Monocyte-derived alveolar macrophage apolipoprotein E participates in pulmonary fibrosis resolution
Huachun Cui, Dingyuan Jiang, Sami Banerjee, Na Xie, Tejaswini Kulkarni, Rui-Ming Liu, Steven R. Duncan, Gang Liu
Huachun Cui, Dingyuan Jiang, Sami Banerjee, Na Xie, Tejaswini Kulkarni, Rui-Ming Liu, Steven R. Duncan, Gang Liu
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Research Article Pulmonology

Monocyte-derived alveolar macrophage apolipoprotein E participates in pulmonary fibrosis resolution

Abstract

Recent studies have presented compelling evidence that it is not tissue-resident, but rather monocyte-derived alveolar macrophages (TR-AMs and Mo-AMs, respectively) that are essential to development of experimental lung fibrosis. However, whether apolipoprotein E (ApoE), which is produced abundantly by Mo-AMs in the lung, plays a role in the pathogenesis is unclear. In this study, we found that pulmonary ApoE was almost exclusively produced by Mo-AMs in mice with bleomycin-induced lung fibrosis. We showed that, although ApoE was not necessary for developing maximal fibrosis in bleomycin-injured lung, it was required for the resolution of this pathology. We found that ApoE directly bound to Collagen I and mediated Collagen I phagocytosis in vitro and in vivo, and this process was dependent on low-density lipoprotein receptor–related protein 1 (LPR1). Furthermore, interference of ApoE/LRP1 interaction impaired the resolution of lung fibrosis in bleomycin-treated WT mice. In contrast, supplementation of ApoE promoted this process in ApoE–/– animals. In conclusion, Mo-AM–derived ApoE is beneficial to the resolution of lung fibrosis, supporting the notion that Mo-AMs may have distinct functions in different phases of lung fibrogenesis. The findings also suggest a potentially novel therapeutic target for treating lung fibrosis, to which effective remedies remain scarce.

Authors

Huachun Cui, Dingyuan Jiang, Sami Banerjee, Na Xie, Tejaswini Kulkarni, Rui-Ming Liu, Steven R. Duncan, Gang Liu

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Figure 5

ApoE directly binds to type I Collagen.

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ApoE directly binds to type I Collagen. (A) Eight-week-old C57BL/6 mice ...
(A) Eight-week-old C57BL/6 mice were i.t. instilled with saline or bleomycin (BLM, 1.5 U/kg in 50 μL saline). Three weeks after treatment, mice were sacrificed and lung slices prepared. Immunofluorescence staining and fluorescence microscopy were performed to determine the expression of ApoE and Collagen I. Nuclei were counterstained with DAPI. Original magnification, ×200. Scale bars: 100 μm. (B) Slices of normal control and IPF lungs were prepared. Immunofluorescence staining and fluorescence microscopy were performed to determine the expression of ApoE and Collagen I. Nuclei were counterstained with DAPI. Original magnification, ×200. Scale bars: 100 μm. (C) Increasing amounts of recombinant human ApoE protein or BSA were dotted on a nitrocellulose membrane. After blocking, the membrane was incubated with the conditioned media from TGF-β1–induced human lung myofibroblasts that are enriched with type I Collagen. Bound proteins on the membrane were determined by immunoblotting. (D) A constant amount of ApoE (1 μg) was incubated with the increasing volumes of the conditioned media from TGF-β1–induced lung myofibroblasts. Collagen I was then immunoprecipitated by anti–Collagen I antibody and ApoE in the immunocomplex was determined by Western blotting. (E) A 96-well high-binding plate was precoated with 10 μg/mL rat tail Collagen I or BSA overnight at 4°C. Wells were washed and incubated with 200 ng/mL BSA or increasing amounts of purified human ApoE protein for 2 hours at room temperature, followed by subsequent incubation with primary ApoE antibody (1:2000 dilution) and HRP-conjugated secondary antibody (1:2000 dilution). Colorimetric development was achieved by TMB reaction. The plate was read at 450 nm in a 96-well plate reader. n = 3; mean ± SD; **P < 0.01 compared with the BSA group. Two-tailed Student’s t test. (F) A 96-well high-binding plate was precoated with 10 μg/mL rat tail Collagen I overnight at 4°C. Wells were washed and incubated with 50 ng/mL BSA or 50 ng/mL ApoE together with increasing amount of soluble Collagen I protein. Solid-binding assays were performed as in E. n = 3; mean ± SD; *P < 0.05, **P < 0.01 by 1-way ANOVA with Bonferroni’s post hoc test.