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`
`FIBROTIC DISEASE AND THE
`
`THl/THZ PARADIGM
`
`Thomas A. Wynn
`
`Tissue fibrosis (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 unsuccessful, 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 mechanisms that initiate, sustain and
`
`suppress the fibrotic process, we will achieve the elusive goal of targeted and effective
`
`BLEOIVIYCIN
`An antineoplastic antibiotic.
`It is active against bacteria and
`fungi, but its cytotoxicity has
`prevented its use as an anti“
`infective agent. 'l‘reatrnent with
`bleomycin is associated with
`significant pulmonary side
`effects —— including fibrosis w
`that limit 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
`mechanisms of fibrosis.
`
`Laboratory ofParasitic
`Diseases, National Institute
`ofAliergy and Infectious
`Diseases, National
`Institutes ofHealth,
`50 South Drive, Room
`6154, MSC 8003, Bethesda,
`Maryland 20892, USA.
`e—mail: twynn@nia1kl.nih.gov
`doi:10.1038/nri1412
`
`therapeutics for fibroproliferative diseases.
`
`Repair of damaged tissues is a fundamental biological
`process that allows the ordered replacement of dead or
`injured cells during an inflammatory response, a mecha~
`nism that is crucial for survival. Tissue damage can
`result from several acute or chronic stimuli, including
`infections, autoimmune reactions and mechanical
`injury. The repair process involves two distinct stages:
`a regenerative phase, in which injured cells are replaced
`by cells of the same type and there is no lasting evidence
`of damage; and a phase known as fibroplasia or fibrosis,
`in which connective tissue replaces normal pa ren chymal
`tissue (HG. i). in most cases, both stages are required to
`slow or reverse the damage caused by an injurious
`agent. However, although initially beneficial, the heal'
`ing process can become pathogenic if it continues
`unchecked, leading to considerable tissue remodelling
`and the formation of permanent scar 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 an d mortality worldwide. Fibro tic 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 and liver. Fibrosis
`is a troubling problem for an increasing number of
`
`individuals and is a common pathological sequela of
`many persistent inflammatory diseases, such as idio—
`pathic pulmonary fibrosis, progressive kidney disease
`and liver cirrhosis (BOX l). Despite their obvious aetio—
`logical and clinical distinctions, most of these 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 normal tissue
`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
`combinations of these strategies (such as BtsOMYCIN or
`schistosomiasis experiments using transgenic mice)
`have been particularly useful in elucidating the molecu~
`lar mechanisms of fibrosis, all of these approaches have
`limitations. The main problem with many of the mouse
`models has been the 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
`few years, particularly in our understanding of the
`immunological mechanisms that regulate fibrogenesis.
`Although severe acute (nonwrepetitive) injuries can also
`
`
`
`
`
`Norm Rrvnw s l
`
`l M M u a o to e v
`
`vows/1r, [l AUGUST 2004 533
`
`Lassen — Exhibit 1010, p. 1
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`Lassen - Exhibit 1010, p. 1
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`

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`RKWHZWFEE
`
`cause marked tissue remodelling, fibrosis that is asso~
`ciated with chronic (repetitive) injury is unique in
`that the adaptive immune response is thought to have
`an important role. So, rather than discussing the basic
`features of wound healing, tissue remodelling and
`fibrosis, which have been reviewed elsewhere], this
`review focuses on b ow the adaptive immune response
`amplifies, sustains and suppresses the fibrotic process,
`particularly in chronic progressive disease.
`
`Cell types involved
`Epithelial or endothelial cell
`
`
`
`
`Epithelial or endothelial
`damage
`
`
`Platelets
`
`
`
`Stages of wound healing
`
`injury phase
`
`Haemostasis phase
`
`inflammation and
`proliferation phase:
`regeneration
`
`Maturation phase:
`remodelling/fibrosis
`
`
`
`
`Clot
`formation
`
`Neutrophils and monocytes
`accumulate
`
`T cells recruited
`
`Collagens and
`fibronectin
`Fibroblast migration and
`proliferation to myofibroblasts
`
`
`EL13 %Angiogenesis
`
`Extracellular-matrix deposition
`
`Figure l l The pathogenesis of fibrotic disease. Healing is the normal reaction of tissues after
`injury. Damaged epithelial and/or endothelial cells release inflammatory mediators that initiate an
`antifibrinolytic—coagulation cascade, which triggers blood—clot formation. Next, epithelial and
`endothelial cells secrete growth factors and chemokines that stimulate the proliferation and
`recruitment of leukocytes that produce profibrotic cytokines, such as interleukin—l 3 (lL—lS) and
`transforming growth factor BUGF— B). Stimulated myofibroblasts and epithelial/endothelial cells
`also produce matrix metalloproteinases (MMPs), which disrupt the basement membrane, allowing
`the efficient recruitment of cells to sites of injury. After this migration, activated macrophages and
`neutrophils ‘clean—up’ tissue debris and dead cells. They also produce cytokines and chemokines
`that recruit and activate T cells, which are important components of granulation tissue as they
`secrete protibrotic cytokines (such as lL—l 3). Fibroblasts are subsequently recruited and activated.
