Transforming growth factor β--at the centre of systemic sclerosis.

REVIEWS Transforming growth factor β—at the centre of systemic sclerosis Robert Lafyatis Abstract | Transforming growth factor β (TGF‑β) has long been implicated in fibrotic diseases, including the multisystem fibrotic disease systemic sclerosis (SSc). Expression of TGF‑β-regulated genes in fibrotic skin and lungs of patients with SSc correlates with disease activity, which points to this cytokine as the central mediator of pathogenesis. Patients with SSc often develop pulmonary arterial hypertension (PAH), a particularly lethal complication caused by vascular dysfunction. Several genetic diseases with vascular features related to SSc, such as familial PAH and hereditary haemorrhagic telangiectasia, are caused by mutations in the TGF‑βsensing ALK‑1 signalling pathway. These observations suggest that increased TGF‑β signalling causes both vascular and fibrotic features of SSc. The question of how latent TGF‑β becomes activated in local SSc tissues is, therefore, central to the understanding of SSc. Both TGF‑β1 and TGF‑β3 can be activated by integrins αvβ6 and αvβ8, whose upregulation in bronchial epithelial cells can activate TGF‑β in SSc lungs. Other αv integrins, thrombospondin‑1 or altered TGF‑β sequestration by matrix proteins might be important in other target tissues. How the immune system triggers this process remains unclear, although links between inflammation and TGF‑β activation are emerging. Together, these observations provide an increasingly secure framework for understanding TGF‑β in SSc pathogenesis. Lafyatis, R. Nat. Rev. Rheumatol. advance online publication 19 August 2014; doi:10.1038/nrrheum.2014.137

Introduction

Systemic sclerosis (SSc) is an autoimmune and/or auto inflammatory disease that affects many different organs and has strikingly pleomorphic clinical features. The clinical features of SSc prominently involve both con nective tissues (leading to skin, lung and heart fibrosis) and vascular tissues, (leading to digital ischaemia, sclero derma renal crisis, pulmonary arterial hypertension [PAH], telangectasias and gastric antral vascular ectasia). The two best-studied tissues in SSc patients are the skin and lung: the lung shows changes leading to pulmonary fibrosis, PAH or both; the skin develops perivascular inflammation associated with deposition of extracellular matrix proteins in the dermis and conversion of subcu taneous fat into fibrotic connective tissue. Expression of TGF‑β-regulated genes in fibrotic (skin and lung) tissues from patients with SSc correlates with disease activity, and suggests that this cytokine is a central mediator of pathogenesis. However, TGF‑β has distinctive pleio tropic activities that affect most cell types in the body, often with disparate or even opposing effects depending on the target cell. Virtually all cell types involved in the

Boston University School of Medicine, E5 Arthritis Centre, 72 E. Concord Street, Boston, MA 02118, USA. [email protected]

Competing interests R.L. declares that he has acted as a consultant for Actelion, Akros, Amira, Biogen, Bristol Myers Squibb, Celdara, Celgene, Celltex, Dart Therapeutics, EMD Serono, Genentech, Genzyme, Idera, Inception, Intermune, Lycera, Medimmune, Novartis, Precision Dermatology, PRISM, Promedior, Regeneron, Roche, Sanofi, Aventis, Shire, UCB and Zwitter, and that he has received grants from Genentech, Genzyme, Human Genome Sciences, Regeneron, Sanofi, Shire, and UCB.

pathogenesis of SSc respond to TGF‑β (including vascu lar, immune and connective tissue cells), and the patho genetic effects of TGF‑β in these cell types are, therefore, complex and hard to predict. This Review posits that the basic defect in SSc is dif fusely increased TGF‑β activity, leading to the vascular and connective tissue changes seen in these patients. New insights into matrix structure, vascular biology, TGF‑β signalling and specific TGF‑β-related genetic disorders are discussed that provide a mechanistic understanding of how TGF‑β could lead to such heterogeneous clini cal features. As most evidence supports the involvement of TGF‑β itself rather than other TGF‑β superfamily members in the pathogenesis of SSc, this Review focuses on the role of TGF‑β. However, two inherited diseases with clinical features related to SSc—hereditary haemor rhagic telangiectasia (HHT) and familial PAH—are caused in most patients by mutations in genes encoding proteins involved in bone morphogenetic protein (BMP) sig nalling. Thus, BMPs and their interactions with TGF‑β signalling will also be considered in the context of SSc.

The TGF‑β superfamily

TGF‑β is part of a superfamily of signalling proteins that includes BMPs, activins, inhibins, growth differentiation factors and myostatin.

TGF‑β The three TGF‑β isoforms are encoded by different genes: TGFB1, TGFB2 and TGFB3. All three isoforms are

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REVIEWS Key points ■■ Transforming growth factor β (TGF‑β) superfamily members are pleotropic cytokines that regulate fibrosis, inflammation and vascular biology, all key aspects of systemic sclerosis (SSc) pathogenesis ■■ Emerging data have shown that integrins, proteases and altered connective tissue sequestration can regulate activation of latent TGF‑β and provide possible mechanisms for TGF‑β-mediated fibrosis in SSc ■■ SSc is characterized by prominent vascular features also seen in familial pulmonary arterial hypertension and hereditary haemorrhagic telangiectasia, both of which are associated with mutations in proteins involved in TGF‑β and bone morphogenetic protein signalling ■■ Immune cells such as dendritic cells and macrophages can activate TGF‑β in SSc through surface integrins or upon ingestion of apoptotic or necrotic cells, respectively ■■ Effects of TGF‑β activation on immune cells include dampened T helper 1 and T helper 2 responses, increased regulatory T cell and T helper 17 cell differentiation and augmented leukocyte infiltration

secreted as latent proteins, interact with the same recep tor heterodimers, TGFR‑1 (TGF-β receptor type-1, also known as ALK‑5) and TGFR‑2 (TGF-β receptor type-2), and activate the same canonical mothers against decap entaplegic homologue (SMAD)-2–SMAD3 signalling pathway (Figure 1). However, the different isoforms are clearly involved in distinct developmental processes, as seen in knockout mice and in homeostatic as well as pathological processes in humans. For example, mice with deletion of TGFB1 show prenatal lethality (around 50% at 10.5 days post coitus) or die shortly after birth of an autoimmune inflammatory disease, strongly impli cating this isoform in immune regulation.1 However, prenatal lethality in these mice is due to defective devel opment of the yolk sac vasculature, indicating that TGF‑β1 has an important role in vascular development as well.2 Mice with deletion of TGFB2 or TGFB3 show multiple developmental defects of the skeleton, heart and genitourinary tract, or of the palate and lungs, respec tively.3–5 TGFB2 mutations are also associated with famil ial aortic aneurysms in humans, and similar defects are found in mice with heterozygous deletion of TGFB2.6,7 These studies emphasize the broad roles of TGF‑β in

TGFR-1 SMAD2/3

BMPs BMPs are a large family of cytokines which interact with a broad array of receptors (Figure 1).8 Similarly to the three TGF‑β isoforms, BMPs have important effects on vascu lar biology, and some interact with TGF‑β signalling. For example, BMP‑7 was previously found to antagonize the effects of TGF‑β.9

