Senescence and tissue fibrosis: opportunities for therapeutic targeting.

Cellular senescence is a key hallmark of aging. It has now emerged as a key mediator in normal tissue turnover and is associated with a variety of age-related diseases, including organ-specific fibrosis and systemic sclerosis (SSc). This review discusses the recent evidence of the role of senescence in tissue fibrosis, with an emphasis on SSc, a systemic autoimmune rheumatic disease. We discuss the physiological role of these cells, their role in fibrosis, and that targeting these cells specifically could be a new therapeutic avenue in fibrotic disease. We argue that targeting senescent cells, with senolytics or senomorphs, is a viable therapeutic target in fibrotic diseases which remain largely intractable.

Emerging therapeutic targets in systemic sclerosis.

Systemic sclerosis is an autoimmune connective tissue disease which is characterised by vascular perturbations, inflammation, and fibrosis. Although huge progress recently into the underlying molecular pathways that are perturbed in the disease, currently no therapy exists that targets the fibrosis element of the disease and consequently there is a huge unmet medical need. Emerging studies reveal new dimensions of complexity, and multiple aberrant pathways have been uncovered that have shed light on disturbed signalling in the disease, primarily in inflammatory pathways that can be targeted with repurposed drugs. Pre-clinical animal models using these inhibitors have yielded proof of concept for targeting these signalling systems and progressing to clinical trials. This review will examine the recent evidence of new perturbed pathways in SSc and how these can be targeted with new or repurposed drugs to target a currently intractable disease.

S100A4 a classical DAMP as a therapeutic target in fibrosis.

Fibrosis regardless of aetiology is characterised by persistently activated myofibroblasts that are contractile and secrete excessive amounts of extracellular matrix molecules that leads to loss of organ function. Damage-Associated Molecular Patterns (DAMPs) are endogenous host-derived molecules that are released from cells dying or under stress that can be triggered by a variety of insults, either chemical or physical, leading to an inflammatory response. Among these DAMPs is S100A4, part of the S100 family of calcium binding proteins that participate in a variety of cellular processes. S100A4 was first described in context of cancer as a pro-metastatic factor. It is now appreciated that aside from its role in cancer promotion, S100A4 is intimately involved in tissue fibrosis. The extracellular form of S100A4 exerts its effects through multiple receptors including Toll-Like Receptor 4 and RAGE to evoke signalling cascades involving downstream mediators facilitating extracellular matrix deposition and myofibroblast generation and can play a role in persistent activation of myofibroblasts. S100A4 may be best understood as an amplifier of inflammatory and fibrotic processes. S100A4 appears critical in systemic sclerosis pathogenesis and blocking the extracellular form of S100A4 in vivo in various animal models of disease mitigates fibrosis and may even reverse established disease. This review appraises S100A4's position as a DAMP and its role in fibrotic conditions and highlight therapeutically targeting this protein to halt fibrosis, suggesting that it is a tractable target.

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.

Elevated interleukin-11 in systemic sclerosis and role in disease pathogenesis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease in which there is elevated inflammation, aberrant cytokine expression, and subsequent fibrosis. Interleukin-11 (IL-11) is a recently described profibrotic cytokine that can mediate fibrosis in the heart, lungs, and skin and is upregulated by transforming Growth Factor-ß (TGF-ß1). The objective of this study was to quantify the serum levels of IL-11 in early diffuse SSc patients. Also, if IL-11 could regulate the alarmin IL-33 in dermal fibroblasts was quantified. Early diffuse SSc patient sera was isolated and IL-11 was quantified by specific commercial ELISA compared to healthy control (n = 17). Healthy dermal fibroblasts were cultured in vitro and then serum starved and incubated with or without recombinant IL-11. At specific early and late time points the supernatant was quantified for the alarmin IL-33 by specific ELISA. In early diffuse SSc patients it was demonstrated that they have elevated IL-11 in their sera. In a subgroup of SSc patients with interstitial lung disease (ILD) this elevation was particularly pronounced compared to those devoid of fibrotic lung disease. In vitro incubation of healthy dermal fibroblasts led to a significant induction of IL-33 cytokine release into the cell media. IL-11 is a profibrotic cytokine that is elevated in early diffuse SSc and is particularly elevated in those with ILD. This suggests that IL-11 could be a possible biomarker of ILD in SSc. It was also found that IL-11 led to release of the cytokine alarmin IL-33 in fibroblasts at earlier time points but not late time points, suggesting early stimulation elicits an inflammatory response in the local microenvironment but prolonged stimulation leads to fibrosis.

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.

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.

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.

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.

Advances in epigenetics in systemic sclerosis: molecular mechanisms and therapeutic potential.

