Interleukin-11 and its eminent role in tissue fibrosis: a possible therapeutic target.

Interleukin-11 is a cytokine from the IL-6 family of cytokines that includes IL-6 and oncostatin-M. Initially described for its role in platelet generation, it is now appreciated that this cytokine has multiple functions. Recently it has been found that IL-11 is critical in fibrosis in multiple different organ systems and systemically as in the autoimmune disease systemic sclerosis. Animal models of fibrosis have determined that animals with IL-11 receptor deletions have retarded fibrosis and that in wild-type animals IL-11 is found at the organ of fibrosis. Recent evidence suggests that IL-11 may be a master regulator of fibrosis regardless of end target organ. With the development of neutralizing antibodies targeting the cytokine in pre-clinical models this could be a possible therapeutic, in a disease in which no specific therapies exist. This review appraises the evidence of the role of IL-11 in tissue fibrosis, its signalling properties, and therapeutic targeting. The review ends with an appraisal of indications for which IL-11 modulation is targeted.

Epigenetics as a versatile regulator of fibrosis.

Fibrosis, a process caused by excessive deposition of extracellular matrix (ECM), is a common cause and outcome of organ failure and even death. Researchers have made many efforts to understand the mechanism of fibrogenesis and to develop therapeutic strategies; yet, the outcome remains unsatisfactory. In recent years, advances in epigenetics, including chromatin remodeling, histone modification, DNA methylation, and noncoding RNA (ncRNA), have provided more insights into the fibrotic process and have suggested the possibility of novel therapy for organ fibrosis. In this review, we summarize the current research on the epigenetic mechanisms involved in organ fibrosis and their possible clinical applications.

Toll-like receptor triggering in systemic sclerosis: time to target.

SSc is an autoimmune disease that has features of vascular abnormalities, inflammation and skin and lung fibrosis. Toll-like receptors (TLRs) are sentinel receptors that serve to recognize pathogens or internal danger signals leading to downstream signalling pathways that ultimately lead to inflammation and modification of adaptive immunity. Inflammation and fibrosis appear intricately connected in this disease and TLR ligation on fibroblasts can directly activate these cells to produce copious amounts of collagen, a hallmark of disease. The presence of damage-associated molecular patterns in association with fibrosis has been highlighted. Given their prominent role in disease, this review discusses the evidence of their expression and role in disease pathogenesis and possible therapeutic intervention to mitigate fibrosis.

Metabolic perturbations in systemic sclerosis.

PURPOSE OF REVIEW: The aim of this review is to evaluate the recent evidence of the role of metabolism in systemic sclerosis (SSc), highlighting specific aberrations and to appraise the feasibility of targeting these therapeutically. RECENT FINDINGS: SSc is an autoimmune disease that is characterised by three facets: vascular problems, inflammation, and fibrosis. The fibrosis primarily affects the skin and lungs and currently, no antifibrotic treatment has been found effective. In recent years a renaissance in metabolism research has begun with renewed vigour in the role of metabolism in disease, particularly in the immune system. Alterations in glycolysis and utilisation of specific metabolic pathways in specific cell types have been associated with specific diseases. Most recently alterations in glycolysis and glutaminolysis have been determined in SSc fibroblasts mediating fibrosis. Reduced nicotinamide adenine dinucleotide levels have also been described in SSc. SUMMARY: Specific metabolic aberrations have been described in SSc and this may lead to novel therapeutic targets in this disease.

Interleukin-36α is elevated in diffuse systemic sclerosis and may potentiate fibrosis.

Systemic sclerosis (SSc) is an autoimmune prototypical connective tissues disease that results in alterations in vasculature, inflammation and fibrosis of the skin. Interleukin-1 family cytokines has been implicated in the disease including IL-1. IL-36α is an IL-1 family member that is clearly implicated in psoriatic skin disease but its role in systemic sclerosis disease is not clear. The aim of this work is to evaluate the levels and role of IL-36α in systemic sclerosis. Early diffuse SSc patients sera was isolated along with healthy controls and IL-36 levels quantified by ELISA. In vitro analysis was also undertaken with primary dermal fibroblasts with recombinant IL-36α and keratinocyte cells were also incubated with IL-36α. Cytokines were measured by ELISA. Serum IL-36 was significantly elevated compared to healthy controls. Elevated neutrophil elastase was also elevated in the matched sera. IL-36 was not directly pro-fibrotic in dermal fibroblasts but did induce pro-inflammatory cytokines that were dependant on the MAPK pathway for their release. IL-36α also led to release of CCL20 and CCL2 in keratinocytes which may potentiate fibrosis. IL-36α is elevated in SSc serum and whilst not directly pro-fibrotic it may through keratinocytes, potentiate fibrosis through keratinocyte-immune fibroblast cross-talk.

