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`FIBROTIC DISEASE AND THE
`T,1/T,2 PARADIGM
`| ThomasA. Wynn
`| Tissuefibrosis (scarring) is a leading cause of morbidity and mortality. Current treatments
`| for fibrotic disorders, such as idiopathic pulmonary fibrosis, hepatic fibrosis and systemic
`| sclerosis, target the inflammatory cascade, but they have been widely unsuccesstul, largely
`| because the mechanisms that are involved in fibrogenesis are now known to be distinct
`| from those involved in inflammation. Several experimental models have recently been
`| developed to dissect the molecular mechanisms of wound healing and fibrosis. It is hoped
`| that by better understanding the immunological mechanismsthatinitiate, sustain and
`| suppressthe fibrotic process, we will achieve the elusive goal of targeted and effective
`| therapeutics for fibroproliferative diseases.
`
`BLEOMYCIN
`An antineoplastic antibiotic.
`It is active against bacteria and
`fungi, but its cytotoxicity has
`prevented its use as an anti-
`infective agent. Treatment with
`bleomycinis associated with
`significant pulmonary side
`effects — including fbrosis —
`that Hmit its use. Bleomycin was
`first noted to cause pulmonary
`fibrosis in the initial clinical
`trials in which it was tested.
`Since that time, it has been used
`extensively in experimental
`models to dissect the
`mechanismsoffibrosis.
`
`Laboratory ofParasitic
`Diseases, National Institute
`ofAllergy and Infectious
`Diseases, National
`Institutes ofHealth,
`50 South Drive, Room
`6154, MSC 8003, Bethesda,
`Maryland 20892, USA.
`e-mail: twynn@niaid.nih.gov
`doi:10.1038/nril 412
`
`Repair of damaged tissuesis a fundamental biological
`processthat allows the ordered replacementof dead or
`injured cells during an inflammatory response, a mecha-~
`nismthat is crucial for survival. Tissue damage can
`result fromseveral acute or chronic stimuli, including
`infections, autoimmune reactions and mechanical
`injury. The repair process involves twodistinct stages:
`a regenerative phase, in which injuredcells are replaced
`by cells of the same type and there is no lasting evidence
`of damage; and a phase known as fibroplasia orfibrosis,
`in which connective tissue replaces normal parenchymal
`tissue (FIG. 1). In most cases, both stages are required to
`slowor reverse the damage caused by an injurious
`agent. However, althoughinitially beneficial, the heal-
`ing process can become pathogenic if it continues
`unchecked, leading to considerable tissue remodelling
`and the formation of permanentscar tissue. In some
`cases, it might ultimately cause organ failure and death.
`Fibrotic scarring is often defined as a wound-healing
`response that has gone awry.
`Fibroproliferative diseases are an important cause of
`morbidity and mortality worldwide. Fibrotic changes
`can occur in various vascular disorders, including
`cardiac disease, cerebral disease and peripheral vascular
`disease, as well as in all the main tissues and organ sys-
`tems, including the skin, kidney, lung andliver. Fibrosis
`is a troubling problemfor an increasing number of
`
`individuals andis a common pathological sequela of
`manypersistent inflammatorydiseases, such as idio-
`pathic pulmonaryfibrosis, progressive kidney disease
`and liver cirrhosis (Box 1). Despite their obviousaetio-
`logical and clinical distinctions, most ofthese fibrotic
`diseases have in common a persistent inflammatory
`stimulus and lymphocyte-monocyte interactions that
`sustain the production of growth factors, proteolytic
`enzymes and fibrogenic cytokines, which together
`stimulate the deposition of connective-tissue elements
`that progressively remodel and destroy normaltissue
`architecture.
`
`As mechanistic studies of fibrogenesis are difficult to
`carry out in humans, several animal models have been
`developed over the past few years (BOX 2). Although
`combinationsofthese strategies (such as BLEOMYCIN or
`schistosomiasis experiments using transgenic mice)
`have been particularly useful in elucidating the molecu-
`lar mechanismsoffibrosis,all of these approaches have
`limitations. The main problem with manyof the mouse
`models has beenthe difficulty in duplicating the pro-
`gressive tissue remodelling and fibrosis that is seen in
`some of the chronic human diseases. Nevertheless,
`considerable progress has been made over the past
`fewyears, particularly in our understanding of the
`immunological mechanisms that regulate fibrogenesis.
