Rationally-based therapeutic disease modification in systemic sclerosis: Novel strategies

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Review

Rationally-based therapeutic disease modification in systemic sclerosis: Novel strategies Yoshihide Asanoa, John Vargab,* a b

Department of Dermatology, University of Tokyo, Japan Northwestern Scleroderma Program, Feinberg School of Medicine, Northwestern University, Chicago, United States

A R T I C LE I N FO

A B S T R A C T

Keywords: Systemic sclerosis Therapy Precision medicine Fibrosis Vasculopathy Inflammation Autoimmunity TGF-beta Aging Senescence TLR Myofibroblast Matrisome

Systemic sclerosis (SSc) is a highly challenging chronic condition that is dominated by the pathogenetic triad of vascular damage, immune dysregulation/autoimmunity and fibrosis in multiple organs. A hallmark of SSc is the remarkable degree of molecular and phenotypic disease heterogeneity, which surpasses that of other complex rheumatic diseases. Disease trajectories in SSc are unpredictable and variable from patient to patient. Diseasemodifying therapies for SSc are lacking, long-term morbidity is considerable and mortality remains unacceptably high. Currently-used empirical approaches to disease modification have modest and variable clinical efficacy and impact on survival, are expensive and frequently associated with unfavorable side effects, and none can be considered curative. However, research during the past several years is yielding significant advances with therapeutic potential. In particular, the application of unbiased omics-based discovery technologies to large and well-characterized SSc patient cohorts, coupled with hypothesis-testing experimental research using a variety of model systems is revealing new insights into SSc that allow formulation of a more nuanced appreciation of disease heterogeneity, and a deepening understanding of pathogenesis. Indeed, we are now presented with numerous novel and rationally-based strategies for targeted SSc therapy, several of which are currently, or expected to be shortly, undergoing clinical evaluation. In this review, we discuss promising novel therapeutic targets and rationally-based approaches to disease modification that have the potential to improve long-term outcomes in SSc.

1. Introduction Systemic sclerosis is a complex chronic multi-system orphan disease that most commonly affects women in the 4-5th decades of life [1]. While genetic factors undeniably play an important (albeit poorly understood and characterized) role in disease susceptibility, SSc is very uncommonly familial, and thus environmental exposures are thought to be particularly important in pathogenesis (Fig. 1). Of interest, SSc occurs in all races, but individuals of African ancestry appear to be at a greater risk, with SSc developing at an earlier age, showing greater propensity for severe internal organ complications particularly interstitial lung disease (ILD), and increased risk of premature death. SSc is highly heterogeneous in its presentation, patterns and tempo of organ involvement and outcomes, it is commonly associated with increased mortality and considerable morbidity. The most characteristic clinical presentation of SSc is Raynaud phenomenon, a manifestation of vascular injury, which must be differentiated from Raynaud Disease, a generally benign, non-progresive

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and isolated form of episodic vasospasm. In many patients, SSc may precede other manifestations of the disease, sometimes by years. Careful clinical investigation of SSc patients even in a relatively early stage of their disease will commonly reveal evidence of ILD (abnormal chest CT scan), myocardial fibrosis (abnormal cardiac MRI), esophageal involvement (abnormal manometry with dysregulated esophageal motility) or microangiopathy (abnormal nailfold capillary pattern), generally accompanied by humoral autoimmunity (positive ANA and distinct autoantibodies), indicating that the processes underlying disease SSc pathogenesis are progressing synchronously (and often silently) in multiple organs. Over time, patients may demonstrate indolent, or rapidly progressing, disease progression, leading to organ failure such as pulmonary fibrosis and heart failure, and premature death. In other patients with SSc, the disease shows long period of inactivity culminating in late-onset pulmonary hypertension; yet others show remarkable stability in disease over time. While not common, such virtual disease remission can be seen in both treated and untreated SSc patients. Thus it is of particular significance to recognize that the field

Corresponding author. E-mail address: [email protected] (J. Varga).

https://doi.org/10.1016/j.semcdb.2019.12.007 Received 3 December 2019; Received in revised form 12 December 2019; Accepted 12 December 2019 1084-9521/ © 2019 Published by Elsevier Ltd.

Please cite this article as: Yoshihide Asano and John Varga, Seminars in Cell and Developmental Biology, https://doi.org/10.1016/j.semcdb.2019.12.007

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Fig. 1. The pathomechanistic triad underlying the clinical manifestations of SSc. In a genetically-predisposed host, as-yet undefined environmental exposure triggers a series of events that culminate in autoimmunity and inflammation, vascular injury and fibrosis. These three processes are tightly interlinked, and occur systematically, but may vary from one organ to the next, and follow distinct temporal trajectories. Potentially causal environmental exposures might include viral infections, lifestyle factors, dietary exposures, microbiome alterations and other factors that influence the epigenome, while SSc-associated genetic risk factors predominantly implicate the immune system. Fig. 2. The pathogenesis of fibrosis in SSc: an integrated evidence-based hypothetical scenario. Vascular injury, conceivably the earliest and primary event in disease pathogenesis, triggers or promotes inflammation and autoimmunity. The influx and activation of a variety of cellular components of the immune system directly, and indirectly via secreted factors, induce fibroblast activation and the transdifferentiation of various stromal progenitor cell types into activated and apoptosis-resistant myofibroblasts. Persistent accumulation of activated myofibroblasts within the target tissue, such as the skin, lungs and heart, contributes to the excessive production of fibrillar collagens and other extracellular matrix macromolecules. These molecules associate into a mechanically-stressed rigid matrix that over time displaces normal tissue architecture and disrupts normal organ function. The components of the fibrotic matrix themselves signal to resident stromal cells via both mechanical and biochemical cues to augment matrix generation and evasion of apoptosis.

currently lacks biomarkers that can robustly identify quiescent versus active disease, progression versus regression, or predict disease trajectories or outcomes [1]. It has long been appreciated that the clinical and pathological manifestations of SSc reflect the defining triad of inflammation/autoimmunity, disseminated microangiopathy and multi-organ fibrosis (Fig. 1). The combination of these three features is unique to the pathogenesis of SSc, distinguishing it from other fibrotic and autoimmune diseases. There is still debate as to which of these three interrelated pathomechanistic processes might be the primary or initiating event in the pathogenesis of SSc, with evidence that implicates either vascular injury or immune system activation and break in immune self-tolerance. Fibrosis developing synchronously in multiple organs, most prominently in the skin, lungs, heart, gut and skeletal muscle, is the distinguishing hallmark of SSc and ultimately accounts for much of its associated mortality [2]. Accordingly, much of the interest in identifying and developing disease-modifying SSc therapies during the past decade focused on approaches to slow, halt, or reverse fibrosis.

matricellular molecules tenascin-C and fibronectin, which are normally only transiently detected in injured tissue during wound healing, but are persistently elevated in SSc and other forms of chronic fibrosis [4–6]. In fact, several of these molecules can perpetuate fibrosis by serving as “damage-associated molecular pattern” endogenous ligands (DAMPs) recognized by toll-like receptors (TLR) on resident stromal fibroblasts and endothelial cells. Binding of such DAMPs to their cognate TLR receptors can elicit potent and long-lived fibrotic responses in stromal cells. In this manner, aberrant in situ generation of such molecules during injury and repair and their tissue accumulation can set the stage for autocrine amplification (so called “feed-forward”) loops that sustains persistent fibrosis via TLR signaling (Fig. 2). Such pathological fibrosis amplification pathways can be selectively targeted for therapy. Indeed, antagonists of tenascin-C or fibronectin-EDA, or targeted pharmacological blockade of the TLR4-DAMP ligand interactions, can attenuate and reverse fibrosis in preclinical disease models, suggesting potential novel strategies for disease-modifying fibrosis therapy in SSc [7–9]. Recent approaches have utilized unbiased proteomics in order to survey the ECM. The aim of these on-going endeavors is to generate an unbiased, comprehensive, and quantitative atlas of the biochemical composition of the stroma (the “matrisome”) of normal and diseased solid organs [10]. Precise definition of the healthy and

