Unifying mechanism for different fibrotic diseases

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.

Keywords: anti-CD47 antibody therapy; c-JUN; fibrotic disease; scleroderma; signaling pathways.

Fig. 1.

Elevated c-JUN expression in various human fibrotic diseases. (A) SMA⁺ fibroblasts in lung fibrosis strongly expressed nuclear c-JUN (red) in fibrotic plaques as revealed by trichrome, Collagen1, and SMA staining. Shown are representative patient biopsies of lung fibrosis (n = 43) stained for indicated markers. (Scale bar: 100 µm.) (B) Example micrographs showing high c-JUN expression in trichrome⁺, fibrotic plaques of various indicated fibrotic conditions. Arrows indicate examples of nuclear c-JUN expression. (C) Quantification of c-JUN expression in SMA⁺ fibroblasts in various fibrotic conditions, including scar tissue (49 patient biopsies), scleroderma (104 patient biopsies of scleroderma, diffuse proliferative type), and lung fibrosis (43 patient lung biopsies) and in marrow fibrosis (57 patient marrow biopsies) but not in normal skin, lung, and marrow. ****P < 0.0001; ANOVA test. We quantified coexpression of c-JUN and SMA in the entire tissue of each sample. (D) Nuclear c-JUN is strongly expressed in mesenchymal cells in fibrotic plaques in lung fibrosis but only weakly in a small subset of bronchoepithelia (CK7 in green) and endothelia (CD31 in green). DAPI, nuclear counterstain in gray. Asterisks indicate auto fluorescence; one out of seven lung fibrosis and one out of six normal lung patient biopsies are shown. Three biological replicates for each stain were performed. (Scale bar: 100 μm.) (E) Lung fibroblasts from lung fibrosis patients require c-JUN to proliferate. Filled bars show cell numbers at indicated time points after c-JUN hairpin induction with doxycycline, open bars after doxycycline of control infections. (F) Normal lung fibroblasts do not require c-JUN to proliferate, unlike lung fibrosis fibroblasts. Shown are relative cell numbers before and after doxycycline-induced c-JUN knockdown normalized to the condition before knockdown. Patient lung fibroblast cultures were infected with one of two lentiviral shRNA hairpins also expressing RFP or a control vector expressing GFP. Infected cells were sorted by FACS and plated at a density of 6,000 cells per well, and the number of red and green fluorescent cells was counted 24, 48, and 72 h after plating. Data (mean ± SEM) represent four replicates (Student’s t test). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. S1.

c-JUN is highly expressed in most human fibrotic conditions and coexpressed with FOS in lung fibrosis. (A) Representative histology of patient biopsies of indicated fibrotic conditions out of 454 biopsies total were stained with c-JUN (brown nuclear staining) and trichrome (blue staining) and show increased collagen-producing fibroblasts with high level expression of c-JUN in densely fibrotic areas. (B) Immunohistochemical stains of JUNB, JUND, and FRA-1 (red) were negative in normal lung and lung fibrosis tissues. (C) Representative histology of normal lung and pulmonary arterial hypertension (PAH) lung tissue stained with H&E and trichrome with minimal perivascular fibrosis (blue on trichrome stain) and a pathological thickened vessel (PAH-lung, Lower Right). (D) c-FOS and FOSB (red), SMA (green), and DAPI (gray/merge) staining of lung tissue from normal, PAH, and lung fibrosis showed a subset of fibroblasts in lung fibrosis expressing nuclear c-FOS and FOSB. The arrows highlight representative nuclear staining. (E) c-JUN (red) costaining with cell type–specific markers SMA, CK7, CD31, and CD68 (green) showed coexpression of c-JUN in a subset of bronchoepithelial cells (CK7), but not in the majority of smooth muscle cells (SMA), endothelial cells (CD31), or macrophages (CD68). For B–E, one of seven idiopathic pulmonary fibrosis patient biopsies, one of five PAH patient biopsies, and one of six normal lung biopsies are shown. Three biological replicates for each stain were performed. (Scale bars: 100 μm.)

Fig. S2.