`Fibroblasts can be derived from local mesenchymal cells or recruited from the bone marrow
`(known as fibrocytes). Epithelial cells can undergo epithelial—mesenchymal transition, providing a
`rich renewable source of fibroblasts. Revascularization of the wound also occurs at this time. After
`activation, myofibroblasts cause wound contraction, the process in which the edges 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 completes the healing
`process. However, when repeated injury occurs, chronic inflammation and repair can cause an
`excessive accumulation of extracellular—matrix components, such as the collagen that is produced
`by fibroblasts and lead to the formation of a permanent fibrotic scar. Pro—fibrotic mediators, such
`as H.— 13 and TGF— B. amplifvthese processes. The net amount of collagen deposited by fibroblasts
`is regulated by continued collagen synthesis and collagen catabolism. The degradation of collagen
`is controlled by MMPs and their inhibitors (such as tissue inhibitors of matrix metalloproteinases,
`TllvlF‘s), and the net increase in collagen within a wound is controlled by the balance of these
`opposing mechanism
`
`584 l AUGUST 2004 VOLUME 4
`
`Polarized T cells regulate organ fibrosis
`in contrast to acute inflammatory reactions, which are
`ch ara cterized by rapidly resolving vascular cha nges,
`oedema and neutrophilic infiltration, chronic inflammaw
`tion is defined as a reaction that persists for several
`weeks or months and in which inflammation, tissue
`destruction and repair processes occur simultaneously.
`W’hen chronic injuries occur, inflammation is charac—
`terized by a large infiltrate of mononuclear cells, which
`include macrophages, lymphocytes, eosinophils and
`plasma cells. In these cases, lymphocytes are mobi—
`lized and stimulated by contact with antigen to pro—
`duce lymphokines that activate macrophages.
`Cytokines from activated macrophages, in turn, stim—
`ula te lymphocytes, thereby setting the stage for per—
`sisten ce of the inflammatory response. So, there is
`considerable a ctivation of the a daptive immune
`response in chronic inflammatory diseases. However,
`although inflammation typically precedes fibrosis,
`results from several experimental models show that
`the amount of fibrosis 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
`own studies of schistosomiasis—induced liver fibrosis
`
`strongly support this hypothesis. In this model, fibrosis
`develops progressively in response to schistosome eggs
`that are deposited in the liver, which induce a CHRONIC
`GILANULOIVLATOUS RESPONSE. Similar to most experimental
`models of fibrosis, (Ll)? T cells have an important role
`in the progression of the disease. in particular, the type
`of CD4‘1 T—cell response that develops is crucial.
`Studies using various cytokine—defi cient mice showed
`that fibrogenesis is strongly linked with the develop—
`ment of a 'r HELPER 2 (er) cpzitrcmt, RESPONSE, involving
`interleukin-4 (ll—4), ll.”5 and ll; 13 (REEZ). Although
`an equally potent inflammatory response develops
`when THl CD4+ T cells, which produce in terferon—y
`(lliN—y), dominateS, under these circumstances, the
`development of tissue fibrosis is almost completely
`attenuated. These studies sh ow that chronic inflamm a—
`
`tion does not always induce the deposition of connective—
`tissue elements and that the magnitude of fibrosis is
`tightly regulated by the phenotype of the developing
`TH—cell response.