Synthesis and sequestration of TGF‑β

TGF‑β is synthesized as a propeptide that is cleaved in the Golgi apparatus to produce a dimeric mature peptide. Within the cell, the mature peptide is held in a latent form by noncovalent binding to LAP (reviewed previously else where10). This TGF‑β–LAP complex is termed the small latent complex (SLC), which is further bound to latent TGF‑β-binding proteins (LTBPs); TGF‑β–SLC–LTBP is referred to as the large latent complex (LLC). TGF‑β is secreted as a LLC by a wide array of cells in vitro, but only a small percentage of TGF‑β found in cell culture supernatants is typically active. LTBPs are matrix proteins structurally related to fibril lin; however, TGF‑β does not bind directly to fibrillin. Each molecule of LTBP‑1, LTBP‑3 or LTBP‑4 is able to bind one SLC through an eight-cysteine domain. LTBP‑1 and LTBP‑3 might be the primary proteins responsible for binding to SLCs, as LTBP‑4 binds only to TGF‑β1– LAP, and more weakly than the other two LTBPs do. Considerable amounts of TGF‑β are held in connective tissues as LLCs, providing a reservoir that makes tran scriptional and translational regulation of production of the latent protein relatively unimportant for tissue availability of TGF‑β (Figure 2).

BMP-2/ BMP-4

BMP-7

BMPR-2/ ALK-2/ ACTR-IIA/ BMPR-1A/ ACTR-IIB BMPR-1B SMAD1/5/8

BMPR-2/ ALK-2/ ACTR-IIA/ BMPR-1A/ ACTR-IIB BMPR-1B SMAD1/5/8

BMP-9/ BMP-10

TGF-β

TGFR-2

vascular pathology and developmental processes, as well as immune system function. Another important differ ence between TGF‑β isoforms is in their latency-associ ated propeptide (LAP). TGF‑β1–LAP and TGF‑β3–LAP contain an Arg–Gly–Asp (RGD) motif important in integrin-mediated activation, whereas TGF‑β2–LAP does not (Figure 2).

BMPR-2/ ACTR-IIA

ALK-1

SMAD1/5/8

Figure 1 | TGF‑β superfamily receptors. The receptor heterodimer mediating signals for TGF-β1, TGF-β2 and TGF-β3 in most cell types is composed of TGFR-2 and TGFR-1. Several receptor heterodimer combinations mediate intracellular signals for the large family of bone morphogenetic proteins: BMP-9 (also known as growth/differentiation factor 2) and BMP-10 interact with heterodimers composed of either BMPR-2 or ACTR-IIA and ALK-1 (serine/threonine-protein kinase receptor R3); BMP-2 and BMP-4 interact with heterodimers composed of BMPR-2 and ALK-2 (also known as ACTR-1), BMPR-1A (also known as ALK-3) or BMPR-1B (previously known as ALK-6); and BMP-7 interacts with heterodimers composed of BMPR-2 or ACTR-IIA and ALK-2, BMPR-1A or BMPR-1B. Abbreviations: ACTR, activin receptor; BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; SMAD, mothers against decapentaplegic homologue; TGF-β, transforming growth factor-β; TGFR, TGF-β receptor.

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REVIEWS Intact cytoskeletal tension and binding to αvβ1 inte grins on the cell surface are required for assembly of a fibronectin matrix, which in turn guides deposition of the fibrillin matrix.11 Since LLCs bind to both fibronec tin and fibrillin in the extracellular matrix through noncovalent interactions with LTBPs,12–14 LLC deposi tion also requires the presence of these elements. These matrix interactions are also a critical component of the orchestrated process of activation of latent TGF‑β, which is discussed further below. In some circumstances, disruption of either LTBPs or the fibrillin matrix leads to reduced TGF‑β sequestra tion and, consequently, increased TGF‑β activity.12 The role of fibrillin in TGF‑β sequestration will be considered further below, in the context of stiff skin syndrome (SSS).

Furin RGD

LAP

ProTGF-β

RGD

LAP

Latent TGF-β

TGF-β

TGF-β Non-covalent interaction

TGF-β

LTBP LAP SLC

The role of TGF‑β in SSc

Immunohistochemical analyses show that expression of all three isoforms of TGF‑β is increased in SSc skin, but as the anti-TGF‑β antibodies used to detect this cytokine bind to both active and latent protein, it is impossible to tell whether increased staining reflects an increase in active TGF‑β levels or simply increased amounts of latent protein.15–17 Thus, whether increased levels of the latent protein have any effect on TGF‑β bioavailability remains uncertain. Changes in matrix composition might also lead to altered sequestration of TGF‑β in the connective tissue of patients with SSc, but surprisingly little is known about LTBP expression in skin or other tissues from such patients. The presence of a large reservoir of latent TGF‑β also considerably complicates the assessment of TGF‑β activ ity in vivo. The process of extracting latent TGF‑β from tissues activates it, which has hindered investigations of its role in the pathogenesis of SSc. TGF‑β levels in SSc sera are similarly difficult to interpret, since latent TGF‑β is released during platelet activation, and consequently is abundant in normal sera. In one study, researchers meas ured both active and latent protein in sera from patients with SSc and found an unexpected inverse correlation between levels of active TGF‑β and the modified Rodnan skin score (MRSS), a clinical measure of the severity of skin disease, despite some earlier studies showing increased plasma TGF‑β levels in patients with diffuse cutaneous SSc.18 Further, a study using the anti-TGF‑β1 monoclonal antibody CAT‑192 failed to show efficacy in patients with SSc, discouraging consideration of TGF‑β as a driver of pathology in SSc. However, the limitations of this seemingly negative observation are important in light of the data that strongly implicate TGF‑β in SSc pathogenesis. The CAT‑192 antibody used in this clinical trial19 has relatively low affinity for TGF‑β1 (Kd ~150 nM) and does not interact with the other two TGF‑β isoforms. Several hallmarks of TGF‑β activity, such as the pres ence of myofibroblasts and expression of TGF‑β target genes, are greatly increased in SSc skin and lungs.20–22 Furthermore, dermal tissues from patients with SSc show increased deposition of both fibronectin and fibrillin matrices,23,24 processes that are both stimulated by TGF‑β.25

LLC

Figure 2 | TGF‑β1/3 maturation and tissue sequestration. Pro-TGF‑β is cleaved intracellularly by a furin protease, producing a noncovalently-bound dimeric complex of LAP and TGF‑β, referred to as the SLC. These complexes are bound to LTBPs forming the LLC for secretion and subsequent incorporation into the matrix. Abbreviations: LAP, latency-associated propeptide; LLC, large latent complex; LTBP, latent TGF-β binding protein; RGD, Arg–Gly– Asp motif; SLC, small latent complex; TGF-β, transforming growth factor-β.

The huge reservoir of latent TGF‑β in serum and con nective tissues and the importance of its local activation is consistent with the notion that SSc pathogenesis is driven by an unidentified process of local TGF‑β acti vation. In this context, intriguing results were reported from a study of TGF‑β secretion by peripheral blood mononuclear cells (PMBCs) in vitro, which showed that high levels of active TGF‑β are spontaneously secreted by PBMCs from patients with SSc. Analysis of leukocyte subsets showed that spontaneous production of TGF‑β (active plus latent) was highest in cultured peripheral monocytes and macrophages.26 Monocytes, macrophages or other inflammatory cells might, therefore, either produce an activator of TGF‑β or induce its expression in other cells. Activation of the cytokine probably occurs locally and leads to cell-associated autocrine or paracrine TGF‑β signalling.

