Significance Severe human fibrotic diseases are devastating and without effective treatments. We found that c-JUN expression is increased in many human fibrotic diseases and that systemic induction of c-Jun in mice resulted in development of fibrosis of multiple organs. These results suggest that many fibrotic diseases share a common pathomechanism that converges on c-Jun induction. Thus, common treatment strategies could potentially be developed for these seemingly different fibrotic disease entities. Moreover, the in vivo c-Jun induction represents a mouse model for these devastating diseases that could be used for preclinical evaluation of candidate antifibrotic treatments. Indeed, we show that blockade of the antiphagocytotic signal CD47 and the AKT and VEGF receptor pathways reverses tissue fibrosis in mice.

Abstract Fibrotic diseases are not well-understood. They represent a number of different diseases that are characterized by the development of severe organ fibrosis without any obvious cause, such as the devastating diseases idiopathic pulmonary fibrosis (IPF) and scleroderma. These diseases have a poor prognosis comparable with endstage cancer and are uncurable. Given the phenotypic differences, it was assumed that the different fibrotic diseases also have different pathomechanisms. Here, we demonstrate that many endstage fibrotic diseases, including IPF; scleroderma; myelofibrosis; kidney-, pancreas-, and heart-fibrosis; and nonalcoholic steatohepatosis converge in the activation of the AP1 transcription factor c-JUN in the pathologic fibroblasts. Expression of the related AP1 transcription factor FRA2 was restricted to pulmonary artery hypertension. Induction of c-Jun in mice was sufficient to induce severe fibrosis in multiple organs and steatohepatosis, which was dependent on sustained c-Jun expression. Single cell mass cytometry revealed that c-Jun activates multiple signaling pathways in mice, including pAkt and CD47, which were also induced in human disease. αCD47 antibody treatment and VEGF or PI3K inhibition reversed various organ c-Jun–mediated fibroses in vivo. These data suggest that c-JUN is a central molecular mediator of most fibrotic conditions.

The fibrotic response is an important component of normal repair processes that, if uncontrolled, can lead to various life-threatening conditions, like idiopathic pulmonary fibrosis (IPF), primary myelofibrosis, and scleroderma (1⇓⇓⇓–5). It is not known whether similar molecular mechanisms are responsible for the fibrotic response in different diseases, and some studies came to conflicting conclusions (6). The molecular processes driving fibrogenesis are not well-understood, involving but not limited to transforming growth factor B (TGFB), platelet-derived growth factor (PDGF), connective-tissue growth factor (CTGF), vasoactive peptide, integrin signaling, and increased tissue stiffness (7, 8). At the cellular level, efforts have been made to characterize fibroblasts by molecular markers (9), and fibrosis is thought to involve the cross-talk of hematopoietic and mesenchymal stroma cells (4, 6).

Although the few currently available animal models are useful, all of them have certain limitations: e.g., bleomycin-induced lung or skin fibrosis develops acutely in response to chemical injury and is self-resolving; however, human diseases such as idiopathic pulmonary fibrosis or scleroderma are not (10). A more recent genetic mouse model showed some fibrosis features but predominantly exhibited vasoocclusive alterations more reminiscent of pulmonary artery hypertension (PAH) (11).

On the other hand, the genetic basis of fibrotic diseases is just emerging but already promises to gain fundamental insights into pathomechanisms: e.g., FAN1 mutations have been associated with kidney fibrosis, PNLAP3 with liver fibrosis, mutations in JAK2, MPL, or calreticulin with bone marrow fibrosis in myeloproliferative neoplasm (12), and telomerase reverse transcriptase (TERT) and mucin 5B (MUC5B) with lung fibrosis, as well as alterations in DNA methylation, and some microRNAs (5) have been shown to play a role in lung fibrosis. Some of these molecular findings have guided current standard of care treatments in, e.g., lung fibrosis and primary myelofibrosis.