`Although severe acute (non-repetitive) injuries can also
`
`
`
`
`
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`cause markedtissue remodelling,fibrosis that is asso-
`ciated with chronic (repetitive) injury is unique in
`that the adaptive immuneresponseis thought to have
`an importantrole. So, rather than discussing the basic
`features of wound healing, tissue remodelling and
`fibrosis, which have been reviewed elsewhere’, this
`review focuses on howthe adaptive immune response
`amplifies, sustains and suppressesthe fibrotic process,
`particularly in chronic progressive disease.
`
`Cell types involved
`Epithelial or endothelial cell
`
`
`
`
`
`
`Epithelial or endothelial
`damage
`
`Platelets
`
`
`Damaged
`
`Stages of woundhealing
`
`Injury phase
`
`Haemostasis phase
`
`inflammation and
`proliferation phase:
`regeneration
`
`Maturation phase:
`remodelling/ibrosis
`
`site Clot
`
`formation
`
`Neutrophils and monocytes
`accumulate
`
`T cells recruited
`
`
`
`fibronectin
`
`Collagens and
`
`Fibroblast migration and
`proliferation to myofibroblasts
`
`=XAngiogenesis
`
`Polarized T cells regulate organ fibrosis
`In contrast to acute inflammatoryreactions, whichare
`characterized byrapidly resolving vascular changes,
`oedema and neutrophilic infiltration, chronic inflamma-
`tion is defined as a reaction that persists for several
`weeks or months and in which inflammation, tissue
`destruction and repair processes occur simultaneously.
`Whenchronic injuries occur, inflammation is charac-
`terized bya large infiltrate of mononuclearcells, which
`include macrophages, lymphocytes, eosinophils and
`plasmacells. In these cases, lymphocytes are mobi-
`lized and stimulated by contact with antigen to pro-
`duce lymphokines that activate macrophages.
`Cytokines fromactivated macrophages, in turn,stim-
`ulate lymphocytes, thereby setting the stage for per-
`sistence of the inflammatoryresponse. So, there is
`considerable activation of the adaptive immune
`response in chronic inflammatorydiseases. However,
`although inflammation typically precedesfibrosis,
`results from several experimental models showthat
`the amount offibrosis is not necessarily linked with
`the severity of inflammation, indicating that the mech-
`anisms that regulate fibrogenesis are distinct from
`those that regulate inflammation. Findings from our
`ownstudies of schistosomiasis-induced liver fibrosis
`
`strongly support this hypothesis. In this model, fibrosis
`develops progressively in response to schistosome eggs
`that are deposited inthe liver, which induce a cHronic
`GRANULOMATOUSRESPONSE. Similar to most experimental
`models offibrosis, CD4* T cells have an important role
`in the progression ofthe disease. In particular, the type
`of CD4* T-cell response that develops is crucial.
`Studies using various cytokine-deficient mice showed
`that fibrogenesisis strongly linked with the develop-
`ment of a T HELPER 2 (T,2) CD4* T-CELL RESPONSE, involving
`interleukin-4 (IL-4), 1L-5 and IL-13 (er. 2). Although
`an equally potent inflammatory response develops
`when T,,1 CD4* T cells, which produce interferon-y
`(IEN-~y), dominate’, under these circumstances, the
`developmentoftissue fibrosis is almost completely
`attenuated. These studies show that chronic inflamma-
`
`tion does not always induce the deposition of connective-
`tissue elements and that the magnitude offibrosisis
`tightly regulated by the phenotypeof the developing
`Tycell response.