Mechanisms of TGF‑β activation αv Integrins The α v integrins are the best-defined activators of TGF‑β1 and TGF‑β3, but do not activate TGF‑β2, since TGF‑β2–LAP lacks an RGD integrin-binding site. The key importance of integrin binding during TGF‑β acti vation is supported by the fact that mice engineered to express a mutant RGD integrin binding site in TGF‑β1 LAP show a phenotype very similar to mice with deletion of TGF‑β1.27 Several integrins containing an αv subunit



REVIEWS Box 1 | Activators of latent TGF‑β ■■ αvβ6 integrin: expression restricted to epithelial cells, binds to TGF‑β1–LAP and TGF‑β3–LAP ■■ αvβ8 integrin: expression on dendritic cells and other cell types, binds to TGF‑β1–LAP and TGF‑β3–LAP ■■ αvβ3 and αvβ5 integrins: implicated in TGF‑β activation by fibroblasts ■■ Integrin β-1: implicated in TGF‑β activation by fibroblasts ■■ Plasmin: degrades and activates TGF‑β small latent complex ■■ Thrombin: activates TGF‑β in combination with αvβ6 integrin, through the PAR‑1 receptor ■■ Thrombospondin‑1: increased in SSc skin and upregulated by TGF‑β; implicated in autocrine activation of latent TGF‑β ■■ MMP‑2: mechanism possibly through proteolysis ■■ MMP‑9: binds CD44 antigen; surface MMP‑9 activates TGF‑β proteolytically ■■ MMP‑14 and MME: linked to αvβ8-mediated activation of latent TGF‑β ■■ Kallikrein‑1: upregulated expression by TNF activates TGF‑β ■■ Periostin: expression induced by IL‑13 and associated with increased TGF‑β activation60 Abbreviations: LAP, latency-associated peptide; MME, macrophage metalloelastase; MMP, matrix metalloproteinase; PAR-1, proteinase-activated receptor 1; SSc, systemic sclerosis; TGF, transforming growth factor.

can bind to the TGF‑β1–LAP, but αvβ6 and αvβ8 have been most strongly associated with TGF‑β activation (Box 1). For instance, integrins β6 and β8 seem to be collectively responsible for all activation of TGF‑β that occurs during lung development.28 Integrin αvβ6 was first shown to activate TGF‑β in the lungs; in mice, deletion of Itgb6 (which encodes inte grin β6) leads to lung inflammation and resistance to bleomycin-induced pulmonary fibrosis.29 TGF‑β1–LAP binds to integrin β6, and overexpression of integrin β6 results in increased activation of latent TGF‑β, a process that requires LTBP‑1. 30 Thus, integrin α vβ 6 activates TGF‑β only when it is part of the LLC. Notably, inte grin β6 is expressed primarily on epithelial cells and is highly upregulated in pneumocytes from patients with SSc-associated interstitial lung disease.31 However, this integrin is not highly upregulated in skin from patients with SSc (R. Lafyatis, S. Violette, unpublished work). Moreover, as epidermal cells are located distantly from the main site of skin fibrosis in the deep dermis, altered α vβ 6 expression is unlikely to drive skin fibrosis in patients with SSc. Integrin αvβ8 is more widely expressed than αvβ6 and can also activate latent TGF‑β (Figure 3).32 Integrin αvβ8 is expressed by mesenchymal, immune, neural and epi thelial cells and can be induced by IL‑1β, which provides a link between inflammation and TGF‑β activation.33 Mice with deletion of Itgb8 (encoding integrin β8) show lethal perinatal defects in vasculogenesis,34 particularly in the brain, where β8 localizes to perivascular cells.35 A similar phenotype to that induced by Itgb8 deletion is also seen in mice with hypomorphic mutations in Tgfb1 or deletion of Tgfb3,36 suggesting that integrin β8

is involved in the activation of these TGF‑β isoforms. Targeted deletion of Itgb8 in dendritic cells (DCs) results in colitis and autoimmune disease (anti-DNA antibodies and adenopathy) thought to be caused by loss of TGF‑βmediated regulatory T (TREG) cell development,37 and thus similar to the autoi mmunity seen in mice with deletion of Tgfb1. Currently, no published data indicate whether expression of integrin β8 is altered in SSc tissues or blood. The RGD motif in TGF‑β1-LAP and TGF‑β3-LAP can bind to αv-containing integrins other than αvβ6 and αvβ8, and these other integrins might be more important than αvβ6 and αvβ8 in settings such as in SSc skin. Several lines of evidence support the involvement of integrin β5 in TGF‑β activation during the development of fibrosis in SSc: fibroblasts from patients with localized scleroderma show increased αvβ5 expression; β5-overexpressing cells show increased activation of latent TGF‑β and con version of fibroblasts into myofibroblastic cells, which is consistent with autocrine activation.38,39 The same investigators showed that fibroblasts from patients with SSc show increased expression of integrin αvβ3, and that fibroblasts engineered to overexpress integrin β3 have an increased capacity to activate latent TGF‑β.40 Data obtained using inducible deletion of Itgav in plateletderived growth factor receptor β (PDGFR‑β)-expressing cells suggest that expression of αv-containing integrins other than β6 and β8 on hepatic stellate cells activates latent TGF‑β in animals with carbon-tetrachlorideinduced liver fibrosis.41 Deletion of several individual αv partners (β3, β5, β6 and β8) in this study did not block fibrosis, suggesting that either multiple integrins are important, or that αvβ1 has a key role in this setting. The role of integrin αvβ1 in TGF‑β activation is particularly difficult to ascertain, since both αv and β1 combine with other integrin partners. Nevertheless, the fact that dele tion of integrin β1 in mouse fibroblasts blocks TGF‑β activation42 and confers resistance to bleomycin-induced dermal fibrosis,43 together with the data implicating αv integrins in fibrosis, suggests that αvβ1 might be critical in fibroblast-mediated activation of latent TGF‑β.