`in addition to the system developed in our own lab—
`oratory, several other experimental systems have been
`used to document the potent antifibrotic activity of
`lFN—y. in the case of schistosomiasis'indu ced fibrosis,
`although treatment with lFN—y or ll: 12 has no effect on
`the establishment of infection, collagen deposition asso—
`ciatedwith chronic granuloma formation is substan—
`tially reducedz. Similar results were obtained in models
`of pulmonary, liver and kidney fibrosis“"7. These find—
`ings led to the development of an experimental anti—
`fibrosis vaccination strategy that involves the use of lL—lZ
`or cpocourArNrNc OLlGODEOXXNUCLEOTIDES as adj uvants to
`switch off pro~fibrotic TH2~cell responses in favour of
`less damaging TH] —cell responses”. The opposing
`effects of THl— and THZ—cytokine responses in fibrosis
`have also been substantiated by recent microarray
`
`www. nature. cam/réviews/immunol
`
`Lassen — Exhibit 1010, p. 2
`
`Lassen - Exhibit 1010, p. 2
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`

`

`REVlfiWfii
`
`
`Box 1
`I Important fibroproliferative diseases of humans
`
`The United States government estimates 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 are listed.
`
`Major-organ fibrosis
`~ Interstitial lung disease (ILD) — includes a wide range of distinct disorders in
`which pulmonary inflammation and fibrosis are the final common pathways of
`pathology. There 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 sear the kidneys, which leads to a
`progressive loss of function. Untreated hypertensive diseases can also contribute.
`° Heart disease —— scar tissue 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 — scar tissue can form between internal organs, causing
`contracture, pain and, in some cases, infertility
`° Chemotherapeutic drug-induced fibrosis
`' Radiation—induced fibrosis
`
`~ Accidental injury
`‘ Burns
`
`eitperimentsgw: studies investigating the gene—expression
`profiles (transcriptomes) of diseased tissues found that
`markedly different programmes of gene expression are
`induced when chronic inflammatory responses are
`dominated by THl or THZ cytokinesg’m. Not surprisingly,
`the transcription of many genes that are associated with
`lliN—y activity is upregulated in the tissues of THl—
`polarized mice, with no evidence of significant activa—
`tion of the fibrotic machinery in this setting)”. Instead,
`two main groups of genes were identified in Tle
`polarized mice: those that are involved in the acute—
`phase reaction and those that are involved in apoptosis,
`which might explain the large amount of cell death and
`tissue damage that is observed when THl—cell responses
`continue unrestra ined“. By contrast, the transcription of
`several genes that are known to be involved in the mech—
`anisms of wound healing and fibrosis is upregulated by
`THZ cytokinesg’m. The regulation and function of a few of
`these genes, including those that encode procollagen—l,
`procollagen—l 11, arginase”, lysyl oxidase”, matrix metal—
`loproteinase 2 (M MP2) (REP 14) , M MP9 (REF. 15) and tis—
`sue inhibitor of matrix metalloproteinase 1 (T1 M 1’1 )
`(REFS 16,17), have been investigated in some detail.
`Moreover, several additional THZ—li nked genes9"0,inc1ud—
`ing those that encode haem oxygenase, procolla genelll,
`secreted phosphoprotein l, procolla geirV, reticulocalbin
`
`CHRONlC GRANULOlWATOUS
`RESPONSE
`Granulomas are localized
`inflammatory reactions that
`contain T cells and are a form of
`delayed-type hypersensitivity.
`They have common features
`involving persistent antigenic
`stimulation that is not easily
`cleared by phagocytic cells. The
`cellular conglomerate is shielded
`from the healthy tissue by
`exn'acellular matrix. Granuloma
`formation and the fibro tic
`scarring that follows can cause
`progressive organ damage.
`
`and fibrillin—l , are also induced in the fibrotic lungs of
`bleomycin—treated mice18 and in carbon tetrachloride
`(CClQ—stimulated rat hepatic stellate cells (collagen—
`producing cells in the liver)”, providing further proof
`that fibrogenesis is intimately linked with THLZvcytokine
`production (FIG. 2).
