Technical advance: The use of tree shrews as a model of pulmonary fibrosis

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease with a high morbidity and mortality. Some of the mechanisms of fibrosis development have been described using rodent models; however, the relevance of findings in these animal models is difficult to assess. New innovative models are needed that closely mimic IPF disease pathology.

Methods: To overcome this unmet need of investigating IPF with a relevant model, we utilized tree shrews, which are genetically, anatomically, and metabolically similar to primates and humans. Using human antibodies and primers, we investigated the role of macrophage phenotypic switching in normal and IPF subjects and bleomycin-injured tree shrews.

Results: Bronchoalveolar lavage (BAL) cells from tree shrews expressed human markers, and there was recruitment of monocyte-derived macrophages (MDMs) to the lung in IPF subjects and bleomycin-injured tree shrews. MDMs were polarized to a profibrotic phenotype in IPF and in bleomycin-injured tree shrews. Resident alveolar macrophages (RAMs) expressed proinflammatory markers regardless of bleomycin exposure. Tree shrews developed bleomycin-induced pulmonary fibrosis with architectural distortion in parenchyma and widespread collagen deposition.

Conclusion: The profibrotic polarization of macrophages has been demonstrated to be present in IPF subjects and in fibrotic mice. Although the lung macrophages have long been considered to be homogeneous, recent evidence indicates that these cells are heterogeneous during multiple chronic lung diseases. Here, we show new data that indicate a critical and essential role for macrophage-fibroblast crosstalk promoting fibroblast differentiation and collagen production. in the development and progression of fibrosis. The current data strongly suggest development of therapeutics that attenuate of the profibrotic activation of MDMs may mitigate macrophage-fibroblast interaction. These observations demonstrate that tree shrews are an ideal animal model to investigate the pathogenesis of IPF as they are genetically, anatomically, and metabolically closer to humans than the more commonly used rodent models.

Fig 1. Recruited monocyte-derived macrophages from tree shrews are increased during fibrosis.

(A) Cell differential of normal (n = 3) and IPF (n = 5) subjects. (B) Representative flow cytometry plots of monocyte-derived macrophages (MDM, CD11b⁺HLA-DR⁺⁺CD206⁺⁺CD169⁺CD163⁺) and resident alveolar macrophages (RAM, CD11b⁺HLA-DR⁺⁺CD206⁺⁺CD169⁺CD163⁺⁺) of normal (n = 3) and IPF (n = 5) subjects. (C) Total cell number of RAM and MDM from BAL of normal (n = 3) and IPF (n = 5) subjects expressed as fold change. (D) Cell differential of saline- (n = 5) and bleomycin-exposed (n = 4) tree shrews. (E) Representative flow cytometry plots of monocyte-derived macrophages (MDM, CD11b⁺HLA-DR⁺⁺CD206⁺⁺CD169⁺CD163⁺) and resident alveolar macrophages (RAM, CD11b⁺HLA-DR⁺⁺CD206⁺⁺CD169⁺CD163⁺⁺) of saline- (n = 3) and bleomycin-exposed (n = 3) tree shrews. (F) Total cell number of RAM and MDM from BAL saline- (n = 3) and bleomycin-exposed (n = 3) tree shrews. Representative H & E staining of lung tissue from (G) saline- (n = 6) and (H) bleomycin-exposed (n = 5) tree shews. Masson’s Trichome staining of lung tissue from (I) saline- (n = 6) and (J) bleomycin-exposed (n = 5) tree shews. Scale bar = 100 μm for G-J. (K) Hydroxyproline analysis of saline- (n = 6) and bleomycin-exposed (n = 5) tree shrews. **, p < 0.001; ***, p < 0.0001. Values shown as mean ± S.E.M. Two-way ANOVA with Tukey post-test was utilized for (A), (C), (D), and (F). Two-tailed t-test statistical analysis was utilized for (K).

Fig 2. BAL cells are polarized to a profibrotic phenotype.

(A) Arginase 1, (B) VEGF, (C) CCL18, (D) iNOS, and (E) TNF-α mRNA expression in BAL cells from normal (n = 5–7) or IPF subjects (n = 5–6). BAL cells were isolated and mRNA expression was determined from saline- (n = 6) and bleomycin-exposed (n = 4) tree shrews for (F) arginase 1, (G) VEGF, (H) TGF-β, (I) PDGF-B, (J) iNOS, and (K) TNF-α. *, p < 0.01; ***, p < 0.0001. Values shown as mean ± S.E.M. Two-tailed t-test statistical analysis was utilized.

Fig 3. Recruited monocyte-derived macrophages are polarized to a profibrotic phenotype.

Tree shrews were exposed to saline (n = 3) or bleomycin (n = 3) and BAL cells were isolated. FACS-sorted RAMs and MDMs were analyzed for (A) arginase 1, (B) VEGF, (C) TGF-β, (D) PDGF-B, (E) iNOS, and (F) TNF-α mRNA expression. ***, p < 0.0001. Values shown as mean ± S.E.M. One-way ANOVA with Tukey post-test was utilized.

Fig 4. Macrophage-derived profibrotic gene expression regulates fibroblast differentiation.

(A) Immunoblot analysis of IPF fibroblasts cultured in BAL fluid from saline- (n = 3) or bleomycin-exposed (n = 3) tree shrews. (B) Quantification of α-SMA expression in A (n = 3 per group). mRNA analysis of (C) collagen 1a and (D) fibronectin in IPF fibroblasts cultured in BAL fluid from saline- (n = 3) or bleomycin-exposed (n = 3) tree shrews. **, p < 0.001; ***, p < 0.0001. Values shown as mean ± S.E.M. Two-tailed t-test statistical analysis was utilized.