Don't-eat-me signal CD47 and calreticulin (eat-me signal) are expressed in lung fibrosis. (A) Representative lung sections from pulmonary arterial hypertension (PAH) and lung fibrosis lungs showed pathologically thickened vessels comprising FRA-2–positive smooth muscle cells. One of seven idiopathic pulmonary fibrosis and one of five PAH patient biopsies are shown. (Scale bars: 100 μm.) (B) Continuous sections of biopsies of lung fibrosis patients were costained as indicated and demonstrated expression of don’t-eat-me signal CD47 (green) in c-JUN⁺ (red) fibroblasts also expressing Collagen1 (green). Trichrome costains highlighted a fibrotic plaque with a small bronchus (Top Right) and otherwise highly abnormal lung histology. Costaining of c-JUN (red) and calreticulin (green) suggested expression of the eat-me signal in bronchoepithelium and macrophages (CD68, green). (Scale bars: 100 μm.) (C) We demonstrated efficient knockdown of c-JUN with hairpin no. 2 and no. 3 with quantitative RT-PCR in primary lung fibroblasts derived from lung fibrosis patient. (D) Western blot of c-JUN antibody revealed the loss of the specific 43-kDa band upon c-JUN knockout with CRISPER and a c-JUN–specific 43-kDa band upon doxycycline–mediated c-JUN induction in primary lung fibroblasts, supporting the specificity of the c-JUN antibody used for immunostaining.

Fig. 2.

Development of multiple organ fibrosis in mice after induction of c-Jun in vivo. (A) c-Jun–induced lethality in mice in a strain-dependent manner. B6, F1 129/C57BL/6; Bdf1, F1 129/BDF1. The red curve represents c-Jun B6 mice (n = 10, P < 0.0001), and the blue curve c-Jun Bdf1 mice (n = 7 mice, P < 0.0001 by Kaplan–Meyer survival analysis and two independent experiments). (B) Mice (B6) released many pro- and antiinflammatory cytokines in the blood 48 h after dox-mediated ubiquitous c-Jun induction. We quantified 38 mouse cytokines/chemokines by multiplex assay and found 13 to be increased significantly (**P < 0.01; ns, not significant) as indicated (n = 3 animals per group, repeated once, P values have been calculated by Student's t test). (C) Dox-mediated c-Jun expression in the lung led to “honeycomb” fibrosis reminiscent of human idiopathic lung fibrosis. Fibrotic plaques with extensive interstitial collagen (blue stained areas on trichrome) and intermixed with interstitial macrophages (CD68⁺, Inset) were identified peribronchial and subpleural and represented 34% of the surface area. We quantified the trichrome⁺ areas of 10 high power fields (40x), n = 3 animals per group (***P < 0.001). (D) Dox-mediated ubiquitous c-Jun expression led to thickening and fibrosis of the dermal skin and the gastroesophageal junction, a pattern of fibrosis reminiscent of human scleroderma. We quantified fibrosis in the trichrome⁺ areas of 10 high power fields (40x) of trichrome-stained sections (Insets) and detected 89% in the dermal skin and 67% in the stomach wall, n = 5 animals. (E) Kidney-restricted c-Jun expression using a Pax8-rtTA strain resulted in interstitial fibrosis and tubular atrophy (quantified at 30 to 40%) with elevated kidney enzymes as indicated in the table reminiscent of a primary tubulointerstitial nephropathy in patients. (Insets) High power views (40x) of the abnormal areas. A PASd stain (Top) labels intact basement membranes of glomeruli and tubuli, the H&E stain demonstrates increased interstitial fibrosis (Middle), and a trichrome stain (blue stain) shows abundant abnormal extracellular collagen matrix deposition (Bottom). n = 4 animals. (F) Systemic c-Jun expression caused fatty liver changes of the micro- and macrovesicular type in mice as shown by the small and large intrahepatic orange deposits on Oil-red O and the intracellular vesicles on H&E stains. We quantified 400 hepatocytes for intracellular lipid droplets in representative areas of Oil-Red O-stained liver sections as indicated. ****P < 0.0001; paired Student's t test. All data (Fig. 2 C–F) (mean ± SEM) represent two replicates of two independent experiments. Representative histology at 20x magnification, n = 4 animals. (Scale bars: 100 μm.)

Fig. S3.

c-Jun but not Junb causes skin, visceral, and marrow fibrosis in adult mice. (A) Targeting construct of c-Jun doxycycline-inducible mice. (B) Southern blot showing positively targeted ES cells with c-Jun. (C and D) Protein expression of c-Jun in mouse marrow by Western plot after 48 h of induction with doxycycline and after 7 d by intracytoplasmic flow cytometric analysis. (E) c-Jun expression and antibody specificity were confirmed by Western blot and immune staining in primary marrow-derived fibroblasts from c-Jun rtTA mouse after three passages in culture on day 2 of doxycycline induction and control (without doxycycline). (F) Southern blot showing positively targeted ES cells with Junb. (G) Protein expression of Junb by Western plot after 48 h of induction with doxycycline. (H) Representative histology of the marrow of Junb-expressing mice (n = 5). (I) Single femur counts revealed normocellular marrows for age. Data (mean ± SEM) are from five independent replicates. P = not significant (ns); paired Student’s t test was used to determine significant changes. (Scale bars: 100 μm.)

