Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity

Abstract

The transcription factor JUN is highly expressed in pulmonary fibrosis. Its induction in mice drives lung fibrosis, which is abrogated by administration of anti-CD47. Here, we use high-dimensional mass cytometry to profile protein expression and secretome of cells from patients with pulmonary fibrosis. We show that JUN is activated in fibrotic fibroblasts that expressed increased CD47 and PD-L1. Using ATAC-seq and ChIP-seq, we found that activation of JUN rendered promoters and enhancers of CD47 and PD-L1 accessible. We further detect increased IL-6 that amplified JUN-mediated CD47 enhancer activity and protein expression. Using an in vivo mouse model of fibrosis, we found two distinct mechanisms by which blocking IL-6, CD47 and PD-L1 reversed fibrosis, by increasing phagocytosis of profibrotic fibroblasts and by eliminating suppressive effects on adaptive immunity. Our results identify specific immune mechanisms that promote fibrosis and suggest a therapeutic approach that could be used alongside conventional anti-fibrotics for pulmonary fibrosis.

Fig. 1. Systems-level analysis of pulmonary fibrosis patients.

a Outline of our “Omics” approach in human fibrotic lung integrating proteomics, secretomics and genomics technology platforms to study the contribution of leukocytes and pathologic fibroblasts and to identify therapeutic targets. b Single-cell force-directed layout of fibrotic lung tissues. Shaded regions indicate the location of manually gated cell populations. c Frequencies of cell populations in the lung detected by mass cytometry (CyTOF). Data are displayed as mean ± SD of 11 fibrotic and 3 normal control-lung samples. d PCA was computed on fibroblast clusters from 11 individual pulmonary fibrosis patients (PF) and 3 normal donors (NC) mass cytometry datasets demonstrating that fibrotic and the normal fibroblasts were distinct from each other. e ViSNE maps of fibroblast mass cytometry data demonstrating that the abundance of fibroblasts differed. The data demonstrate a representative example per group and each point in the viSNE map represents an individual cell. f ViSNE analysis of mass cytometry data of fibrotic lung, normal lung and normal PBMCs revealed increased activation of the JUN and AKT pathways in fibrotic lung fibroblasts. Schematic diagram of the location of the indicated cell types on the viSNE map are based on the expression of lineage specific markers. Red indicates high and blue low protein expression. g Representative mass cytometry plots of the pro-fibrotic fibroblast population in fibrotic lung compared with normal lung. h Immune fluorescent stains confirmed increased CD47 and PD-L1 co-expression in lung fibroblasts from fibrotic lungs but not in normal controls (activated fibroblasts expressing FSP1⁺Collagen1⁺ and SMA⁺). The arrow indicates the blood vessel. (Scale bars, 100 μm). i RNA expression analysis of JUN, PD-L1 and CD47 in fibrotic and normal lung fibroblasts are detected by Taqman assay. Data are expressed as mean ± SD of 5 fibrotic fibroblasts and 3 normal fibroblasts and representative of at least three experiments. Data were analyzed by two-tailed unpaired t-test, *P < 0.05; **P < 0.01. See Supplementary Data 2 for statistical details. Source data are provided as a Source Data file.

Fig. 2. Lung fibrotic condition converts macrophages into an immunosuppressive phenotype.

a Main cluster frequencies of CD45⁺ leukocytes (T cells, macrophages, B cells, NK cells, dendritic cells and other inflammatory cells such as neutrophils/eosinophils/plasma cells) contained in the lungs of pulmonary fibrosis patients and normal controls were quantified by mass cytometry. Data are expressed as mean ± SD of 11 fibrotic and 3 normal lung samples. b Computational analysis of mass cytometry data of leukocytes derived from fibrotic lungs with a single-cell, force-directed algorithm demonstrated that the different inflammatory subsets segregated as indicated on the map: CD4⁺ T cells (blue), CD8⁺ T cells (purple), macrophages (green), B cells (orange), NK cells (yellow) and the dendritic cell subset (white). c Principal component analysis (PCA) of manually gated macrophages (CD45⁺ CD68⁺ nonB nonT, nonNK live cells) indicating that macrophages derived from the pulmonary fibrosis lung (PF) clusters are distinct from those in normal lungs (NC). d A refined viSNE analysis of mass cytometry data demonstrating that macrophages derived from normal lungs (orange) have a distinct profile from fibrotic lungs (blue: black dotted circle). e ViSNE analysis of macrophages isolated from normal lungs and fibrotic lungs demonstrating decreased activation of HLA-DR, CD169 and CD206 expression in fibrotic lungs relative to controls. Each point represents a single cell, and the samples are color coded as indicated: blue colors represent low expression and yellow to red represent high protein expression. f The corresponding ratio of interstitial macrophages (HLA-DR⁺CD206⁺CD169⁻, IM) versus alveolar macrophages (HLA-DR⁺⁺CD206⁺⁺CD169⁺, AM) is displayed with mean ± SD of 11 fibrotic and 3 normal lung samples and analyzed by two-tailed unpaired t-test, **P < 0.01. g Representative images of immune fluorescent stains highlighted increased PD-1 expression in macrophages from fibrotic lung tissues (scale bars, 100 μm). See Supplementary Data 2 for statistical details. Source data are provided as a Source Data file.