Thrombospondin‑1 In certain contexts, soluble factors seem to be suffi cient for TGF‑β activation. Several studies have impli cated one such factor, thrombospondin‑1, in TGF‑β activation and SSc pathogenesis. Thrombospondin‑1 and peptides derived from it can activate latent TGF‑β in vitro,44 and mice with deletion of Thbs1 show several phenotypic similarities to mice lacking Tgfb1.45 In the context of SSc, expression of thrombospondin‑1 is increased in both fibroblasts and endothelial cells in SSc skin.46 Expression of thrombospondin‑1 is also strongly induced by TGF‑β in vitro and correlates strongly and positively with the extent of skin disease in patients with SSc.20 Dermal fibroblasts from individuals with SSc produce increased levels of thrombospondin‑1, which have been implicated in the autocrine activation of latent TGF‑β, since treatment of SSc fibroblasts with either a thrombospondin‑1 blocking peptide or a neutralizing



REVIEWS

Fibrillin

LTBP TGF-β LAP

Active TGF-β TGF-β activator TGF-βR

TGF-β activation αvβ8

Endothelium DC

Fibroblast αvβ6 Epithelium

Figure 3 | Integrin-mediated activation of TGF‑β. Active TGF‑β bound to LAP forms a complex with LTBP, which is bound noncovalently to fibrillin. Activation of TGF‑β occurs by binding of TGF‑β-LAP to integrins αvβ6 on epithelial cells, and/or αvβ8 on DCs and endothelial cells. A soluble TGF‑β activator is shown, which might represent inflammatory cytokines that can upregulate these two integrins or other co-factors required for integrin-mediated TGF‑β activation. Abbreviations: DC, dendritic cell; LAP, latency-associated propeptide; LLC, large latent complex; LTBP, latent TGF-β binding protein.

antibody that targets TGF‑β reduces the overexpression of thrombospondin‑1.46 Collectively, the results of these studies suggest that thrombospondin‑1 might have a key role in TGF‑β activation in SSc.

Proteases Proteases are implicated at several steps in TGF‑β bio logy, including propeptide processing, TGF‑β activation by direct cleavage of latent TGF‑β, or by degradation of LTBPs. For example, furin processes the TGF‑β pro peptide into mature (latent) TGF‑β inside the cell,47,48 and is part of a larger family of preprotein convertases impli cated in neoplasia.49 A furin-like protease also activates latent TGF‑β upon platelet stimulation by thrombin.50 Plasmin was the first protease identified as capable of activating latent TGF‑β in vitro by partially degrad ing LAP,51 but several studies have failed to show a con vincing role for plasmin in activation of latent TGF‑β in vivo.52 However, plasmin might only exert this func tion in concert with other factors, such as cathepsin B (which is needed for the plasmin-dependent activa tion of latent TGF‑β seen in a breast carcinoma cell line treated with 12‑O-tetradecanoylphorbol‑13-acetate).53 Thrombin also contributes to activation of latent TGF‑β

through proteinase-activated receptor 1, in concert with αvβ6.54 Several matrix metalloproteinases (MMPs) have also been implicated in TGF‑β activation, particularly MMP‑2 and MMP‑9.55,56 BMP‑1 (an endopeptidase that is not a TGF‑β family member) can cleave LTBPs in the matrix, and MMP‑2 can activate TGF‑β from this liber ated complex.57 A number of other proteases have been implicated in TGF‑β activation, namely MMP‑14 (also known as MT1-MMT), which seems in some cases to be linked to αvβ8 activation;32,58,59 periostin (following induction by IL‑13);60 and kallikrein (induced by TNF).61 Levels of kallikrein and MMP‑9 are upregulated in SSc sera,62,63 and thus possibly contribute to TGF‑β activa tion in SSc. Despite all these links, it remains uncertain whether proteases activate TGF‑β in SSc. However, it is interesting to note that plasmin-dependent activation of TGF‑β has been implicated in the cardiac fibrosis that occurs in patients with heart block associated with Sjögren’s syndrome.64

Tension in latent TGF‑β activation Another key feature in TGF‑β activation is the require ment for cellular tension on the latent protein.65 Models of how intracellular tension acts in concert with integrin engagement suggest that cells harbouring αv integrins on their surface are able to place traction on matrixbound TGF‑β. Traction, either alone or in concert with a proteolytic step, separates active TGF‑β from the latent complex, whereupon the active molecule is immediately bound by the TGF‑β receptor. 66 These observations further suggest that increased matrix stiffness in patho logical states of fibrosis might increase TGF‑β activa tion by increasing the tension in interactions between matrix fibroblasts and matrix-bound LLC. The increase in tension and consequent TGF‑β activation might aggravate fibrosis, as it sets up a positive-feedback loop.

Vascular pathology: links with TGF‑β SSc-associated vasculopathy The vasculopathy in SSc can be observed in several affected organs: lungs in patients with PAH, kidneys from patients with scleroderma renal crisis, heart in those with cardiac fibrosis, and in digital arteries. Most pathology manifests in small and medium arteries, which show intimal hyperplasia and obliteration, medial thick ening, and sometimes microthrombi.67–70 Perivascular inflammation has been described in the lungs of patients with SSc and PAH, as well as in SSc skin, though not in the affected kidneys of patients with scleroderma renal crisis. In most patients, scleroderma renal crisis responds dramatically to inhibition of angiotensin II, suggesting that the renal vasculature has important differences from other vascular beds (in which SSc-associated vasculo pathy does not respond to this approach). In addition to arteries, vasculopathy in SSc also affects capillaries as can be seen by nailbed and nailfold capillaroscopy, which reveals capillary dilatation in early stages and capillary loss in later stages, possibly as a parallel to the intimal obliteration seen in small arteries in other



REVIEWS Table 1 | Hereditary diseases with features that overlap with SSc Disease

Mutant gene

Protein product

Function

Clinical features shared with SSc

Type 1 HHT

ENG

Endoglin

TGF‑β co-receptor

PAH, telangiectasia

Type 2 HHT

ACVRL1

ALK-1

Alternative TGF‑β receptor, receptor for BMP‑9 and BMP‑10

PAH, telangiectasia

Familial PAH

BMPR2

BMPR‑2*

BMPR

PAH

SSS

FBN1

Fibrillin-1

Microfibril protein that binds the TGF‑β large latent complex

Fibrotic skin

*Accounts for 70% of all cases. Abbreviations: BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; HHT, hereditary haemorrhagic telangiectasia; PAH, pulmonary arterial hypertension; SSc, systemic sclerosis; SSS, stiff skin syndrome.

tissues.71 Despite these differences, it is tempting to con sider these multiorgan manifestations of vasculopathy as having closely related initiating factors that simply show modified expression in various organs. Several lines of evidence indicate that TGF‑β could be respon sible for the vasculopathy observed. Indeed, TGF‑β is critical to vascular homeostasis, as its overexpression and underexpression both lead to vascular pathology.

Pleiotropic effects on endothelial cells The pathogenesis of vasculopathy in SSc is probably com plex, involving endothelial as well as perivascular cells. In this Review I focus on endothelial cells, but given that pericyte biology in SSc is increasingly understood, these cells might be crucial to both vasculopathy and fibrotic pathology. The potential role of pericytes is discussed further in the section on myofibroblasts. TGF‑β can have multiple effects on endothelial cells. It downregulates the vasodilator inducible nitric oxide synthase and upregulates endothelial nitric oxide syn thase.72 TGF‑β also stimulates the expression of vascular endothelial growth factor (VEGF) as well as the splice variant of VEGF, VEGF165b.73,74 High levels of VEGF cause retinal neovascularization and thus might be responsible for the dilated capillaries seen in nailfolds of patients with SSc.75 By contrast, the splice variant VEGF165b is antiangiogenic and might contribute to vascular dropout, which is also seen in patients with SSc. 74 TGF‑β also stimulates endothelin‑1 (ET‑1), a cytokine that is strongly upregulated in SSc.73,76 ET‑1 seems to have an important role in the vasoconstric tion seen in patients with SSc who have PAH and digital ischaemia, as ET‑1 inhibitors prevent vasoconstriction in these settings.77,78 However, TGF‑β also blocks the pro liferative and motility-enhancing effects of fibroblast growth factor on endothelial cells.79 Thus, the complex effects of TGF‑β on pro-angiogenic and anti-angiogenic factors might explain the complex vascular phenotype seen in patients with SSc, who can sometimes show proliferative and obliterative vascular lesions occurring simultaneously. Integrin-mediated TGF‑β activation seems to be a key event in the involve ment of TGF‑β1 (and probably also TGF‑β3) in vas culogenesis, since targeted deletion of the RGD motif of TGF‑β–LAP in mice with deletion of Tgfb3 leads to

defective brain vasculature,27 and a similar phenotype is seen in mice with deletion of Itgb8.34

