BCL9 inhibition promotes fibroblast lipogenesis by regulating macrophage–fibroblast interactions to attenuate pulmonary fibrosis

Figures

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  Fig. 1. Increased BCL9 levels correlated with IPF progression. Based on a publicly available scRNA-seq dataset: a BCL9 expression levels across various types of ILD. b BCL9 expression profile across myeloid cell subtypes in IPF patients. c–g Reanalysis of the GSE136831 dataset focused on IPF macrophages. To control for age as a confounder, only subjects aged ≥40 years were included in both the IPF and control groups. c Identification of macrophage subsets in IPF. d Density distribution of BCL9 expression within the macrophage population. e BCL9 expression levels across individual macrophages. f Average BCL9 expression levels in macrophages. g Proportion of BCL9-expressing macrophages. h Immunofluorescence staining for BCL9 (red) and CD68 (green) in human lung sections, Scale bar: 25 μm. i Immunofluorescence staining for BCL9 (red) and F4/80 (green) in mouse lung sections, Scale bar: 25 μm. P value by Wilcoxon rank-sum test (e, g). IPF idiopathic pulmonary fibrosis, ILD interstitial lung disease, COPD chronic obstructive pulmonary disease, scRNA-seq single cell RNA sequencing

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  Fig. 2. Genetic depletion of Bcl9 in macrophages attenuates pulmonary fibrosis in mice. a Scheme for the generation of mice with macrophage-specific Bcl9 knockout (Bcl9fl/fl Lyz2Cre). b Lung coefficients of WT and Bcl9fl/fl Lyz2Cre mice (n = 3). qPCR was performed to detect c Ccn4, d Ccn1, and e Ccn2 expression in lung tissues (n = 3). f Representative whole-lung sections from mice of each group (n = 3). g Representative image of H&E and Masson staining in lungs (n = 3). Scale bar: 500 μm. h Quantification of the degree of fibrosis based on pathological sections (n = 3). i Flow cytometric analysis of M2 macrophage infiltration in the lungs (n = 3). Data are derived from three independent experiments, and are represented as the mean ± SD. * P < 0.05, *** P < 0.001 by two-way ANOVA. BLM bleomycin, IPF idiopathic pulmonary fibrosis, i.t. intratracheal administration, H&E hematoxylin-eosin, WT wild-type

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  Fig. 3. Macrophage-intrinsic BCL9 drives M2-like reprogramming and promotes fibroblast-to-myofibroblast transdifferentiation. a Schematic representation of the generation of WT, Bcl9-KO, Bcl9-OE and Vector-OE PMs. Macrophages were polarized toward the M1 phenotype with LPS and IFN-γ or toward the M2 phenotype with IL-4. b Gating strategy for M2 macrophages by flow cytometry. Flow cytometry quantification of M2 macrophage percentages in c WT versus Bcl9-KO and d Vector-OE versus Bcl9-OE PMs after stimulation (n = 3). e TGF-β1 levels in culture supernatants from the indicated M2 macrophage groups, measured by ELISA (n = 3). f Illustration of a coculture system containing PMs and MLFs. qPCR analysis of fibrotic gene expression, including g Acta2, h Col1a1, and i Col3a1, in MLFs cocultured with WT or Bcl9-KO PMs (n = 3). qPCR analysis of fibrotic gene expression, including j Acta2, k Col1a1, and l Col3a1, in MLFs cocultured with Vector-OE or Bcl9-OE PMs (n = 3). Data are derived from three independent experiments, and are represented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by two-way ANOVA (c, d), or Student’s t test, unpaired, two-tailed (e, g–l). WT wild-type, KO knockout,…

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  Fig. 4. hsBCL9Z96 administration attenuates pulmonary fibrosis in mice. a Experimental scheme of BLM-induced pulmonary fibrosis and therapeutic hsBCL9Z96 intervention. b Representative whole-lung sections from each group of mice (n = 6–10). c Representative images of H&E and Masson staining in lungs (n = 6–10). Scale bar: 625 μm. d Lung coefficient of mice (n = 6–10). Quantification of the e degree of fibrosis and f degree of inflammation based on pathological sections (n = 6–10). qPCR detection was used to determine the expression of genes, including g Ccn4, h Ccn1, i Ccn2, j Col1a1, k Col3a1, l Tgfb1, and m Arg1, in lung tissues (n = 4). n Flow cytometric quantification of the percentage of M2 macrophages in lung tissues (n = 4). Data are derived from three independent experiments, and are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s, unpaired, two-tailed t test. BLM bleomycin, i.t. intratracheal administration, i.p. intraperitoneal injection, H&E hematoxylin-eosin

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  Fig. 5. hsBCL9Z96 alters macrophage differentiation trajectories during pulmonary fibrosis. a Schematic of the experimental workflow of scRNA-seq (n = 3). b t-SNE plot showing the distribution of nine cell clusters identified in the scRNA-seq data. c Dot plot showing the expression levels of fibrotic genes in the lungs. d Proportion of M2 macrophages within the total macrophage population. e Dot plot showing the expression levels of profibrotic genes in M2 macrophages. f Pseudotime analysis of the macrophage differentiation trajectories. Arrows indicate the inferred direction of differentiation. scRNA-seq, single-cell RNA sequencing