Gremlin: a complex molecule regulating wound healing and fibrosis.

Gremlin-1 is part of the TGF-ß superfamily and is a BMP antagonist that blocks BMP signalling to precisely control BMP gradients. Gremlin-1 is primarily involved in organogenesis and limb patterning however, has recently been described as being involved in fibrotic diseases. Initially described as a key factor involved in diabetic kidney fibrosis due to being induced by high glucose, it has now been described as being associated with lung, liver, eye, and skin fibrosis. This suggests that it is a key conserved molecule mediating fibrotic events irrespective of organ. It appears that Gremlin-1 may have effects mediated by BMP-dependent and independent pathways. The aim of this review is to evaluate the role of Gremlin-1 in fibrosis, its mechanisms and if this can be targeted therapeutically in fibrotic diseases, which currently have very limited treatment options and are highly prevalent.

IL11 is elevated in systemic sclerosis and IL11-dependent ERK signalling underlies TGFß-mediated activation of dermal fibroblasts.

OBJECTIVES: Interleukin 11 (IL11) is highly upregulated in skin and lung fibroblasts from patients with systemic sclerosis (SSc). Here we tested whether IL11 is mechanistically linked with activation of human dermal fibroblasts (HDFs) from patients with SSc or controls. METHODS: We measured serum IL11 levels in volunteers and patients with early diffuse SSc and manipulated IL11 signalling in HDFs using gain- and loss-of-function approaches that we combined with molecular and cellular phenotyping. RESULTS: In patients with SSc, serum IL11 levels are elevated as compared with healthy controls. All transforming growth factor beta (TGFß) isoforms induced IL11 secretion from HDFs, which highly express IL11 receptor α-subunit and the glycoprotein 130 (gp130) co-receptor, suggestive of an autocrine loop of IL11 activity in HDFs. IL11 stimulated ERK activation in HDFs and resulted in HDF-to-myofibroblast transformation and extracellular matrix secretion. The pro-fibrotic action of IL11 in HDFs appeared unrelated to STAT3 activity, independent of TGFß upregulation and was not associated with phosphorylation of SMAD2/3. Inhibition of IL11 signalling using either a neutralizing antibody against IL11 or siRNA against IL11RA reduced TGFß-induced HDF proliferation, matrix production and cell migration, which was phenocopied by pharmacological inhibition of ERK. CONCLUSIONS: These data reveal that autocrine IL11-dependent ERK activity alone or downstream of TGFß stimulation promotes fibrosis phenotypes in dermal fibroblasts and suggest IL11 as a potential therapeutic target in SSc.

Wnt antagonist DKK-1 levels in systemic sclerosis are lower in skin but not in blood and are regulated by microRNA33a-3p.

BACKGROUND: Systemic sclerosis is an autoimmune skin disease which is associated with inflammation and resulting skin fibrosis. Myofibroblasts are the key cell type associated with the fibrosis but how they are differentiated is not clear. DKK-1 is a Wnt antagonist that blocks Wnt-mediated fibrosis and is reduced in fibrotic conditions. Thus, DKK-1 is a clear negative regulator of fibrosis in systemic sclerosis and its regulation is unknown. The aim of this work is to determine the levels of DKK-1 in serum and tissues of SSc and its regulation. METHODS: Skin biopsies were taken from early diffuse systemic sclerosis patients and healthy controls and DKK-1 measured by ELISA; serum was also isolated and DKK-1 quantified. DKK-1 was also measured by qRT-PCR. MicroRNA33a-3p was measured by TaqMan PCR. miR mimics and controls were transfected into dermal fibroblasts. Bleomycin mouse model was employed and compared to vehicle control treated mice, and gene expression was employed for DKK-1 and various extracellular matrix genes. RESULTS: DKK-1 is reduced in SSc skin and fibroblasts but is not reduced in the circulation in patients. MicroRNA33a-3p regulates DKK-1 levels epigenetically and is significantly reduced in SSc cells and whole tissue. DKK-1 is also reduced in the bleomycin mouse model and pro-fibrotic genes elevated. CONCLUSION: DKK-1 is reduced in SSc cells and is regulated by miR33a-3p, and restoring DKK-1 levels through epigenetic means could be a therapeutic target in systemic sclerosis.