Fig. S4.

c-Jun expression in stroma cells caused severe marrow and visceral fibrosis that was mildly suppressed by hematopoietic cells. (A) Histological analysis revealed c-Jun–mediated dense fibrosis in the marrow in c-Jun–expressing mice (Left) quantified at 80% (graph, Far Right). H&E stains (Top) demonstrated that hematopoietic cells are significantly decreased and replaced by fibroblasts. Trichrome stains (Middle) highlighted the densely fibrotic areas with pathologic deposition of extracellular collagen (representative images, n = 17 mice analyzed) and fibroblasts coexpressed smooth muscle actin (SMA, brown cytoplasmic staining, Bottom). Trichrome stains label cross-linked collagen and are standard of care in clinical practice to grade organ involvement by fibrosis. n = 17 mice. (B) Histological analysis revealed fibrosis of the bladder wall 38 d after dox-mediated c-Jun induction on H&E and trichrome (Inset) stain and quantified at 82%. (C) Histological analysis revealed dense fibrosis of the uterus (82%) on H&E and trichrome 8 d after dox-mediated c-Jun induction. The black frames indicate the areas shown on higher magnification to the right. C-Jun staining demonstrated increased expression of c-Jun 8 d after c-Jun induction. (D and F) c-Jun–induced fibrosis was partially rescued by WT hematopoietic cells after bone marrow transplantation and also in c-Jun–induced mice parabiosed to WT syngeneic mice (n = 10 transplanted mice, n = 2 parabiosed mouse pairs). (E and G) Extent of fibrosis was quantified at 10.3% in sections of c-Jun–induced animals transplanted with WT bone marrow and at 29% in sections of parabiosed c-Jun–induced animals. To assess the percent involvement by fibrosis, we quantified the trichrome⁺ areas of 10 high power fields (40x) in all cases. **P < 0.01, ***P < 0.001, Student’s t test. (Scale bars: 100 μm.)

Fig. S5.

c-Jun–mediated fibrosis is specific to c-Jun induction in vivo. (A) Single femur counts revealed severe cytopenia in the bone marrow. Data (mean ± SEM) are from four independent replicates. ***P < 0.001 (paired Student’s t test). (B) 7AAD/annexin V staining demonstrated increased apoptosis in c-Jun–expressing hematopoietic precursors. Data (mean ± SEM) represent three replicates and two independent experiments. *P < 0.05, **P < 0.01; paired Student’s t test. (C) Lung-specific c-Jun expression resulted in interstitial fibrosis in the lung, and, as a consequence, CO accumulated in the respiratory air. CD47 antibody resolved interstitial fibrosis and thus normalized CO diffusion in the lung. Data (mean ± SEM) represent four replicates and two independent experiments; **P < 0.01; Student's paired t test. (D) Long-term intratracheal aerosol treatment of control mice with doxycycline and control or c-Jun mice with PBS did not result in any fibrosis in the lung. (E) Established c-Jun–mediated lung fibrosis (after 21 d of intratracheal dox treatment) normalized 300 d after c-Jun was switched off, and we show normal lung histomorphology with only minimal fibrosis on trichrome stain. (F and G) Representative kidney and liver sections of control mice stained with trichrome to highlight fibrosis. (H and I) c-Jun stains highlight increased c-Jun expression in all cell types and most organs 48 h after dox-mediated c-Jun induction in c-Jun mice (+dox) but not controls. Similarly, c-Jun is expressed in many different hematopoietic cell types in the bone marrow. (Scale bars: 100 μm.)

Fig. 3.