`
`lL-13 is the main pro-fibrotic mediator
`Each of the main THZ cytokines —— 111—4, 111—5 and 11.43
`~— has a distinct role in the regulation of tissue remod—
`elling and fibrosis. lL—4 is found at increased concen—
`trations in the BRONCHO ALVEOLAR LAVAGE fluids of patients
`with idiopathic pulmonary fibrosiszo, in the pul~
`monary interstitium of individuals with cararoomic
`naaosmo Armorms21 and in the peripheral blood
`mononu clear cells of those suffering from periportal
`fibrosis”. Development of post—irradiation fibrosis is
`also associated with increased concentrations of 111—4
`
`(REP. 23). Although the extent to which lL—4 participates
`in the progression of fibrosis can vary in each disease,
`it has long been considered an effective pro—fibrotic
`mediator. 1n fact, some studies have indicated that 111—4
`is nearly twice as efficient at mediating fibrosis as
`transforming growth factor'li (TOP—B)“, another
`potent pro—fibrotic cytokine that has been wi dely stu d—
`ied25 (discussed later). Receptors specific for 11.4 are
`found on many mouse25 and human27 fibroblast sub—
`types, and in vitro studies showed that the extracellular
`matrix (ECM) proteins, types 1 and 111 collagen and
`fibronectin, are synthesized after stimulation with lL—4
`(REFS 24,27,28). Although studies with fibroblasts showed
`that 1L4 can directly stimulate collagen synthesis
`in vitro, blocking studies were required to confirm its
`role in viva. One of the first such reports to investigate
`the contribution of lL—4 was a study of schistosomiasis
`in mice. In this report, a consistent reduction in hepatic
`collagen deposition was observed when infected mice
`were treated with neutralizing antibodies specific for 11.4
`(REF. 29). Inhibitors of11.—4 also reduced the development
`of dermal fibrosis in a chronic skin —graft rejection model
`and in a putative mouse model of SCIERODERMASO’m.
`However, because 11:13 production decreases in the
`absence of 1L—4 (REP. 29), it was not possible to discern the
`specific contributions ofllr/t and 1L—13 in these early
`1L—4—blocking stitches.
`lL—l3 shares many functional activities with 11.41
`because both cytokines use the same 111—4 receptor
`(it—chain (ll.—4Ro:)—signal transducer and activator of
`transcription protein 6 (STA 1‘6) signalling pathway 2.
`However, the development of [1—13wtransgenic and
`—knockout mice33’34, as well as 11:13 antagonistsii“, has
`revealed unique and non—redundant roles for 11:13 and
`11:4 in host immunity. Experiments in which 11.4 and
`lL—13 were inhibited independently identified 111—13 as
`the dominant effector cytokine of fibrosis in several
`modelsss’is. 1n schistosomiasis, although the egg—induced
`inflammatory response was unaffected by lL—l3 block—
`ade, collagen deposition decreased by more than 85% in
`chronically infected animals36’39, despite continued and
`undiminished produ ction of 1 [:4 (REFS 36,40). Related
`studies have also shown a dominant role for 11:13 in the
`
`
`
`NA‘i‘URe REVIEWS I immumotoar
`
`" VOLUME. 4 1 AUGUST 2004 l585
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`Lassen — Exhibit 1010, p. 3
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`Lassen - Exhibit 1010, p. 3
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`

`

`RfiWlfiWfi‘i
`
`T HELPER 2 (THE) CD4‘ 'l‘rCliLl.
`RESPONSE
`CD4" T cells are classified
`according to the cytokines that
`they secrete. THZ cells secrete
`large amounts of interleukindl
`(TL—4), lL-S and 11.43, which
`promote antibody production
`by B cells and collagen synthesis
`by fibroblasts, Whereas THl cells
`secrete large amounts of
`interferon-Y and associated
`pro~inflammatory cytokines.
`TE 1 —type and THZ-type
`cytokines can cross-regulate
`each other’s responses. An
`imbalance of THl/THZ
`responses is thought to
`contribute to the pathogenesis
`of various infections, allergic
`responses and autoimmune
`diseases.
`
`pathogenesis of pulmonary fibrosis. Overexpression of
`ll.—13 in the lung induced considerable subepithelial
`airway fibrosis in mice in the absence of any additional
`inflammatory stimulus“, whereas treatment with ll.—l3—
`specific antibodies markedly reduced collagen deposi—
`ti on in the lungs of animals that were challenged with
`Aspergillus fimzigams conidia37 or bleomycin’“. By
`contrast, transgenic mice that overexpressed lira
`showed little evidence of subepi thelial airway fibrosis,
`despite developing an intense intla mmatory response
`in the lung”.
`Given that IL—4 and IL—13 use similar signalling
`pathways“, it was not immediately clear why ll.—l3
`should have greater fibrogenic activity than ll.—4.