We previously developed mouse models of primary myelofibrosis associated with myeloproliferative disease (13, 14) and wished to investigate their molecular downstream effectors. Gene expression analysis suggested the dysregulation of cJUN, an AP-1 transcription factor that is a well-established regulator of critical cell biological processes and involved in cancer and other human diseases (15, 16). We subsequently investigated the expression of AP-1 transcription factors in most human fibrotic diseases and found increased c-JUN expression in SMA+ fibroblasts. In addition, c-JUN was responsible for the pathologically increased proliferation of fibroblasts of patients with idiopathic pulmonary fibrosis. Based on our observations in patients with various clinical pictures of fibrosis, we generated a mouse model with inducible c-Jun. In these mice, we were able to induce fibrosis via the c-JUN pathway, which closely resembled the various organ manifestations in patients. Remarkably, we found that fibroblasts selectively responded to c-Jun despite ubiquitous c-Jun expression. Single cell mass cytometry analysis in mouse fibrosis revealed that transcriptional effects of c-Jun lead to a profound rewiring of active signaling pathways, which we exploited for effective therapeutic intervention in mice.

SI Results c-Jun, but Not JunB, Induces Severe Marrow Fibrosis in Mice. To dissect the functional relevance of AP-1 transcription factor up-regulation that we observed in patients, we generated c-Jun and JunB doxycycline (dox)-inducible mice by gene targeting in mouse embryonic stem cells, followed by blastocyst injection of the transduced cells (Fig. S3 A, B, and F). In this expression system, the reverse tetracycline transactivator (rtTA) is ubiquitously expressed from the Rosa26 locus, and the inducible cassette is targeted downstream of the Col1a1 locus, leading to robust and reliable drug-dependent transgene induction in most tissues (25). In both mouse strains, c-Jun and JunB were expressed in all cell types and organs (Fig. S5 H and I), and proteins were readily induced after 2 d of dox administration in the drinking water (Fig. S3 C, D, and G). Surprisingly, only 4 d after induction of c-Jun, hybrid F1 129/C57BL/6 mice became moribund and died between 6 and 8 d due to systemic cytokine release (Fig. 2B). In the BDF1 genetic background, the mice also died almost synchronously, but survival was prolonged to about 38 d (Fig. 2A). Before death, the mice exhibited scruffy fur and stiff skin, difficulties breathing, and prolonged bleeding. Initially, we focused our histopathologic analysis on the marrow, which revealed the lack of hematopoietic cells and striking fibrosis with collagen deposition replacing 87% of the marrow space, fulfilling all clinico-pathological criteria of grade 3 marrow fibrosis (Fig. S4A). Initially, we focused our histopathologic analysis on the bone marrow, which architecture was completely effaced by dense fibrosis with collagen deposition by trichrome stain. There were only a few pockets with residual trilineage hematopoiesis and mild megakaryocytic dysplasia. These findings are compatible with the clinico-pathological criteria of grade 3 marrow fibrosis, replacing 87% of the marrow space. (Fig. S4A). Time course analysis of the bone marrow demonstrated that pancytopenia and fibrosis in c-Jun–induced animals were progressive between days 2 through 6. The fibrotic infiltrate comprised spindle shaped cells that stained positive with trichrome and smooth muscle actin (Fig. S4A, Middle and Bottom). Gross morphologic paleness and single femur cell counts revealed severe cytopenia in the marrow, with an over ninefold reduction in the total number of bone marrow cells (2.5 × 106 versus 15 × 106, P < 0.01) in c-Jun–induced mice (Fig. S5A). Annexin V/7AAD stains indicated distinct c-Jun–mediated cell type–specific response 4 d after induction in vivo, which was a significant increase in the apoptosis in c-Jun-expressing hematopoietic precursors, suggesting cell