DOI: 10.1126/scitranslmed.3001510, 57ra82 (2010);2 Sci Transl Med

, et al.Glenda TrujilloPulmonary FibrosisTLR9 Differentiates Rapidly from Slowly Progressing Forms of Idiopathic

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I D IOPATH IC PULMONARY F I BROS I S

TLR9 Differentiates Rapidly from Slowly ProgressingForms of Idiopathic Pulmonary FibrosisGlenda Trujillo,1* Alessia Meneghin,1 Kevin R. Flaherty,2 Lynette M. Sholl,3 Jeffrey L. Myers,1

Ella A. Kazerooni,4 Barry H. Gross,4 Sameer R. Oak,1 Ana Lucia Coelho,1 Holly Evanoff,1

Elizabeth Day,5 Galen B. Toews,2 Amrita D. Joshi,1 Matthew A. Schaller,1 Beatrice Waters,5

Gabor Jarai,5 John Westwick,5 Steven L. Kunkel,1 Fernando J. Martinez,2† Cory M. Hogaboam1†

(Published 10 November 2010; Volume 2 Issue 57 57ra82)

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Idiopathic pulmonary fibrosis is characterized by diffuse alveolar damage and severe fibrosis, resulting in asteady worsening of lung function and gas exchange. Because idiopathic pulmonary fibrosis is a generally pro-gressive disorder with highly heterogeneous disease progression, we classified affected patients as either rapidor slow progressors over the first year of follow-up and then identified differences between the two groups toinvestigate the mechanism governing rapid progression. Previous work from our laboratory has demonstratedthat Toll-like receptor 9 (TLR9), a pathogen recognition receptor that recognizes unmethylated CpG motifs inbacterial and viral DNA, promotes myofibroblast differentiation in lung fibroblasts cultured from biopsies ofpatients with idiopathic pulmonary fibrosis. Therefore, we hypothesized that TLR9 functions as both a sensor ofpathogenic molecules and a profibrotic signal in rapidly progressive idiopathic pulmonary fibrosis. Indeed,TLR9 was present at higher concentrations in surgical lung biopsies from rapidly progressive patients thanin tissue from slowly progressing patients. Moreover, fibroblasts from rapid progressors were more responsiveto the TLR9 agonist, CpG DNA, than were fibroblasts from slowly progressing patients. Using a humanizedsevere combined immunodeficient mouse, we then demonstrated increased fibrosis in murine lungs receivinghuman lung fibroblasts from rapid progressors compared with mice receiving fibroblasts from slowly progress-ing patients. This fibrosis was exacerbated by intranasal CpG challenges. Furthermore, CpG induced the differ-entiation of blood monocytes into fibrocytes and the epithelial-to-mesenchymal transition of A549 lungepithelial cells. These data suggest that TLR9 may drive the pathogenesis of rapidly progressive idiopathicpulmonary fibrosis and may serve as a potential indicator for this subset of the disease.

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INTRODUCTION

Idiopathic pulmonary fibrosis (IPF) is a chronic, generally progressivelung disease with high mortality and inadequate therapeutic options.It is widely accepted that IPF is initiated by an unknown insult to thelung that leads to irreversible scarring marked by severe alveolar de-struction, variable inflammation accompanied by excessive depositionof extracellular matrix (ECM), and ultimate loss of normal lung func-tion (1). The pathogenesis of IPF is not completely understood, al-though persistent fibroblast activation and proliferation are consideredto be targetable mechanisms for therapeutic intervention.

Fibroblasts produce ECM proteins, which are critical for tissue ho-meostasis and normal wound repair. In fibrosing diseases such as IPF,fibroblasts exhibit unregulated proliferation, differentiate into myofi-broblasts, and produce excessive ECM, all of which lead to destructionof normal interstitial architecture. There is growing evidence that, inaddition to the proliferation of resident fibroblasts, disease-associatedfibroblasts also arise from other cellular sources such as bone marrow–derived fibrocytes and epithelial cells (2). Moreover, fibrocytes can en-

1Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109–2200, USA. 2Division of Pulmonary and Critical Care Medicine, Department of InternalMedicine, University of Michigan Health System, Ann Arbor, MI 48109–5360, USA.3Brigham and Women’s Hospital, Department of Pathology, Harvard Medical School,Boston, MA 02115, USA. 4Department of Radiology, University of Michigan MedicalSchool, Ann Arbor, MI 48109–5030, USA. 5Novartis Institutes of Biomedical Research,Respiratory Disease Area, Horsham, RH12 5AB West Sussex, UK.*To whom correspondence should be addressed. E-mail: gtrujillo@notes.sunysb.edu†These authors contributed equally to this work.

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ter damaged tissue through chemokine-dependent mechanisms andmature into collagen-producing myofibroblasts (3–6). In addition,epithelial structures can differentiate into myofibroblasts throughepithelial-mesenchymal transition (EMT) (7–10). Further investiga-tion of these pathways may lead to novel treatments for various formsof fibrotic diseases.

The disease course of IPF patients is extremely variable, with somepatients exhibiting disease stability for prolonged periods of time,whereas others exhibit rapid disease progression (11). Although someIPF patients exhibit gradual physiological decline, others experience arapid deterioration as a result of acute exacerbation of IPF (AE-IPF)(12). Disease progression in IPF patients has been defined by a com-posite approach; however, a method to predict the disease course dur-ing an initial evaluation would have great clinical value.