Metabolic reprogramming of glycolysis and glutamine metabolism are key events in myofibroblast transition in systemic sclerosis pathogenesis.

Systemic Sclerosis (SSc) is a rare fibrotic autoimmune disorder for which no curative treatments currently exist. Metabolic remodelling has recently been implicated in other autoimmune diseases; however, its potential role in SSc has received little attention. Here, we aimed to determine whether changes to glycolysis and glutaminolysis are important features of skin fibrosis. TGF-ß1, the quintessential pro-fibrotic stimulus, was used to activate fibrotic pathways in NHDFs in vitro. Dermal fibroblasts derived from lesions of SSc patients were also used for in vitro experiments. Parameters of glycolytic function were assessed using by measuring extracellular acidification in response to glycolytic activators/inhibitors, whilst markers of fibrosis were measured by Western blotting following the use of the glycolysis inhibitors 2-dg and 3PO and the glutaminolysis inhibitor G968. Succinate was also measured after TGF-ß1 stimulation. Itaconate was added to SSc fibroblasts and collagen examined. TGF-ß1 up-regulates glycolysis in dermal fibroblasts, and inhibition of glycolysis attenuates its pro-fibrotic effects. Furthermore, inhibition of glutamine metabolism also reverses TGF-ß1-induced fibrosis, whilst glutaminase expression is up-regulated in dermal fibroblasts derived from SSc patient skin lesions, suggesting that enhanced glutamine metabolism is another aspect of the pro-fibrotic metabolic phenotype in skin fibrosis. TGF-ß1 was also able to enhance succinate production, with increased succinate shown to be associated with increased collagen expression. Incubation of SSc cells with itaconate, an important metabolite, reduced collagen expression. TGF-ß1 activation of glycolysis is a key feature of the fibrotic phenotype induced by TGF-B1 in skin cells, whilst increased glutaminolysis is also evident in SSc fibroblasts.

Bi-directional communication: Conversations between fibroblasts and immune cells in systemic sclerosis.

Systemic Sclerosis (SSc) is an autoimmune idiopathic connective tissue disease, characterized by aberrant fibro-proliferative and inflammatory responses, causing fibrosis of multiple organs. In recent years the interactions between innate and adaptive immune cells with resident fibroblasts have been uncovered. Cross-talk between immune and stromal cells mediates activation of stromal cells to myofibroblasts; key cells in the pathophysiology of fibrosis. These cells and their cytokines appear to mediate their effects in both a paracrine and autocrine fashion. This review examines the role of innate and adaptive immune cells in SSc, focusing on recent advances that have illuminated our understanding of ongoing bi-directional communication between immune and stromal cells. Finally, we appraise current and future therapies and how these may be useful in a disease that currently has no specific disease modifying treatment.

cRel expression regulates distinct transcriptional and functional profiles driving fibroblast matrix production in systemic sclerosis.

OBJECTIVES: NF-κB regulates genes that control inflammation, cell proliferation, differentiation and survival. Dysregulated NF-κB signalling alters normal skin physiology and deletion of cRel limits bleomycin-induced skin fibrosis. This study investigates the role of cRel in modulating fibroblast phenotype in the context of SSc. METHODS: Fibrosis was assessed histologically in mice challenged with bleomycin to induce lung or skin fibrosis. RNA sequencing and pathway analysis was performed on wild type and Rel-/- murine lung and dermal fibroblasts. Functional assays examined fibroblast proliferation, migration and matrix production. cRel overexpression was investigated in human dermal fibroblasts. cRel immunostaining was performed on lung and skin tissue sections from SSc patients and non-fibrotic controls. RESULTS: cRel expression was elevated in murine lung and skin fibrosis models. Rel-/- mice were protected from developing pulmonary fibrosis. Soluble collagen production was significantly decreased in fibroblasts lacking cRel while proliferation and migration of these cells was significantly increased. cRel regulates genes involved in extracellular structure and matrix organization. Positive cRel staining was observed in fibroblasts in human SSc skin and lung tissue. Overexpression of constitutively active cRel in human dermal fibroblasts increased expression of matrix genes. An NF-κB gene signature was identified in diffuse SSc skin and nuclear cRel expression was elevated in SSc skin fibroblasts. CONCLUSION: cRel regulates a pro-fibrogenic transcriptional programme in fibroblasts that may contribute to disease pathology. Targeting cRel signalling in fibroblasts of SSc patients could provide a novel therapeutic avenue to limit scar formation in this disease.

Interleukin-31 promotes pathogenic mechanisms underlying skin and lung fibrosis in scleroderma.

