Myeloid Heterogeneity Mediates Acute Exacerbations of Pulmonary Fibrosis

Abstract

Epidemiological evidence indicates that exposure to particulate matter is linked to the development of idiopathic pulmonary fibrosis (IPF) and increases the incidence of acute exacerbations of IPF. In addition to accelerating the rate of lung function decline, exposure to fine particulate matter (particulate matter smaller than 2.5 μm [PM2.5]) is a risk factor for increased mortality in subjects with IPF. In this article, we show that exposure to PM2.5 mediates monocyte recruitment and fibrotic progression in mice with established fibrosis. In mice with established fibrosis, bronchoalveolar lavage cells showed monocyte/macrophage heterogeneity after exposure to PM2.5. These cells had a significant inflammatory and anti-inflammatory signature. The mixed heterogeneity of cells contributed to the proinflammatory and anti-inflammatory response. Although monocyte-derived macrophages were recruited to the lung in bleomycin-injured mice treated with PM2.5, recruitment of monocytes expressing Ly6Chi to the lung promoted progression of fibrosis, reduced lung aeration on computed tomography, and impacted lung compliance. Ly6Chi monocytes isolated from PM2.5-exposed fibrotic mice showed enhanced expression of proinflammatory markers compared with fibrotic mice exposed to vehicle. Moreover, IPF bronchoalveolar lavage cells treated ex vivo with PM2.5 showed an exaggerated inflammatory response. Targeting Ly6Chi monocyte recruitment inhibited fibrotic progression in mice. Moreover, the adoptive transfer of Ly6Chi monocytes exacerbated established fibrosis. These observations suggest that enhanced recruitment of Ly6Chi monocytes with a proinflammatory phenotype mediates acute exacerbations of pulmonary fibrosis, and targeting these cells may provide a potential novel therapeutic target to protect against acute exacerbations of IPF.

Figure 1.. Fibrotic progression occurs in AE-PF

WT mice were exposed to saline or bleomycin (Bleo, 1.75 U/kg i.t.). At day 14, mice were exposed to saline (vehicle) or PM_2.5 (8.25 μg in saline, i.t.) daily. Lungs were excised and BAL was performed on day 21. (A) Representative Masson’s trichrome staining of lung tissues. n = 8. Bar = 200 μm. (B) Hydroxyproline analysis of lung homogenates. n = 8. (C) Cell differential using Wright-Giemsa stain to identify mononuclear (mono), neutrophil (PMN), and lymphocytes (lymph). n = 9. (D) Total number of BAL cells. n = 5. (E) Kaplan-Meier survival curve. n = 5. WT mice were exposed to saline or bleomycin. At day 21, mice were exposed to vehicle or PM_2.5 daily. Lungs were excised and BAL was performed on day 28. (F) Representative Masson’s trichrome staining of lung tissues. n = 5. Bar = 200 μm. (G) Hydroxyproline analysis of lung homogenates. n = 8-10. (H) Cell differential of BAL cells. n = 9-10. (I) Total number of BAL cells. n = 8-10. (J) Kaplan-Meier survival curve. n = 8. **, p < 0.005; ***, p < 0.0001. Values shown represent means ± S.E.M. One-way ANOVA followed by Tukey’s multiple comparison test was used for B-D, G-I and a log-rank (Mantel-Cox) test was used for E and J. Data shown were pooled from three independent experiments.

Figure 2.. BAL cells in mice have a mixed phenotype in AE-PF

WT mice were exposed to saline or bleomycin. At day 21, mice were exposed to vehicle or PM_2.5 daily. BAL was performed on day 28. mRNA expression for (A) Tnf, (B) Nos2, (C) Tgfb1, and (D) Arg1 in BAL cells. n = 9-10. (E) TNF-α and (F) active TGF-β1 levels in BAL fluid. n = 8-10. IPF BAL cells were obtained by BAL and exposed ex vivo to PM_2.5 (10 μg/cm²). mRNA expression of (G) TNF, (H) NOS2, (I) TGFB1, and (J) ARG1 levels in BAL cells. n = 9. *, p < 0.05; ***, p < 0.0001. Values shown represent means ± S.E.M. One-way ANOVA followed by Tukey’s multiple comparison test was used for A-F. Two-tailed t-test statistical analysis was utilized for G-J. Data shown were pooled from three independent experiments.