Several hypotheses have been proposed for the etiology of IPF dis-ease progression, but no consensus has been reached. An acceleratedvariant of IPF appears to clinically distinguish rapid progressors fromIPF patients who experience a slower or more stable progressive clinicalcourse, but it is not known whether rapid progression equates withhaving a history of an acute exacerbation (13). Viral infections, espe-cially herpesviruses, have been associated with IPF and may be linkedto AE-IPF. Specifically, the prototypic gammaherpesvirus, Epstein-Barrvirus (EBV), has been consistently detected in IPF patients (14–16),and gammaherpesvirus exacerbates established pulmonary fibrosis ina fluorescein isothiocyanate (FITC) murine model of pulmonary fi-brosis (17). Toll-like receptor 9 (TLR9) is a primary innate immune

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sensor mechanism that recognizes unmethylated CpG DNA motifspresent in bacterial and viral DNA and interacts with gammaherpes-virus to mediate host immunity (18). Our laboratory has recently re-ported that enhanced TLR9 expression on pulmonary fibroblastsfrom lung biopsies of IPF patients can drive the differentiation ofmyofibroblasts in response to CpG in vitro (19). Therefore, we hypoth-esized that TLR9 may be a predictor of the rapidly progressive form of

IPF and may render these patients suscep-tible to acute exacerbations.

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RESULTS

IPF patients were stratified intorapidly and slowly progressive formsTwenty-three patients were diagnosedwith IPF using a multidisciplinary, clin-ical, radiological, and pathological mecha-nism (20). The physiological criteria usedto validate disease progression during thefirst year of follow-up included a forcedvital capacity (FVC) decrease of greaterthan or equal to 10% and a diffusing ca-pacity for carbon monoxide (DLCO) de-crease of greater than or equal to 15%based on baseline physiological abnormali-ty. Baseline data for each patient in thestudy included detailed clinical assess-ment, physiological studies, high-resolutioncomputed tomography (HRCT), and sur-gical lung biopsies (SLBs). Patients weretreated with a variety of treatment regimensand followed closely with physiologicalstudies and capture of clinical informationduring acute events. Acute exacerbationsof IPF were defined with criteria recentlyproposed by our group (21) or all-causemortality.

Ten IPF patients in our cohort exhib-ited disease progression during the initialfirst year of follow-up after initial diag-nosis, whereas 13 did not; the mean timeof follow-up for the patients was 1154 ±603 days. Of the 10 patients experiencingprogressive disease during the first year offollow-up, 8 were characterized as rapidprogressors on the basis of physiologicalparameters (FVC decrease greater than orequal to 10%, DLCO decrease greater thanor equal to 15%), 1 experienced an AE-IPF, and 1 died of respiratory causes overa time frame longer than usually used todefine an acute exacerbation. Overall sur-vival was significantly reduced in patientswith rapidly progressing disease over thefirst year of follow-up (P = 0.003) (Fig. 1A).Table 1 enumerates the clinical, physio-logical, imaging, and histological features

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at initial diagnosis baseline.No statistically significant differences werenoted in demographics, physiological severity, or HRCT and histolog-ical semiquantitative abnormality as a function of progressive disease.Figure 1B demonstrates representative histology for slow (panels 1 and2) and rapid progressors (panels 3 and 4). In both groups of patients, theSLBs showed heterogeneous interstitial fibrosis with architectural dis-tortion (panels 1 and 3) and multifocal fibroblast foci (panels 2 and 4)

Fig. 1. Clinical features of patients with rapidly and slowly progressive forms of IPF. (A) Survival of IPFpatients classified as rapid (red line) or slow progressors (black line). (B) Representative lung histology of

IPF in slow (1 and 2) and rapid (3 and 4) progression shown at ×20 and ×40 magnification. (C) QuantitativeTaqMan PCR analysis of TLR9 expression in upper lobe surgical lung biopsies (SLBs) from rapid and slowprogressors. The data shown are the mean of all the combined upper lobe mRNA values compared to themean of normal SLB mRNA values [standardized to glyceraldehyde-3-phosphate dehydrogenase(GAPDH)]. Statistical analysis of the data in the rapid (n = 10) and slow (n = 13) progressor patient groupswas determined by the unpaired t test with Welch correction. (D) Immunohistochemical staining of TLR9in SLBs from slow (1) and rapid (3) progressors shown at ×20 magnification (representative samples fromtotal of seven slow and five rapid patients). Corresponding fields stained with isotype control [immuno-globulin G (IgG)] shown in (3) and (4). Arrows in (1) and (3) indicate TLR9-positive staining.

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characteristic of usual interstitial pneumonias (UIP). None of the patientshad evidence of AE-IPF (diffuse alveolar damage) at the time of SLB.

Because TLR9 is highly expressed in IPF lungs andCpG-oligodeoxy-nucleotide (ODN) can drive a myofibroblast differentiation of IPF lungfibroblasts in vitro (19), we tested whether TLR9 expression differed be-tween rapidly and slowly progressive IPF. TLR9 gene expression waselevated in lung tissue from rapidly progressive IPF patients comparedto slow progressors (Fig. 1C). These results were confirmed by immu-nohistochemical analysis of TLR9 in SLBs, where expression was evidentin the interstitial areas of the lung from rapid progressors (Fig. 1D), but theslow progressors showed TLR9 staining that was localized to immunecells (Fig. 1D, panel 1).

CpG-ODN induces a fibroblast-like phenotypein primary human blood monocytes in vitro in thepresence of transforming growth factor–bBased on our previous findings that CpG induces myofibroblast dif-ferentiation of IPF fibroblasts, the question of whether CpG could alsodrive a fibroblast-like phenotype in additional IPF-relevant cell typeswas investigated. Other laboratories have reported that fibroblast-likecells (“fibrocytes”) can arise from purified human CD14+ monocytesunder serum-free conditions after 4 days in culture (22–24). This is incontrast to other reports demonstrating a fibrocyte population devoidof CD14 in human peripheral bloodmononuclear cell (PBMC) culturesafter 7 days in serum (24, 25). In these studies, the addition of trans-forming growth factor–b (TGFb) to PBMCcultures promoted fibrocyte