`Tn addition to the system developed in our own lab-
`oratory, several other experimental systems have been
`used to documentthe potent antifibrotic activity of
`IFN-y. In the case of schistosomiasis-inducedfibrosis,
`although treatment with IFN-y or IL-12 has no effect on
`the establishment of infection,collagen deposition asso-
`ciated with chronic granuloma formationis substan-
`tially reduced’. Similar results were obtained in models
`of pulmonary,liver and kidneyfibrosis’. These find-
`ings led to the developmentof an experimental anti-
`fibrosis vaccination strategy that involves the use of IL-12
`OF CpG-CONTAINING OLIGODEOXYNUCLEOTIDES as adjuvants to
`switch off pro-fibrotic T,,2-cell responses in favour of
`less damaging Tyl-cell responses*®. The opposing
`effects of T,,1- and T,,2-cytokine responses in fibrosis
`have also been substantiated by recent microarray
`
`Extracellular-matrix deposition
`
`Figure 1 | The pathogenesis offibrotic disease. Healing is the normal reaction oftissuesafter
`injury. Damaged epithelial and/or endothelial cells release inflammatory meciators thatinitiate an
`antifiprinolytic-coagulation cascacle, which triggers blood-ciot formation. Next, epithelial anc
`endothelial cells secrete growth factors and chemokines that stimulate the proliferation anc
`recruitmentof leukocytes that produce pro-fibrotic cytokines, such as interleukin-13 (L-13) and
`transforming growth factor-B (TGF-B}. Stimulated myofibroblasts and epithelial/endothelial cells
`also produce matrix metalloproteinases (MMPs), which cisrupt the basement membrane,allowing
`the efficient recruitment of cells to sites of injury. After this migration, activatecl macrophages anc
`neutrophils ‘clean-up’ tissue debris and cead cells. They also produce cytokines and chemokines
`that recruit and activate T cells, which are important components of granulation tissue as they
`secrete pro-fibrotic cytokines (such as IL-13). Fibroblasts are subsequently recruited and activated.
`Fibroblasts can be clerived from local mesenchymal cells or recruited from the bone marrow
`(knownasfibrocytes). Epithelial cells can undergo epithelial-mesenchymaltransition, providing a
`rich renewable source of fibroblasts. Revascularization of the wound also occursat this time. After
`activation, myofibroblasts cause wound contraction, ihe process in which the eciges of the wound
`migrate towards the centre. Last, epithelial and/or endothelial cells divide and migrate over the
`basal layers to regenerate the epithelium or endothelium, respectively, which cormpietes the nealing
`process. However, wher repeated injury occurs, chronic inflarmmation and repair can cause an
`excessive accumulation of extracellularmatrix components, such as the collagen that is produced
`by fibroblasts, and lead to the formation of a perrnanentfibrotic scar. Pro-fiorotic mediators, such
`as IL-13 and TGF-B, amplify these processes. The net amount of collagen deposited by fibroblasts
`is reguiated by conlinued collagen synthesis and collagen catabolism. The degraclation of collagen
`is controlled by MMPs and their inhibitors (such as tissueinhibitors of matrix metalloproteinases,
`TIMPs}, and the net increase in collagen within a woundis controlled by the balance of these
`opposing mechanism:
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`
`Box |
`| important fibroproliferative diseases of humans
`
`‘The United States governmentestimates that 45% of deaths in the United States can
`be attributed to fibrotic disorders. Fibrosis affects nearly all tissues and organ systems.
`Disorders in which fibrosis is a major cause of morbidity and mortality arelisted.
`
`Major-organfibrosis
`* Interstitial lung disease (ILD) — includes a wide rangeof distinct disorders in
`which pulmonary inflammation and fibrosis are the final common pathways of
`pathology. [here are more than 150 causes of ILD, including sarcoidosis,silicosis,
`drug reactions, infections and collagen vascular diseases, such as rheumatoid
`arthritis and systemic sclerosis (also known as scleroderma). Idiopathic pulmonary
`fibrosis, which is by far the most common type of ILD, has no known cause.
`* Liver cirrhosis — has similar causes to ILD, with viral hepatitis, schistosomiasis and
`chronic alcoholism being the main causes worldwide.
`* Kidney disease — diabetes can damage and scar the kidneys, which leads to a
`progressive loss of function. Untreated hypertensive diseases can also contribute.
`* Heart disease — scartissue can impair the ability of the heart to pump.
`* Diseases of the eye — macular degeneration and retinal and vitreal retinopathy can
`impair vision.