death rather than migration as the primary mechanism accounting for the rapid disappearance of hematopoietic cells from the fibrotic bone marrow (Fig. S5B). In contrast, none of the JunB-inducible mice showed any signs of disease after up to 8 mo of doxycycline treatment. Histopathological analysis revealed bone marrow cellularity and composition indistinguishable from control mice of the same age (Fig. S3 H and I). Thus, fibrogenesis is specifically induced by c-Jun and not by the related AP-1 transcription factor JunB. c-Jun–Induced Marrow Fibrosis Is Primarily Mediated by Stroma Cells, with Some Contribution from Hematopoietic Lineage Cells. We next sought to address whether c-Jun induction in the hematopoietic or mesenchymal cell population is responsible for fibrogenesis and whether the effects are mediated in a cell-autonomous manner as could be expected from a transcription factor. First, we transplanted whole bone marrow of c-Jun–inducible mice into WT recipients. Despite nearly 100% blood chimerism, no fibrosis was detected after dox treatment, suggesting that the hematopoietic compartment alone is insufficient to induce fibrogenesis. Next, we injected 1 × 106 CD45.1 WT whole bone marrow into c-Jun–inducible mice expressing CD45.2. After 25 d, the bone marrow was nearly completely reconstituted with WT donor cells. Upon dox treatment, fibrosis formation occurred but was substantially suppressed compared with nontransplanted c-Jun–inducible mice (Fig. S4 F and G, compare with B). To independently confirm these results, we performed a parabiosis experiment allowing blood exchange after anastomosis of the circulation of c-Jun–inducible and littermate control mice for 3 wk, a time point at which peripheral blood chimerism is known to be established. One week after dox treatment, the bone marrow fibrosis was present but markedly suppressed in c-Jun mice parabiosed to WT mice (Fig. S4 F and G, compare with A). To understand which blood cell subsets are contributing to fibrosis, we investigated c-Jun–mediated fibrogenesis in NOD-SCID IL2R-gamma and RAG2-gamma knockout mice lacking B, T, and NK cells. Fibrosis developed in all conditions, demonstrating that other blood cell types, such as macrophages, modulate the fibrotic process. To understand whether cytokine release contributed to c-Jun–mediated fibrosis and rapid mortality of B6 mice, we quantified cytokines in mouse serum 48 h after dox-mediated c-Jun expression by multiplex assay. We found that cJun mice released many pro- and antiinflammatory cytokines at high levels, leading to death between 6 and 8 d (Fig. 2B). In summary, these observations establish that fibrogenesis can result from the direct induction of c-Jun in mesenchymal stroma cells, which leads to a systemic release of potent proinflammatory chemo- and cytokines, and that induction in the hematopoietic compartment alone is insufficient. However, WT transplanted or circulating hematopoietic cells, likely macrophages, can partially suppress fibrosis in c-Jun marrows. Thus, the fibrosis is driven in a cell-autonomous manner by tissue fibroblasts but is modulated in a non cell-autonomous manner by hematopoietic cells compatible with the well-characterized interaction of inflammatory and fibrotic mechanisms in sclerotic disease. Additional Results: Blockade of the VEGF and PI3K Pathways Reverses Marrow and Skin Fibrosis in Vivo. Any effects on gastroesophageal or bladder were not assessed because the fibrosis in these organs takes longer to develop. Both Rosa26-rtTA control mice were treated with dox, and c-Jun–inducible mice were treated with PBS. No fibrosis developed in either control condition in lung or other organs over the same time span (Fig. S5D). Thus, the PI3K and VEGFR pathways are critical mediators of c-Jun–induced fibrosis in mice and may also be involved in the development of human fibrotic conditions. We note that a recent study showed initial clinical benefit to idiopathic pulmonary fibrosis patients of VEGFR/FGFR/PDGFR pathway inhibition (1).