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differentiation, which isminimally defined by spindle-shapedmorphol-ogy and collagen 1 expression. Thus, we testedwhether CpG can drive afibrocyte-like phenotype in purified CD14+ monocytes derived fromthe peripheral blood of healthy human donors. Purified CD14+ cellswere plated in serum-free media in the presence or absence of TGFbfor 3 days, after which they were treated for an additional day with noth-ing, control nonstimulatory CpG-ODN (non-CpG), CpG-ODN, or aTLR3 agonist [polyinosinic–polycytidylic acid (poly IC)] (Fig. 2A).Morphological assessment by phase-contrast microscopy revealedthat monocytes cultured inmedia alone or in combination with TGFbmaintained a round shape typical of a monocytic phenotype (Fig. 2B,panels 1 and 2). The samephenotypewas observed inmacrophages stim-ulated with non-CpG (Fig. 2B, panels 3 and 4) and poly IC (Fig. 2B,panels 7 and 8) in the presence and absence of TGFb. In contrast,monocytes stimulated with either CpG alone (Fig. 2B, panel 5) orCpG in the presence of TGFb (Fig. 2B, panel 6) exhibited a distinctpopulation of elongated, spindle-shaped cells resembling fibrocytes.To determinewhether the differences in the cultures correspondedwiththe induction of fibrocyte markers, we isolated and purified RNA fromthe adherent cells and subjected it to gene expression analysis by quan-titative TaqMan real-time polymerase chain reaction (PCR). aSMA (asmoothmuscle actin) is a protein marker expressed primarily onmes-enchymal cells and is usually absent in blood or epithelial cells. Up-regulation has been linked to myofibroblast differentiation and, morerecently, fibrocyte differentiation. Induction ofaSMA gene transcriptswas observed only inmonocytes stimulatedwith CpG and not in thosetreated with poly IC or untreated monocytes (Fig. 2C, left panel), sug-gesting that up-regulation of aSMA gene expression in our culture sys-tem is specific to CpG. Furthermore, and in agreement with previousstudies fromother laboratories,monocytes demonstrated up-regulationof collagen1whencultured in thepresenceofTGFb (Fig. 2C, right panel).Unstimulated monocytes also express collagen 1, which is consistentwith previous reports that macrophages express the entire repertoireof collagens (26). Although we did not observe any differences in colla-gen expression that correlated with the CpG-induced effects, these dataconfirm that TGFb increases collagen expression in CD14+ monocytes,an effect that may be limited to TGFb.

The effect of CpGon collagen protein production in culturedCD14+

monocytes was determined by immunofluorescence and flow cytome-try. Collagen staining was increased in CD14+ monocytes that werecultured with TGFb alone or stimulated with CpG alone in media(Fig. 2D). Treatment with both CpG and TGFb enhanced collagen1 staining (panel 4), which is consistent with the change inmorphologydemonstrated in Fig. 2B (panel 6). Furthermore, flow cytometry analy-sis of collagen-positiveCD14+CD45+monocytes indicates thatCpG en-hances collagen 1 protein production in TGFb-cultured cells (panel 6).Moreover, fibrocyte-like monocytes were characterized with flow cy-tometry to analyze the forward and side scatter properties. CD14+

monocytes stimulated with CpG in the presence of TGFb have a dom-inant population (72.3% of total cells) composed of cells with increasedforward scatter and side scatter, indicative of increased cellular size andcomplexity (Fig. 2E, second panel), whereas most monocytes culturedin TGFb alone appeared smaller in size (Fig. 2E, first panel).

Monocyte-derived fibrocytes arewidely reported asCD14−, andPBMCsloseCD14 expression upon differentiation into fibrocytes (27, 28). CD14,a cell surface co-receptor on macrophages for lipopolysaccharide (LPS),is shed along with TLR4 and MD-2 during bacterial infections (29, 30).We tested whether TGFb alters the CD14+monocyte population during

Table 1. Clinical features of patients with rapid versus slowly progres-sive IPF. NA, not available.

Feature

Rapid progressor

(n = 10)

Slow progressor

(n = 13)

P

Demographic

Age

64 ± 7 64 ± 5 0.9

Gender (M/F)

6/4 9/4 0.69

Physiological

FVC (% predicted)

63 ± 14 73.2 ± 17.5 0.17

DLCO (% predicted)

44 ± 18 51 ± 26 0.09

HRCT

Alveolar

1.53 ± 0.79 1.60 ± 0.71 0.85

Interstitial

1.31 ± 0.60 0.97 ± 0.38 0.11

Histological

Honeycomb changescore (median)

1.1

1.0 0.55

Maximum HC score (median)

2.0 2.0 0.54

Disease progression*

FVC >10%

6 NA NA

DLCO >15%

2 NA NA

AE-IPF

1 NA NA

Death

1 NA NA

FVC slope

−0.05 ± 0.06 0.01 ± 0.04 0.13

DLCO slope

−0.14 ± 0.17 0.10 ± 0.27 0.14

*Time to first event during first year of follow-up.

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fibrocyte differentiation in our culture system. After 4 days, >95% of thetotal cells were CD14− when cultured in media alone, and CpG did notaffect the population (Fig. 2E, third panel). In media containing TGFb orTGFb plus CpG, CD14+ monocytes comprise almost 100% of the cellpopulation (Fig. 2E, fourth panel). These results demonstrate that CD14expressiononmonocytes is dynamic and that loss ormaintenanceofCD14expression does not necessarily correlate with fibrocyte differentiation.

Next, we analyzed the effects of CpG on the CD14+ and CD14−mono-cyte population for up-regulation of fibrocyte markers by flow cytome-try. We found that in CD14− cells, CpG alone or in combination withTGFb induced expression of CD45, a hematopoietic marker widely used

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to phenotype fibrocytes (Fig. 2E, fifth and sixth panels). Up-regulation ofCD45 by CpG was also observed in CD14+ monocytes that were culturedwith TGFb (Fig. 2E, sixth panel). No effect on CD45 expression was ob-served in CD14+ cells cultured in media alone (Fig. 2E, fifth panel). Col-lectively, these data suggest that CpG induces a fibrocyte-like phenotypein CD14+ monocytes as defined by induction of an elongated, spindle-shaped morphology, and up-regulation of aSMA, collagen 1, and CD45.