Altered BMP signalling The observations regarding the potential role of TGF‑β in vascular pathology in SSc imply a close relationship with ALK‑1–SMAD1–SMAD5 signalling, which is con sidered to be a BMP signalling pathway. SSc-associated PAH is currently the second leading cause of death in patients with SSc, and other closely related types of PAH show genetic associations that clearly implicate perturba tions in BMP receptor signalling, which in endothelial cells is closely integrated with TGF‑β signalling. BMPR‑2 in PAH Although the availability of samples from patients with early stage PAH is limited, familial PAH, idiopathic PAH and SSc-associated PAH seem to share most pathological features.70 Loss-of-function mutations in BMPR‑2 cause ~70% of familial PAH and also ~20% of idiopathic PAH (Table 1),80,81 strongly suggesting that BMP signalling is crucial for normal pulmonary vascular homeostasis. This phenotype can be largely reproduced in mice with endothelial cell deletion of BMPR‑2.82 Unfortunately, the specific BMPs that maintain normal pulmonary vascular homeostasis remain unknown and difficult to ascertain, since BMP‑2, BMP‑4, BMP‑6, BMP‑7, BMP‑9, BMP‑10, BMP‑13, BMP‑14 and BMP‑15 all interact with BMPR‑2. Despite this complexity, mice with deletion of Bmp4 show increased susceptibility to hypoxia-induced PAH, indicating the possible involvement of BMP‑4 in this pathology.83 In addition to developing PAH, mice with targeted deletion of Bmpr2 in the endothelium, but also have perivascular inflammation, a feature of both PAH and of vascular disease in patients with SSc, in whom perivascular inflammation is also easily detected in the skin. Whether vascular inflammation in patients with SSc is a cause or consequence of the vascular injury remains a central conundrum in SSc. ALK‑1–ALK‑5 signalling in endothelial cells In most cell types, TGF‑β signals primarily by binding to TGFR‑2, which then associates with TGFR‑1 (also known as ALK‑5), a serine/threonine kinase that phos phorylates SMAD2 and SMAD3. These two proteins then bind to SMAD4, forming a complex that directly activates transcription of target genes. However, TGF‑β can also activate ALK‑1 (also known as serine/threonine- protein kinase receptor R3),84 which was first identified as a receptor for BMP‑9 and BMP‑10, and mediates downstream signals generally associated with SMAD1, SMAD5 and SMAD8 (Figure 4).85 TGF‑β has complex effects on vascular angiogenesis exerted through these receptors, stimulating angiogenesis and endothelial cell proliferation through ALK‑1, SMAD1 and SMAD5, but blocking angiogenesis through ALK‑5, SMAD2 and SMAD3.84 Further, the action of TGF‑β on endothelial cells is strongly modulated by the membrane glyco protein endoglin, a co-receptor for TGF‑β1 and TGF‑β3. Endoglin stimulates endothelial cell proliferation and



REVIEWS Box 2 | Fibrotic diseases associated with TGF‑β signalling

TGF-β

TGF-β

TGF-β

Endoglin Mutation

TGFR-1

TGFR-2

ALK-1 SMAD2/3

SMAD2/3

SMAD1/5 P

Inhibits angiogenesis Stimulates profibrotic genes

SMAD1/5

P

Stimulates angiogenesis

Figure 4 | TGF‑β signalling mediated by TGFR-1 and ALK‑1. Endoglin shifts binding of TGF‑β to the ALK‑1–TGFR‑2 co-receptor, which mediates proangiogenic signals. Mutations (yellow stars) in ENG or ACVRL1 (which encode endoglin and ALK‑1, respectively) cause HHT. An imbalance in TGF‑β signalling toward binding to TGFR-1 in endothelial cells might also lead to anti-angiogenic effects on endothelial cells. Abbreviations: HHT, hereditary haemorrhagic telangiectasia; SMAD, mothers against decapentaplegic homologue; TGF-β, transforming growth factor-β; TGFR, TGF-β receptor.

modulates TGF‑β delivery by favouring TGF‑β binding to ALK‑1 rather than ALK‑5.86 ALK‑1 in HHT HHT is an autosomal dominant disease caused by muta tions in ACVRL1 (which encodes ALK‑1) that cause type 2 HHT, or mutations in ENG (which encodes endoglin) that cause type 1 HHT. Both forms of the disease are characterized by arteriovenous malfor mations, skin telangectasia and bleeding in the nose, gastrointestinal tract, lung, liver and brain. The skin tel angiectasia seen in patients with SSc clinically resembles that in patients with HHT and the two conditions are pathologically indistinguishable, showing postcapillary venule dilatation and mononuclear cell infiltrates.87 In a few patients, HHT can also be complicated by a form of PAH that is pathologically indistinguishable from primary PAH.88 Also, ACVRL1 mutations are commonly seen in patients with childhood-onset PAH.89 Thus we again see an intriguing link between a genetic disease caused by dysfunctional TGF‑β signalling and vascular pathology that clinically overlaps with SSc. BMPs are also implicated in both HHT and familial PAH: in particular, BMP‑9 and BMP‑10 bind to both ALK‑1 and BMPR‑2, inhibiting angiogenesis and basic fibroblast growth factor-mediated endothelial cell pro liferation.90 However, the fibrotic features seen in SSc suggest that BMPs are much less likely candidates than TGF‑β in single-cytokine-mediated models of SSc pathogenesis.

■■ Liver cirrhosis (in patients with viral or drug-induced hepatitis) ■■ Renal fibrosis (the pathological feature that leads to many forms of end-stage renal disease) ■■ Cardiac fibrosis (in patients with hypertension or after myocardial infarction) ■■ Pulmonary fibrosis (either idiopathic or associated with a variety of other diseases or environmental exposures)

Despite the intriguing clinical overlaps between HHT, familial PAH and SSc, genetic studies have failed to reveal associations between mutations in ENG, ACVRL1, BMPR2 and either SSc or SSc-associated PAH.91 The lack of associations found is not entirely surprising, given that twin studies have not suggested a strong genetic component to SSc; the overactivity of TGF‑β in patients with SSc is likely to be acquired, probably triggered by an infectious or environmental stimulus. Nonetheless, evidence of perturbed ALK‑1 signalling in patients with SSc can be inferred from the elevated levels of soluble endoglin in patients with limited SSc and telangiectasia or PAH.92 In addition, the results of several studies have reinforced the potential for increased TGF‑β signalling to mediate vascular pathology similar to that seen in SSc, as seen by the development of intimal and medial hyper plasia following TGF‑β overexpression in pig arteries,93 or the development of PAH in a mouse model with global increased activation of TGFR‑2 after treatment with a VEGF inhibitor. These studies highlight that a balance of cytokines is apparently responsible for maintaining vascular stability, and place TGF‑β in a central position in the development of vascular pathology.94

Thrombospondin‑1 in SSc In addition to its function as a TGF‑β activator discussed above, thrombospondin‑1 might contribute directly to vascular pathology in SSc. Thrombospondin‑1 inhibits both basal and VEGF-mediated endothelial cell prolif eration and migration, and induces apoptosis via CD36. In vivo thrombospondin‑1 inhibits angiogenesis in the rat cornea,95 and blocks proangiogenic signals down stream of nitric oxide (such as soluble guanylate cyclase and cyclic-GMP-dependent protein kinase I) through CD36 and CD47. Thus, both direct and indirect effects of thrombospondin‑1 can block angiogenesis and induce endothelial cell apoptosis.