`Presumably, both cytokines bind the same signalling
`receptor (ill—4RDL—llx13lttrl ) that is expressed by
`fibroblasts“. indeed, studies carried out using several
`fibroblast subtypes sh owed potent collagenwinduci n g
`activity for both le/l and Iii—13 (REFS 36,44,45). So,
`these cytokines are equally capable of functioning as
`
`
`Box 2 | Experimental models commonly used to study 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)
`
`Toxins and drugs
`- Bleomycin, asbestos, silica or ovalbumin (pulmonary fibrosis)
`° Acetaldehyde, carbon tetrachloride or concanavalinA (liver cirrhosis)
`° Vinyl chloride (liver and lung fibrosis)
`' Trinitrobenzene sulphonic acid or oxazolone (gut)
`- Cerulein (pancreas)
`
`Autoimmune disease or malfunctioning immune—mediated processes
`° Antibody and immune—complex disease models (kidney)
`~ Organ—transplant rejection (skin, heart and multiple organs)
`- Tight skin (Tsk)-mouse model (progressive systemic sclerosis)
`° Ischaemia—reperfusion injury (liver)
`° Various models of rheumatoid arthritis (joints)
`
`Chronic infectious diseases
`
`° Schistosoma species or chronic viral hepatitis (liver)
`‘ Aspergillusfumigatus (lung)
`° Mycobarterium tuberculosis (lung and liver)
`' Trypanosoma cmzz' (heart or gut)
`
`Genetically engineered mice
`- Transforming growth factor-B (TGIF—B) or TGF-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 TGF—B activation: for example, al-integrin or
`matrix metalloproteinase 9
`- Cytokine—gene transgenic and knockout mice: for example, tumour-necrosis factor,
`interleukin—4 (IL-4), IL—13 or IL— 10
`
`pro~fibrotic mediators in vitro. Results from several
`disease models indicate that differences in ligand density
`might provide at least one explanation for the differen—
`tial activities of IL—4 and ll,.—l3 (REFS 36,4648). When the
`production of [Let and ll.-13 are compared, the concen—
`trations of lL—l 3 often exceed those of iii—4 by a factor of
`10—100. Therefore, lL—l3 might be the dominant effec—
`tor cytokine simply because greater concentrations are
`produced in viva. Nevertheless, this finding alone
`might not fully explain the differential activities
`because IZ—4— and ll—l3—transgenic mice develop distinct
`forms of pulmonary pathology, even though both
`types of animal express high concentrations of
`cytokine34’“. identical cell—specific promoters were
`used in ea ch study, yet fibrosis was more marked in the
`lungs of [LB—transgenic mice. Consequently, a more
`important role for HA3 in tissue remodelling could be
`inferred. Interestingly, two recent studies showed that
`ill—lSMregulated responses“, including lung fibrosis“,
`can develop in the absence oflL—AlRtx or STAT6 sig—
`nalling molecules. So, lL—l 3 might use a signalling
`pathway that is in some way distinct from that used
`by lL—4,which could be an additional mechanism to
`augment its fibrogenic potential.
`in contrast to ll.—13, the extent to which ll.—5 and
`eosinophils participate in fibrotic processes varies
`greatly, with no clear explanation for the widely diver—
`gent findings. The differentiation, activation and
`recruitment of eosinophils is highly dependent on ll.—5,
`and eosinophils could be an important source of fibro—
`genic cytokines (such as TGFWB and IL"13). lL—S and
`tissue eosinophils have been linked with tissue remod—
`elling in several diseases, including skin all ograft rejec—
`tion and pulmonary fibrosism’so. Nevertheless, studies
`using neutralizing lL—S—specific antibodies and lL—5—
`deficient mice have yielded conflicting results. Early
`experiments using lL—S'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 some of the skin
`and lung fibrosis models51’52, in other studies, signifi
`cant reductions in tissue fibrosis were observed after Ill—5
`
`activity was al)lated31’53’55. interestingly, a recent study
`showed that, although bleomycin—induced fibrosis is
`exacerbated in transgenic mice that overexpress ill—5,
`Iii—5+ mice remain highly susceptible to fibrosis“,
`indicating that ll.—5 and/or eosinophils function as
`amplifiers rather than as indispensable mediators of
`fibrosis. In mice that are deficient in ll.»5 and CC—
`
`chemokine ligand 1 l (CCLll; also known as eotaxin),
`tissue eosinophilia is abolished and the ability of
`CD4" TH2 cells to produce the pro—fibrotic cytokine
`llrl 3 is impaired“. In addition, Iii—5 was recently
`sh own to regulate “1le expression in the lungs of
`mice that were chronically challenged with ovalbu—
`minSS. So, one of the key functions of Iii—5 and
`eosinophils might be to facilitate the production of
`pro—fibrotic cytokines, including HA3 and/or TGF—B,
`which then function as the main mediators of tissue
`
`remodelling.