CpG-ODN induces EMT in A549 cellsBased on the CpG-mediated effects observed in monocytes (Fig. 2), wepostulated that CpG may induce EMT in epithelial cells. The human

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Fig. 2. Induction by CpG of CD14+ humanmonocytes to differentiate into fibrocyte-like cells. (A) Experimental scheme for thein vitro differentiation of CD14+ monocytes.(B) Photomicrographs of monocytes on day3 cultured in serum-free media alone orcontaining TGFb (10 ng/ml) with no stim-ulus (1 and 2), non-CpG (50 mg/ml) (3 and4), CpG (50 mg/ml) (5 and 6), or poly IC (50mg/ml) (7 and 8). Arrows indicate alteredmorphology. (C) (Left panel) QuantitativeTaqMan reverse transcription PCR (qRT-PCR) analysis of aSMA expression in mono-cytes cultured for 3 days in serum-freemedia ± CpG or poly IC for 24 hours. (Rightpanel) Collagen 1 gene expression in mono-cytes cultured for 3 days in serum-free mediaor TGFb, ±CpG, or poly IC. (D) Fluorescentimmunocytochemistry for collagen 1 inmonocytes (×40 magnification) cultured inserum-free media (1) or TGFb (2), serum-freemedia + CpG (3), or TGFb + CpG (4). Isotypecontrol for monocytes cultured in TGFb +CpG (5). Representative (n = 3) flow cytom-etry (FC) for collagen 1 protein as percent ofCD14+ cells in CD45+ gate from monocytescultured in serum-free media or TGFb, andserum-free media + CpG or TGFb + CpG.(E) Forward and side scatter FC of mono-cytes cultured in serum-free media containingTGFb or TGFb + CpG. Representative (n = 3)FC for CD14 as percent of total cells frommonocytes cultured in serum-free mediaor monocytes cultured in serum-free mediacontaining TGFb stained with antibodies toCD14. Representative (n = 3) FC for CD45 aspercent of CD14− cells from monocytescultured in serum-free media or monocytescultured in serum-free media containingTGFb stained with antibodies to CD45 andgated with respect to CD14 expression.

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adenocarcinoma type II alveolar epithelial cell line A549, widelyused to investigate TGFb-driven EMT (31–33), was used to test thishypothesis. A549 cells undergo EMT and exhibit cell spreading andelongation, loss of epithelial cell markers such as E-cadherin, and ex-pression of mesenchymal proteins including aSMA, collagen 1, andvimentin. A549 cells in culture media maintained a cobblestone epi-thelial morphology and growth pattern after 96 hours (Fig. 3A, panel 1).

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Figure 3A (panel 2), however, is a repre-sentative image of A549 cells cultured withTGFb (5 ng/ml) for 96 hours and showscell spreading and a fibroblast-like mor-phology. CpG treatment induced similarmorphological changes associated withEMT (cell spreading and elongated, spindle-shaped cells) in a concentration-dependentmanner during a 96-hour treatment (Fig.3A, panels 3 to 7). Changes in cell morphol-ogy assessed under phase-contrast lightmicroscopy were observed as early as 24hours with the lowest concentration ofCpG; however, the most marked effects oc-curred after 72 and 96 hours. To confirmwhether the morphological changes ob-served with CpG corresponded with EMT,we next isolated RNA from A549 cells andmeasured gene expression of EMTmarkers.CpG stimulated expression of aSMA, withan optimal effect of CpG at 200 mg/ml(Fig. 3B, first panel). CpG treatment ofA549 cells also resulted in a concentration-dependent induction of vimentin, with anoptimal effect of CpG at 200 mg/ml (Fig.3B, second panel), which is also accompa-nied by a loss of E-cadherin expression(Fig. 3B, third panel). To determine wheth-er CpG can also induce an innate immuneresponse from A549 cells (34), we mea-sured interferon-a (IFN-a) gene expressionafter increasing concentrations of CpG.We detected optimal IFN-a gene transcriptin cells treated with 200 mg/ml (Fig. 3C),indicating that the EMT effects observedat this concentration also correlated withan innate immune response. In addition,fluorescent immunocytochemistry revealeda dose-dependent induction of collagen1 by CpG in A549 cells after 96 hours(Fig. 3D, panels 1 to 4). These data showthat CpG induces EMT in lung epithe-lial cells.

To determine whether CpG DNAinduction of EMT in A549 cells is TLR9-dependent, we targeted TLR9 protein ex-pression by RNA interference. A549 cellswere treated with a small interfering RNA(siRNA) pool of four different sequencesspecific for TLR9, the reference proteincyclophilin B, or an irrelevant nontarget

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control and then were lysed after a 96-hour treatment. TLR9 pro-tein expression was ablated in cells treated with TLR9 siRNA whencompared with the nontarget or cyclophilin B siRNA-treated cells,in which TLR9 protein levels were similar (Fig. 3E). Moreover, A549cells at this time point appeared similar to those cultured in treatmentmedia plus transfection reagent alone (Fig. 3E, panel 5) and showed noindication of stress response or changes in morphology microscopically