SI Materials and Methods General Statement Regarding Human Samples and Animal Studies. Deidentified patient specimens in paraffin and discarded fresh patient tissues were used for our studies as approved in IRB11177. Experiments were conducted on c-Jun tetracycline-inducible transgenic mice in accordance with guidelines established by the Stanford University Administrative Panel on Laboratory Animal Care (SU-APLAC 30911, 30912, 31026). Generation of Tetracycline-Inducible Transgenic Mice. The tetracycline-inducible system was used to generate transgenic mice overexpressing c-Jun and JunB in vivo as described exactly in ref. 25. Briefly, it consists of two components, one encoding the tetracycline-controllable transactivator (rtTA) and the other consisting of the tetracycline operator minimal promoter (tetOP) driving the gene of interest. We targeted the ColA1 locus that encodes the type I collagen protein, and transgenic tetracycline-inducible mice were generated. ES cells carrying both the R26-M2rtTA allele and the flp-in tetO-c-Jun or flp-in tetO-JunB alleles were screened by Southern blot. Subsequently, targeted ES cells were injected into blastocysts and viable chimeras were generated; alternatively, mice were derived by tetraploid embryo complementation. Mice were genotyped by PCR for the presence of both alleles, and 6- to 8-wk-old mice were treated with doxycycline administered in the drinking water (1 mg/mL) for 7 d up to 36 d, and tissues were harvested and analyzed by immunocytochemistry. Transgenic mice that were not exposed to doxycycline as well as mice harboring the rtTA only showed no detectable c-Jun or JunB expression, and those were used as controls as indicated. Bone Marrow Transplantation and Parabiosis. Transplant studies and parabiosis surgery were performed exactly as previously described (14) and in accordance with the guidelines established by the Stanford University for the humane care and use of animals (APLAC). Tissue Processing, Immunostaining, and Apoptosis Assessment. Animals were killed at times indicated based on an APLAC-approved protocol that includes assessment of morbidity by >10% loss of weight, scruffy appearance, and lethargy. For in vivo studies, tetracycline-inducible transgenic mice were put on doxycycline, and, consecutively, tissues from all major organ systems were collected, fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin or, to assess for fibrosis, stained with reticulin, trichrome, anti-SMA, and c-Jun. Images of histological slides were obtained on a Nikon Eclipse E400 microscope (Nikon) equipped with a SPOT RT color digital camera (model 2.1.1; Diagnostic Instruments). Images were analyzed in Adobe Photoshop (Adobe Systems). For flow cytometry, cells were washed in PBS, washed in 2% FBS, blocked with Fc-Block (BD PharMingen) for 10 min on ice, and stained with primary antibodies in PBS and 2% FCS for 30 min on ice. A comprehensive list of antibodies used, including brand names and dilutions, is included in Dataset S1. For assessment of apoptosis, marrow-derived cells were flushed from the leg bones of c-Jun mice. The cells were then washed, and the red cells were lysed on ice with RBC lysis buffer (Gentra). Pooled progenitor populations were sorted and analyzed as previously reported, and apoptosis analysis was performed using the Annexin V Apoptosis Detection Kit (BD Pharmingen). Flow cytometry was performed on a FACS Aria cytometer (BD Biosciences), at least 10,000 events were acquired, and data were analyzed using FlowJo software. The results are presented as graphs and representative dot plots of viable cells selected on the basis of scatter and 7-AAD staining. Tissue sections (4 μm thickness) were cut from tissue blocks of archival deidentified human biopsies using a microtome for immunofluorescence staining. The sections were baked at 65 °C for 20 min, deparaffinized in xylene, and rehydrated via a graded ethanol series. The sections were then immersed in epitope retrieval buffer (10 mM sodium citrate, pH 6) and placed in a pressure cooker for 45 min. The sections were subsequently rinsed twice with dH 2 O and once with wash buffer (TBS, 0.1% Tween, pH 7.2). Residual buffer was removed by gently touching the surface with a lint-free tissue before incubating with blocking buffer for 30 min. Blocking buffer was subsequently removed, and the sections were stained overnight at 4 °C in a humidified chamber. The following morning, the sections were rinsed twice in wash buffer, and secondary antibody (Invitrogen) was used for visualization of signal. Images of histological slides were obtained on a Leica Eclipse E400 microscope (Leica) equipped with a SPOT RT color digital camera (model 2.1.1; Diagnostic Instruments). We validated all of the antibodies we used for immunostaining first by staining positive and negative controls of healthy human