Systemic sclerosis (SSc) is a prototypical inflammatory fibrotic disease involving inflammation, vascular abnormalities and fibrosis that primarily affect the skin and lungs. The aetiology of SSc is unknown and its pathogenesis is only partially understood. Of all the rheumatic diseases, SSc carries the highest all-cause mortality rate and represents an unmet medical need. A growing body of evidence implicates epigenetic aberrations in this intractable disease, including specific modifications affecting the three main cell types involved in SSc pathogenesis: immune cells, endothelial cells and fibroblasts. In this Review, we discuss the latest insights into the role of DNA methylation, histone modifications and non-coding RNAs in SSc and how these epigenetic alterations affect disease features. In particular, histone modifications have a role in the regulation of gene expression pertinent to activation of fibroblasts to myofibroblasts, governing their fate. DNA methyltransferases are crucial in disease pathogenesis by mediating methylation of DNA in specific promoters, regulating expression of specific pathways. We discuss targeting of these enzymes for therapeutic gain. Innovative epigenetic therapy could be targeted to treat the disease in a precision epigenetics approach.

The Cell-Permeable Derivative of the Immunoregulatory Metabolite Itaconate, 4-Octyl Itaconate, Is Anti-Fibrotic in Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease that leads to skin fibrosis. Altered metabolism has recently been described in autoimmune diseases and SSc. Itaconate is a product of the Krebs cycle intermediate cis-aconitate and is an immunomodulator. This work examines the role of the cell-permeable derivative of itaconate, 4-octyl itaconate (4-OI), in SSc. SSc and healthy dermal fibroblasts were exposed to 4-OI. The levels of collagen Nrf2-target genes and pro-inflammatory cytokines interleukin 6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) were determined. Levels of reactive oxygen species (ROS) as well as the gene expression of collagen and Cellular Communication Network Factor 2 (CCN2) were measured after transforming growth factor beta 1 (TGF-ß1) stimulation in the presence or absence of 4-OI. Wild-type or Nrf2-knockout (Nrf2-KO) mouse embryonic fibroblasts (MEFs) were also treated with 4-OI to determine the role of Nrf2 in 4-OI-mediated effects. 4-OI reduced the levels of collagen in SSc dermal fibroblasts. Incubation with 4-OI led to activation of Nrf2 and its target genes heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). 4-OI activated antioxidant response element (ARE)-dependent gene expression, reduced inflammatory cytokine release and reduced TGF-ß1-induced collagen and ROS production in dermal fibroblasts. The effects of 4-OI are dependent on Nrf2. The cell-permeable derivative of itaconate 4-OI is anti-fibrotic through upregulation of Nrf2 and could be a potential therapeutic option in an intractable disease.

Bone Morphogenetic Protein Antagonist Gremlin-1 Increases Myofibroblast Transition in Dermal Fibroblasts: Implications for Systemic Sclerosis.

OBJECTIVE: Systemic Sclerosis is an autoimmune connective tissue disease which results in fibrosis of the skin and lungs. The disease is characterized by activation of myofibroblasts but what governs this is unknown. Gremlin-1 is a BMP antagonist that is developmentally regulated and we sought to investigate its role in Systemic Sclerosis. METHODS: Dermal fibroblasts were transfected with Grem1pcDNA3.1 expression vectors or empty vectors. Various markers of myofibroblasts were measured at the mRNA and protein levels. Scratch wound assays were also performed. Media Transfer experiments were performed to evaluate cytokine like effects. Various inhibitors of TGF-ß signaling and MAPK signaling were used post-transfection. siRNA to Gremlin-1 in SSc dermal fibroblasts were performed to evaluate the role of Gremlin-1. Different cytokines were incubated with fibroblasts and Gremlin-1 measured. Bleomycin was used as model of fibrosis and immunohistochemistry performed. RESULTS: Overexpression of Gremlin-1 was achieved in primary dermal fibroblasts and lead to activation of quiescent cells to myofibroblasts indicated by collagen and α-Smooth muscle actin. Overexpression also led to functional effects. This was associated with increased TGF-ß1 levels and SBE luciferase activity but not increased Thrombospondin-1 expression. Inhibition of Gremlin-1 overexpression cells with antibodies to TGF-ß1 but not isotype controls led to reduced collagen and various TGF-ß pathway chemical inhibitors also led to reduced collagen levels. In SSc cells siRNA mediated reduction of Gremlin-1 reduced collagen expression and CTGF gene and protein levels in these cells. IL-13 did not lead to elevated Gremlin-1 expression nor did IL-11. Gremlin-1 was elevated in an animal model of fibrosis compared to NaCl-treated mice. CONCLUSION: Gremlin-1 is a key regulator of myofibroblast transition leading to enhanced ECM deposition. Strategies that block Gremlin-1 maybe a possible therapeutic target in fibrotic diseases such as SSc.

The emerging role of metabolism in fibrosis.

The metabolic shift that cancer cells undergo towards aerobic glycolysis was identified as a defining feature in tumours almost 100 years ago; however, it has only recently become apparent that similar metabolic reprogramming is a key feature in other diseases - with fibrosis now entering the fray. In this perspective, an overview of the recent evidence implicating increased glycolysis and glutaminolysis as mediators of fibrosis is presented, with a particular emphasis on the novel therapeutic possibilities this introduces. Furthermore, the impact that metabolic reprogramming has on redox homeostasis is discussed, providing an insight into how this often-overlooked mechanism may drive the pathogenesis.

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.