`
`Fibroproliferative disorders
`* Systemic and local scleroderma
`« Keloids and hypertrophic scars
`Atherosclerosis and restenosis
`
`Scarring associated with trauma(can be severe when persistent)
`+ Surgical complications — scartissue can form between internal organs, causing
`contracture, pain and, in somecases, infertility
`* Chemotherapeutic drug-induced fibrosis
`* Radiationinduced fibrosis
`
`* Accidental injury
`° Burns
`
`experiments”!*: studies investigating the gene-expression
`profiles (transcriptomes)of diseased tissues found that
`markedly different programmes of gene expression are
`induced when chronic inflammatoryresponses are
`dominated by Ty] or T2 cytokines””’. Not surprisingly,
`the transcription of many genesthat are associated with
`IFN-yactivity is upregulated in the tissues of Ty1-
`polarized mice, with no evidenceof significant activa-
`tion of the fibrotic machineryin this setting®’°. Instead,
`two main groups of genes were identified in T,,1-
`polarized mice: those that are involvedin the acute-
`phase reaction and those that are involvedin apoptosis,
`which might explain the large amount ofcell death and
`tissue damage that is observed whenT,,1-cell responses
`continue unrestrained”. By contrast, the transcription of
`several genes that are knowntobe involved in the mech-
`anisms of woundhealing and fibrosis is upregulated by
`Ty2 cytokines”””. The regulation and function of a few of
`these genes,including those that encode procollagen-I,
`procollagen-IIJ, arginase™,lysyl oxidase’, matrix metal-
`loproteinase 2 (MMP2) (RER14), MMP9(REE15) andtis-
`sue inhibitor of matrix metalloproteinase 1 (TIMP1)
`(REFS 16,17), have been investigated in some detail.
`Moreover, several additional 'T,,2-linked genes”, includ-
`ing those that encode haem oxygenase, procollagen-I],
`secreted phosphoprotein 1, procollagen-V,reticulocalbin
`
`CHRONIC GRANULOMATOUS
`RESPONSE
`Granulomasare localized
`inflammatory reactions that
`contain Tcells and are a form of
`delayed-type hypersensitivity.
`They have commonfeatures
`involving persistent antigenic
`stimulation thatis noteasily
`cleared by phagocytic cells. The
`cellular conglomerateis shielded
`from the healthytissue by
`extracellular matrix. Granuloma
`formation andthefibrotic
`scarring that follows can cause
`progressive organ damage.
`
`and fibrillin-1, are also induced in the fibrotic lungs of
`bleomycin-treated mice’® and in carbon tetrachloride
`(CCl,)-stimulated rat hepatic stellate cells (collagen-
`producingcells in the liver)”, providing further proof
`that fibrogenesis is intimately linked with T,2-cytokine
`production (FIG. 2).
`
`IL-13 is the main pro-fibrotic mediator
`Eachof the main T,2 cytokines — IL-4, IL-5 and IL-13
`—— has a distinct role in the regulation of tissue remod-
`elling and fibrosis. IL-4 is foundat increased concen-
`trations in the BRONCHOALVEOLAR LAVAGEfluids of patients
`with idiopathic pulmonary fibrosis”, in the pul-
`monary interstitium of individuals with ceyverocenic
`FIBROSING atvEouTis’! and in the peripheral blood
`mononuclearcells of those suffering from periportal
`fibrosis’. Developmentof post-irradiationfibrosis is
`also associated with increased concentrations of IL-4
`
`(REF, 23). Although the extent to which IL-4 participates
`in the progression of fibrosis can vary in each disease,
`it has long been considered an effective pro-fibrotic
`mediator. In fact, some studies have indicated that IL-4
`is nearly twice as efficient at mediating fibrosis as
`transforming growth factor-B (TGF-B)*4, another
`potent pro-fibrotic cytokine that has been widely stud-
`ied* (discussed later). Receptors specific for IL-4 are
`found on many mouse” and human”fibroblast sub-
`types, and in vitro studies showed that the extracellular
`matrix (ECM) proteins, types I and II collagen and
`fibronectin, are synthesized after stimulation with IL-4
`(REFS 24,27,28). Although studies with fibroblasts showed
`that IL-4 can directly stimulate collagen synthesis
`in vitro, blocking studies were required to confirmits
`role in vive. One of thefirst such reports to investigate
`the contribution of IL-4 was a study ofschistosomiasis
`in mice. In this report, a consistent reductionin hepatic
`collagen deposition was observed wheninfected mice
`were treated with neutralizing antibodies specific for IL-4
`(REE29). Inhibitors of IL-4 also reduced the development
`of dermal fibrosis in a chronic skin-graft rejection model
`and in a putative mouse model of scrzroprrma*>”.
`However, because [L-13 production decreases in the
`absence ofIL-4 (REE29), it was notpossible to discern the
`specific contributions of IL-4 and IL-13 in these early
`IL-4-blocking studies.