`
`
`wviiilmaturefcorm/ reviews/ inirriunol
`
`586 I AUGUST 2004 {VOLUME 4
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`Lassen — Exhibit 1010, p. 4
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`Lassen - Exhibit 1010, p. 4
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`REVifiWfii
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`Cooperation between TGF-fi and lL-13
`TGPB is undoubtedly the most intensively studied reg—
`ulator of the ECM, and production of TGFMB has been
`linked with the development of fibrosis in several dis—
`ea sesSS’“. There are three isotypes of "[‘GIi—B found in
`mammals M TGF—Bl, WBZ and 433 —— all of which have
`similar biological activities“. Although various cell
`types produce and respond to TGF—B”, tissue fibrosis is
`mainly attributed to the TGF—Bl isoform, with circulat—
`ing monocytes and tissue macrophages being the main
`cellular source. In macrophages, the main level of cone
`trol is not in the regulation of expression of the mRNA
`that encodes TOP—Bl but in the regulation of both the
`secretion and activation oflatent TGF—BI. TGF—BI is
`stored in the cell in an inactive form, as a disulphide—
`bonded homodimer that is non—covalently bound to a
`la tencywassociated protein (LAP). Binding of the
`cytokine to its receptors (type I and type II serine/
`threonine—kinase receptors) requires dissociation of the
`LAP, a process that is catalysed in viva by several
`agents, including cathepsins, plasmin, calpain, thromw
`bospondin, %fi6-illtegfill and MMPsZS’SZ’“. After activa—
`tion, TGF—B signals through transmembrane receptors
`that stimulate the production of signalling intermediates
`known as SMAD (mothers—against—decapentaplegic
`homologue) proteins, which modulate the transcription
`of target genes, including those that encode the ECM
`proteins procolla gen—I and —III54. Dermal fibrosis after
`irradiation65 and renal interstitial fibrosis induced by
`unilateral ureteral obstructionSS are both reduced in
`
`SMADS—deficient mice, confirming an important role
`for the TGF—B signalling pathway. So, macrophage—
`derived TGF—Bl
`is thought to promote fibrosis by
`directly activating resident mesenchymal cells, which
`then differentiate into collagen—producing myofibro—
`blasts. In the bleomycin model of pulmonary fibrosis,
`alveolar macrophages are thought to produce nearly
`all of the active TGF—B that is involved in the patho—
`logical matrix—remodelling process“. Nevertheless,
`TGF—Bl—SMAD3—independent mechanisms of fibro—
`sis have also been proposedm’“, indicating that addi—
`tional pro—fibrotic cytokines (for example, IL—4 or
`ILwl3) can function separately or together with the
`TGF—fi—SMADwsignalling pathwayto stimulate the
`collagen—producing machinery.
`Interestingly, in addition to inducing the production
`of latent TGF—Bl , IL—13 also indirectly activatates TGF—fi
`by upregulating the expression of MMPs that cleave the
`I.AI’~TGF—Bl complexm’“. Indeed, IL—l 3 is a potent
`stimulator of MMP and cathepsin'based proteolytic
`pathways in the lung and liver‘m. So, the tissue remod—
`elling that is associated with polarized THZ responses
`might involve a pathway in which IL— 13wproducing
`CD4" THZ cells stimulate macrophage production of
`TGF—Bl, which then functions as the main stimulus for
`fibroblast activation and collagen (leposition34’70. In
`support of this hypothesis, when TGF—B] activity was
`neutralized in the lungs of [1—1 3~transgenic mice, devel—
`opment of subepithelial fibrosis was markedly reduced”.
`However, related studies observed enhanced pulmonary
`pathology when the TGF—B—SMAD signalling pathway
`
`Cpti CONTAINING
`OLIGODEOXYNUCLEOTIDES
`
`DNA oligodeoxynucleotide
`sequences that include a
`cytosine—guanosine sequence
`and certain flanking
`nucleotides. They have been
`found to induce innate
`immune responses through
`interaction with Toll—like
`receptor 9.
`
`BRONCHOAIVEOIAR LAVAGE.