2. Targeted disease-modifying therapies: conventional targets 2.1. Fibrotic pathway 2.1.1. Fibrosis: a mechanistic overview (Fig. 2) Fibrosis in multiple organs developing more or less synchronously is a defining feature of SSc, and is characterized by the accumulation of excessive extracellular matrix (ECM) that disrupts normal tissue architecture and ultimately impairs tissue function. A multiplicity of tissue-resident and bone marrow-derived cell types play unique roles in the pathogenesis of fibrosis (Fig. 2). The ECM of all somatic tissues, including the skin, is composed of fibrillar collagens (such as Type I), but also variable amounts of elastin, proteoglycans, fibrillin, fibulin, periostin, cartilage oligomeric matrix protein (COMP),matricellular proteins such as SPARC, as well as a large number of matrix-sequestered growth factors including transforming growth factor-ß (TGF-ß), connective tissue growth factor, Wnt ligands, latent TGF-β binding protein and thrombospondin [3]. Particularly prominent of the matrix molecules implicated in fibrosis are alternately-spliced variants of the large 2

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disease mechanism in SSc, since fibrotic tissue shows markedly increased stiffness. Mechanotransduction pathways involve focal adhesion kinase (FAK) and Rho-associated kinase, Yes-associated protein (YAP) and myocardin-related transcription factor (MRTF), generally operating in networks linked to TGF-ß [26]. These signaling intermediates can each be targeted for therapy using small molecules currently under development [27]. An intirguing recent study demonstrated that activation of the DRD1 dopamine receptor, which shows upregulation selectively on fibrotic fibroblasts, is associated with attenuation of YAP/TAZ signaling and prevention of fibrosis in disease models [28]. While these observations highlight the feasibility of pharmacological YAP/TAZ blockade for fibrosis, the relative role of YAP/TAZ as a therapeutic target in SSc remains to be established.

disease matrisome in particular organs, and its changes across the lifespan and disease progression and regression will be of great potential relevance for understanding the pathogenesis and for monitoring of therapeutic responses in SSc and potentially other fibrotic conditions. The combination of factors contributing to pathological matrix accumulation in SSc includes excessive accumulation of myofibroblasts with a matrix-producing secretory phenotype and the ability to evade apoptosis; crosslinking of secreted ECM molecules catalyzed by lysyl oxidases (LOX), transglutaminases and other enzymes; and impaired ECM turnover [11]. The pathways that trigger fibrotic cellular responses, and prevent fibrosis resolution in the chronic stages of the disease, appear to be shared across affected organs, and represent viable therapeutic targets in SSc. Myofibroblasts are considered to be the pivotal cellular components of fibrosis in all organs and all forms of fibrosis. These fibroblast-like stromal cells endowed with prominent contractile and biosynthetic capacity arise from diverse stromal progenitor cell types, including fibroblasts, pericytes and endothelial cells, which sequentially undergo differentiation into proto-myofibroblasts and then myofibroblasts [12,13]. The origins of myofibroblasts have been intensively investigated using both lineage-tracing approaches in mice, and increasingly, transcriptome analysis of human tissues at single-cell resolution [14]. Recent evidence indicates that dermal white adipocytes, residing deep to the reticular dermis, are highly plastic cells that can readily give rise to myofibroblasts [15]. In fact, a significant proportion of activated myofibroblasts within the fibrotic dermis in patients with SSc appears to arise from adipocytes normally residing in the dermal white adipose tissue [16,17]. Pharmacological targeting the adipocyte-mesenchymal transition (AMT), whereby quiescent adipocytes transform into activated myofibroblasts, is feasible and represents an exciting potential novel therapeutic approach in SSc. In light of the myofibroblast’s pivotal roles in ECM biosynthesis, deposition, persistence and remodeling, the cellular origins, factors and mechanisms controlling the differentiation, metabolism, survival and senescence of these hybrid cell types are of considerable interest [18]. Reprogramming of mesenchymal progenitor cells to a fibrotic phenotype is triggered by members of the TGF-ß superfamily, along with the developmental morphogens Notch and Hedgehog, Wnt ligands as well as soluble cytokines such as IL-6, IL-13 and oncostatin M [19]. Recent studies identified IL-11, an additional member of the IL-6 cytokine family, as a major driver of fibrotic responses across multiple organs [20]. Cytokines, including members of the IL-6 family, are highly upregulated in SSc fibroblasts, and have the ability to directly or in a relay reprogram quiescent fibroblasts into persistently activated myofibroblasts [21]. Consequently, selective targeting using antibodies directed against individual cytokines or their receptors, represents an appealing therapeutic strategy in SSc (Table 1). Indeed, a small number of randomized clinical trials provide evidence for partial efficacy of IL-6 targeting in mitigating skin and/or lung disease [22]. Evidence shows that treatment of SSc patients with IL-6 antagonist might mitigate fibrotic TGF-ß signaling in the skin [23]. However, in view of the potential redundancy among pathogenic cytokines implicated in SSc fibrosis, targeting any single cytokine might be insufficient, raising the question whether combinatorial blockade of multiple profibrotic factors may be required to effectively mitigate fibroblast activation [24]. Thrombin is generated and hypoxia can develop at sites of tissue injury, and are prominent in skin biopsies in SSc patients. Both of these stimuli can elicit sustained myofibroblast activation, potentially implicating them in SSc pathogenesis. Moreover, cell-intrinsic metabolic alterations such as increased glycolytic flux, oxidative stress and depletion of the cellular co-substrate NAD (see below), might also be important in fibrosis pathogenesis, although their precise roles and mechanisms of action are just beginning to become untangled [25]. Additionally, resident fibroblasts can both sense and respond to mechanical cues emanating within their microenvironment. This represents a potentially fundamental, but until recently understudied,

2.1.2. Therapeutic targeting fibrotic mediators in SSc: TGF-ß and its signaling The earliest identified trigger for fibrosis, the quintessential profibrotic factor TGF-ß, is widely considered to be a potential target for antifibrotic therapy in a variety of conditions, including SSc [29,30]. TGF-ß is a member of a large growth factor superfamily, which signal in many cells via the canonical Smad pathways, as well as non-canonical pathways involving Egr-1, MAP kinases and other signal transducers. Originally considered to be a “growth factor” TGF-ß is, in fact a highly pleiotropic signaling system with fundamental physiological roles in development, immune regulation and tissue remodeling, and is implicated in cancer, autoimmunity and fibrosis. There are three known isoforms of TGF-ß with largely overlapping biological activity (ß1, ß2 and ß3), but most evidence to date implicates TGF-ß1 as the most important driver of pathological fibrosis [31]. TGF-ß induces myofibroblast differentiation, an extensive profibrotic reprogramming of not only fibroblasts, but also pericytes, endothelial, epithelial cells,preadipocytes, and even bone marrow-derived monocytes. Moreover, TGF-ß stimulates reactive oxygen species (ROS) generation via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4), and induces the full panoply of genes involved in collagen processing, secretion and maturation (HSP47, FKBP10, PLOD2, P4HA3, LOX and LOXL1-4, periostin and fibronectin) [32]. Importantly, TGF-ß1 promotes both survival and senescence of myofibroblasts [33]. Many of these fibrotic cellular responses are associated with, or mediated via, epigenetic mechanisms [34]. As an example, the persistently activated metabolic phenotype of SSc skin fibroblasts was shown to be associated with cell-autonomous constitutitve up-regulation of TGF-ß2, which is attributable to chromatin modifications in the TGF-ß2 gene regulatory enhancer region [34]. It is therefore not surprising that TGF-ß is viewed as a pivotal driver of pathological fibrosis in multiple diseases, including SSc. Elevated levels of TGF-ß1 can be detected in SSc biopsies, and SSc fibroblasts constitutively express TGF-ß [35]. Moreover, many of the genes activated in SSc are known TGF-ß targets [36]. As noted above, whether all three TGF-ß isoforms are equally implicated in SSc pathogenesis remains an area of some uncertainty, with distinct therapeutic approaches focusing on pan-TGF-ß inhibition versus selectively targeting individual TGF-ß isoforms. For instance, a synthetic trap for TGF-ß ligand that binds selectively to TGF-ß1 and TGF-ß3 while not TGF-ß2, is currently under development as a potential fibrosis therapy [37]. Regulation of TGF-ß activation is another potential therapeutic target in fibrosis [38]. Cell type-specific expression of integrins (avß6 on epithelial cells, avß5 on fibroblasts, and avß8 on dendritic cells) accounts for latent TGF-ß activation in situ, and avß5 integrins are constitutively upregulated on SSc fibroblasts [39]. A recent study reported that metformin (dimethyl biguanide) inhibited TGF-ß activity by direct binding, potentially explaining its anti-fibrotic activity [40]. Intracellular TGF-ß signaling involves both Smad-dependent (canonical) and Smad-independent (non-canonical) pathways which show extensive cross-talk with each other, and with myocardin-related transcription factor (MRTF) and mechanotransduction mediated via FAK 3

