Myeloid-mesenchymal crosstalk drives ARG1-dependent profibrotic metabolism via ornithine in lung fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive lung remodeling and collagen deposition that leads to respiratory failure. Myeloid cells are abundant in IPF lung and in murine lung fibrosis, but their functional effects are incompletely understood. Using mouse and human lung models, we show that ornithine produced by myeloid cells expressing arginase 1 (ARG1) serves as a substrate for proline and collagen synthesis by lung fibroblasts. The predominant ARG1-expressing myeloid cells in mouse lung were macrophages, but in IPF lung, high-dimensional imaging revealed ARG1 was expressed mainly in neutrophils. Small-molecule ARG1 inhibition suppressed both ornithine levels and collagen expression in cultured, precision-cut IPF lung slices and in murine lung fibrosis. These results were confirmed in macrophage-specific Arg1-KO mice. Furthermore, we found that this pathway is regulated by cell-to-cell crosstalk, starting with purinergic signaling: extracellular ATP receptor P2RX4 was necessary for fibroblast IL-6 expression, which, in turn, was necessary for ARG1 expression by myeloid cells. Taken together, our findings define an immune-mesenchymal circuit that governs profibrotic metabolism in lung fibrosis.

Keywords: Amino acid metabolism; Fibrosis; Immunology; Pulmonology.

Figure 1. ARG1-expressing cells localize to the fibrotic niche in murine and human lung.

(A) Volcano plot for macrophages (mac) from scRNA-Seq of macrophage-fibroblast cocultures for WT macrophages and fibroblasts (fibs) versus WT macrophages cocultured with P2rx4 KO (Pdgfrb-Cre P2rx4^fl/fl) fibroblasts. Significance was determined by Wilcoxon’s rank-sum test corrected for multiple comparisons by the Bonferroni method. Color-labeled genes had P < 0.05 and absolute value of log₂(fold change) > 0.75. Data represent 2 separate cocultures. DGE, differential gene expression. (B) Analysis of macrophages from bleomycin lung injury (data reanalyzed from Strunz et al., ref. 6). Top left: Annotation of macrophages from multiple time points, according to C1, C2, or C3 macrophage annotation (as described by Aran et al., ref. 2): C1, alveolar macrophages; C2, transitional monocyte-derived macrophages; C3, monocyte-derived macrophages). Top right: Proportions of C1, C2, and C3 across time. Bottom left: Feature plot of Arg1 expression. Bottom right: Violin plot of Arg1 expression according to cluster (C1, C2, and C3). ***P < 0.001 by Student’s t test. (C) Immunofluorescence imaging of tamoxifen-induced Arg1-RFP-CreERT2 LSL-tdTomato-Col1a1-GFP mice at 14 days after bleomycin injury. Quantification shows total Arg1⁺ cells inside and outside contiguous Col1a1-GFP⁺ areas. n = 3 mice per condition. *P < 0.05 by Student’s t test. (D) ARG1 expression by microarray analysis of gene expression of BAL cells from healthy patients (n = 20) and patients with IPF (n = 112) from GSE70867 (9). ****P < 0.0001 by Mann-Whitney test. (E) Representative immunofluorescence of human healthy control and IPF lung sections (n = 4 and 6, respectively) for ARG1. Scale bars: 50 μm. The quantification shows the proportion of ARG1⁺ cells (i.e., ARG1⁺/total DAPI count) for each sample. *P < 0.05 by Student’s t test. (C–E) Data are reported as mean ± SEM.

Figure 2. ARG1 is expressed predominantly by neutrophils in IPF.

(A) Schematic of MIBI and a heatmap of scaled marker expression from MIBI of sections from 5 IPF explanted lungs. (B) MIBI UMAPs by cluster and individual markers. (C) MIBI images for individual markers within a representative FOV of IPF lung. Scale bars: 50 μm (left panel), 5 μm (right panels).

Figure 3. ARG1 regulates lung collagen via ornithine production.