`A diagnostic procedure
`conducted by placing a fibre-
`optic scope into the lung of
`a patient and injecting sterile
`saline into the lung to llush
`out free material. The sterile
`material removed contains
`secretions, cells and proteins
`from the lower respiratory tract.
`
`CRYIJTOGENIC FIBROSING
`AIVEOLITIS
`Together with various other
`chronic lung disorders,
`cryptogenic fibrosing alveolitis
`is known as intersn'tial 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
`becomes stiff.
`
`SCILRODERIVTA
`A chronic autoimmune disease
`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.
`
`N ATURE REVIEWS
`
`lMMUNOi.QGY
`
`
`lL-l3 filMPs
`MMPs\lFl\l—y
`
`
`
`Collagen
`degradation
`
`Collagen
`synthesis
`
`.
`.
`Fibrosis
`
`
`
`Tissue
`breakdown
`
`Figure 2 i Opposing roles for TH‘l and TH2 cytokines
`in fibrosis. The T helper i (THU—cell cytokine interieron—yilFN—y)
`directly suppresses collagen synthesis by fibroblasts. it achieves
`this through regulating the balance of matrix metalloproteinase
`(MMP) and tissue inhibitor of matrix metalloproteinase (TIMP)
`expression, thereby controlling the rates of collagen degradation
`and synthesis, respectively, in the extracellular matrix. lFN—y
`and/or interleukin—i 2 ilL—i 2) might also indirectly inhibit fibrosis
`by reducing pro—tibrotic cytokihe expression by TH2 cells. The
`main TH2 cytokines (lL—4,
`lL—5 and lL—i 3) enhance collagen
`deposition by various mechanisms; however, lL—iS seems to
`be the crucial mediator.
`
`was blockedflfi, indicating that TGF—B might suppress,
`rather than induce, tissue remodelling in some settings.
`The source of TGF—[Sl might be crucial to these differ,
`ent effects —— macrophagemderived TGF—Bl is often
`pro—fibrotic”, whereas T—cell—derived TGF—Bl seems to
`be suppressive”. A recent study investigating the mech—
`anisms of IL—1 3—dependent fibrosis found no reduction
`in infection—indu ced liver fibrosis in MMP9—, SMAD3—
`or TGF—BI—deficient mice, indicating that IL—13 can
`function independently of TGFwi’)“; however, the extent
`to which lL—I3 must act through TGF~Bl to induce
`fibrosis remains unclear. Given that many antifibrotic
`therapies are focused on inhibiting TGF—Bl (REF 25), it
`will be important to deterntine whether the collagen—
`inducing activity of IL—13 is mediated solely by the
`downstream actions of TGFMB and MMPs or whether
`lL—l 3 and other pro—fibro tic mediators44 have direct
`pro—fibrotic activity, as has been indicated by some
`studies35r44r69 (FIG. 3).
`The timing, dose and source ofIL—13 and TGF—B
`might also affect their individual contributions to tissue
`remodelling and fibrosis. Because both mediators
`might stimulate collagen deposition directly“, in site
`nations in which IL—13 production exceeds TGF—B
`production, IL—l3 could be the main pro—fibrotic
`mediator. This might explain the unexpected failure of
`TGFMB/SMAD inhibitors in some blocking studiesmrfig.
`We speculate that ill—13 might be the key driver of an
`‘adaptive’ healing programme that is induced during
`persistent inflammatory responses and is perhaps stimu—
`lus specific“, whereas the TGF—b pathway of fibrosis
`might be more of an ‘innate’, and possibly indispense
`able”, mechanism of tissue remodelling. IL—13 is pro—
`duced mainly by cells of the adaptive immune response
`(CD1? THZ cells)33, whereas TGF—ii is produced by
`
`vomit/iii; i Atrousr 2004 l587
`
`Lassen — Exhibit 1010, p. 5
`
`Lassen - Exhibit 1010, p. 5
`
`

`

`RfiWiEWfii
`
`nearly all haematopoietic cell populations, which
`might support such a hypothesis”. In addition, 11:13'
`deficient mice are fertile and show no obvious devel—
`
`opmental problems until an invading pathogen or
`persistent irritant induces damage to tissues and the
`development of an immune response33’36. By contrast,
`TGF—Bl ”deficient mice are considerably impaired
`
`
`
` Active TGF—B
`
`homodimer
`
`
`
` asee
`
`
`
`during embryonic development and at birth”, which
`also supports an intrinsic