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Table 1 New therapeutic targets driving fibroblast activation. Target molecules/cells

Drugs

The phase of clinical trials

Results

TGF-β1, 2, 3

Fresolimumab [43]

Phase I

IL-6 receptor

Tocilizumab*

Phase III

Oncostatin M

GSK2330811 (Humanized monoclonal antibody) Dabigatran [196]

Phase II

Rapid inhibition of TGF-β-regulated gene expression with parallel reduction of mRSS A trend of improvement in mRSS compared with placebo A statistically significant inhibition of ILD progression compared with placebo No results available

Phase II

BAFF

SAR100842 [67] (Antagonist) Belimumab [133]

Phase II

IFN-α

Anifrolumab [94]

Phase I

IL-4, IL-13

SAR156597 (Chimeric bispecific monoclonal antibody) Rituximab [125,126,127,128,129,130] (Anti-CD20)

Phase II

Thrombin LPA-1, LPA-3

B cells

T cells JAK-STAT signaling Unknown (TGF-β, TNF-α, etc.) PPARs CB2 Receptor tyrosine kinases (PDGF, bFGF, VFGF receptors) and Src family non-receptor tyrosine kinases

Phase I

Phase III Other studies

Safe and well tolerated in patients with SSc‐ILD Decreased or stable BAL fluid thrombin activity No significant clinical benefit or change in biomarkers Reduced LPA-regulated gene expression in the skin Decreased mRSS in early SSc patients recently started on MMF (statistically not significant compared with patients treated with placebo and MMF) Safe and well tolerated in patients with SSc Reduced type I IFN signature in whole blood within 1 day and in skin after 7 days No results available

Inebilizumab [197] (Anti-CD19) Abatacept [150]

Phase II

Tofacitinib (JAK1/3 inhibitor) Pirfenidone [198]

Phase I/II Phase II

Lanifibranor (Agonist) Lenabasum** (Agonist) Nintedanib [48]

Phase II

No results available Still controversial, but effective for mRSS and/or ILD in several studies Positive correlation between baseline plasma cell gene signature with MRSS improvement No statistically significant change in mRSS compared with placebo No results available Safe and tolerated in patients with SSc‐ILD, even in combination with MMF No results available

Phase II Phase III Phase III

Satistically significantCRISS score improvement No results available Reduced annual rate ofFVC decline vs placebo

Phase I

TGF-β, transforming growth factor-β; mRSS, modified Rodnan total skin thickness score; ILD, interstitial lung disease; BAL, bronchoalveolar lavage; LPA-1, lysophosphatidic acid receptor 1; LPA-3, lysophosphatidic acid receptor 3; BAFF, B cell activating factor; MMF, mycophenolate mofetile; PPAR, peroxisome proliferatoractivated receptors; CB2, cannabinoid receptor type 2; PDGF, platelet-derived growth factor; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; FVC, functional vital capacity, CRISS: Combined Response Index of the diffuse cutaneous Systemic Sclerosis. * Clinicaltrial.gov identifier: NCT02453256. ** Clinicaltrial.gov identifier: NCT02465437.

suppression, a widely-shared concern of TGF-ß blockade is that global inhibition could be complicated by considerable toxicity [29]. A theoretically attractive alternate strategy to block TGF-ß is to exploit the tissue-restricted distribution of the TGF-ß-activating integrins. Indeed, a humanized antibody that targets integrin avß1, which is selectively up-regulated in injured tissue, blocked latent TGF-ß activation, and is currently in clinical trial (ClinicalTrial.gov NCT01371305). A recent study additionally suggested that the IL-6 cytokine family member IL11, a putative downstream mediator for the profibrotic effects of TGF-ß, could be effectively targeted for fibrosis therapy using antibodies [20].

[41]. There is also evidence that TGF-ß, PDGF and other profibrotic signals can activate intracellular PI3 kinase pathways that are mediated via the mTOR signaling complex. Indeed, inhibiting the mTOR complex activity using rapamycin has been evaluated in preclinical models of SSc and fibrosis, and shows modest efficacy, with reduced cellular proliferation and ECM production. Small clinical trials of rapamycin have shown good tolerability in SSc [42]. In view of elevated expression and activity of mTOR kinases demonstrated in SSc, mTOR inhibition, possibly by targeting both mTORC1 and mTORC2 complexes, appears to be a reasonable therapeutic strategy, and randomized controlled trials in this setting are awaited. Since as discussed above, most of these intracellular relay factors for TGF-ß responses can be blocked using pharmacological agents, TGF-ß signal transduction represents a potential therapeutic target in SSc. In contrast to intracellular TGF-ß, fresolimumab is a humanized anti-TGF-ß antibody that binds to and neutralizes all three TGF-ß isoforms. In a small open-label short-term clinical trial, fresolimumab treatment in early diffuse cutaneous SSc (dcSSc) was associated with skin improvement accompanied by a decrease in tissue levels of thrombospondin (TSP) and cartilage oligomeric matrix protein (COMP) [43]. Novel approaches to pharmacologically blocking TGF-ß ligand or signaling in both isoform-specific and pan-TGF-ß manners are under development. In view of the well-known homeostatic roles of TGF-ß in immunity, wound healing and tumor

2.1.3. Kinase inhibition Since a number of receptor and nonreceptor tyrosine kinases are implicated in SSc, inhibiting their activity might be beneficial. However, clinical trials of the c-Abl inhibitor imatinib, and other tyrosine kinase inhibitors such as nilotinib and dasatinib, showed only modest clinical efficacy and poor tolerability [44–47]. Of considerable interest is the multi-kinase inhibitor nintedanib (Vargatef) which blocks receptors for fibroblast growth factor, vascular endothelial cell growth factor (VEGF) and PDGF. Nintedanib treatment was recently shown to slow the decline in forced vital capacity in patients with SSc-associated interstitial lung disease [48]. Based on encouraging results from this large randomized clinical 4

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Pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) is a synthetic antifibrotic that was recently approved by the FDA for the treatment of IPF. Pirfenidone inhibits myofibroblast differentiation and blocks TGFß and HH signaling and STAT3 activation [61,62]. A randomized clinical trial is currently underway to evaluate the efficacy of pirfenidone (in combination with mycophenolate) in patients with SSc-ILD.