Fig. 3. T cells present in fibrotic lungs mitigate a suppressive immune response in Lung fibrotic.

a, b Representative CyTOF plots and quantification of CD4⁺ (top) and CD8⁺ (bottom) naive T cells (CCR7⁺ CD45RA⁺) demonstrating decreased naive T cells in fibrotic lungs. Data represent mean ± SD of 11 fibrotic and 3 normal samples and are analyzed by two-tailed unpaired t-test, ***P < 0.001; ****P < 0.0001. c, d Representative CyTOF analysis showing increased frequency of regulatory CD4 T cells (T_reg: CD4⁺ Foxp3⁺ CD25⁺) in fibrotic lungs. Data represent mean ± SD of 11 fibrotic and 3 normal samples and are analyzed by two-tailed unpaired t-test, ***P < 0.001. e, f Representative CyTOF plots and quantitative analysis indicating increased percentage of exhausted T cells (Tex: CD8⁺ PD-1⁺ TIM3⁺) in fibrotic lungs. Data represent mean ± SD of 11 fibrotic and 3 normal samples and are analyzed by two-tailed unpaired t-test, *P < 0.05. g Representative images of immune fluorescent stains for PD-1 on T cells (CD3⁺) highlighting increased percentage of PD-1⁺ T cells in fibrotic lung samples (scale bars, 100 μm). See Supplementary Data 2 for statistical details. Source data are provided as a Source Data file.

Fig. 4. Accessibility of PD-L1 (CD274) and CD47 depended on JUN.

a Heatmap demonstrating dynamic chromatin changes in fibrotic lung fibroblasts with (JUN-KO) or without (Control) JUN deletion and normal lung fibroblasts with (TetO-JUN Dox⁺) or without (TetO-JUN Dox⁻) JUN activation. b Representative genome browser tracks comparing ATAC-seq signal in fibrotic lung fibroblasts (with (JUN-KO) or without (Control) JUN-knockout) and also ChIP-seq signal in normal lung fibroblasts (with (TetO-JUN Dox⁺) or without (TetO-JUN Dox⁻) JUN overexpression) with A549, MCF-7, h1-hESC, HepG2 and K562 from published data at JUN, CD47 and CD274 loci. The red boxes highlight ATAC-seq and ChIP-seq peaks in the promoter sites of JUN, CD47 and CD274 (and enhancer is shown in green). We also compared our peaks with H3K4me3 or H3K27Ac (=histone mark for open chromatin), H3K9me3 or H3K27me3 (=histone mark for closed chromatin), ChIP-seq data generated from normal human-lung fibroblast is from published data, which highlighted the same areas respectively. c Gene expression changes in primary lung fibroblasts by comparing JUN knockout (KO) or overexpression (OE). Taqman assay were normalized to the value in JUN knockout. Data are expressed as mean ± SD from four experimental repeats. Two-tailed ratio paired t-test, **P < 0.01; ****P < 0.0001. d Representative flow cytometry histograms showing reduced expression of pJUN, PD-L1 and CD47 after JUN overexpression (OE) or knockout (KO). Yellow plot: JUN overexpression; Black plot: JUN knockout. e Vector maps of the control and CD47 enhancer constructs used to engineer reporter cell lines. f, g CD47 enhancer reporter assays demonstrating that doxycycline induced JUN expression initiated CD47 enhancer expression, which disappeared when JUN expression was turned off (f) or JUN was knocked out (g). Data are expressed as mean ± SD from three independent experiments, Ordinary one-way ANOVA (Tukey’s multiple comparisons test), n.s. non-significant; **P < 0.01; ***P < 0.001; ****P < 0.0001. See Supplementary Data 2 for statistical details. Source data are provided as a Source Data file.