OBJECTIVES: Cytokines released by infiltrating T cells may promote mechanisms leading to fibrosis in scleroderma. The aim of this study was to investigate the role of the Th2 cytokine IL-31, and its receptor IL-31RA, in scleroderma skin and lung fibrosis. METHODS: IL-31 was measured by ELISA of plasma, and by immunochemistry of fibrotic skin and lung tissue of scleroderma patients. The receptor, IL-31RA, was assayed by qPCR of tissue resident cells. Next-generation sequencing was used to profile the responses of normal skin fibroblasts to IL-31. In wild-type Balb/c mice, IL-31 was administered by subcutaneous mini pump, with or without additional TGFß, and the fibrotic reaction measured by histology and ELISA of plasma. RESULTS: IL-31 was present at high levels in plasma and fibrotic skin and lung lesions in a subset of scleroderma patients, and the receptor overexpressed by downstream cells relevant to the disease process, including skin and lung fibroblasts, through loss of epigenetic regulation by miR326. In skin fibroblasts, IL-31 induced next generation sequencing profiles associated with cellular growth and proliferation, anaerobic metabolism and mineralization, and negatively associated with angiogenesis and vascular repair, as well as promoting phenotype changes including migration and collagen protein release via pSTAT3, resembling the activation state in the disease. In mice, IL-31 induced skin and lung fibrosis. No synergy was seen with TGFß, which supressed IL-31RA. CONCLUSION: IL-31/IL-31RA is confirmed as a candidate pro-fibrotic pathway, which may contribute to skin and lung fibrosis in a subset of scleroderma patients.

Current and Potential New Targets in Systemic Sclerosis Therapy: a New Hope.

PURPOSE OF REVIEW: Systemic sclerosis (SSc) is an autoimmune connective tissue disease in which there is an activation of fibroblast to a myofibroblast that secretes huge amounts of extracellular matrix. Currently, no treatment exists that modifies the fibrosis elements and new therapeutic targets are badly needed. This review examines the current state of treatments and emerging therapeutics. RECENT FINDINGS: Nintedanib was found to significantly reduce the rate of decline in SSc associated FVC, although it has no benefit on skin fibrosis. New cannabinoid receptor2 agonist has shown superb effects in phase II and results in phase III are anticipated. Other targets are currently being tested in clinical trials and new targets that are yet to be tested are increasing in the SSc literature. Nintedanib is now licenced for SSc interstitial lung disease but this does not modify the skin fibrosis. Current ongoing trials will determine the role of various targets. New targets are emerging as we gain a deeper understanding of disease pathogenesis.

Epigenetic modulation as a therapy in systemic sclerosis.

SSc is an autoimmune idiopathic disease in which there is an inflammatory component driving fibrosis. The chief cell involved is the myofibroblast, which when activated secretes copious amounts of extracellular matrix that forms deposits, leading to stiffness and fibrosis. The fibrosis is most prevalent in the skin and lungs. In recent years epigenetic modifications have been uncovered that positively and negatively regulate the genesis of the myofibroblasts and that can be activated and regulated by a variety of cytokines and hormones. The epigenetic contribution to these cells and to SSc is only now really coming to light, and this opens up a new therapeutic target for the disease for which many epigenetic drugs, such as miRNA replacements, are beginning to be developed. This review will examine the epigenetic regulators in the disease and possible targeting of these.

Methyl cap binding protein 2: a key epigenetic protein in systemic sclerosis.

OBJECTIVE: SSc is an autoimmune connective tissue disease that results in skin fibrosis and currently has no effective treatment. Epigenetic modifications have been described and these may be key in initiating and driving fibroblast activation. Among these epigenetic modifications methylation may be of central importance. The aim of this study was to examine the role of methyl cap binding protein-2 (MeCP2) in SSc fibrosis. METHODS: We used healthy and SSc dermal fibroblasts to examine the role of MeCP2, using both small interfering RNA silencing and lentiviral overexpression to determine its effects. We also examined the expression of MeCP2 in SSc fibroblasts by immunoblotting. miRNA132 was quantified by Taqman real time PCR. RESULTS: We demonstrated that TGF-ß1 induced the expression of MeCP2 in normal cells, and showed that SSc fibroblasts expressed high levels of MeCP2 under basal conditions. MeCP2 positively regulated the expression of extracellular matrix through epigenetic repression of the Wnt antagonist sFRP-1, leading to enhanced Wnt signalling. This mediated fibrosis through glycolysis, as the glycolysis inhibitor 2-deoxyglucose diminished the Wnt-mediated collagen expression. MiR132 expression was reduced in SSc fibroblasts. CONCLUSION: The results suggest that an epigenetic loop exists mediating fibrosis. Targeting of MeCP2, as a key epigenetic regulator, may be a promising therapeutic approach, as would targeting the metabolic reprogramming that occurs through aerobic glycolysis.