trial, nintedanib was recently granted FDA approval, thus becoming the first anti-fibrotic therapy in SSc. However, in light of its cost and associated side effects, particularly diarrhea, the precise role, timing and duration of nintedanib therapy in the management of patients with SSc remain to be clarified. 2.1.4. Additional targets for anti-fibrotic therapy in SSc Additional soluble mediators implicated in SSc include IL-6 and oncostatin M, both of which signal via gp130 receptors and the JAKSTAT pathway, PDGF, and the matricellular growth factor previosuly called connective tissue growth factor (CTGF) nut more recently called CCN2) [49]. An on-going randomized clinical trial is evaluating oncostatin M blockade in SSc (NCT03041025), while a clinical trial using an anti-CTGF antibody showed efficacy in retarding the decline in FVC in patients with idiopathic pulmonary fibrosis (IPF) (Clinicaltrials.gov NCT01890265). Cell-intrinsic factors can serve as rheostats to regulate fibrotic signal amplitude or duration in a cell-autonomous manner. One such factor is the nuclear receptor and transcription factor peroxisome proliferator activated receptor-gamma (PPAR-gamma). The activity of PPARgamma is potently induced by anti-diabetic drugs, as well as cannabinoid receptor 2 (CB2) agonists. Pharmacological PPAR-gamma activation has anti-fibrotic effects in vitro, and can reverse organ fibrosis in preclinical models of SSc [28]. SSc is associated with genetic variants of PPAR-gamma, and the expression of PPAR-gamma is reduced in SSc patient biopsies [50,51]. A small molecule that activates all three isoforms of PPAR (IVA337, lanifibranor) was shown to mitigate fibrosis in multiple preclinical models of SSc [52]. The serine protease thrombin stimulates fibroblast proliferation and myofibroblast transition through protease-activated receptor 1 (PAR-1) [53]. Thrombin levels are elevated in SSc patients [53], and the thrombin inhibitor dabigatran attenuated organ fibrosis in mice [54]. A pilot study to test safety and potential efficacy of non-hemostatic low-dose dabigatran in SSc is underway (Clinicaltrials.gov NCT02426229).

2.1.7. Additional profibrotic mediators as targets in SSc The IL-6 cytokine family includes IL-6 oncostatin M and IL-11.IL-6, a central regulator of acute phase responses, signals through the gp130 IL-6 receptor heterodimer directly or in trans, leading to activation of the intracellular JAK/STAT pathways. IL-6 levels are elevated in SSc and correlate with the extent of skin involvement. Mice that are IL-6deficient, or treated with anti-IL-6R antibody, showed reduced inflammation and fibrosis [63,64]. IL-6 blockade using tocilizumab in patients with early-stage SSc improved skin scores and reduced the loss of lung function [22,23,65]. Levels of circulating and tissue IL-4 and IL13, which are type 2 cytokines, are also elevated in SSc patients. Mice with genetic deletion of IL-13, are protected from fibrosis [66]. These observations provide a rationale for future clinical trials using bispecific antibodies targeting both IL-4 and IL-13 in SSc. Lysophosphatidic acid (LPA) is a pleiotropic bioactive lipid mediator generated via hydrolysis of membrane phospholipids. At sites of injury LPA accumulates and signals through G-protein-coupled receptors, with LPA1 relatively specific for fibroprogenitor cells. LPA stimulates mesenchymal cell migration, ECM production, differentiation and survival, and additionally may also promote latent TGF-ß activation. A small molecule LPA1 receptor antagonist showed no significant clinical benefit or change in biomarkers in a short-term trial in early-stage SSc [67]. However, transcriptome analysis of the skin showed reduced LPA-regulated gene expression, which implies effective target engagement [67]. 2.1.8. Therapeutic targeting the cannabinoid receptor axis in SSc Cannabinoids comprise a family of pleiotropic arachidonic acidderived small molecules. There are three types of cannabinoids: endocannabinoids, botanical (phytocannabinoid), and synthetic small molecules [68]. Cannabinoids signal via two distinct G-protein-coupled transmembrane receptors. The CB1 receptors are expressed mainly on neurons and elicit psychoactive effects. In contrast, CB2 receptors are expressed on both immune and tissue-resident stromal cells and are associated with both anti-inflammatory and anti-fibrotic effects. The synthetic cannabinoids ajulemic acid and quinol are specific for CB2 and show minimal psychoactive effect. These compounds promote proresolution mediators PGJ2 and LXA4 at low doses, and PPAR-gamma at higher doses, which might explain their anti-fibrotic effect [69,70]. A Phase 2 clinical trial of ajulemic acid (aka resunab or lenabasum) in SSc showed clinical efficacy (clinicaltrials.gov NCT02465437), and a large clinical trial is underway (clinicaltrials.gov. NCT03398837).

2.1.5. Reactive oxygen species Oxidative stress results from the imbalance of oxidant and antioxidant factors and directly contributes to fibrosis via DNA damage and amplification of TGF-ß and Wnt responses [32,55]. Explanted SSc fibroblasts express elevated levels of the oxidative enzyme NOX4, a direct TGF-ß target, which together with mitochondrial activity generate ROS. The small molecule GKT137831 blocks NOX4 activity and attenuates fibrotic responses in SSc fibroblasts. Additionally, both the synthetic triterpenoid CDDO (bardoxolone methyl) and pirfenidone have anti-oxidant activity, which might contribute to their therapeutic anti-fibrotic activity [56]. These observations suggest that attenuating ROS generation and mitigating oxidative stress in SSc might have beneficial effects. 2.1.6. Developmental pathways and fibrosis Developmental pathways mediated via the morphogens Wnt, Notch and Sonic hedgehog (SHH) appear to be (re)activated in SSc, suggesting their direct pathogenic roles [49]. Nuclear (activated) β-catenin and its target gene Axin2, markers for activated Wnt signaling, were prominent in skin and lung biopsies from SSc patients, as well as in animal models of disease [57,58]. In a recent randomized placebo-controlled pilot study, short-term treatment of SSc patients with a topical ß-catenin inhibitor C82 was associated with changes in adipogenic gene expression in the lesional skin, suggesting effective blockade of Wnt/ß-catenin activity (target engagement), but was not accompanied by a significant reduction in the skin score [59]. The morphogen Hedgehog (HH) signals via the patched (PTC) membrane receptor localized to primary cilia to activate Smoothended and Gli-mediated fibrotic transcription [60]. Demonstration of aberrant HH signaling in SSc biopsies suggests that this pathway represents a potential therapeutic target in SSc [49].

2.2. The role of the immune system/inflammation in SSc fibrosis Fibrosis generally emerges as a consequence of chronic inflammation [71]. Therefore, the pathways leading from tissue damage to inflammation and from inflammation to fibrosis represent potential therapeutic targets. Currently available anti-fibrotic therapies mainly focus on reducing the proinflammatory behavior of cellular mediators and/or inhibiting cell proliferation using non-specific immunosuppressive agents, but recent studies have drawn attention to new therapeutic targets driving fibrosis-linked innate and adaptive immune responses (Table 1). 2.2.1. Innate immune system The innate immune system comprises a network of cells including monocytes/macrophages, neutrophils, natural killer (NK) cells, natural killer T (NKT) cells, innate lymphoid cells (ILCs) and dendritic cells 5

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in both tissue fibrosis and repair needs further study, these clinical and basic findings suggest that targeting mast cells and related signaling pathways may be a potential therapeutic approach against SSc.

(DCs) that together mediate the earliest interactions with pathogens [72]. Innate immunity is also responsible for non-specific responses to endogenous host molecules, DAMPs, generated at the site of tissue injury, which include proteins such as high-mobility group box 1, HSPs, S100 proteins and ECM fragments such as EDA-fibronectin, tenascin-C and hyaluronan, and non-protein molecules such as adenosine triphosphate, uric acid, heparin sulfate and self-nucleic acids. DAMPs are recognized by a variety of cell surface, endosomal and cytosolic pattern recognition receptors (PRRs). These include Toll-like receptors, Nodlike receptors, RIG-I-like receptors and others. Persistence of a sterile inflammation can convert a self-limited repair response to non-resolving pathological fibrosis, suggesting a critical role of DAMP-induced innate immune signaling in fibrosis development and perhaps even more important, fibrosis persistence [73]. Supporting this idea, inflammatory cytokines produced by innate immune cells are abundantly expressed in the affected organs of fibrotic disorders, and expression of their receptors, including TLRs, is elevated. Although the initial events triggering sterile inflammation in pathological fibrosis remain unclear, an imbalance in DAMP release and/or PRR signaling is implicated in sustained production of inflammatory mediators by innate immune cells, such as type I interferon (interferon [IFN]-α/β, IFN-I) from plasmacytoid dendritic cells (pDCs) and TGF-ß,IL-4, IL-6 and IL-13from macrophages and mast cells.