c-Jun rewires transcriptional and signaling pathways in fibroblasts in vivo. (A) Genome-wide transcriptional profiling in mouse marrow after 24 h of dox-mediated c-Jun expression in vivo demonstrated up-regulation of c-Jun and Fos and indicated genes, many of which were previously associated with fibrotic conditions. We selected 1,144 probes out of 45,101 based on greater than ±2-fold change. Gene expression studies were performed in triplicate per each time point, and bone marrow of three mice was pooled per array. (B) Fibrosis signature genes are significantly enriched by gene set enrichment analysis (GSEA) in the up-regulated genes 24 h after dox-mediated c-Jun expression. (C) ViSNE maps of primary, marrow-derived fibroblast cultures before (Ctrl, 0 h dox) and after c-Jun induction (48 h dox) were generated by considering all 12 surface markers and represent its degree of F4_80 (Upper) or Dusp1 (Lower) expression; blue colors represent low expression and yellow to red colors high expression. (D) Conditional density visualization (DREVI plots) of the relationship between Phospho-c-Jun (pc-Jun) and Phospho-Akt (pAkt) (Top), pc-Jun and pErk (Middle), and pc-Jun and Dusp1 (Bottom) in F4_80-negative (subset B) versus F4_80-positive (subset A) marrow-derived adherent cells 48 h after c-Jun induction. The visualization method described how the y axis molecule changes as a function of the x axis molecules. Dark red (maximal color) represents the most likely y axis molecule value in the corresponding x axis molecular value. A response function (white curve) is fit to the region of highest conditional density. Representative data of two independent series are shown.

Fig. S6.

c-Jun modifies wiring of signaling and transcriptional response in fibrosis. (A) We performed gene expression studies and selected the top 100 differentially expressed genes of 45,101 based on greater than ±2-fold change 24 h after c-Jun induction in vivo. Gene expression studies were performed in triplicate per each time point, and bone marrow of three mice was pooled per array. (B) Ingenuity analysis revealed activation of MAPK pathway 24 h after c-Jun induction. (C) viSNE map of bone marrow-derived primary fibroblast cocultures before (Ctrl, 0 h dox) and after (“48hr Dox”) c-Jun induction showed distinct subsets based on macrophage lineage marker F4_80 and CD172a expression (A-F4_80-positive and B-F4_80-negative subsets). Each point in the viSNE map represents an individual cell, and color represents the expression level of indicated markers from high (red) to low (blue). (D) DREVI plots in all panels represent the relationship between pc-Jun on the x axis and the indicated genes on the y axis. (C and D) Representative data of one out of four experiments.

Fig. 4.

Anti-CD47, PI3K and VEGF inhibitors block c-Jun–mediated fibrosis. (A) Assessment of the doubling time (in days) of primary bone marrow fibroblasts with or without c-Jun induction revealed an about 10-fold increased proliferation rate upon c-Jun induction. Data (mean ± SEM) represent three replicates and two independent experiments; ****P < 0.0001; paired Student’s t test. (B) Transwell migration of primary marrow fibroblasts from c-Jun–inducible mice increased about 100-fold upon dox treatment at both 2 and 24 h after seeding. Data (mean ± SEM) represent three replicates and two independent experiments; **P < 0.01; paired Student’s t test. (C) c-Jun–mediated transwell migration of primary marrow fibroblasts in the presence of indicated small molecule inhibitors. Migration was impaired by PI3K, Vegf, Pdgfr, and Tgfb pathway inhibitors. Data (mean ± SEM) represent two replicates and two independent experiments; ***P < 0.001; paired Student’s t test. (D) In vitro phagocytosis assay of primary c-Jun–expressing fibroblasts and primary macrophages in the presence or absence of anti-CD47 antibodies. Anti-CD47 increased the phagocytosis rate. Data (mean ± SEM) represent two replicates and two independent experiments; **P < 0.01; paired Student’s t test. (E) H&E stain of lung sections of anti-CD47–treated (Left) and untreated (Right) mice after airway-restricted doxycycline delivery, which resulted in c-Jun–induced honeycomb-type lung fibrosis. The fibrosis was histomorphologically reversed with anti-CD47 antibody (MIAP 410, also known as clone 3). (F) Quantification of the extent of fibrosis in lung sections revealed that the residual fibrosis in the treatment group was 5%. n = 3 mice per experiment, two independent experiments; **P < 0.01, paired Student’s t test. (G and J) H&E histological analysis revealed that c-Jun–mediated bone marrow fibrosis was eliminated and replaced with normal hematopoietic cells after systemic treatment of PI3K and VEGF pathway antagonists. (H) Quantification of marrow fibrosis of PI3K inhibitor-treated and control mice revealed almost complete absence of fibrosis in treated animals. N = 3 animals, two independent experiments; ***P < 0.001. BM, bone marrow. (K) Quantification of marrow fibrosis of VEGF inhibitor-treated and control mice. n = 3 animals, two independent experiments; **P < 0.01. (I) c-Jun–mediated scleroderma-type dermal skin thickening was substantially reduced with PI3K inhibitor treatment. Data (mean ± SEM) represent n = 3 replicates, two independent experiments; *P < 0.05; paired Student’s t test. (Scale bars: 100 μm.)