The Transferrin Receptor CD71 Delineates Functionally Distinct Airway Macrophage Subsets during Idiopathic Pulmonary Fibrosis

Abstract

Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating progressive disease with limited therapeutic options. Airway macrophages (AMs) are key components of the defense of the airways and are implicated in the pathogenesis of IPF. Alterations in iron metabolism have been described during fibrotic lung disease and in murine models of lung fibrosis. However, the role of transferrin receptor 1 (CD71)-expressing AMs in IPF is not known. Objectives: To assess the role of CD71-expressing AMs in the IPF lung. Methods: We used multiparametric flow cytometry, gene expression analysis, and phagocytosis/transferrin uptake assays to delineate the role of AMs expressing or lacking CD71 in the BAL of patients with IPF and of healthy control subjects. Measurements and Main Results: There was a distinct increase in proportions of AMs lacking CD71 in patients with IPF compared with healthy control subjects. Concentrations of BAL transferrin were enhanced in IPF-BAL, and furthermore, CD71^- AMs had an impaired ability to sequester transferrin. CD71⁺ and CD71^- AMs were phenotypically, functionally, and transcriptionally distinct, with CD71^- AMs characterized by reduced expression of markers of macrophage maturity, impaired phagocytosis, and enhanced expression of profibrotic genes. Importantly, proportions of AMs lacking CD71 were independently associated with worse survival, underlining the importance of this population in IPF and as a potential therapeutic target. Conclusions: Taken together, these data highlight how CD71 delineates AM subsets that play distinct roles in IPF and furthermore show that CD71^- AMs may be an important pathogenic component of fibrotic lung disease.

Keywords: airway macrophages; idiopathic pulmonary fibrosis; transferrin receptor.

Figure 1.

Proportions of CD71⁻ airway macrophages (AMs) are increased in idiopathic pulmonary fibrosis (IPF) BAL compared with healthy controls. (A) Gating strategy for flow cytometric analysis of BAL CD71-expressing AMs. (B) Wright-Giemsa staining of sorted CD71⁻ AMs (upper left) and CD71⁺ AMs (lower left) and immunofluorescence staining for CD71 in sorted CD71⁻ AMs (upper right) and CD71⁺ AMs (lower right). (C and D) Numbers of CD71⁺ (C) and CD71⁻ (D) AMs in healthy and IPF BAL. (E and F) Proportions of CD71⁺ (E) and CD71⁻ (F) AMs in healthy and IPF BAL. n = 11 healthy controls; n = 97 IPF. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ****P < 0.0001, Mann-Whitney U test. FSC = forward scatter; SSC = side scatter.

Figure 2.

Defective transferrin uptake in CD71⁻ airway macrophages (AMs). (A) Concentrations of transferrin in the BAL of patients with idiopathic pulmonary fibrosis (IPF) or of healthy control subjects (n = 7 healthy control subjects; n = 31 patients with IPF). (B) Correlation of proportions of BAL CD71⁺ AMs with concentrations of transferrin in the airways (n = 67 patients with IPF). (C) Comparison of transferrin uptake in IPF-CD71⁻ and IPF-CD71⁺ AMs. (D) Staining (Prussian blue) for iron in sorted CD71⁻ or CD71⁺ AMs. Arrows indicate iron-positive AMs. (E and F) Gene expression analysis of sorted CD71⁻ or CD71⁺ AMs is shown for ACO1 (n = 10 patients with IPF) (E) and IREB2 (n = 6 patients with IPF) (F). GAPDH was used as a housekeeping gene. Data are presented as mean ± SD. *P < 0.05 and ****P < 0.0001, Mann-Whitney U test. ns = not significant.

Figure 3.

CD71⁺ and CD71⁻ airway macrophages (AMs) are phenotypically distinct. (A) Flow cytometric gating strategy for the analysis of AM phenotypic markers. Data are shown as a percentage of either CD71⁺ AMs expressing a marker of interest (highlighted in red) or CD71⁻ AMs expressing a marker of interest (highlighted in blue). (B–E) Proportions of idiopathic pulmonary fibrosis (IPF)-CD71⁺ and IPF-CD71⁻ AMs expressing CD163 (n = 17 patients with IPF) (B), CD86 (n = 12 patients with IPF) (C), HLA-DR (n = 38 patients with IPF) (D), and CD14 (n = 38 patients with IPF) (E). Data are presented as mean ± SD. ****P < 0.0001 by Mann-Whitney U test. SSC = side scatter.

Figure 4.

CD71⁺ and CD71⁻ airway macrophages (AMs) are functionally distinct. (A) Comparison of nitric oxide (NO) expression in idiopathic pulmonary fibrosis (IPF)-CD71⁺ and IPF-CD71⁻ AMs (n = 23 patients with IPF). (B) Phagocytosis assay of IPF-CD71⁺ and IPF-CD71⁻ AMs (n = 23 patients with IPF). (C) Representative staining of AMs after phagocytosis assay showing phagocytosed bacteria (green) and DAPI (blue). (D–F) Proportions of IPF-CD71⁺ and IPF-CD71⁻ AMs expressing CD206 (n = 39 patients with IPF) (D), MARCO (n = 7 patients with IPF) (E), and SR-A (n = 7 patients with IPF) (F). (G and H) Expression of TLR2 (n = 10 patients with IPF) (G) and TLR3 (n = 10 patients with IPF) (H) in CD71^+/− AMs. GAPDH was used as a housekeeping gene. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, Mann-Whitney U test.

Figure 5.

CD71⁻ airway macrophages (AMs) have profibrotic characteristics. (A) Heat map representation of fibrosis array of sorted CD71^+/− idiopathic pulmonary fibrosis (IPF)-AMs. (B) Scatterplot highlighting differentially expressed genes in CD71⁺ and CD71⁻ IPF-AMs. Plot shows fibrosis-related genes upregulated in CD71⁻ AMs compared with CD71⁺ cells in green and those downregulated in CD71⁻ AMs compared with CD71⁺ AMs in red. Genes with statistically significant and greater than twofold change compared with control are highlighted. (C) Kaplan-Meier survival plot for subjects with IPF dichotomized above (High) and below (Low) the median for proportion of CD71⁻ AMs. Individuals with an increased proportion of CD71⁻ AMs have worse survival than patients with a low proportion of CD71⁻ AMs (n = 97 patients with IPF).