2.2.1.3. Conventional and plasmacytoid dendritic cells and the Interferon Response. DCs are stratified into 2 subsets; conventional DCs (cDCs) and plasmacytoid DCs (pDCs).Conventional DCs take up internal and external antigens, migrate to lymph nodes, and present antigens to naïve T cells. Thus, cDCs serve to bridge innate and adaptive immunity. Under physiological conditions, cDCs continuously undergo homeostatic maturation, which transport cutaneous self-antigen to the T cell zones of draining lymph nodes and activate abortive program of autoreactive T cells escaping from central tolerance [87]. In this manner, cDCs play a protective role in autoimmune inflammatory conditions including fibrotic disorders, but their exact contributions to SSc pathogenesis are still largely unknown. pDCs are specialized in IFN-I secretion, and play a key role in the initiation of antiviral immune responses as well as sterile inflammation. In host defence IFN-I production by pDCs requires recognition of viral nucleic acids, such as single stranded RNA by TLR7 and unmethylated DNA with CpG-motifs by TLR9. pDCs also produce IFN-I in response to selfnucleic acids and consequently contribute to the development of autoimmune and inflammatory disorders. In SSc, genome-wide association studies have identified polymorphisms in genes involved in the regulation of IFN-I expression in pDCs, in particular IFNregulatory factor (IRF)-5, IRF-7 and IRF-8, and∼50 % of SSc patients harbor an IFN-I signature in their peripheral blood mononuclear cells and fibrotic skin [88]. pDCs are prominent in SSc skin and lung, which coincides with reduced frequency in the circulation [89,90]. Importantly, TLR9 is implicated in the pathogenesis of IPF, particularly in patients with aggressive disease [91], and lung tissue of SSc patients with interstitial lung disease (ILD) exhibits increased expression of IFN-I and IFN-I-related genes [92]. Furthermore, circulating pDCs from SSc patients secrete elevated levels of IFN-I, at least partially due to aberrant TLR8 expression [93]. Of interest, a recent phase I SSc clinical trial of anti-IFN-I receptor antibody, anifrolumab, showed reduction of IFN-related gene signature in involved skin [94]. Since pDC depletion ameliorates established bleomycin (BLM)-induced skin fibrosis, pDCs are critical even in the maintenance of skin fibrosis [93]. Overall, pDCs play a critical role in SSc pathogenesis, thus the restoration of aberrant pDC function may be a novel approach for preventing disease progression in SSc. However, the molecular mechanisms through which pDCs contribute to the disease require further investigation.

2.2.1.1. Macrophages. Macrophages play a major role in sterile inflammation by serving as key cellular mediators of tissue injury and repair [74]. Macrophages are highly plastic cells that undergo differentiation into either a pro-inflammatory M1 subtype (a classically activated subtype) or an anti-inflammatory M2 subtype (an alternatively activated subtype) according to their microenvironment. M1 macrophages are induced by the Th1 cytokines (IFN-γ) and TNFα,promoting inflammation, while M2 macrophages are induced by the Th2 cytokines (IL-4 and IL-13),inhibiting inflammation and healing wounds by producing anti-inflammatory cytokines such as IL-10 and TGF-β. Macrophages are the major source of TGF-β as well as myofibroblasts, and actively participate in matrix remodeling and collagen recycling, thus involved in the development of tissue fibrosis [75]. In SSc, a prominent M2 macrophage signature is observed in the skin and lung [76,77], and circulating levels of soluble CD163, a putative marker of M2 macrophages, are elevated and associated with poor clinical outcomes [78]. Importantly, IL-6 receptor blockade by tocilizumab reduces M2 macrophage signature in biopsied SSc skin samples [22]. Thus, macrophages themselves and related signaling pathways are potential candidates for new therapeutic targets. However, the mechanisms underlying aberrant M2 macrophage polarization and the precise pathways through which M2 macrophages contribute to tissue fibrosis remain unclear. In addition, the safety and efficacy of targeting a broad range of resident and recruited macrophages and/or their specific polarized subset and functions still remain unknown. Therefore, further basic and clinical studies are required to apply macrophage-targeting therapies in the clinical setting.

2.2.1.4. Other innate immune cells. Other innate immune cells such as NK and NKT cells and neutrophils display altered properties and phenotypes in the blood of SSc patients. NK cells from SSc patients possess a highly cytotoxic property against endothelial cells, and CD56+ cells (NK and NKT cells) from SSc patients with different stages differentially respond to TLR stimulation [95–98]. Neutrophils from SSc patients abundantly release neutrophil extracellular trap (NET) by-products [99]. With respect to ILCs, a heterogeneous group of cellular subsets that produce large amounts of T helper cell-related cytokines in response to innate stimulation, the ILC2 subset that produces Th2 cytokines is of particular interest. In a murine model of pulmonary fibrosis, IL-13 released by ILC2s is sufficient for collagen deposition by promoting the activation and differentiation of fibroblasts and macrophages toward a profibrotic phenotype [100]. In patients with SSc, it is still controversial whether ILC2 is increased in both the peripheral blood and the affected skin [101,102]. Regarding ILC1s and ILC3s, a previous study has demonstrated that the NKp44+ ILC3 population that produces IL-17 and IL-22 and the CD4 + ILC1 population that is potent producers of TNF and GM-CSF are increased in the peripheral blood of SSc patients compared to healthy controls [101]. At this moment, the data on the role of ILCs in SSc are limited,

2.2.1.2. Mast cells. Mast cells secrete histamine, cytokines and chemokines, and have attracted much attention as drivers of fibrosis. Studies in patients and preclinical models of SSc have demonstrated that mast cells infiltrate the fibrotic skin, and the degree of mast cell infiltration is positively associated with disease severity [79,80]. A major role of mast cells in the pathological tissue fibrosis is likely to serve as a potent source of TGF-β,IL-6, IL-4 and IL-13 [80–83]. This notion is supported by the following findings; (I) mast cells play a major role in inducing inflammation of the skin and the production of extracellular matrix by fibroblasts in transgenic mice that spontaneously develop skin fibrosis [81,84], (II) inhibitors of mast cell degranulation ameliorate skin fibrosis in tight-skin mice and graftversus-host disease model mice [85,86]. Although the role of mast cells 6

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patients, and implicated in collagen gene overexpression by fibroblasts [124]. Additionally, numerous autoantibodies targeting G-proteincoupled receptors have been detected in patients with SSc, and elucidation of their precise role in disease pathogenesis is eagerly awaited. Consistent with alteration of homeostasis and function in SSc B cells, B cell depletion therapy with rituximab, a chimeric anti-CD20 antibody, have been shown to improve or stabilize skin sclerosis and ILD in several open label studies and case-control cohorts [125–131]. There are currently ongoing randomized control trial (RCT)s of rituximab for SSc patients with polyarthritis, ILD and pulmonary arterial hypertension. Another option of B cell depletion therapy is anti-CD19 antibody, inebilizumab, the phase I clinical trial of which demonstrated the improvement of skin score at 12 weeks in SSc patients who presented with an elevated baseline plasma cell signature in the skin [132]. Also, the blockade of BAFF by anti-BAFF antibody, belimumab, revealed the improvement of skin score at one year in an RCT with early SSc patients recently started on mycophenolate mofetil, although the effect was not statistically significant [133]. Thus, the depletion or inactivation of aberrantly activated B cells is a potential therapeutic strategy of SSc.