Fig. 5. IL-6 mediates the pro-fibrotic response of JUN involvement.

a The secreted proteins in the lung bronchoalveolar lavage (BAL) of 4 fibrotic lung patients were quantified by Luminex assay, showing IL-6 as the highest expressed cytokine across all fibrotic patient BAL samples. Data were normalized by protein levels of the BAL of 3 normal lungs, and presented as mean ± SD. b Cytokines and chemokines in the fibrotic mouse bronchoalveolar lavage (BAL) after JUN induction were quantified by Luminex assay, and detected IL-6 was consistently among the most highly expressed cytokines in JUN-induced mouse fibrotic lungs indicative of IL-6-JAK-STAT pathway activation. Data were normalized by normal lung expression, and presented as mean ± SD, n = 3. c The cytokines/chemokines released from JUN-induced lung fibrotic mice-derived whole bone marrow (n = 4), fibroblasts (n = 4) and monocytes/macrophages (n = 3) in the medium after 48 h of Dox-initiated JUN induction were quantified by Luminex assay, demonstrating that whole bone marrow and fibroblasts are secreting increased IL-6 in response to JUN. Data were presented as mean ± SD. d Increased IL-6 expression levels were detected by Taqman assay and Flow cytometry in primary lung fibroblasts with JUN knockout (KO) or overexpression (OE). Data are expressed as mean ± SD from 4 experimental repeats. Two-tailed ratio paired t-test, ***P < 0.001. e, f IL-6 increased CD47 enhancer activity at concentrations as low as 1 ng/ml (e) and protein expression at 10 ng/ml (f) in a dose-dependent fashion. Data are expressed as mean ± SD from three independent experiments, ordinary one-way ANOVA with multiple comparisons test, n.s. non-significant; **P < 0.01; ***P < 0.001. See Supplementary Data 2 for statistical details. Source data are provided as a Source Data file.

Fig. 6. Inhibition of immune checkpoints together with IL-6 resolves lung fibrosis.

a Whole-lung scaffold map for Bleomycin-induced lung fibrosis in mice. Each node represents unsupervised cell clusters. b Representative mass cytometry plot demonstrating increased expression of immune-checkpoint proteins-CD47⁺ PD-L1⁺ in fibroblasts, an expansion of CD11b⁺ F4/80⁺ macrophages, regulator T cells (CD3⁺ CD4⁺ CD25⁺ FOXP3⁺) and exhausted T cells (CD3⁺ CD8⁺ PD-1⁺ TIM3⁺) in mouse model after fibrosis induction with bleomycin for 2 weeks. c, d Representative images of Micro CT scans of wildtype and B6.129S2-Il6^tm1Kopf/J (IL-6KO) mice highlighting increased fibrosis in the lung after fibrosis induction (wildtype and IL-6KO mice) and much improved fibrosis after treatment with HAC (anti-PD-L1) alone or combined with a blocking antibody against CD47 or/and IL-6. Data are expressed as mean ± SD of five animals and analyzed by using one-way ANOVA followed by Tukey’s multiple comparisons test for multiple comparison. n.s. non-significant; *P < 0.05; ****P < 0.0001. e Trichrome of lung sections of control mice, mice after fibrosis induction with bleomycin (wildtype and IL-6KO mice) and mice after treatment with blocking antibodies against IL-6 and CD47 and HAC (the blocking reagent against PD-L1) demonstrating markedly improved fibrosis (significantly decreased blue stained areas on Masson’s trichrome stain which correspond to cross-linked collagen) and diminished PD-L1 expression in FSP1⁺ fibroblasts after treatment. Scale bar, 100 μm. See Supplementary Data 2 for statistical details. Source data are provided as a Source Data file.

Fig. 7. Schematic diagram of the proposed mechanisms of fibrosis clearance.

Left: In fibrotic lung, we find persistent myofibroblast activation in fibrotic plaques and JUN upregulation. JUN expression in fibrosis-associated fibroblasts (FAFs) appears to directly control the promoters and enhancers of CD47 and CD274 (PD-L1). The direct consequence is increased expression of these immune-checkpoint proteins in fibroblasts and dormant macrophages which do not phagocytose, but continue to release chronic inflammatory cytokines. JUN also directly regulates IL-6 at the chromatin level. The increased expression and secretion of this potent cytokine leads to a suppressive adaptive immune response-chiefly T-cell exhaustion and upregulation of regulatory T cells. Right: Disrupting the suppression of the innate and adaptive immunity with CD47 and PD-L1 inhibitors as well as the proinflammatory IL-6 cytokine pathway stimulated phagocytic removal of pro-fibrotic fibroblasts and T-cell activation leading to clearance of the fibrosis in the lung.