and mouse tissues, but also lung cancer, breast cancer, adrenal, placenta, tonsils, peripheral blood mononuclear cells, and spleen. To find the optimal antibody concentration and decrease nonspecific staining, we subsequently titrated the respective antibodies by serial dilution from 1:50, 1:100, and 1:200 to 1:500. We included positive and negative control tissues and sections stained with isotype control with each subsequent immunostain. In addition, to proof specificity of the c-JUN antibody, we engineered mouse and human c-JUN–inducible cell lines and generated a c-JUN knockout cell line by crisper/cas9, as shown in Figs. S2D and S3 C and E. Expression Arrays and Computational Methods. Time course experiments of c-Jun protein expression in vivo were performed by harvesting marrow from mice exposed to doxycycline on day 0 and day 1 and applied to standard affymetrix mouse arrays. We processed the raw gene expression values with the robust multiarray analysis (RMA) algorithm using BioConductor software. We then filtered out unchanging genes that had an absolute change less than 70 and minimum fold change less than 3 across any two samples. We set thresholds of a minimum of 20 and a maximum of 20,000. Using these preprocessed data, we then identified differentially expressed genes by performing permutation testing with the Comparative Marker Selection algorithm using GenePattern software. We used the signal-to-noise ratio to rank-order the genes that distinguish two classes of gene expression samples. In addition to finding differentially expressed genes, we also ran gene set enrichment analysis to measure enrichment of fibrosis gene sets in our data. Single Cell Mass Cytometry. Samples were processed as described. Briefly, primary bone marrow-derived adherent cells from c-Jun mice were induced with doxycycline for 48 h, labeled with IdU to assess cell proliferation as previously described, washed once with PBS, treated with 25 μM cisplatin for 1 min for live–dead cell discrimination, washed once with RPMI medium containing 10% FBS, treated with 1× TrypLE (Invitrogen) for 5 min at 37 °C, dissociated into single-cell suspension by trituration, and washed with PBS containing 0.5% BSA. The cell samples were then fixed with 2% paraformaldehyde at room temperature for 20 min, followed by two washes with PBS containing 0.5% BSA. Formaldehyde-fixed cell samples were incubated with metal-conjugated antibodies against surface markers for 1 h, washed once with PBS containing 0.5% BSA, permeabilized with methanol on ice for 15 min, washed twice with PBS containing 0.5% BSA, and then incubated with metal-conjugated antibodies against intracellular molecules for 1 h. Cells were washed once with PBS containing 0.5% BSA and then incubated at room temperature for 20 min with an iridium-containing DNA intercalator (Fluidigm) in PBS containing 2% paraformaldehyde. After intercalation/fixation, the cell samples were washed once with PBS containing 0.5% BSA and twice with water before measurement on a CyTOF mass cytometer (Fluidigm). Normalization for detector sensitivity was performed as previously described. After measurement and normalization, the individual FCS files were analyzed by first gating out doublets, debris, and dead cells based on cell length, DNA content, and cisplatin staining. viSNE maps were generated with software tools available at https://www.cytobank.org/ by considering all surface markers. The F4/80 and CD172a positive or negative subpopulations were gated on a viSNE map, and the events in each gated population were exported for a DREVI plot using a software package available at www.c2b2.columbia.edu/danapeerlab/html/dremi.html. Isolation of Marrow-Derived Fibroblasts. Primary mouse bone marrow-derived fibroblasts were generated as such. The femoral and tibial bones of the donor mice were collected, and the adherent soft tissue was removed. Both ends of the bones were cut away from the diaphysis with bone scissors, the bone marrow plugs were then hydrostatically expelled from the bones, and the dispersed cells were plated into 10-cm polystyrene tissue culture dishes (Corning, Inc.) and cultured in alpha DMEM (alpha DMEM; Gibco Laboratories) containing selected lots of 10% FCS (FCS; JR Scientific Inc.) and antibiotics (penicillin G, 100 U/mL; streptomycin, 100 μg/mL; amphotericin B, 0.25 μg/mL; Gibco Laboratories) at 37 °C in a humidified atmosphere of 5% CO 2 . Three days later, the medium was changed, and nonadherent cells were discarded and cultured until confluence was reached. Transwell Migration and Proliferation Assays. For transwell migration assays, Costar transwell chambers were used according to the manufacturer's directions, and transmigrated adherent cells were fixed and stained and counted by microscopy after periods of 2 h and 24 h. Alternatively, a cytoselect 24-well cell migration