and further studies are required to evaluate their roles in the pathogenesis of SSc. 2.2.2. Adaptive immune system The aberrant activation of adaptive immune system has caught attention in the pathogenesis of fibrotic disorders earlier than the innate immune system, yet the exact mechanisms driving this process are not fully understood. Increasing evidence demonstrate the abnormal activation of B cells and T cells in fibrotic disorders. 2.2.2.1. B cells. The role of aberrantly activated B cells in the pathology of fibrosis has been well studied in SSc. Numerous studies have demonstrated the alteration of B cell subsets in SSc, such as the decrease in memory B cells, plasmablasts and regulatory B cells, the increase in naïve B cells, the decrease in transitional B cells, as well as the functional abnormality of regulatory B cells [103,104]. As for cell surface markers, overexpression and hyperactivity of a positive regulator CD19 and low surface expression and functional impairment of a negative regulator CD22 are characteristic of SSc B cells [105,106], and the up-regulated expression of CD80 and CD86 on SSc memory B cells suggests their chronic activation [107]. B cell infiltration is prominent in affected organs, including the skin and lungs [108–110]. SSc patients with higher expression of CD19 in the bronchoalveolar lavage fluid are more likely to experience ILD progression [111]. Consistent with these clinical findings, a critical role of aberrantly activated B cells has been implicated in the development of SSc-like features in murine animal models. Cd19 transgenic mice exhibit hypergammaglobulinemia and autoantibody production due to the abnormal activation of B cells [112]. Tight-skin mice show hypodermal fibrosis, hypergammaglobulinemia, positivity of anti-nuclear antibody and anti-topoisomerase I antibody, and imbalance of mRNA expression profiles of Th1 and Th2 cytokines, such as the increase in IL-4, IL-6 and IL-10 and the decrease in interferon-γ, but both CD19 loss and B cell depletion by anti-CD20 antibody result in the reduction of these abnormalities [113,114]. In addition to antibody production, B cells play multifaceted roles in immune system, such as cytokine production, antigen presentation, macrophage differentiation and activation, and lymphoid tissue development [115]. B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), two important factors regulating B cell survival, maturation and activation, are type II transmembrane proteins produced by B cells that are proteolytically cleaved and released as soluble forms. Circulating levels of BAFF and APRIL are increased and associated with disease severity in SSc [116]. In SSc animal models, BAFF antagonists attenuate skin and lung fibrosis possibly by restoring the balance of IL10-producing regulatory and IL-6-producing effector B cells [117]. Although the contribution of disease-specific autoantibodies in SSc pathogenesis remains uncertain, potential pathogenic roles have been demonstrated for anti-topoisomerase I and anti-nucleolar antibodies. Both antibodies react with nuclear antigens derived from apoptotic cells and these immune complexes induce IFN-I production from pDCs [118]. Since IFN-I induces maturation of immature pDCs and development of autoreactive T cells and B cells, these anti-nuclear antibodies may be involved in the process of autoimmune activation in SSc. The other group of autoantibodies with putative pathogenic roles includes anti-endothelial cell antibodies, which are estimated to occur in 44–84 % of SSc patients and may induce apoptosis of endothelial cells [119]. Nuclear antigens derived from apoptotic endothelial cells may lead to an autoimmune reaction through the production of IFN-I from activated pDCs [120,121]. Anti-fibrillin-1 antibodies are detectable in more than 50 % of SSc patients and can activate fibroblasts and stimulate release of TGF-β [122]. Antibodies to matrix metalloproteinase-3 may also occur in a high proportion of patients, preventing the degradation of excessive collagen [123]. A putative pathogenic autoantibody to PDGF receptor (PDGFR) has been recognized in SSc

2.2.2.2. T cells. In the early stage of SSc, a number of T cells accumulate in lesional skin, the majority of which express activation markers, such as CD69 [134]. CD69 seems to regulate interaction of T cells with other cells, including fibroblasts and monocytes, by promoting T cell migration. Thus activated T cells may affect the activation status of SSc fibroblasts by regulating the release of pro- and anti-fibrotic cytokines. A series of studies have revealed the immune polarization skewed towards Th2/Th17 responses during the early and progressive stage of dcSSc [77,79,135,136]. In the early stage of dcSSc, serum IL-6 and IL10 levels are significantly elevated, while decreased to normal levels in the late stage of dcSSc characterized by the improvement of skin sclerosis [137]. IL-4 keeps normal levels in the early stage of dcSSc, but decreases with the resolution of skin sclerosis. In contrast, serum IL-12 levels are decreased in the early stage of dcSSc, then gradually increase in parallel with disease duration and finally reach significantly higher levels than normal controls in the late stage of dcSSc [135]. Thus, immune polarization generally shifts from Th2 to Th1 in parallel with the resolution of skin sclerosis in SSc. With respect to Th17 cytokines, the expression levels of IL-17A, IL-21, and IL-22, but not IL-17 F, are increased in the lesional skin of early dcSSc [138,139]. Furthermore, the percentage of circulating Th17 cells and IL-17 production are elevated in peripheral blood mononuclear cells of SSc patients and the number of Th17 cells correlates with disease activity [136]. Of note, Th17 cell infiltration and IL-17A expression in the skin are increased and serum IL-17A levels correlate with the severity of skin fibrosis in BLM-treated mice [140]. Furthermore, loss of IL-17A, but not IFN-γ and IL-4, results in the reduction of BLM-induced skin fibrosis and IL-17A stimulation induces the expression of TGF-β and CTGF in NIH3T6 fibroblasts [141]. Therefore, IL-17A serves as a potent pro-fibrotic cytokine in the pathological skin fibrosis. Overall, the Th2/Th17-skewed immune polarization is likely associated with tissue fibrosis in SSc. Regulatory T cells (Treg) have also been implicated in the pathogenesis of SSc. For instance, skin-infiltrating Treg cells produce high amounts of IL-4 and IL-13, but not IL-17A [142]. Despite the controversy of Tregs in SSc, with reports of decreased, increased, or equal proportions of Tregs in the blood of SSc patients, most reports suggest that SSc Treg cells have impaired production of inhibitory cytokines and impaired suppression of effector T cells [143–146]. Importantly, elevated percentages of circulating Treg cells in SSc have been linked to more severe skin and lung disease, possibly reflecting impaired regulatory function of Tregs [145,147]. Based on data, selective T cell-targeted therapies may lead to clinical improvement in SSc. A small open-label study of basiliximab, a chimeric anti-IL-2 receptor CD25α chain antibody that blocks T and B 7

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remains to be met. The precise mechanism of action underlying effective immune reprogramming leading to clinical benefit in these interventions remains under investigation. Of note, substantial changes of adaptive immunity are reported; these include increase in T cell receptor diversity and expression of cytotoxic T-lymphocyte-associated protein 4 and glucocorticoid-induced tumor-necrosis-factor-receptorrelated protein (GITR) on Treg cells, and the restoration of Treg suppressive function [165,166]. A potential cellular therapy for SSc and other fibrotic disorders is local or systemic administration of mesenchymal stromal cells (MSCs) isolated from bone marrow or adipose tissue. MSCs modulate proliferation, activation and effector function of all immune cells through juxtacrine or paracrine mechanisms, and have potential to differentiate into various cell types required for tissue repair. Transplanted MSCs significantly reduce the total number of effector T cells at the site of tissue injury and attenuate Th1-, Th2-, or Th17-driven inflammation through the induction of anergy by affecting maturation and function of DCs, and/or by providing the exogenous programmed death-ligand 1 to engage the programmed death-1 expressed on effector T cells [167,168]. Previous case series demonstrated the efficacy of systemic administration of bone-marrow derived MSCs for SSc patients, such as the improvement of skin score, digital ulcers and gangrenes [169–171]. Adipose-derived stem cells (ASCs) are a subset of MSCs obtained easily from adipose tissues and possess many of the same regenerative properties as other MSCs [172]. A recent RCT disclosed that ASC injection into the fingers was well tolerated and associated with a trend toward improvement of hand function at 48 weeks, although the change was not statistically significant compared with placebo-treated patients [173]. Exciting recent findings indicate a novel potential cell-therapybased approach to fibrosis. The investigators generated chimeric antigen receptor T cells (CAR-T) specifically recognizing FAP, a marker of activated fibroblasts [174]. Infusion of CAR-T cells in mice mitigated experimental cardiac fibrosis, presumably via elimination of FAP + activated fibroblasts in the damaged tissues. Thus, various cellular therapy approaches are promising options to improve or stabilize disease, but further clinical studies are required to confirm their efficacy and safety in SSc.