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  Fig. 6. BCL9 inhibition suppresses M2 macrophage polarization through the MerTK-ERK-SPP1 axis. a Heatmap of differentially expressed genes in M2 macrophages. b Correlation analysis of Ctnnb1 and Spp1 expression dynamics along the pseudotime trajectory. c qPCR was used to detect the Spp1 expression in the lungs (n = 4). ELISA measured SPP1 protein levels in culture supernatants from d PMs treated with or without hsBCL9Z96 or e BMDMs from WT or Bcl9fl/fl Cre-ERT2 mice (n = 3). f MFI of the M2 marker CD206 on PMs (n = 3). g TGF-β1 content in the supernatant of PMs was measured by ELISA (n = 3). Flow cytometric analysis of MerTK and p-ERK MFI in h PMs treated with or without hsBCL9Z96 and i BMDMs from the indicated genotypes (n = 3). j Flow cytometric analysis of CD206 MFI in PMs from the indicated groups (n = 3). k SPP1 and TGF-β1 content in the supernatant of PMs (n = 3). Flow cytometric analysis of l CD206 MFI and m p-ERK MFI in PMs (n = 3). ELISA measurement of n SPP1 and o TGF-β1 levels in PMs supernatants (n = 3). Data are derived from three independent experiments, and are represented as the mean ± SD. ns, no significance. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test,…

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  Fig. 7. hsBCL9Z96 promotes lipogenesis in fibroblasts through macrophage–fibroblast crosstalk. a Cell-cell interaction analysis between M2 macrophages and fibroblasts through the TGFB1 axis. b Pseudotime analysis of fibroblast differentiation trajectories. Arrows indicate the inferred direction of differentiation. c Dot plot showing the expression levels of lipogenic and myogenic genes in myofibroblasts and lipofibroblasts. d Reanalysis of a public spatial transcriptomic dataset from IPF patients. e Immunofluorescence staining of human lung tissues (n = 5). ADFP (red), CD68 (green), CD163 (purple), DAPI (blue), Scale bar: 25 μm. f Lipid droplet staining (LipidTOX, green; DAPI, blue) in MLFs cultured with conditioned media from PMs (n = 3). Scale bar, 130 μm, red arrow: lipid droplet. g Pparg expression in MLFs under the indicated co-culture conditions (n = 3). Flow cytometric analysis of the proportion of h lipofibroblasts and i AT2 cells in lung tissues from WT and Bcl9fl/fl Lyz2Cre mice (n = 3). Flow cytometric analysis of the proportion of j lipofibroblasts and k AT2 cells in lung tissues from mice treated with or without hsBCL9Z96 (n = 3). l Proportion of AT2 cells within the total…

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  Fig. 8. hsBCL9Z96 demonstrates translational potential by attenuating human macrophage-driven fibrosis. a Flow cytometric analysis of the proportion of M2 macrophages in THP-1-derived macrophages (n = 3). Representative immunofluorescence images showing b MerTK (red) and c p-ERK (green), in treated THP-1-derived macrophages; nuclei were counterstained with DAPI (blue) (n = 3), scale bar: 70 μm. d SPP1 levels in THP-1 supernatant (n = 3). e qPCR was used to detect ACTA2 expression in correspondingly treated HFL1 cells (n = 3). f Lipid droplet staining (LipidTOX, green; DAPI, blue) in HFL1 cells (n = 3). Scale bar, 70 μm, red arrow: lipid droplet. g Proportion of M2 macrophages in PBMC-derived macrophages from IPF patients (n = 5). qPCR detected the expression of h ACTA2, i COL1A1, j COL3A1, k PPARG, l SREBF2, and m PLIN2 in HFL1 cells cultured with conditioned media from the patient-derived macrophages described in g (n = 5). Data in a–f were derived from three independent experiments. Data are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA (a, d, e) or Student’s t test, unpaired, two-tailed (g–m). PBMCs peripheral blood mononuclear cells, IPF…

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  Fig. 9. Schematic representation of the mechanism of BCL9 inhibition-mediated resolution of pulmonary fibrosis. BCL9 inhibition, achieved either by genetic knockout or via the peptide inhibitor hsBCL9Z96, suppresses the pro-fibrotic phenotype of macrophages, thereby reprogramming macrophage–fibroblast crosstalk. This reprogramming drives a phenotypic switch in fibroblasts from a myogenic (fibrosis formation) to a lipogenic (fibrosis resolution) state. As a result, this transition not only attenuates pathological extracellular matrix (ECM) deposition but also promotes the expansion of alveolar epithelial type II (AT2) cells, collectively leading to the resolution of pulmonary fibrosis. Mechanistically, disruption of the BCL9/β-catenin interaction inhibits the MerTK–ERK–SPP1 axis in macrophages, leading to reduced secretion of TGF-β1. The ensuing attenuation of TGF-β1 signaling from macrophages to fibroblasts upregulates lipogenic genes (e.g., PPARG, PLIN2, SREBF2) while downregulating myofibroblast-associated genes (e.g., ACTA2, COL1A1, COL3A1) in fibroblasts. Together, these findings delineate a novel macrophage–fibroblast–AT2 cell communication axis through which targeting BCL9…