assay (12-μm fluorometric format) was purchased from Cell Biolabs, Inc. and used according to the manufacturer's instructions. Briefly, 1 × 104 cells per milliliter for mouse fibroblasts and 2 × 104 cells per milliliter for human idiopathic lung fibrosis and WT fibroblasts were prepared in 300 μL of serum-free medium containing DMEM with 0.5% BSA, 2 mM CaCl 2 , and 2 mM MgCl 2 and added to the inside of each insert. Then, 500 μL of DMEM containing 20% FBS or respective inhibitor were added to the lower well of the migration plate and incubated for 4 and 72 h at 37 °C. Subsequently, the migrated cells were treated with cell detachment solution and lysis buffer and stained, and the fluorescence was assessed with a fluorescence plate reader at 480 nm/520 nm according to the manufacturer's instructions or manually counted under the microscope. Briefly, cell proliferation was assessed in cell lines derived from lung biopsies of human lung fibrosis patients or normal control lungs. We purchased two hairpins directed against human c-JUN and control hairpins in a pTRIPZ vector-based system engineered to be Tet-On from Thermo Scientific. Primary fibroblastic cell lines were infected with hairpin no. 2 or no. 3 directed against human c-JUN or hairpin controls, and 6 × 103 cells per well were plated in duplicate. We quantified cell proliferation every 24 h up to 72 h by manually counting the total cell numbers and performed Edu staining to assess cell division as outlined in the click-iT EdU Alexa Fluor 488 Imaging Kit. Subsequently, we acquired images of proliferating and migrating cells at the Stanford Microscopy Core Facility. In Vitro Inhibitory Screen. Inhibitors such as EGFR/ErbB-2 inhibitor (324673) (10 μM), PDGFR inhibitor (10 μM and 4 μM), wortmannin/PI3K (1 μM), GSK/lithium (1 μM), rapamycin (1 μM), Dapt/Notch (1 μM), Cyclopamin (1 μM), gamma-secretase inhibitor (1 μM), U0126 Mek inhibitor (10 μM), SB203580 inhibitor (1 μM), SU5402/FGF1/VEGF (10 μM), PI3 kinase inhibitor Ly294002 (10 μM), and JAK2 Inhibitor II (420132) (10 μM) were purchased from Calbiochem/Selleckchem and dosed as indicated, and anti-mouse CD47 clone 3 was used as described previously (21, 24). Anesthesia and CO Diffusion Testing. Mice were anesthetized with isoflurane gas or avertin (0.025 mL/g mouse of a 20 mg/mL filter sterilized solution) in saline via i.p. injection. For the measurement of pulmonary diffusing capacity for carbon monoxide (DFCO), a gas mixture of CH 4 /CO/air was used that is similar to the one used in humans for CO diffusion capacity measurements. Anesthetized mice were intubated with a 22-gauge stub needle cannula for the administration of intratracheal doxycycline or DFCO measurements. In a 3-mL syringe, 0.8 mL from the commercially available gas mixture was withdrawn, connected with a syringe to the tracheal cannula, and inflated in the lung. After 9 s, 0.8 mL of gas mixture was withdrawn and further analyzed with mass spectrometry. Cytokine/Chemokine Multiplex Assay. The following 38 mouse cytokines/chemokines have been quantified by cytokine/chemokine multiplex assay by the Stanford core facility: G-SCF/CSF-3, IL10, IL-3 LIF IL-1B, IL-2, M-CSF, IP-10, VEGF-A, IL4, IL-5, IL-6, TGFB, IFN-a, IL-22, IL-9, IL-13, IL-27, IL-23, IFN-g, IL-12P70, GM-CSF, GRO-a, RANTES, TNF-a, MIP-1a, MCP-3, MCP-1, IL-17A, IL-15/IL-15R, MIP-2, IL-1a, LIX, EOTAXIN, IL-28, IL-18, MIP-1b, IL-31; the mean fluorescent intensity has been measured and the concentrations of each cytokine/chemokine has been quantified by the standard curve method in pg/mL; the experimental details can be found at iti.stanford.edu/himc.html, the Stanford Human Immune Monitoring Center. In Vivo Drug Application. Six- to 12-wk-old c-Jun transgenic and control mice were either maintained on doxycycline containing water or induced intratracheally and concomitantly systemically treated with anti-CD47 antibody (100 μL i.p.), VEGF inhibitor PD173074 (2 mg/kg once per day i.p.), and a PI3K inhibitor wortmannin (2 mg/kg 3x/wk i.p.). Intratracheal intubation was performed under isoflurane anesthesia using a 22-gauge catheter, a light source, and an intubation platform with the administered volume not exceeding >125 μL per mouse. Protein Lysates and Western Blot Analysis. For protein assays, protein extracts were prepared by cell lysis in buffer containing protease inhibitors, subjected to SDS/PAGE, and analyzed by Western blot using primary antibodies directed against c-Jun and JunB as indicated throughout. Statistics. The results are expressed as the mean ± SEM for n given samples. Data were analyzed using the two-tailed Student’s t test or ANOVA with any P value less than or equal to 0.05 being considered significant. Survival was monitored and analyzed by Kaplan–Meyer analysis. Numbers of recipient mice are indicated, and the P value was derived by log-rank test.