cell activation, demonstrated significant improvements in skin disease and lung function [148]. Abatacept, a soluble recombinant fusion protein that binds to CD80 and CD86 and blocks T cell co-stimulation by preventing their interaction with CD28, improved SSc-related arthropathy, but not skin score, muscle involvement and ILD in an observational study based on the EUSTAR database [149]. Another phase II RCT of abatacept revealed no significant effect on skin score at 52 weeks in early dcSSc [150]. Considering the predominance of Th2/ Th17-skewed immune polarization in early SSc, antibodies targeting IL4, IL-13, IL-17A, IL-23 and their receptors are interesting new candidate targets for the treatment of SSc. Finally successful autologous hematopoietic stem cell transplantation in SSc has been associated with an increased in the number of Tregs post-transplant, as well as an increase in the diversity of T cell receptor repertoires. 2.2.3. Chemokines and Janus kinase signaling Chemokines recruit innate and adaptive immune cells to the site of tissue injury to promote inflammation and tissue repair [151]. Several chemokines are elevated in affected organs and may contribute to the development of SSc. Serum and/or tissue levels of CCL2, CCL3, CCL5, CCL7, CCL18, CXCL4, CXCL6, CXCL8, CXCL9, CXCL10, CXCL13 and CX3CL1 are elevated in SSc and some of them correlate with disease severity [152–156]. The inhibition of chemokines and their receptors may therefore represent a unique therapeutic strategy for preventing the development and/or progression of SSc fibrosis. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is the principal signaling mechanism for a wide range of cytokines and growth factors. In mammals, the JAK tyrosine kinases comprise of four members, JAK1, JAK2, JAK 3 and Tyk2. JAK activation occurs upon ligand-mediated receptor multimerization, and the activated JAKs subsequently prompt the phosphorylation, dimerization and nuclear translocation of STATs that function as transcription factors regulating the expression of target genes. There is considerable pharmacological interest in JAK inhibitors that target the IFNI pathway in a variety of inflammatory diseases [157]. In SSc, JAK2 is activated in the affected skin of SSc patients, and the blockage of JAK2 attenuates the pro-fibrotic effects of TGF-β on fibroblasts [158]. STAT-3 plays a pivotal role in TGF-β-induced myofibroblast differentiation and collagen production [159], and single nucleotide polymorphisms of STAT-4 are associated with SSc [160,161]. Thus, the JAK/STAT signaling is likely a potential therapeutic target in SSc. Indeed, a recent case report documented an efficacy of tofacitinib, the JAK1/3 inhibitor, for skin disease, digital ulcers and arthritis in a patient with SSc [162]. We have recently shown that JAK1/2/3 are activated in SSc skin and lung biopsies, as is STAT3. Targeting JAK signaling using tofacitinib has potent anti-fibrotic effect on multiple mouse models of SSc [163]. Based on these and the results, clinical trials of tofacitinib and similar JAK inhibitors are under way in SSc.

3.2. Targeting the pathological mechanobiology of fibrosis Emerging data highlight the critical pathogenic role of the physical tissue microenvironment in SSc [26]. Fibrosis is accompanied by increasing stiffness of the ECM, for example from 1 kPa in healthy lung up to 100 kPa, approximating the stiffness of bone, in fibrotic lungs [175]. Fibroblasts can sense and respond to mechanical cues from their microenvironment via integrins and the focal adhesion complex, FAK, Rho-associated protein kinase (ROCK), and YAP and TAZ. These pathways elicit extensive transcriptional reprogramming and activation of resident fibroblasts. In a stiff microenvironment, integrins promote activation of latent TGF-ß, driving further fibrogenesis. The mechanotransduction pathways underlying these responses represent potential therapeutic targets. Clinical trials of monoclonal antibodies blocking integrins and small molecules to block ROCK2, FAK and MRTFA/B are under investigation for fibrotic conditions.

3. Emerging targets and potential therapies 3.1. Cellular therapies Cellular therapies such as administration of autologous or allogeneic stem cells is emerging as potential strategies to achieve a therapeutic effect in selected SSc patients. Given that SSc has an autoimmune-related pathogenesis, particularly in its early stage, immunoablation by high-dose immunosuppressive therapy accompanied with autologous hematopoietic stem cell transplantation (HSCT) is a plausible potential therapy. Three RCTs were conducted to assess the clinical efficacy of autologous HSCT in SSc patients with rapidly progressive skin and internal organ involvement [164]. In each of these clinical trials, HSCT was associated with improvement of both overall and progression-free survival, as well as skin score, lung function and other indicators of disease activity. To date, however, consensus on optimal patient selection, pre-transplant evaluation and post-transplant management

3.2.1. Targeting cellular aging biology, senescence and metabolic dysregulation Increasing evidence highlights the potential pathogenic role of cellular aging and metabolic reprogramming in a variety of fibrotic conditions as well as in SSc [25,176]. Cellular senescence is prominent in age-associated chronic disease states, and is the biological hallmark of aging [177]. Senescent cells have permanently exited the cell cycle, and during biological aging are efficiently cleared by the immune system. Transient cellular senescence has homeostatic functions, including resolution of fibrosis [178]. However, as senescent cells increasingly accumulate in aging (or damaged) tissue due to increased production or 8

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Table 2 Anti-fibrotic therapies targeting epigenetic mechanisms – experimental data with SSc fibroblasts. Types of therapies

Reagents

The types of fibroblasts

Mode of action

DNMT inhibitor

5-aza-2′-deoxycytidine 5-aza-2′-deoxycytidine Trichostatin A Resveratrol DZNep GSKJ4

Lung fibroblasts Dermal fibroblasts Dermal fibroblasts Dermal fibroblasts Dermal fibroblasts Dermal fibroblasts

PTGS2 upregulation [199] Up-regulation of PARP1 and KLF5 [200,201] WIF1 upregulation [202] Decreased expression of collagen, α-SMA and fibronectin extracellular domain A [191,203] LRRC16A up-regulation [204] Suppression of TGF-β-induced FRA2 expression and cell migration [205]

HDAC inhibitor Sirtuin (HDAC) activator HMT inhibitor HDM inhibitor

DNMT, DNA methyltransferase; HDAC, histone deacetylase; HMT, histone methyltransferase; HDM, histone demethylase; DZNep, 3-Deazaneplanocin A; PTGS2, Prostaglandin-Endoperoxide Synthase 2; PARP1, Poly[ADP-ribose] polymerase-1; KLF5, Krüppel-like factor 5; WIF1, WNT inhibitory factor 1; FRA2, Fos-related antigen 2.

3.3. Epigenetics

diminished clearance, they exert potentially deleterious effects. Most prominently, they secrete a suite of proinflammatory and profibrotic cytokines such as IL-6 and TGF-ß (a secretome called senescence associated secretory pattern, SASP), which promotes persistent inflammation, elicits tissue damage and triggers further senescence in neighboring cells. Clearing senescent cells via pharmacological targeting is called senolysis, and has been shown to mitigate pathological fibrosis, where aberrant accumulation and impaired clearance of senescent cells have been identified [179]. A variety of senolytic agents with distinct mechanisms of action are currently being developed or already under investigation. Prominent are inhibitors of the antiapoptotic protein B-cell lymphoma 2,and the tyrosine kinase inhibitor dasatanib [180]. When combined with the flavonoid quercetin in a senolytic “cocktail”, dasatinib reduced inflammation and attenuated experimental lung and cardiac fibrosis in mice. A recent small and open-label clinical trial of dasatinib in patients with SSc-ILD showed that improvement in skin and lung fibrosis in some patients that was accompanied by a significant decrease in a consensus senescence gene signature in the skin [46]. These observations, albeit preliminary, provide theoretical support for therapies targeting cellular senescence to mitigate fibrosis in SSc. Metabolic alterations are increasingly recognized in fibrosis and implicated in pathogenesis [25]. Prominent metabolic changes associated with fibrosis include the switch to aerobic glycolysis (the Warburg effect), oxidative stress, dysregulated fatty acid oxidation and synthesis, organismal NAD depletion and mitochondrial dysfunction. Depletion of NAD, a critical co-factor for sirtuins and other cellular enzymes, is prominent in biological aging, and is due primarily to elevated expression of NAD hydrolase CD38. Similar decline in NAD is seen in fibrosis and in SSc [181]. These observations highlight existing parallels between the biological processes driving aging and fibrosis in SSc, and suggest novel avenues for targeted therapies aiming to restore NAD homeostasis [182]. Boosting organismal NAD by dietary supplementation using NAD precursors such as nicotinamide riboside (NR), and/or by blocking CD38 NADase activity using monoclonal antibodies or small molecule is highly effective in restoring metabolic healthy and attenuating age-related pathologies including fibrosis, in rodent models [183]. Therefore, targeting the NAD metabolism and boosting NAD levels represent potential anti-fibrotic treatment strategies, with the added advantage of potentially low cost and favorable safety profiles. One of the most widely used drugs targeting metabolic pathways is the antidiabetic agent metformin, discovered over 50 years ago. Metformin has broad effects on cellular metabolism that are mediated via AMP-activated protein kinase (AMPK), as well as AMPK-independent pathways. By transiently inhibiting the mitochondrial electron transport chain, metformin induces a decrease in cellular energy state and ATP production, causing AMP kinase activation. In turn AMP kinase has potent anti-inflammatory and anti-fibrotic activities. Recent studies highlight the beneficial effects of metformin and AMP kinase including broad anti-fibrotic activities [184–187]. Furthermore, by inducing myofibroblast differentiation into quiescent lipofibroblasts, metformin promotes resolution of fibrosis.