`TL-13 shares manyfunctional activities with IL-4
`because both cytokines use the same IL-4 receptor
`o-chain (L-4Ra)—-signal transducer and activator of
`transcription protein 6 (STATS) signalling pathway”.
`However, the development of H-13-transgenic and
`-knockout mice, as well as IL-13 antagonists*”®, has
`revealed unique and non-redundant roles for IL-13 and
`IL-4 in host immunity. Experiments in which IL-4 and
`IL-13 were inhibited independentlyidentified IL-13 as
`the dominant effector cytokine offibrosis in several
`models®™. In schistosomiasis, although the egg-induced
`inflammatory response was unaffected by IL-13 block-
`ade, collagen deposition decreased by more than 85%in
`chronically infected animals’,despite continued and
`undiminished production of IL-4 (REFs 36,40). Related
`studies have also shown a dominantrole for IL-13 in the
`
`
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`T HELPER 2 (7,2) CD4* T-CELL
`RESPONSE
`CD4*Tcells are classified
`according to the cytokines that
`they secrete. T,,2 cells secrete
`large amounts of interleukin-4
`(IL-4), IL-5 and IL-13, which
`promote antibody production
`byB cells and collagen synthesis
`by fibroblasts, whereas Ty] cells
`secrete large amounts of
`interferon-y and associated
`pro-inflammatory cytokines.
`Tyl-type and T,2-type
`cytokines can cross-regulate
`each other’s responses. An
`imbalance of Ty1/T,2
`responsesis thought to
`contribute to the pathogenesis
`of various infections, allergic
`responses and autoimmune
`diseases,
`
`pathogenesis of pulmonary fibrosis. Overexpression of
`IL-13 in the lung induced considerable subepithelial
`airwayfibrosis in mice in the absence of any additional
`inflammatorystimulus™, whereas treatment with IL-13-
`specific antibodies markedly reduced collagen deposi-
`tion in the lungs of animals that were challenged with
`Aspergillus fumigatus conidia’ or bleomycin". By
`contrast, transgenic mice that overexpressed IL-4
`showedlittle evidence of subepithelial airway fibrosis,
`despite developing an intense inflammatory response
`in the lung”.
`Given that IL-4 and IL-13 use similar signalling
`pathways’, it was not immediately clear why IL-13
`should have greater fibrogenic activity than IL-4.
`Presumably, both cytokines bind the samesignalling
`receptor (L-4Ra-IL-13Ra1) that is expressed by
`fibroblasts*’. Indeed, studies carried out using several
`fibroblast subtypes showed potent collagen-inducing
`activity for both IL-4 and IL-13 (REFs 36,44,45). So,
`these cytokines are equally capable of functioning as
`
`
`Box 2 | Experimental models commonly used to stucly fibrosis
`Trauma
`
`+ Surgical trauma or organ transplantation (multiple organs and tissues)
`* Burns (skin)
`* Bile-duct occlusion (liver)
`
`* Irradiation (skin, lungs and other organs)
`* Traumatic aorto-caval fistula or rapid ventricular pacing(heart)
`
`Joxins and drugs
`* Bleomycin, asbestos,silica or ovalbumin (pulmonary fibrosis)
`« Acetaldehyde, carbon tetrachloride or concanavalin A (liver cirrhosis)
`* Vinyl chloride (liver and lung fibrosis)
`* [rinitrobenzene sulphonic acid or oxazolone (gut)
`* Cerulein (pancreas)
`
`Autoimmunedisease or malfunctioning immune-mediated processes
`+ Antibody and inimune-complex disease models (kidney)
`* Organ-transplant rejection (skin, heart and multiple organs)
`* light skin (1sk)-mouse model (progressive systemic sclerosis)
`* ischaemiareperfusion injury (liver)
`* Various models of rheumatoid arthritis (joints)
`
`Chronic infectious diseases
`* Schistosoma species or chronic viral hepatitis (liver)
`* Aspergillusfurnigatus (lung)
`* Mycobacterium tuberculosis (lung andliver)
`* [rypanosoma cruzi (heart or gut)
`
`Genetically engineered mice