Fig. 3. CpG-induced EMT in human A549 cells. (A) Representative photos (n = 5) of A549 cells culturedin media [DMEM + 10% fetal calf serum (FCS)] (1), TGFb (2), and increasing CpG concentrations for 96

hours: 5 mg/ml (3), 10 mg/ml (4), 50 mg/ml (5), 100 mg/ml (6), and 200 mg/ml (7). (B) qRT-PCR analysis ofaSMA, vimentin, and E-cadherin in A549 cells cultured with increasing concentrations of CpG for 96hours. (C) qRT-PCR analysis of IFN-a in A549 cells cultured with increasing concentrations of CpG for96 hours. (D) Fluorescent immunocytochemistry for collagen 1 in A549 cells (×40 magnification) thatwere cultured for 96 hours in media (1), CpG (10 mg/ml) (2), CpG (50 mg/ml) (3), and CpG (100 mg/ml)(4). Isotype control for collagen 1 antibody using cells cultured with CpG (100 mg/ml) (5). (E) siRNAknockdown of TLR9 in A549 cells: Western blot analysis of TLR9 protein and b-actin loading controlin A549 cell lysates after siRNA treatment as indicated (1 to 4). A549 cells before CpG DNA treatmentin media and transfection agent alone (5), with nontarget siRNA (6), and with TLR9 siRNA (7). A549 cellsafter siRNA treatment and stimulated with media and transfection agent alone (8), nontarget siRNA +CpG (75 mg/ml) (9), and TLR9 siRNA + CpG DNA (75 mg/ml) for 72 hours (10). Representative of n = 4.qRT-PCR analysis of vimentin and E-cadherin in siRNA-treated A549 cells cultured with CpG (75 mg/ml)for 72 hours. Data are mean ± SD.

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in cells cultured with nontarget siRNA (Fig. 3E, panel 6) or TLR9siRNA (Fig. 3E, panel 7). After TLR9 protein silencing was confirmedby Western blot, siRNA-treated A549 cells from the same experimentwere stimulated with CpG DNA for an additional 72 hours and moni-tored throughout for changes in morphology. The morphology ofA549 cells cultured in treatment media plus transfection reagent ap-peared unaltered (panel 8). Nontarget siRNA had no effect on inhibit-ing CpG-mediated EMT, as indicated by cell spreading and elongated,spindle-shaped cells (panel 9). In contrast, A549 cells treated with TLR9siRNA failed to demonstrate similar morphological changes (Fig. 3E,panel 10). These cells appeared stressed and apoptotic, which may in-dicate that ablation of TLR9 may drive alternative innate immune re-sponses in alveolar epithelial cells in the presence of CpG DNA. Tofurther demonstrate that CpG induces EMT in a TLR9-dependent man-ner, we isolated RNA from the siRNA- and CpG-treated cultured A549cells and measured gene expression of EMT markers. TLR9 silencingby siRNA inhibited CpG-mediated induction of vimentin (eleventhpanel) and down-regulation of E-cadherin expression (twelfth panel).

TLR9 expression and response to CpG-ODN are increasedin rapidly progressive IPFLung fibroblasts derived from SLBs of IPF patients were cultured invitro with media alone or with the profibrotic stimulus interleukin-4(IL-4) to determine induction of TLR9 messenger RNA (mRNA). Stim-ulation of fibroblast 204A (rapid progressor) with CpG resulted in in-creased TLR9 expression (Fig. 4A) compared to that response observedwith fibroblast 100A (slow progressor) (Fig. 4B). In vitro cytokine pro-duction by these IPF fibroblasts was measured in cultured cell super-natants to compare the effect of CpG in the presence or absence ofIL-4. Because type I IFNs are secreted by cells after TLR9 signaling (35),IFN-a in culture supernatants was measured. The rapidly progressivecell line 204A (Fig. 4C) demonstrates enhanced production of IFN-acompared to the slowly progressive line 100A (Fig. 4D)when stimulatedwithCpG in the presence of IL-4. This observation is consistentwith theheightened expression of TLR9 by 204A in the presence of both CpG-ODN and IL-4 (Fig. 4A). The rapidly progressive cell line 204A alsodemonstrates increased secretion of the profibrotic cytokines platelet-derived growth factor (PDGF) (Fig. 4E), monocyte chemoattractantprotein-1 (MCP-1)/chemokine (C-C motif) ligand 2 (CCL2) (Fig. 4G),and MCP-3/CCL3 (Fig. 4I) when stimulated with both CpG andIL-4. This is in contrast to the response observed with the slowly pro-gressing line 100A, which does not show a comparable effect on theproduction of profibrotic cytokines with CpG in the presence of IL-4(Fig. 4, F, H, and J). Together, these data show a differential expressionpattern of TLR9 and response to CpG between lung fibroblasts fromrapid and slowly progressive IPF lungs.

Rapidly progressing human IPF fibroblasts show increasedfibrogenicity in a humanized SCID model of IPFWe used a previously described humanized severe combined immu-nodeficient (SCID) mouse model to test the fibrogenic potential ofhuman lung fibroblasts from rapid versus slow progressors in vivo(36). Lung fibroblasts cultured from rapid or slow progressors were ana-lyzed in vitro (Fig. 4) and intravenously transferred into C.B-17SCID/bgmice. On day 35 after transfer, mice were intranasally challenged with50 mg of CpG or saline, and fibrosis was assessed on day 63 after trans-fer (Fig. 5A). No pulmonary histopathology was observed in C.B-17SCID/bg mice that received normal pulmonary fibroblasts (Fig.

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5B, panel 1), and no effect was observed in these mice when chal-lenged with CpG on day 35 (Fig. 5B, panel 2). Histological assessmentof mouse lungs by trichrome stain on day 63 after transfer revealedthat rapidly progressive human IPF fibroblasts exhibited collagendeposition and disruption of the alveolar space as a result of severe

Fig. 4. TLR9 expression in rapidly and slowly progressive IPF lung fibro-blasts in response to CpG. (A and B) qRT-PCR analysis of TLR9 gene expres-

sion in representative rapid progressor IPF (n = 5 to 8) (A) and slowprogressor IPF (n = 5 to 8) fibroblasts (B) treated for 24 hours without (un-treated) or with CpG-ODN (10 mg/ml) in the presence or absence of IL-4 (10ng/ml). Fold increase is calculated within each group of disease comparedwith the respective untreated fibroblasts. (C to J) Bioplex analyses of rapid orslow progressor IPF fibroblast conditioned media for IFN-a (C and D), PDGF(E and F), MCP-1/CCL2 (G and H), and MCP-3/CCL3 (I and J). Fibroblastswere treated for 24 hours without (untreated) or with CpG-ODN (10 mg/ml)in the presence or absence of IL-4 (10 ng/ml). Data are representative ofat least five slow progressor IPF and five rapid progressor IPF fibroblasts.Data are mean ± SEM.