TGF‑β-induced repair and fibrosis Fibrosis in SSc Soon after its discovery, TGF‑β was recognized to have potent profibrotic activity.96,97 TGF‑β promotes the syn thesis, secretion, processing and crosslinking of colla gen,98 as well as secretion of other matrix molecules such as fibronectin and thrombospondin‑1.99 Furthermore, TGF‑β is implicated in fibrosis in many other diseases (Box 2).100 TGF‑β stimulates a plethora of secondary mediators, some of which are responsible for downstream signalling,



REVIEWS Box 3 | Accessory mediators of TGF‑β TGF‑β stimulates the production of a number of secondary signals that can mimic the effects of TGF‑β, leading to confusion about which factor is primarily responsible for the observed changes in gene expression or pathological features. Identification of accessory mediators might also provide targets for therapeutic intervention with potentially fewer safety issues than blocking upstream TGF‑β signalling.

CTGF Mice with fibroblast-specific overexpression of Ctgf show skin and lung fibrosis.149 Connective tissue growth factor, the protein encoded by Ctgf, mediates the effect of TGF‑β on palatogenesis,150 promotes conversion of mesenchymal stem cells to a fibroblastic lineage151 and inhibits preadipocyte differentiation.152 Connective tissue growth factor mimics the effect of TGF‑β on branching morphogenesis in the lung,153 and its expression is induced by TGF‑β and regulated through SMAD and MAPK signalling.154 PAI‑1 PAI‑1 (also known as plasminogen activator inhibitor 1) is strongly induced by TGF‑β and blocks the activity of plasminogen activators. Inhibition of PAI‑1 blocks the pulmonary fibrosis induced by TGF‑β by an as yet unknown mechanism,155 and mice with deletion of SERPINE1 (which encodes PAI‑1) develop cardiac fibrosis with ageing.156 NADPH oxidase 4 Connective tissue growth factor induces reactive oxygen species by inducing NADPH oxidase 4 activity, an effect implicated in the proapoptotic activity of TGF‑β.157 NADPH oxidase 4 mediates lung fibrosis in animal models, through hydrogen-peroxide-mediated induction of myofibroblasts. Treatment with an antioxidant can block TGF‑β-induced expression of CTGF and collagen,158 and a NADPH oxidase 4 inhibitor ameliorates lung fibrosis.159 Cadherin-11 Cadherin-11 expression in SSc skin correlates with MRSSs, as does that of other TGF‑β-regulated genes, and can be induced by TGF‑β.160–162 Endothelin‑1 TGF‑β induces expression of endothelin‑1, which in some contexts seems to be essential for TGF‑β-mediated fibrosis. Treatment of lung fibroblasts with the endothelin‑1 inhibitor bosentan blocks the expression of several TGF‑βinduced proteins: collagen α-1(I) chain, TIMP-1 (also known as metalloproteinase inhibitor 1), connective tissue growth factor and α‑actin-2 (also known as Actin, aortic smooth muscle).163 Endothelin‑1 also stimulates TGF‑β and connective tissue growth factor secretion by pulmonary smooth muscle cells,164 but large controlled trials of bosentan in patients with idiopathic pulmonary fibrosis failed to show any benefit.165 Abbreviations: CTGF, connective tissue growth factor; MAPK, mitogen-activated protein kinase; MRSS, modified Rodnan skin score; PAI‑1, plasminogen activator inhibitor‑1; SMAD, mothers against decapentaplegic homologue.

whereas others potentially drive positive feedback of TGF‑β activation (Box 3). Expression of several genes regulated by TGF‑β is increased in fibrotic SSc skin: CTGF, THBS1, SERPINE1 (which encodes plasminogen activator inhibitor‑1, PAI‑1) and COMP (which encodes cartilage oligomeric matrix protein [COMP]). These observations support a direct role of upregulated TGF‑β signalling in fibrotic SSc skin.20 Data from our group have shown tight correlations between MRSS values (the most commonly used clinical measure of fibrotic skin disease) and the extent of myofibroblast infiltration, as well as TGF‑β-responsive gene expression.20,21 Data from animal models also lend strong support to the notion that TGF‑β has a major role in fibrotic mani festations of SSc. For example, a TGF‑β receptor kinase inhibitor blocked bleomycin-induced lung disease;101 in addition, in a different mouse study, administration of LAP (a biological inhibitor of TGF‑β) blocked SSclike graft versus host disease.102 These and many other

observations have shown that TGF‑β mediates fibrosis in many animal models.

The role of myofibroblasts in SSc Myofibroblasts have been implicated as the primary cells that mediate fibrosis in many fibrotic diseases.103 Multiple cell types can act as myofibroblast precursors: resident fibroblasts, cells undergoing epithelial–mesenchymal transition (EMT), circulating bone-marrow-derived fibrocytes, and vascular pericytes (Figure 5). Myofibroblast precursors Cells undergoing EMT are a source of fibroblasts and myofibroblasts in several models of fibrosis. For example, in renal fibrosis, ~36% of FSP1+ fibroblasts are derived from γ‑glutamyltransferase-positive epithelial cell pro genitors,104 and in a TGF‑β1-driven model of lung fibro sis, EMT was an important source of myofibroblasts.105 However, subsequent cell-fate mapping studies point to pericytes as the primary source of myofibroblasts in several fibrotic diseases. In renal fibrosis, pericytes apparently differentiate into myofibroblasts in response to TGF‑β produced by renal epithelial cells. 106,107 In ble omycin-induced lung fibrosis, myofibroblasts are derived from Foxd1+ pericyte progenitors.108 In models of fibrosis following muscle injury or adjuvant-induced skin inflammation, a specific population of mesenchymal cells that express both PDGF‑Rα and ADAM12 reside in the perivascular space. These cells (and not ADAM12– cells) are the source of progenitor cells that differentiate into profibrotic myofibroblasts in response to TGF‑β.109 A subgroup of GLAST‑1-expressing pericytes also seems to be the primary source of myofibroblasts in models of spinal cord injury.110 However, considerable contro versy remains regarding the sources of myofibroblasts in fibrotic diseases. In contrast to the accumulating evi dence implicating pericytes as the source of myofibro blasts in many models of fibrosis, resident fibroblasts that respond to TGF‑β are the predominant source of myofibroblasts in renal fibrosis.111 The source of myofibroblasts in patients with early SSc is still uncertain, but might be perivascular cells. Sup porting this hypothesis, increased numbers of PDGF‑Rβ+ perivascular cells are detected in such patients, which is suggestive of pericyte activation, 112 and skin from patients with SSS shows increased numbers of Thy1+ cells that co-localize with both pericytes and myofibroblasts. Still, these studies do not fully address the question of which cell population is the source of myofibroblasts, in part because Thy1 does not adequately define cells as pericytes. Myofibroblast differentiation Several common themes have emerged from these studies. First, cells that express the primary marker of myofibroblasts, smooth muscle actin (SMA), can dif ferentiate from different cell types. Some of the discrep ancies in the findings of studies that aimed to identify the sources of these cells relate to the choice of fibrotic model and method used for lineage tracing. Nevertheless,