Figure 3.. Recruitment of Ly6C expressing monocytes in AE-PF

WT mice were exposed to saline or bleomycin. At day 21, mice were exposed to vehicle or PM_2.5 daily. BAL was performed on day 28. (A) Representative flow cytometry plots of resident alveolar macrophages (RAM, CD45⁺CD11b^+/−Ly6G⁻CD64⁺Ly6C⁻Siglec F^hi) and monocyte-derived macrophages (MDM, CD45⁺CD11b^+/−Ly6G⁻CD64⁺Ly6C⁻Siglec F^low). (B) Total cell number of RAM and MDM from BAL. n = 9-10. (C) CCL2 levels in BAL fluid. n = 8-10. (D) Representative confocal images of Ly6C positive BAL cells counterstained with DAPI. Bar = 50 μm (n = 5). (E) mRNA expression of Ly6c1 in isolated BAL cells. n = 9-10. (F) Representative flow cytometry plots of Ly6C^lo (CD45⁺CD11b⁺Ly6G⁻CD64^+/−MHC II⁻Ly6C^loSiglec F⁻) and Ly6C^hi (CD45⁺CD11b⁺Ly6G⁻CD64^+/−MHC II⁻Ly6C^hiSiglec F⁻) monocytes. (G) Total cell number of Ly6C^lo and Ly6C^hi monocytes from BAL. n = 9-10. Data shown were pooled from three independent experiments. (H) Number of Ly6C^lo and Ly6C^hi monocytes from BAL in bleomycin injured mice exposed to PM_2.5 for indicated days. n = 6. (I) Representative confocal images of ZO-1 and DAPI staining in mouse lung tissue. Bar = 10 μm (n = 5). (J) Mean fluorescent intensity of ZO-1 staining in lung tissue. n = 8. (K) Albumin levels in BAL fluid. n = 7-8. Data shown are representative from three independent experiments. *, p < 0.05; **, p < 0.001; ***, p < 0.0001. Values shown represent means ± S.E.M. One-way ANOVA followed by Tukey’s multiple comparison test was used. ns = not significant.

Figure 4.. PM_2.5 induces recruitment of inflammatory Ly6C^hi monocytes to mediate AE-PF

WT mice were exposed to saline or bleomycin. At day 21, mice were exposed to vehicle or PM_2.5 daily. BAL was performed on day 28. (A) Heat map of mRNA expression of FACS-sorted RAMs and MDMs for (B) Tnf, (C) Nos2, (D) Tgfb1, and (E) Arg1. n = 8-10. (F) Heat map of mRNA expression of FACS-sorted Ly6C^lo and Ly6C^hi monocytes for (G) Tnf, (H) Nos2, (I) Tgfb1, and (J) Arg1. n = 9-10. Data shown were pooled from three independent experiments. (K) Representative micro-CT images of mouse lung parenchyma identifying ≤−500 Hounsfield units (HU, green, aerated lung) and >−500 HU (red, non-aerated lung) using 3D-Slicer imaging software. Quantification of lung density in (L) HU and (M) percentage of aerated lung tissue. n = 6-7. Data shown were pooled from two independent experiments. mtROS generation measured by mitoSOX in (N) isolated BAL cells from exposed mice (n = 6) and (O) FACS-sorted Ly6C^hi monocytes from bone marrow exposed to exposed to PM_2.5 (10 μg/cm²) and treated with EDTA (5mM). n = 6. Data shown are representative from three independent experiments.*, p < 0.05; **, p < 0.001;***, p < 0.0001. Values shown represent means ± S.E.M. One-way ANOVA followed by Tukey’s multiple comparison test was used. ns = not significant.

Figure 5.. Ly6C^hi monocytes mediate fibrotic progression in mice with advanced fibrosis

(A) Schematic indicating Ly6C^hi monocytes derived from bone marrow (1 x 10⁶) were adoptively transferred by i.t. into bleomycin-exposed WT mice 21 days after exposure. (B) Total number of BAL cells. n = 8. mRNA expression in BAL cells for (C) Tnf, (D) Nos2, (E) Tgfb1, and (F) Arg1. n = 7-8. (G) Representative Masson’s trichrome staining of lung tissues. n = 7. Bar = 250 μm. (H) Hydroxyproline analysis of lung homogenates. n = 8. Data shown are representative from two independent experiments. ***, p < 0.0001. Values shown represent means ± S.E.M. Two-tailed t-test statistical analysis was utilized.

Figure 6.. Ly6C^hi monocytes are required for AE-PF

WT mice were exposed to saline or bleomycin. At day 21, mice were exposed to vehicle or PM_2.5 daily. On day 23, mice were administered IgG control or anti-CCR2, clone MC-21 antibody (20μg, i.p. daily). BAL was performed on day 28. Representative flow cytometry plots of (A) RAMs and MDMs and (B) Ly6C^lo Ly6C^hi monocytes. (C) Total cell number of Ly6C^lo and Ly6C^hi monocytes from BAL. n = 5. mRNA expression for (D) Tnf, (E) Nos2, (F) Tgfb1, and (G) Arg1 in BAL cells. n = 4-5. (H) Representative Masson’s trichrome staining of lung tissues. n = 5. Bar = 250 μm. (I) Hydroxyproline analysis of lung homogenates. n = 8. Data shown were pooled from two independent experiments. (J) Compliance was determined by respiratory mechanics analysis. n = 5-6. **, p < 0.005; ***, p < 0.0001. Values shown represent means ± S.E.M. One-way ANOVA followed by Tukey’s multiple comparison test was used. ns = not significant.