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interstitial thickening and remodeling (Fig. 5B, panel 3). Furthermore,fibrosis was markedly enhanced in those lungs that received a CpGchallenge on day 35 (Fig. 5B, panel 4). This is in contrast to the degreeof fibrosis observed in mouse lungs that received slowly progressinghuman IPF fibroblasts and a CpG challenge. Slowly progressing IPFhuman lung fibroblasts induce a modest fibrotic response in mouselungs as assessed on day 63 after transfer (panel 5) that was not en-hanced by a CpG stimulus (panel 6). Hydroxyproline, a marker of denovo collagen synthesis in experimental models of fibrosis, was mea-sured on day 35 in half-lung samples from C.B-17SCID/bg mice thathad received IPF human fibroblasts. CpG challenge significantly in-creases hydroxyproline content only in mouse lungs transplanted withfibroblasts from rapidly progressive IPF patients (Fig. 5C, first panel),correlating with the histological assessment of increased collagen depo-sition in lungs frommice adoptively transferred with rapidly progressiveIPF fibroblasts. Moreover, panel 2 confirms the histology in Fig. 5B(panels 5 and 6): CpG challenge does not result in an increase in hy-droxyproline content in mouse lungs transplanted with fibroblasts fromslowly progressive IPF patients.

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DISCUSSION

Here, we propose that an innate immuneresponse to infections via TLR9 may aug-ment the underlying fibrotic process inthe lung. Viral infections, primarily EBV,have been identified in the lungs of IPFpatients (14–16) and may be a mediatingfactor in the rapid progression of IPF.Previously, we have demonstrated sig-nificantly elevated amounts of the innateimmune sensor TLR9 in SLBs from IPFpatients (19). Therefore, we investigatedherein whether expression of TLR9 differsbetween IPF patients who demonstrate ra-pid or slow disease progression. Evidenceis presented that shows that TLR9 func-tions as both a pathogenic sensor and aprofibrotic mediator in IPF. Our data indi-cate that elevated TLR9 is a potential pre-dictor of a rapidly progressive diseasevariant of IPF and may be a potential ther-apeutic target.

To explore the mechanism by whichTLR9 functions as both a pathogenic sen-sor and a profibrotic mediator in IPF, weconducted studies in cell types implicatedin the fibrotic response. Previous reportshave demonstrated that bone marrow–derived cells (fibrocytes) promote repairby migrating to wound sites and servingas a contributing source of myofibroblastsin fibrotic disease. Circulating fibrocytes(defined as CD45+Col1+) increase to an av-erage of 15% of peripheral blood leukocytesin IPF patients who were evaluated duringepisodes of AE-IPF (37). Our current studyextends the examination of fibrocytes andidentified them as pathogenic sensors of

CpG DNA. Whether fibrocytes arise from circulating monocytes re-mains controversial, although TGFb induces the in vitro differentiationof CD14+ monocytes into CD14−/collagen 1+ fibrocytes and CpG in-duces myofibroblast differentiation in cultured lung fibroblasts (19).Moreover, CD14+ monocytes express high amounts of TLR9 gene tran-scripts, in contrast to a previous report that demonstrated expression ofTLR7, but not TLR9, in fibrocytes (38). Here, we tested the hypothesisthat CpG may also induce the differentiation of CD14+ monocytes intofibrocytes. Because we did not have access to blood monocytes from IPFpatients undergoing an acute exacerbation, we used naïve blood mono-cytes from healthy donors to investigate the agonistic potential of CpGin the context of fibrosis. Our data demonstrate that CpG treatmentresults in a hybrid monocyte phenotype, having both fibrocyte markers(spindle-shaped morphology, CD45, collagen 1, and aSMA expression)and CD14. We also showed that CpG enhanced TGFb-driven differen-tiation, as demonstrated by increased cell size and immunostaining forcollagen. These data confirm that monocytes can respond to CpG in aprofibrotic manner and may be a cellular source, separate from fibro-cytes, for contributing to the myofibroblast population in the lung.

Fig. 5. Exacerbation of fibrosis by CpG in a human-SCID mouse model of IPF induced by rapidly progres-sive human lung fibroblasts. (A) Experimental scheme for establishing a human-SCID model of AE-IPF. (B)

Representative mouse lung sections stained with Masson’s trichrome to depict degree of fibrosis frommice that received normal human lung fibroblasts and intranasally challenged on day 35 with saline(1) or CpG (2), rapid IPF human lung fibroblasts intranasally challenged on day 35 with saline (3) orCpG (4), and slow IPF human lung fibroblasts intranasally challenged on day 35 with saline (5) or CpG (6).(C) Amount of hydroxyproline in half-lung homogenates from saline- or CpG-challenged mice that re-ceived rapid IPF human lung fibroblasts and slow IPF human lung fibroblasts. Data are mean ± SEM from fivemice at each time point.