REVIEWS

Epithelial cells

Fibroblasts

Fibrosis

Myofibroblast Fibrocytes

Vascular pericytes

Figure 5 | Myofibroblast progenitors. Myofibroblasts can be derived from several different sources: resident fibroblasts, epithelial cells, pericytes, and/or circulating fibrocytes.

disparate results have been obtained even for studies using the same disease model and lineage-tracing metho dology, suggesting that multiple cell types act as myo fibroblast progenitors. Second, and more important for this Review, TGF‑β can induce a myofibroblast pheno type in all the progenitor cell types mentioned above. TGF‑β induces myofibroblast differentiation from fibro blasts,113 but also pericytes,106 and can induce expression of SMA (as well as other morphological and genetic features of mesenchymal cells) in epithelial cells.114 Two different fibroblast subpopulations induced by TGF‑β are present in SSc skin: SMA+ myofibroblasts and COMP-expressing fibroblasts.115 This observation suggests that TGF‑β stimulates heterogeneous fibroblast and/or pericyte subpopulations in SSc, and supports a primary role for TGF‑β in SSc skin pathology in general.

Fibrillin and TGF‑β sequestration Fibrillin is the major protein component of microfibrils and seems to have a key role in organizing extracellular matrices.116 Mutations in fibrillin lead to several pheno types in humans, which have been largely replicated in mice. For example, Marfan syndrome is characterized by bone overgrowth, lens dislocation and aortic root dilatation, which is caused by a variety of mutations in fibrillin. Studies in mouse models of Marfan syndrome suggest that the aortic dilatation is not simply due to structural weakness resulting from altered fibrillin, but also to increased release of active TGF‑β.117 Mutant fibril lin is thought to release increased amounts of LLC from the matrix; moreover, disruption of the fibrillin matrix affects the stability of LTBP‑1 and is associated with increased TGF‑β activation.12 SSS is caused by mutations in a specific region of fibrillin that harbours an RGD integrin-binding site.118 Mice with knock-in mutations matching those seen in human SSS develop skin fibrosis similar to that seen in SSc, with progressive dermal collagen deposition and

loss of subcutaneous fat.119 Other skin features of these mice also mimic human SSc, such as autoantibodies to topoisomerase and inflammatory cell infiltrates, which include T‑helper‑2 (T H2) and T H17 cells, as well as plasmacytoid DCs. All these pathological changes were reversed by treatment with a neutralizing antibody to TGF‑β.119 Furthermore, fibrosis in this mouse model of SSS did not involve SMAD signalling. TGF‑β-induced collagen expression by fibroblasts from these mice was dependent on MAPK signalling, and an inhibi tor of MAPKK1 and MAPKK2 (also known as MEK1 and MEK2, respectively) blocked the SSS phenotype in vivo.119 The association of SSS with mutant fibrillin reinforces the potential importance of TGF‑β seques tration in SSc pathogenesis. These mice also showed increased TGF‑β1–LAP and TGF‑β2–LAP, suggesting that increased sequestration of TGF‑β is the mechanism behind the elevated TGF‑β activity observed.119 In addi tion, the fibrillin matrix is also stimulated by TGF‑β and increased in SSc skin, which might represent a feedback mechanism for moderating the activity of TGF‑β.24

ALK‑5 and ALK‑1 signalling in fibroblasts Studies in fibroblasts from patients with SSc have high lighted roles for both ALK‑5 and ALK‑1 in generation of TGF‑β-mediated profibrotic signals. SSc fibroblasts show increased surface expression of endoglin and ALK‑1mediated phosphorylation of SMAD1 in response to TGF‑β.120,121 Activation of this pathway is important for expression of at least some of the profibrotic genes regu lated by TGF‑β, in particular CTGF, the expression of which stimulates further ALK‑1 and SMAD1 activation via a positive-feedback loop.122 By contrast, ALK‑5 kinase inhibition blocks the expression of type I collagen and β1 integrin by SSc fibroblasts, but does not block constitu tive CTGF expression or acquisition of a myofibroblast phenotype in SSc fibroblasts.123 SMAD-independent pathways are also important in TGF‑β-mediated profibrotic signalling. For example, early growth response protein 1 (also known as EGR-1) is upregulated through an MAPKK1-dependent pathway and Egr1 deletion attenuates bleomycin-induced fibro sis in mice.124,125 TGF‑β also activates tyrosine-protein kinase ABL1 upstream of EGR‑1, which suggests that this pathway could be targeted using clinically avail able ABL1 inhibitors.126 These and other non-SMAD pathways of TGF‑β activation are discussed further in other reviews.127–129

Immune and autoimmune roles of TGF‑β Autoimmunity in SSc The best-defined associations of specific genes with SSc involve genes implicated in immune responses rather than vascular or connective tissue biology.130 Several of these genes are also associated with other autoimmune diseases, such as IRF5 and STAT3, which are also associ ated with systemic lupus erythematosus (SLE). In addi tion, patients with SSc and SLE show similar interferon gene expression signatures in peripheral blood mono nuclear cells. 131 These observations, along with the



REVIEWS overlap in autoantibody specificities and clinical features observed in SLE and SSc, implicate immune activation in the pathogenesis of both diseases. Despite these simi larities, the pathogenesis of SLE seems to be primarily autoantibody-mediated, whereas little evidence indicates that autoantibodies mediate the pathogenesis of SSc. In fact, most of the effects of TGF‑β dampen rather than aggravate immune activation.