`° Transforming growth factor-3 (1GE-B) or 1GF-B-receptor transgenic and
`knockout mice
`
`¢ Signalling-molecule-deficient mice: for example, mothers-against-decapentaplegic
`homologue 3 (SMAD3)-deficient mice
`* Mice deficient in molecules that affect [GEF-B activation: for example, 0, integrin or
`niatrix metalloproteinase 9
`* Cytokinegene transgenic and knockout mice: for example, tumour-necrosis factor,
`interleukin-4 (IL-4), 1L-13 or [L-10
`
`pro-fibrotic mediators in vitro. Results from several
`disease models indicate that differences in ligand density
`might provide at least one explanationfor the differen-
`tial activities of IL-4 and IL-13 (R&Fs 36,46-48). When the
`production of IL-4 and IL-13 are compared, the concen-
`trations of IL-13 often exceed those of IL-4 bya factor of
`10-100. Therefore, IL-13 might be the dominanteffec-
`tor cytokine simply because greater concentrationsare
`produced in vive. Nevertheless, this finding alone
`might not fully explain the differential activities
`because //-4- and fl-13-transgenic mice develop distinct
`forms of pulmonary pathology, even though both
`types of animal express high concentrations of
`cytokine***”, Identical cell-specific promoters were
`used in eachstudy,yet fibrosis was more marked in the
`lungsof I/-13-transgenic mice. Consequently, a more
`important role for IL-13 in tissue remodelling could be
`inferred.Interestingly, two recent studies showed that
`IL-13-regulated responses®, including lung fibrosis”,
`can develop in the absence of IL-4Ra or STAT6 sig-
`nalling molecules. So, IL-13 might use a signalling
`pathwaythat is in some waydistinct fromthat used
`byIL-4, which could be an additional mechanismto
`augmentits fibrogenic potential.
`In contrast to IL-13, the extent to which IL-5 and
`eosinophils participate in fibrotic processes varies
`greatly, with no clear explanationfor the widely diver-
`gent findings. The differentiation, activation and
`recruitment of eosinophils is highly dependent on IL-5,
`and eosinophils could be an important source of fibro-
`genic cytokines (such as TGE-B and IL-13). IL-5 and
`tissue eosinophils have been linked with tissue remod-
`elling in several diseases, including skin allograft rejec-
`tion and pulmonaryfibrosis*»**. Nevertheless, studies
`using neutralizing IL-5-specific antibodies and IL-5-
`deficient mice have yielded conflicting results. Early
`experiments using IL-5-specific monoclonal antibodies
`showed no reduction in liver fibrosis after infection
`
`with Schistosoma mansoni, even though tissue-
`eosinophil responses were markedly reduced™. Although
`negative findings were reported for someof the skin
`and lung fibrosis models*!’, in other studies, signifi-
`cant reductions in tissue fibrosis were observed after IL-5
`activity was ablated****. Interestingly, a recent study
`showedthat, although bleomycin-inducedfibrosis is
`exacerbated in transgenic mice that overexpress IL-5,
`Hl-5* mice remain highly susceptible to fibrosis”,
`indicating that IL-5 and/or eosinophils function as
`amplifiers rather than as indispensable mediators of
`fibrosis. In mice that are deficient in IL-5 and CC-
`chemokine ligand 11 (CCL11; also knownas eotaxin),
`tissue eosinophilia is abolished and the ability of
`CD4* T,2 cells to produce the pro-fibrotic cytokine
`IL-13 is impaired*’. In addition, IL-5 was recently
`shownto regulate TGF-B expressionin the lungs of
`mice that were chronically challenged with ovalbu-
`min®. So, one of the key functions of IL-5 and
`eosinophils might be to facilitate the production of
`pro-fibrotic cytokines, including I-13 and/or TGF-B,
`which then function as the main mediators oftissue
`
`remodelling.