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Consistent with these results, we also report a CpG-mediated dif-ferentiation of the A549 human alveolar epithelial cell line to a myofi-broblastic phenotype. A549 cells express functional TLR9 (39), and thesiRNA data suggest that the CpG-mediated EMT is TLR9-dependent,but the precise mechanism that distinguishes innate immune responsesfrom EMT in these cells is unknown. Production of chemokines bylung epithelial cells in response to CpG may also lead to the attractionof immune cells that may further exacerbate the process (39). One lim-itation in our study is that the CpG effects we report are from a trans-formed cancer cell line and not in primary alveolar epithelial cells fromIPF patients. We can nevertheless speculate that alveolar epithelial cellsfrom IPF lungs may be more comparable to a cancer cell line than tonormal alveolar epithelial cells, as evidenced by increased Wnt/b-cateninsignaling shown to drive epithelial cell injury, hyperplasia, and EMT inIPF lungs (40, 41). Indeed, we can conclude from these data that CpGDNA is recognized by TLR9 expressed on an alveolar epithelial cell line,promotes EMT, and may contribute to the pathogenesis of AE-IPF.

The in vitro culture of fibroblasts from SLBs of IPF patients has per-mitted us to establish a humanized mouse model of IPF (36), which weextended to investigate the role of TLR9 activation in rapidly progres-sive IPF. Lung fibroblasts from patients experiencing a rapidly progres-sive course demonstrate a hyperresponsiveness to CpG DNA challengein a SCID model. A single bolus of CpG DNA given intranasally to micetransplanted with rapidly progressive IPF fibroblasts augmented the pul-monary fibrotic response in these mouse lungs compared to those trans-planted with normal or slowly progressive IPF fibroblasts. In vitro studiesconducted with the same IPF fibroblasts indicated that CpG stimulationresults in the enhanced production of profibrotic cytokines from rap-idly progressive fibroblasts. We speculate that in the SCID model, CpGinduces the production of human profibrotic cytokines within themouse lung and promotes an autocrine response from the human fi-broblasts that results in increased fibrosis. Furthermore, these data sug-gest that CpG recognition by TLR9 in fibroblasts is another potentialcomponent of the mechanism by which bacterial or viral constituentsaugment fibrogenesis during rapidly progressive IPF.

TLR9 has recently been implicated in experimental models of otherfibrosing diseases. Studies investigating the role of TLR9 in experimen-tal liver fibrosis have demonstrated that TLR9-deficient mice show aprotective fibrotic effect in the bile duct ligation model of liver fibrosis,indicating a pathophysiological role for bacterial DNA and TLR9 inthe development of hepatic fibrosis (42). CpG-ODN increased renalfibrosis in a study with a murine model for lupus nephritis, as mea-sured by the amount of interstitial fibroblast proliferation in MRLlpr/lpr

mice (43). Moreover, CpG promotes cellular invasion in malignantbreast cancer and prostate cells via a TLR9-dependent mechanism, sug-gesting that certain cancers may be susceptible to infectious exacerba-tions (44–46). These studies are consistent with our assertion that TLR9is a major mediator of the rapidly progressive forms of IPF.

The variable disease course of IPF is an obstacle to understandingdisease pathogenesis and, consequently, the development of successfultherapies. In addition, it has been very challenging to identify predictorsof disease progression because the cause of IPF is not known. Therefore,rigorous studies aimed at understanding the etiology, risk factors, andpathogenesis of disease progression are essential for accurate manage-ment of IPF. Our study demonstrates that the therapeutic design ofspecific TLR9 antagonists is warranted to potentially improve the treat-ment of IPF patients and serve as a preventative approach for minimiz-ing susceptibility to acute exacerbations.

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MATERIALS AND METHODS

MiceAll procedures described below were performed in a sterile, laminar en-vironment and were approved by the Institutional Animal Care and UseCommittee at the University of Michigan Medical School. We used adultage-matched female C.B-17-scid-beige (C.B-17SCID/bg) mice (TaconicFarms). SCID mice were housed in a specific pathogen–free (SPF) facil-ity for immunocompromised mice at the University of Michigan.

Human-SCID model of AE-IPFSingle-cell preparations of IPF (from clinically classified rapid or slowprogressors) and normal fibroblasts were obtained after trypsinizationfrom culture flasks and labeled with PKH26 dye (Sigma). Each labeledfibroblast line was diluted to 2 × 106 cells/ml of phosphate-bufferedsaline (PBS), and 0.5 ml of this suspension was injected via a tail vaininto groups of 5 to 10 SCID mice. Thirty-five days later, all groups ofmice were mildly anesthetized and received a single bolus of CpG-ODN (dissolved in sterile saline) or saline by intranasal delivery. Micewere euthanized by cervical dislocation 63 days after the intravenoushuman pulmonary fibroblast transfer. Whole-lung tissue was dissectedfor histological and biochemical analysis (see below).

IPF patientsTwenty-three patients diagnosed with IPF using a multidisciplinary,clinical/radiological/pathological mechanism (20) were included. Base-line data included detailed clinical assessment, physiological studies,HRCT, and SLBs. Semiquantitative scores of HRCT abnormality weregenerated with validated methodology (47). Patients were treated witha variety of treatment regimens and followed closely with physiologicalstudies and capture of clinical information during acute events. Usingmethodology that has been validated disease progression during thefirst year of follow-up used a composite of physiological deterioration(48), the physiological criteria include an FVC decrease of >10% and aDLCO decrease of >15% based on baseline physiological abnormality.Acute exacerbations of IPF were defined using criteria recently proposedby our group (21) or all-cause mortality. This composite approach isnow common in National Heart, Lung and Blood Institute (ACE IPF)and industry-sponsored trials (BUILD 3, Artemis) (49).