Bioengineering the microanatomy of human skin.

Recreating the structure of human tissues in the laboratory is valuable for fundamental research, testing interventions, and reducing the use of animals. Critical to the use of such technology is the ability to produce tissue models that accurately reproduce the microanatomy of the native tissue. Current artificial cell-based skin systems lack thorough characterisation, are not representative of human skin, and can show variation. In this study, we have developed a novel full thickness model of human skin comprised of epidermal and dermal compartments. Using an inert porous scaffold, we created a dermal construct using human fibroblasts that secrete their own extracellular matrix proteins, which avoids the use of animal-derived materials. The dermal construct acts as a foundation upon which epidermal keratinocytes were seeded and differentiated into a stratified keratinised epithelium. In-depth morphological analyses of the model demonstrated very close similarities with native human skin. Extensive immunostaining and electron microscopy analysis revealed ultrastructural details such as keratohyalin granules and lamellar bodies within the stratum granulosum, specialised junctional complexes, and the presence of a basal lamina. These features reflect the functional characteristics and barrier properties of the skin equivalent. Robustness and reproducibility of in vitro models are important attributes in experimental practice, and we demonstrate the consistency of the skin construct between different users. In summary, a new model of full thickness human skin has been developed that possesses microanatomical features reminiscent of native tissue. This skin model platform will be of significant interest to scientists researching the structure and function of human skin.

Innate immunity and Toll-like receptor signaling in the pathogenesis of scleroderma: advances and opportunities for therapy.

PURPOSE OF REVIEW: Systemic sclerosis (SSc) is an autoimmune connective tissue disease in which inflammation and cytokine dysregulation leads to skin fibrosis. Toll-like receptors (TLRs) are conserved pattern recognition receptors, recognizing pathogens danger-associated molecular patterns (DAMPs) that elicit a cascade of proinflammatory signaling. Recently, TLRs have been found to be critically important in SSc pathogenesis, with increased levels of the TLRs and their ligands present in the disease. Animal models have also been pivotal in delineating the role of these innate immune receptors in SSc. This current review examines the role of TLRs and the most recent evidence of the role of DAMPs and how these may be exploited therapeutically. RECENT FINDINGS: Increasingly, studies have demonstrated the key roles of TLR4 and other intracellular TLRs in mediating fibrosis in SSc patients and animal models. TLR4 activation appears a key point and novel DAMPs, expressed upon tissue damage, appear critical in mediating the profibrotic effect through a downstream enhancement of transforming growth factor ß. Deletion of Tenascin-C or a splice variant of fibronectin ameliorates animal models of skin fibrosis. Intracellular, nucleic acid sensing, TLR8 is critical in activating macrophages to secrete profibrotic molecules. The mechanism involves histone modification through epigenetic modifying enzymes. SUMMARY: TLRs are key therapeutic targets in SSc.

Functional Implications of Cross-Linked Actin Networks in Trabecular Meshwork Cells.

BACKGROUND/AIMS: The Trabecular meshwork (TM) is the tissue responsible for outflow resistance and therefore intraocular pressure. TM cells contain a contractile apparatus that is composed of actin stress fibres which run parallel to the axis of the cell and are responsible for facilitating contraction. Cross-Linked Actin Networks (CLANs) are polygonal arrangements of actin that form a geodesic network found predominantly in TM cells both in situ and in vitro. The aim of this work is to determine the functional significance of CLANs in TM cells and to assess the effect of mechanical stretch stimulation on the induction (or not) of CLANs. METHODS: We used collagen gel contraction models to demonstrate functional impairment of cells when induced to express CLANs in situ. Cyclic mechanical stretch was used to stimulate cells and measure CLANs Results: CLANs inhibited contraction and cyclic mechanical stretch induced CLANs. Furthermore, we also demonstrated that using shape alone we could predict the appearance of CLANs using a simple light microscopy technique. CONCLUSION: Taken together we have now shown, for the first time, a functional deficit In TM cells with CLANs Furthermore that shape alone can predict the appearance of CLAN containing cells. CLANs can now be linked to a functional effect and may underlie the appearance of CLANs with the pathology of primary open angle glaucoma (POAG).