TGF-β activation by the immune system We first consider how the immune system activates TGF-β in SSc. Some of the molecular mechanisms leading to TGF-β activation have already been discussed above, with particular emphasis on integrins. Indeed, αv inte grins seem to be responsible for most observed TGF‑β activation, which is consistent with the RGD-integrinbinding motif in TGF-β1–LAP, and the observed severe autoimmune disease in mice with deletion of Tgfb1. Although αvβ6 integrin expression is restricted to epi thelial cells, αvβ8 integrin is expressed on most leuko cytes. In particular, lack of expression of αvβ8 on CD11c+ DCs mediates most of the immune effects of deleting all αv integrins.37,132 However, αvβ8 expression on DCs of patients with SSc has not yet been reported, and other αv integrins might also be important. Inflammation and latent TGF‑β activation Although TGF‑β activation has been implicated in many inflammatory and/or fibrotic models of disease, the manner in which inflammation leads to fibrosis is in most cases not understood. The best-defined relationship is represented by integrin αvβ6; its inhibition or deletion blocks renal, biliary and lung fibrosis.29,31,133,134 In IL‑1induced asthma, integrin αvβ8 has also been implicated in controlling inflammation and associated fibrosis.135 Additionally, αvβ8 integrin deletion leads to colitis, indicat ing that it also has a role in regulation of inflammation.37 Given the current paradigm of TGF‑β activation by the immune system, upregulation of αvβ6 and/or αvβ8 integrin expression would be critical to activation of TGF‑β1 and TGF‑β3. In this regard, the observation that IL‑1 induces expression of integrin β8 might be crucial for understand ing the link between inflammation and the role of TGF‑β in tissue fibrosis.33 The profibrotic activity of transgenic expression of IL‑13 in the lungs has also been shown to be mediated by TGF-β, in this case through MMP‑9 and a serine protease, possibly plasmin.136 Macrophages activate TGF‑β through a poorly defined mechanism following ingestion of apoptotic or necrotic cells (which is known as efferocytosis).137,138 Activation of TGF‑β by macrophages is enhanced by lipopolysaccha ride (LPS), which is a TLR4 ligand.139 The importance of this mechanism in resolving LPS-induced lung injury has been shown in vivo,140 but little is understood about the mechanisms underlying TGF‑β activation in this setting. Impact of TGF‑β on the immune system Downregulation of immune responses In addition to exploring how the immune system acti vates TGF‑β in SSc, the reverse question—what influence

TGF‑β has on immune-mediated pathogenetic mecha nisms in SSc—is equally important. Deletion of TGFB1 and TGFR‑2 in various leukocyte subsets has partially clarified the roles for TGF‑β in immune regulation.132 TGF‑β downregulates T‑cell activation, which directly inhibits differentiation of both TH1 and TH2 cells and induces differentiation of TREG cells. In contrast to this anti-inflammatory effect, TGF‑β acts in combination with other inflammatory cytokines to promote differen tiation of TH17 cells. Garpin (also known as leucine-rich repeat-containing protein 32) localizes latent TGF‑β on the surface of TREG cells, where it contributes to matura tion of TH17 cells (in combination with IL‑6) and TREG cells (in combination with IL‑2).141 Patients with SSc have increased numbers of circulating TH17 cells and TREG cells,142,143 possibly owing to increased levels of soluble or cell-associated activators of TGF‑β. Stimulation of leukocyte infiltration TGF‑β has long been known to stimulate leukocyte infiltration when injected subcutaneously into mice.97 TGF‑β also has chemotactic effects on monocytes at very low concentrations, and stimulates production of IL‑1β at higher concentrations.144 Transgenic over expression of TGF‑β in skin strongly stimulates leuko cyte infiltration and increases the expression of several chemokines, namely CC-chemokine ligand (CCL) 3 (also known as MIP‑1α), CCL4 (also known as MIP‑1β), CXCchemokine ligand (CXCL) 2 (also known as MIP‑2α), CXCL10 and CCL2 (also known as MCP‑1).145 These effects do not seem to be mediated by upregulation of IL‑17, a cytokine group with chemoattractant prop erties.146 Instead TGF‑β, through as yet incompletely defined mechanisms, directly and indirectly attracts monocytes and other inflammatory cells. Although this list of chemokines upregulated by overexpression of TGF‑β in skin does not overlap strikingly with that of chemokines increased in SSc skin (CCL2, CCL5, CCL18, CCL19, CXCL9, and CXCL13), 147 levels of CCL18, CCL19, CXCL9, and CXCL13 were not assessed in mice that overexpress TGF‑β.146

Conclusions

Several lines of evidence place TGF‑β at the centre of SSc pathogenesis. TGF‑β has a key part in altered vascular and connective tissue homeostasis, the hallmark patho genic features of the disease. SSc also shares features with several genetic diseases in which TGF‑β or BMPs are strongly implicated: HHT, familial PAH, and SSS. Finally, gene expression studies show strongly increased expres sion of TGF‑β-regulated genes in SSc skin, and that the extent of upregulation of these genes is associated with the severity of clinical skin disease. Many questions remain to be answered about how TGF‑β activity is deregulated in SSc and about the relationship of dysfunctional TGF‑β signalling to auto immunity. For example, which TGF‑β isoform(s) pri marily drive fibrosis—TGF‑β1, TGF‑β2 or TGF‑β3 —is not yet known, although activation of TGF‑β1 and TGF‑β3 is fairly well understood to involve αvβ6 and



REVIEWS αvβ8 integrin expression,148 and TGF‑β2 might be acti vated through thrombospondin‑1. Another possibility is that altered sequestration of TGF‑β in the matrix might drive its activation. Despite these questions, our current understanding of the role of TGF‑β in SSc provides many exciting targets for treatment of this disease, and gives great hope that effective treatments can be developed to address the painful and often lethal consequences of this disease. 1.

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Review criteria We searched for original articles in MEDLINE and PubMed published between 1995 and 2014. The search terms we used were “transforming growth factor β”, “fibrosis”, “pulmonary hypertension”, “endothelial cells”, “myofibroblasts”, “immunity” and “systemic sclerosis”. All papers identified were English-language full text papers. We also searched the reference lists of identified articles for further papers.

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Transforming growth factor-β-induced CUX1 isoforms are associated with fibrosis in systemic sclerosis lung fibroblasts.

Increased expression of NAPDH oxidase 4 in systemic sclerosis dermal fibroblasts: regulation by transforming growth factor β.

Transforming growth factor-alpha.

The physiology of transforming growth factor-alpha.

The transforming growth factor-beta family.

Transforming growth factor-beta and transforming growth factor beta-receptor expression in human meningioma cells.

Patients with encapsulating peritoneal sclerosis have increased peritoneal expression of connective tissue growth factor (CCN2), transforming growth factor-β1, and vascular endothelial growth factor.

Purification of transforming growth factor type e.

TβRI complex in systemic sclerosis dermal fibroblasts.

Hepatic transforming growth factor-β 1 stimulated clone-22 D1 controls systemic cholesterol metabolism.

Transforming growth factor-beta complexes with thrombospondin.

Transforming growth factor-β1 and diabetic nephropathy.

Transforming growth factor-β1 in plaque morphea.

[Transforming-growth-factor-β-induced corneal dystrophies].

Human eosinophils express transforming growth factor alpha.

Multidomain binding of transforming growth factor alpha to the epidermal growth factor receptor.

Regulation of expression of transforming growth factor-beta 2 by transforming growth factor-beta isoforms is dependent upon cell type.

Co-localization of transforming growth factor beta 2 with alpha 1(I) procollagen mRNA in tissue sections of patients with systemic sclerosis.

Evolutionary origins of the transforming growth factor-beta gene family.

Modulation of the immune response by transforming growth factor beta.

The solution structure of human transforming growth factor alpha.

The cell biology of transforming growth factor beta.

Characterization of B cell growth in systemic lupus erythematosus. Effects of recombinant 12-kDa B cell growth factor, interleukin 4 and transforming growth factor-beta.

Immunomodulatory effects of transforming growth factor-β in the liver.