(A) Ornithine concentration measured in lysates of mouse lung (n = 5 uninjured and n = 6 injured mice), healthy human donor lung (n = 9), and lung explants of patients with IPF (n = 10). ***P < 0.001, ****P < 0.0001 by Student’s t test. (B) Ornithine concentration with and without CB-1158 treatment of lungs of bleomycin-injured mice (n = 8 in each condition) and cultured IPF PCLS (n = 16 slices from a total of 3 patients). ****P < 0.0001 by Student’s t test. (C) Hydroxyproline assay for collagen content of lungs from injured and uninjured WT mice treated with or without the ARG1 inhibitor CB-1158. Mice were dosed with 100 mg/kg CB-1158 twice daily during days 9–15 after bleomycin (bleo) administration. n = 6, 7, and 8 mice, respectively, left to right. ****P < 0.0001 by 1-way ANOVA with post hoc Šídák’s multiple-comparison test. Median, upper, and lower quartiles indicated by dashed lines. i.t., intratracheal. (D) Representative immunoblot for COL1A1 from lysates of precision-cut IPF lung slices cultured for 24 hours with or without ARG1 inhibitor CB-1158. Quantitation is for slices from a total of 3 patients. ***P < 0.001 by Student’s t test. (E) Hydroxyproline assay for collagen content of lungs from mice 21 days after bleomycin injury. n = 10 and 9 mice, respectively, left to right. *P < 0.05 by Mann-Whitney test. Median, upper, and lower quartiles indicated by dashed lines. (F) Left: COL1A1 immunofluorescence of monocultured mouse lung fibroblasts with or without ornithine treatment. Quantification is for 3 separate cultures each. *P < 0.05 by Student’s t test. Right: COL1A1 immunofluorescence of monocultured human lung fibroblasts with or without ornithine treatment. Quantification is for 3 separate cultures each. *P < 0.05 by Student’s t test. (G) Hydroxyproline assay for collagen content of lungs from injured WT mice treated with or without ornithine (2 g/kg) twice daily by ornithine gavage during days 7 through 20. n = 8 and 7 mice, respectively, left to right. ***P < 0.001, ****P < 0.0001 by 1-way ANOVA with post hoc Šídák’s multiple-comparison test. Median, upper, and lower quartiles indicated by dashed lines. (H) Representative samples of COL1A1 immunofluorescence of mouse lung macrophage-fibroblast cocultures from WT or fibroblast-specific P2rx4-KO (Pdgfrb-Cre P2rx4^fl/fl) mice treated with or without CB-1158 or ornithine. (I) Quantitation of MFI from (H). n = 3 biological replicates per condition. ****P < 0.0001 by 1-way ANOVA with post hoc Šídák’s multiple-comparison test. (J) Representative samples of COL1A1 immunofluorescence of mouse lung macrophage-fibroblast cocultures treated with or without CB-1158. Mac, Arg1^fl/fl control mice; Mac_ΔArg1, macrophages isolated from Lysm-Cre Arg1^fl/fl mice. Quantification is for 3 separate cultures each. **P < 0.01, ***P < 0.0001 by 1-way ANOVA with post hoc Šídák’s multiple-comparison test. (K) Relative quantities of proline and hydroxyproline in lysates of murine primary lung fibroblasts isolated after coculture with macrophages, with or without CB-1158 inhibitor treatment, quantified by liquid chromatography–mass spectrometry. n = 3 biological replicates for macrophages and fibroblasts, respectively. ****P < 0.0001 by 1-way ANOVA with post hoc Šídák’s multiple-comparison test. (A, B, D, F, I–K) Data are reported as mean ± SEM. Spec, spectrometry. Scale bars: 50 μm.

Figure 4. IL-6 is necessary for ARG1 expression after bleomycin injury in mice.

(A) Left: Ingenuity Pathways Analysis of predicted upstream regulators for macrophages cultured with WT relative to P2rx4-KO fibroblasts (fib). Right: CellChat plot comparing WT and P2rx4-KO conditions. Significance was determined by Wilcoxon’s test, with P < 0.05 being statistically significant. Data represent 2 separate cocultures. (B) Violin plot for Il6 expression in WT relative to P2rx4-KO fibroblasts from cocultures. Line shows median values. Data represent 2 separate cocultures. (C) ARG1 ELISA of cell lysates and conditioned media collected from mouse lung macrophage (mac) monoculture 72 hours after IL-6 treatment. n = 3 biological replicates per condition. ***P < 0.001, ****P < 0.0001 by Student’s t test. (D) Left: ARG1 ELISA of BAL fluid normalized to lung CD11B⁺CD64⁺ macrophage count taken at day 14 from bleomycin-injured WT or Il6-KO mice. Right: Arg1 qPCR of CD11B⁺CD64⁺ macrophages. n = 5 mice per condition. **P < 0.01 by Student’s t test. (E) Quantification of Il6 expression by cell type in cells that underwent scRNA-Seq directly after isolation from the lung at steady state and multiple time points after injury: reanalysis of merged data from Strunz et al. (6) and Tsukui et al. (29) normalized by Sctransform (62). (F) UMAP of fibroblast subtypes (left) and Il6 feature plots at steady state (center) and 7 days after bleomycin injury (right), from Tsukui et al. (29). (C, D, and F) Data are reported as mean ± SEM.

Figure 5. IL-6 is necessary for ARG1 expression in IPF lung and in cocultures of human blood–derived neutrophils and lung fibroblasts.