`
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`Cooperation between TGF-f and IL-13
`TGE-Bis undoubtedlythe most intensivelystudied reg-
`ulator of the ECM,and production of TGF-$ has been
`linked with the developmentoffibrosis in several dis-
`eases”*!, There are three isotypes of TGF-B found in
`mammals — TGF-61, -B2 and -B3 — all of which have
`similar biological activities’. Although various cell
`types produce and respond to TGF-B*,tissuefibrosisis
`mainlyattributedto the TGE-B1 isoform,with circulat-
`ing monocytes and tissue macrophages being the main
`cellular source. In macrophages,the mainlevel of con-
`trol is not in the regulation of expression of themRNA
`that encodes TGE-B1 but in the regulation of both the
`secretion and activation of latent TGF-B1. TGF-B1 is
`stored in the cell in an inactive form,as a disulphide-
`bonded homodimerthat is non-covalently boundto a
`latency-associated protein (LAP). Binding of the
`cytokine to its receptors (type I and type I] serine/
`threonine-kinase receptors) requires dissociation ofthe
`LAP, a process that is catalysed in vivo by several
`agents, including cathepsins, plasmin,calpain, throm-
`bospondin, «,8,-integrin and MMPs****?,After activa-
`tion, TGE-Bsignals through transmembranereceptors
`that stimulate the productionofsignalling intermediates
`known as SMAD (mothers-against-decapentaplegic
`homologue) proteins, which modulate the transcription
`of target genes, including those that encode the ECM
`proteins procollagen-I and -III®. Dermalfibrosis after
`irradiation® and renal interstitial fibrosis induced by
`unilateral ureteral obstruction® are both reduced in
`
`SMAD3-deficient mice, confirming an importantrole
`for the TGF-B signalling pathway. So, macrophage-
`derived TGF-B1 is thought to promote fibrosis by
`directly activating resident mesenchymalcells, which
`then differentiate into collagen-producing myofibro-
`blasts. In the bleomycin model of pulmonaryfibrosis,
`alveolar macrophages are thought to produce nearly
`all of the active TGF-B that is involved in the patho-
`logical matrix-remodelling process®. Nevertheless,
`TGE-B1-SMAD3-independent mechanismsoffibro-
`sis have also been proposed”, indicating that addi-
`tional pro-fibrotic cytokines (for example, IL-4 or
`IL-13) can function separately or together with the
`TGF-B-SMAD-signalling pathwayto stimulate the
`collagen-producing machinery.
`Interestingly, in addition to inducing the production
`of latent TGF-B1, IL-13 also indirectlyactivatates TGF-B
`by upregulating the expression of MMPsthat cleave the
`LAP-TGE-B1 complex”! Indeed, IL-13 is a potent
`stimulator of MMP and cathepsin-based proteolytic
`pathways in the lung and liver'””!, So,the tissue remod-
`elling that is associated with polarized T2 responses
`might involve a pathwayin which IL-13-producing
`CD4*T,,2 cells stimulate macrophage production of
`TGE-B1, which then functionsas the main stimulusfor
`fibroblast activation and collagen deposition®**”. In
`support of this hypothesis, when TGF-B1 activity was
`neutralized in the lungsof [-13-transgenic mice, devel-
`opment of subepithelial fibrosis was markedly reduced”.
`However,related studies observed enhanced pulmonary
`pathology when the TGF-B-SMADsignalling pathway
`
`CpG-CONTAINING
`OLIGODEOXYNUCLEOTIDES
`
`DNA oligodeoxynucleotide
`sequences that include a
`cytosine—guanosine sequence
`andcertain flanking
`nucleotides. They have been
`found to induce innate
`immune responses through
`interaction with Toll-like
`receptor 9.
`
`BRONCHOALVEOLAR LAVAGE
`A diagnostic procedure
`conducted by placing a fibre-
`optic scope into the hing of
`a patient and injecting sterile
`saline into the lung to flush
`out free material. Thesterile
`material removed contains
`secretions, cells and proteins
`from the lower respiratorytract.
`
`CRYPTOGENIC FIBROSING
`ALVEOLITIS
`‘Together with various other
`chronic hing disorders,
`cryptogenic fibrosing alveolitis
`is knownas interstitial lung
`disease (ILD). ILD affects the
`lung in three ways:first, the
`tissue is damaged in some
`known or unknown way;
`second, the walls of the air sacs
`become inflamed; and third,
`scarring (or fibrosis) begins in
`the interstitium (tissue between
`the air sacs), and the lung
`becomesstiff.
`
`SCLERODERMA
`Achronic autoimmunedisease
`that causes a hardening of the
`skin, The skin thickens because
`of Increased deposits of collagen.
`There are two types of
`scleroderma. Localized
`scleroderma affects the skin
`in limited areas and the
`musculoskeletal system.
`Systemic sclerosis causes more
`widespread skin changes and can
`be associated with internal organ
`damage to the lungs, heart and
`kidneys.
`
`NATURE REVIEWS
`
`IMMUNOLOGY
`
`L-13 [ow
`
`
`Collagen
`synthesis
`
`:
`.