Discussion Here, we report that c-JUN, a well-characterized AP1 transcription factor, is expressed in many different fibrotic diseases. We found decreased proliferation of patient fibroblasts from fibrotic lungs after knockdown of c-JUN. We detected activated c-Jun and Akt as well as up-regulation of CD47 expression in vivo in endstage fibrosis lungs. We further showed that c-Jun can induce rapid and widespread fibrosis in all organs in mice and is also expressed in fibrotic areas of abdominal adhesions in WT mice. c-JUN is widely expressed in skin epithelium and many other epithelial cells, but not highly in stromal cells. c-JUN is also part of the acute phase response cascade, has a role in bone formation, and has a reputation as an oncogene, and its up-regulation has been shown in various cancers (16). Although c-JUN’s role in cell cycle promotion has been well established primarily in vitro (16, 22), we observed a striking cell context-dependent fibrotic response in vivo. Despite ubiquitous c-Jun induction, we observed primarily fibrotic changes, indicating that the proproliferative and promigratory effects of c-Jun require the specific cellular context of tissue fibroblasts. Systemic induction of c-Jun in hematopoietic precursors caused rapid apoptosis; induction in the liver caused a pronounced hepatosteatosis. This unique c-Jun responsiveness seems to be shared among fibroblasts of many different tissues although fibroblasts are considered highly heterogeneous and tissue-specific (23). The fibrogenic response in multiple tissues and organs also contrasts with previously developed fibrosis models, suggesting that induction of c-Jun could be a common molecular mechanism across different human fibrotic conditions. We further provide evidence that the transcription factor c-Jun, which is a downstream target of MAPK-signaling cascades, can itself rewire and stabilize a specific pattern of multiple signaling pathways. We assume that the remodeling of signaling pathways will be different in different cell types, leading to the opposing cell biological effects of c-Jun in different cell types. Importantly, our mouse model also confirmed the functional relevance of several signaling pathways, some of which were previously associated with fibrosis and were targeted in past clinical trials (23), and idiopathic pulmonary fibrosis is currently treated with a combination of small molecule inhibitors targeting four different pathways: VEGF/FGFR/PDGFR and TGFBR (1). This finding suggests that c-Jun may be a central node controlling these essential pathways. Although combination therapy is in principle an attractive strategy, in practice, it is difficult to identify the right combination of pathways to target. In particular, for clinical trials, it is not feasible to evaluate combination therapy in a systematic manner. Our discovery that c-Jun coordinates several signaling pathways leading to fibrosis in vivo provides a unique opportunity to identify all relevant signaling pathways and predict the most effective therapeutic drug combinations. Moreover, it may be possible to develop therapeutic strategies interfering with the activity of c-Jun directly, which would eliminate the need to search for the most effective combination by eliminating the key disease-driving element. Several other mouse models have been established previously and have served to gain important insight into some specific aspects of disease (4). The most widely used model is a bleomycin-induced lung fibrosis isolated or in combination with the genetic model of Marfan syndrome. This model suggested the involvement of the TGFBR pathway in fibrosis, which we could confirm to also play a role in our c-Jun–induced model. In particular, the genetic model of Marfan syndrome was very instructive regarding the dissection of the contribution of dendritic helper cells toward the pathomechanism of skin and pulmonary fibrosis (4). Another model system uses carbon tetrachloride (CCl4) or bile duct ligation to study fibrosis in the liver, which has been shown to be linked to