(A) ARG1 ELISA of lysates of precision-cut IPF lung slices cultured for 24 hours with or without IL-6R–blocking antibody tocilizumab. Data represent 3 patients, with 3 slices per condition per patient assayed. ****P < 0.0001 by Student’s t test. (B) 10x Xenium spatial analysis: Left: Representative FOV showing cells expressing ARG1 and EPCAM, and IL6⁺ fibroblasts (fibs) (defined by coexpression of either COL1A1 or CTHRC1). Right: Proximity analysis of Xenium data. Data shown are for the probability ratio, P (exp|ARG1⁺)/P(exp), the probability of encountering an IL6⁺ fibroblast, normalized by fibroblast frequency, at the radial distance of 25 μm from ARG1⁺ cells. *P < 0.05 by paired Student’s t test. Data are for 9 separate patients. (C) Correlation scatter plot of tissue-resident neutrophils for IL6R and ARG1 expression. The plot is a reanalysis of integrated scRNA-Seq data for NSCLC samples from 19 data sets from Salcher et al. (30). (D) COL1A1 immunofluorescence of human lung fibroblasts that were either monocultured or cocultured with human blood–derived neutrophils with or without IL-6R–blocking antibody (tocilizumab) treatment. Quantification is for 3 separate cultures each. Donor 1 and donor 2 represent separate donors for both neutrophils and fibroblasts. **P < 0.01 by 2-way ANOVA followed by Šídák’s multiple-comparison test. (E) ARG1 ELISA of conditioned media from the same cocultures as in D. ***P < 0.001, ****P < 0.0001 by 2-way ANOVA followed by Šídák’s multiple-comparison test. (F) Ornithine concentration of conditioned media from the same cocultures as in D. ****P < 0.0001 by 2-way ANOVA followed by Šídák’s multiple-comparison test. (A, B, D–F) Data are reported as mean ± SEM.

Figure 6. P2RX4 is necessary for IL-6 expression in mouse and human lung fibroblasts.

(A) Top left: UMAP plot of scRNA-Seq for macrophage-fibroblast cocultures with SingleR-based cell type annotation (2) shown. Data represent 2 separate cultures for each condition. Top right: UMAP plot for data showing sample of origin. Fib, fibroblast monoculture; Fib + Mac, fibroblast coculture with macrophages. Bottom left: Feature plot showing Il6 expression. Bottom right: Violin plot of Il6 expression. ***P < 0.001 by Wilcoxon’s rank-sum test corrected for multiple comparisons by Bonferroni’s method. (B) Gene set enrichment analysis of fibroblast single cell transcriptomes in coculture with macrophages compared with fibroblast monoculture using Gene Ontology “Response to stimulus” pathways. (C) IL-6 ELISA of conditioned media from macrophage-fibroblast cocultures with or without fibroblast-specific P2rx4 deletion. n = 5 biological replicates per condition. ****P < 0.0001 by 1-way ANOVA with post hoc Šídák’s multiple-comparison test. Data presented as mean ± SEM. (D) IL-6 ELISA of conditioned media from monocultured mouse lung fibroblasts from WT mice, with and without ATPγS and SB203580 (p38 MAP kinase inhibitor) treatment, or from fibroblast-specific P2rx4-KO (Pdgfrb-Cre P2rx4^fl/fl) mice with ATPγS treatment. n = 8, 8, 5, and 3 biological replicates per respective condition. *P < 0.05, ***P < 0.001, ****P < 0.0001 by 1-way ANOVA with post hoc Šídák’s multiple-comparison test. (E) IL-6 ELISA of BAL from mice with or without fibroblast-specific P2rx4 deletion. n = 7, 7, and 6 biological replicates, respectively, left to right. *P < 0.05, **P < 0.01 by 1-way ANOVA with post hoc Šídák’s multiple-comparison test. (F) Left: IL-6 ELISA of conditioned media from human donor lung fibroblast monocultures with P2RX4 siRNA KD or nontargeting control siRNA (NT siRNA), with and without ATPγS treatment (left, n = 3 per condition). ***P < 0.001, ****P < 0.0001 by 1-way ANOVA with post hoc Šídák’s multiple-comparison test. Right: Quantification of KD by qPCR, n = 3 per condition. ***P < 0.001 by Student’s t test. (G) IL-6 ELISA of conditioned media from human donor lung fibroblast monocultures, human blood–derived neutrophil monocultures, or cocultures with or without P2RX4 inhibition with BAY-1797. Conditioned media were collected after 24 hours of culture. Each point represents a separate technical replicate. Neutrophils were derived from 2 separate blood donors, and fibroblasts were derived from 2 separate healthy lung donors. ***P < 0.001, ****P < 0.0001 by 2-way ANOVA followed by Šídák’s multiple-comparison test. Donor 1 and donor 2 represent separate donors for both neutrophils and fibroblasts. (C–F) Data are reported as mean ± SEM.