IL-4 alone were sufficient to induce α-SMA expression in monocytes from SSc patients. Despite the induction of α-SMA expression, monocyte-derived myofibroblasts only had a moderate capability of contraction in functional analyses. Conclusion SSc monocytes display increased maturation towards myofibroblasts demonstrated by their phenotype and α-SMA expression when compared to monocytes from healthy controls, however only with minor functional contraction properties.

Emerging role of epigenetics in systemic sclerosis pathogenesis.

Systemic sclerosis is a connective tissue disease of unknown aetiology characterised by autoimmunity, inflammation, vascular abnormalities and ultimately fibrosis. Although great advances have been made in determining the molecular mechanisms of disease pathogenesis over the last decade, aided by new genetic screens, no current specific disease-modifying treatment is yet available. Epigenetics is defined as heritable changes that are not due to changes in DNA sequence, and there is at present intense research effort to understand the basic mechanisms of epigenetic regulation and how these impact diseases. Epigenetic modifications and dysregulation are associated now with autoimmune disease, inflammatory disease and cancer. In rheumatic diseases all three epigenetic processes are associated with various diseases including rheumatoid arthritis and systemic sclerosis. In systemic sclerosis much focus has been on microRNAs; however, other modifications including DNA methylation are emerging to have a key role. This review examines the role of epigenetics in systemic sclerosis and appraises the contribution of each modification and suggests that modulators of epigenetic changes may be a novel therapeutic option.