Cell culture and monocyte differentiation assayBlood was collected from healthy adult volunteers in accordance with Uni-versity of Michigan Human Research Protection Program (Ann Arbor,MI). PBMCs were isolated from EDTA blood by Ficoll-Paque Plus(GE Healthcare Biosciences). CD14+ monocytes were purified by neg-ative selection with the Human Monocyte Isolation Kit II and MACSLS column separators (Miltenyi Biotec). Briefly, a cocktail of biotin-conjugated antibodies against CD3, CD7, CD16, CD19, CD56, CD123,and CD235a (glycophorin A), as well as Anti-Biotin MicroBeads, yieldshighly pure unlabeled monocytes obtained by depletion of the magnet-ically labeled cells. CD14+monocytes [>97% pure by fluorescence-activatedcell sorting (FACS)] were plated at a density of 2.5 × 106 cells per well ina six-well plate containing EX-CELL Hybri-Max protein-free medium(Sigma) plus 0.5% sterile bovine serum albumin (BSA) with or withoutTGFb (10 ng/ml). After 3 days, monocytes were either unstimulated ortreated with sterile human CpG-ODN (50 mg/ml) (Hycult Biotechnology/Cell Sciences, HC4032), non-CpG (Hycult Biotechnology/Cell Sciences),or poly IC (InvivoGen). Twenty-four hours later, monocyte cultures were

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visualized under phase-contrast microscopy or processed for FACSanalysis as described. For gene expression analysis, Trizol reagent wasadded to each well and RNA extraction was performed according tothe manufacturer’s instructions. RNA was purified with the RNeasy RNACleanup kit (Qiagen) and subjected to on-column DNase digestion(Qiagen). RNA concentration and purity were determined by NanoDropand confirmed by agarose gel electrophoresis. Purified RNA was subse-quently reverse-transcribed into complementary DNA (cDNA) by real-time PCR, and similar treatments were pooled for analysis.

A549 cell culture and EMT assayA549 cells were seeded at a concentration of 40,000 cells per well in12-well culture plates containing Dulbecco’s modified Eagle’s medi-um (DMEM) supplemented with 10% fetal bovine serum, penicillin(100 U/ml), and streptomycin (100 mg/ml). Treatments consisted ofmedia alone, CpG (at 5, 10, 50, 100, or 200 mg/ml), or TGFb (0.1, 0.5,1, 5, or 10 ng/ml). Cells were treated for 72 or 96 hours (as indicated)and then trypsinized for analysis as described.

siRNA knockdown of TLR9A549 cells were seeded at a concentration of 10,000 cells per well in 12-well culture plates containing DMEM supplemented with 5% fetal bo-vine serum. Twenty-four hours later, cells remained untreated or treatedwith 50 nM ON-TARGETplus Non-Targeting siRNA Pool, 50 nM ON-TARGETplus Cyclophilin B Control siRNA Pool, or 50 nM TLR9 ON-TARGETplus siRNA SMARTpool (Dharmacon, Thermo Scientific) inDharmaFECT transfection reagent according to the manufacturer’sinstructions. Cells were incubated for 48 hours for RNA analysis orfor 96 hours for protein analysis to confirm TLR9 knockdown. ForCpG-mediated EMT, CpG at the indicated concentration was addedto the siRNA-treated cells for 72 or 96 hours and then trypsinized foranalysis as described.

Statistical analysisAll results are expressed as mean ± SEM or median as appropriate. Base-line characteristics of patients were contrasted by unpaired t tests orMann-Whitney tests as appropriate. Overall survival characteristics werecontrasted between patients experiencing disease progression during thefirst year of follow-up compared to those that did not with Cox regres-sion analysis. The means between groups at different time points werecompared by two-way analysis of variance (ANOVA) (50). Individualdifferences were further analyzed with unpaired t test with Welch cor-rection where indicated. Where appropriate, data were analyzed withANOVA with a Tukey-Kramer multiple comparisons test. Values ofP < 0.1 (*), P < 0.01 (**), and P < 0.001 (***) were considered significant.

SUPPLEMENTARY MATERIAL

www.sciencetranslationalmedicine.org/cgi/content/full/2/57/57ra82/DC1MethodsReferences

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Lung Tissue Research Consortium and R. Kunkel for her expertise in the preparation of thefinal figures. Funding: This worked was supported, in part, by NIH funding via P50HL56402 (toC.M.H., K.R.F., and F.J.M.) and HL073728 (to C.M.H.). The authors also acknowledge the financialsupport of Novartis Institute of Biomedical Research and Novartis Pharmaceuticals UK Ltd. G.T.received funding from an NIH Institutional Training Grant (T32) for Pulmonary Research at theUniversity of Michigan Medical School. Author contributions: G.T. and C.M.H. designed andanalyzed all the experiments and data. G.T. performed all the experiments except for the TLR9TaqMan assays, which were performed and analyzed by A.M. and S.R.O., the IPF fibroblastenzyme-linked immunosorbent assays (ELISAs), which were performed and analyzed byA.M., and the TLR9 immunohistochemistry, which was performed by A.L.C. F.J.M., K.R.F., L.M.S.,J.L.M., E.A.K., B.H.G., G.B.T., and C.M.H. collected and interpreted the clinical data. H.E., A.D.J.,and B.W. provided technical assistance. E.D., A.D.J., M.A.S., G.J., J.W., and S.L.K. contributed tothe critical review of the study and the interpretation of the data. G.T., F.J.M., and C.M.H. wrotethe paper. Competing interests: Novartis and the University of Michigan have filed a patenton the diagnostic use of TLR9 as a biomarker for fibrosis on which G.T., S.L.K., and C.M.H. areco-inventors. The other authors declare that they have no conflicts of interest.

Submitted 20 July 2010Accepted 21 October 2010Published 10 November 201010.1126/scitranslmed.3001510

Citation: G. Trujillo, A. Meneghin, K. R. Flaherty, L. M. Sholl, J. L. Myers, E. A. Kazerooni,B. H. Gross, S. R. Oak, A. L. Coelho, H. Evanoff, E. Day, G. B. Toews, A. D. Joshi, M. A. Schaller,B. Waters, G. Jarai, J. Westwick, S. L. Kunkel, F. J. Martinez, C. M. Hogaboam, TLR9 differentiatesrapidly from slowly progressing forms of idiopathic pulmonary fibrosis. Sci. Transl. Med. 2,57ra82 (2010).

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