Transient environmental exposures induce cell type-specific epigenetic modifications, resulting in heritable changes in gene expression independent of alterations in DNA sequence (epigenetic reprogramming).These include DNA methylation, chromatin histone modification, and RNA interference by non-coding microRNAs (miRNAs) and long non-coding RNAs (lncRNAs).Each of these processes appears to play a role in fibrosis [188]. As an example, the cell-autonomous sustained elevation of the profibrotic cytokine TGF-ß2 in SSc fibroblasts is due to reversible epigenetic changes in the enhancers of the TGF-ß2 gene, and can be effectively targeted by a variety of strategies to normalize the locusspecific chromatin landscape [34]. Epigenetic modifications are dynamic and reversible, thus targeting the enzymes driving these changes may result in reduced disease activity and promote regression of fibrosis. The key enzymes catalyzing epigenetic modifications are DNA methylases (DNMTs), ten eleven translocation (TET) proteins, histone acetyltransferases (HATs), histone deacetylases (HDACs) including sirtuins, histone methyltransferases (HMTs) and histone demethylases (HDMs). The contribution of DNA methylation and histone modification to SSc development has been extensively investigated in the activation of immune cells and fibroblasts (Table 2). The sirtuins represent an important class of histone deacetylases. These t These ubiquitous and pleiotropic NAD-dependent enzymes have key roles in regulating a number of age-related biological processes [189]. The observation that SIRT expression and/or enzymatic activity is impaired in aging and in fibrosis, point to novel therapeutic strategies targeting SIRT activation. Indeed, such approaches have been amply shown to exert beneficial effects in preclinical models of fibrosis and SSc [190,191]. Numerous agents modulating epigenetic pathways are currently in preclinical studies, or already in clinical use particularly in hematopoietic disorders and solid tumors [192]. However, current chromatinmodifying agents generally lack target or cell type-specificity, thus promoting global demethylation and subsequently causing unwanted deleterious effects. Therefore, gene-specific control of DNA methylation is necessary for the success of DNA methylation-modifying drugs and to avoid side effects. Further comprehensive studies of the dynamic nature and pathogenetic significance of cell type-specific alterations involved in the pathogenesis of SSc will be beneficial in developing novel strategies to prevent, stabilize or even reverse the fibrotic process. A number of miRNAs are dysregulated in SSc fibroblasts (Table 3), as well as endothelial cells and immune cells. Importantly, administration of anti-fibrotic miRNA mimics that are chemically modified improves skin and lung fibrosis in preclinical models [188,193]. Therefore, targeting the reduced anti-fibrotic miRNAs with mimics would be a potential treatment option if the technology to deliver these miRNAs to the target cell type is established. On the other hand, little is known about the function of lncRNAs and data on their expression and potential roles are limited in SSc.

9

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Table 3 Non-coding RNAs implicated in the activation of SSc fibroblasts. Non-coding RNA

Expression level in SSc

Target mRNA

Cells/tissues

Let-7a [206] miR-21 [207] miR-29 [208] miR-30b [209] miR-150 [210] miR-196a [211] miR-202-3p [212] miR-155 [213,214,215] miR-130b [216] miR-483-5p [217] miR-4458 [218] miR-18a [218] miR-135b [219] miR-142-3p [220] miR-150, miR-196a [220] miR-125b [221] miR-542-3p [222] TSIX (lncRNA) [223] OTUD6B-AS1 (lncRNA) [224]

Decreased Increased Decreased Decreased Decreased Decreased Increased Increased Increased Increased Increased Decreased Decreased Increased Decreased Decreased Decreased Increased Decreased

Type I collagen α-SMA, COL1A1, COL1A2, Smad7 COL1A1, COL3A1 COL1A2, PDGFR-β Type I collagen, Smad3, Integrin β3 Type I collagen MMP1 CK1α, SHIP1 PPARγ Fibrosis-related genes Type I collage CTGF STAT6 ND ND BAK1, BMF, BBC3 Survivin Type I collagen ND (increased cyclin D1 expression in a sense gene-independent manner)

Dermal fibroblasts Dermal fibroblasts Dermal fibroblasts Dermal fibroblasts Dermal fibroblasts Dermal fibroblasts Skin tissue and dermal fibroblasts Skin tissue and dermal and lung fibroblasts Skin tissue and dermal fibroblasts Serum and serum exosomes Dermal fibroblasts Dermal fibroblasts Dermal fibroblasts, serum and monocytes Dermal fibroblast exosomes Dermal fibroblast exosomes Skin tissue and dermal fibroblasts Dermal fibroblasts Skin tissue, dermal fibroblasts and serum Skin tissue

4. Conclusions

Acknowledgments

While the complex and incompletely-understood pathogenesis and exceptional degree of clinical and molecular heterogeneity of SSc pose unique challenges for the development of effective therapies, recent advances have considerably deepened our understanding of the biological processes underlying particular disease endotypes. There now exists an unprecedented robust pipeline of promising small molecules, biological and cellular therapies that target the immune system, fibrotic pathways, epigenetic processes, mechanotransduction, altered metabolism and cellular senescence. Additionally, both in silico and in vitro screening of existing drug libraries for repurposing hold significant promise for the development of SSc therapies. Notwithstanding this encouraging progress, numerous unanswered questions remain in the field of SSc therapeutics. Given the variability in the tempo of the development and progression of organ involvement, the precise timing for initiation and duration of disease-modifying therapy is unclear. While traditional immunosuppressive agents are conventionally deemed to be most effective early in the course of the disease, newer agents (e.g., anti-fibrotics) may have a role at later disease stages. Furthermore, as with other chronic rheumatic diseases, such as RA and lupus, concurrent or sequential combination therapies may improve outcomes while limiting toxicity in SSc patients refractory to monotherapy. There is compelling indications for tailoring SSc interventions based on specific patient factors and disease stage that may predict disease severity, activity and progression, treatment response, or risk of adverse effects [19]. Recent advances in basic and clinical science have created innovative opportunities to customize patient-specific therapeutic approaches based on the integration of genetic, clinical and biological data and thereby develop precision medicine for SSc [194]. In addition, tremendous strides have been made in refining clinical trial design and cohort enrichment strategies, and incorporating biomarkers to reduce the risk of failure in clinical trials [195]. In the future, therapeutic SSc trials are expected to incorporate stratification strategies to optimize our ability to detect treatment effects among specific patient subgroups based on clinical characteristics and biomarkers. There is now more hope than ever that effective treatment paradigms that not only reduce mortality and disease burden, but also yield sustained and meaningful improvements in quality of life, might finally be within reach.

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Funding Supported by grants from the NIH (AR074997) and the Rheumatology Research Foundation. 10

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