`Fibrosis
`
`Gollagen
`degradation
`
`
`
`breakdown
`
`Tissue
`
`Figure 2 | Opposing roles for T,,1 and T,,2 cytokines
`in fibrosis. The T helper 1 (T.1)-cell cytokine interferon-y (IFN-y)
`directly suppresses collagen synthesis by fibroblasts. It achieves
`this through regulating the balance of matrix metalioproteinase
`(MMP)andtissueinhibitor of matrix metalloproteinase (TIMP)
`expression, thereby controlling the rates of collagen degraciation
`and synthesis, respectively, in the extraceliular matrix. IFN-y
`and/orinterleukin- 12 (IL-12} might also indirectly inhibit fibrosis
`by reducing pro-fibrotic cylokine expression by 7.2 cells. The
`main 7,2 cytokines(IL-4, IL-5 and IL-13) enhance collagen
`deposition by various mechanisms; nowever, IL-13 seems to
`be the crucial mediator.
`
`was blocked””*, indicating that TGF-B might suppress,
`rather than induce,tissue remodelling in some settings.
`The source of TGE-B1 mightbe crucial to these differ-
`ent effects — macrophage-derived TGF-B1 is often
`pro-fibrotic”, whereas T-cell-derived TGF-B1 seemsto
`be suppressive”’. A recent studyinvestigating the mech-
`anisms of IL-13-dependentfibrosis found no reduction
`in infection-inducedliver fibrosis in MMP9-, SMAD3-
`or TGF-61-deficient mice, indicating that IL-13 can
`function independently of TGF-B®; however,the extent
`to which IL-13 must act through TGF-B1 to induce
`fibrosis remains unclear. Given that manyantifibrotic
`therapies are focused on inhibiting TGE-B1 (REE25), it
`will be important to determine whether the collagen-
`inducing activity of IL-13 is mediated solely by the
`downstream actions of TGF-B and MMPs or whether
`IL-13 and other pro-fibrotic mediatorshave direct
`pro-fibrotic activity, as has been indicated by some
`stucies*®*4°(g]G.3).
`The timing, dose and source of IL-13 and TGE-B
`mightalso affect their individual contributionsto tissue
`remodelling and fibrosis. Because both mediators
`might stimulate collagen deposition directly“, in sit-
`uations in which IL-13 production exceeds TGE-B
`production, IL-13 could be the main pro-fibrotic
`mediator. This might explain the unexpected failure of
`TGE-B/SMADinhibitors in some blocking studies’.
`We speculate that IL-13 might be the keydriver of an
`‘adaptive healing programme that is induced during
`persistent inflammatory responses and is perhaps stimu-
`lus specific”, whereas the TGF-B pathwayof fibrosis
`might be more of an ‘innate’, and possiblyindispens-
`able”, mechanism oftissue remodelling, IL-13 is pro-
`duced mainlybycells of the adaptive immuneresponse
`(CD4* T,2 cells), whereas TGE-B is produced by
`
`VOLUME4 | AUGUST2004 | 887
`
`Lassen - Exhibit 1010, p. 5
`
`Lassen - Exhibit 1010, p. 5
`
`

`

`
`
`
`
`
`
`
`Collagen
`production
`
`
`
`
`
`REVIEWS
`
`nearly all haematopoietic cell populations, which
`might support such a hypothesis”. In addition, IL-13-
`deficient mice are fertile and show no obvious devel-
`
`opmental problems until an invading pathogen or
`persistent irritant induces damage totissues and the
`development of an immune response**. By contrast,
`TGE-Bi-deficient mice are considerably impaired
`
`during embryonic developmentandat birth, which
`also supports an intrinsic role for TGF-B but more of a
`conditional requirementfor TL-13 in tissue remodelling
`and fibrosis.
`
` Active TGF-8
`
`homodimer
`
`
`
`>) secseon Creve se
`; cons) ©
`
` ©ee
`
`
`
`
`
`Chemokines regulate fibrogenesis
`Chemokines are potent leukocyte chemoattractants that
`cooperate with pro-fibrotic cytokines (such as IL-13 and
`'TGE-B) in the development offibrosis, by recruiting
`macrophages and othereffector cells to sites oftissue
`damage. Although numerous chemokinesignalling
`pathways a