loss or constitutive activation of PDGFR-β in stellate cells. Unlike these mouse models, the c-Jun–induced model is a purely genetically driven model. Importantly, it recapitulates many aspects of the respective human disease conditions and is not limited to one organ system (such as only lung or skin) akin to the multiorgan disease of scleroderma. Furthermore, c-Jun is highly expressed in all human fibrotic conditions analyzed, and thus in vivo c-Jun induction is likely more physiologically relevant than chemical or infectious conditions that are not involved in the pathogenesis of human disease. We would argue for these reasons that our mouse model will be an important tool to further dissect the pathomechanisms leading to human disease. One such application was our surprising discovery that endogenous macrophages can be exploited to eliminate pathologic fibroblasts. We showed that fibrogenic cells expressed high levels of the self-protective don’t-eat-me epitope CD47. It had been shown in various solid cancers and hematopoietic malignancies, and most recently in atherosclerosis, that blockade of CD47 by antibodies or artificial, high-affinity Sirpα analogs prevents this repressive signal in macrophages, leading to their activation and active phagocytosis (24). The remarkable low toxicity of anti-CD47 treatments, however, suggested that additional alterations in cancer cells are required to induce phagocytosis (21, 25). Here, we show that this property is not limited to cancer cells because fibrosis was effectively reversed with anti-CD47 treatment by elimination of fibroblasts by macrophages. Studies are needed to identify the common mechanisms between fibrotic cells and cancer cells that allow effectiveness of the anti-CD47 treatment and to identify which other noncancerous diseases may benefit from such a therapy. In conclusion, our study revealed the unexpected role of c-Jun as a key and selective driver of organ fibrosis in most human fibrotic diseases. Our findings suggest that diverse fibrotic syndromes may have different etiologies but share common pathomechanisms centered around activation of c-Jun. The c-Jun mouse model may well be suitable to further dissect the pathogenesis of all types of pathologic fibrotic conditions and to develop new and effective therapies.

Materials and Methods Animal studies were approved by Stanford University Administrative Panels for Lab Animal Care (SU-APLAC 30911, 30912, 31026) and human research under IRB11177. We generated the flp-in tetO-c-JUN or flp-in tetO-JUNB transgenic mice as previously described (25) and induced the mice with tetracycline. CyTOF studies were performed in vivo exactly as described (18, 19). Details can be found in SI Materials and Methods.

Acknowledgments We thank Erika Dobos for pathology expertise, Daniel Haag for ingenuity analysis and GSEA analysis, Samuele Marro for help with heat maps, Norm Cyr for help with digital images, Farnaz Khameneh for help with genotyping PCRs, Patrick Sweeney and Shirley Kwok for help with tissue array assembly, and the Stanford Immuno core facility for help with cytokine/chemokine quantification. For these studies, I.L.W. was funded by the Virginia and D. K. Ludwig Fund for Cancer Research; the National Heart, Lung, and Blood Institute; and the National Cancer Institute of the National Institutes of Health under Grants U01HL099999 and R01CA86017, respectively. G.W. was funded by the Stanford Cancer Institute Fellowship Award; the Stanford Physician Scholar Society Award; and Institute for Immunity, Transplantation and Infection Young Investigator Award grants. The CyTOF studies were supported by NIH Grants U19AI057229, U19AI100627, R33CA183654, R33 CA0183692, R01GM10983601, R01-CA184968, R01CA19665701, R21-CA183660, R01-NS08953301, 5UH2AR067676, and R01HL120724; Northrop-Grumman Corporation; Novartis Grant CMEK162AUS06T; Pfizer Grant 123214; Juno Therapeutics Grant 122401; Department of Defense Grants OC110674 and W81XWH-14-1-0180; Gates Foundation Grant OPP1113682; and Food and Drug Administration Grant BAA-15-00121.