UGRP1-modulated MARCO+ alveolar macrophages contribute to age-related lung fibrosis

Mice

Female C57BL/6 mice were obtained from the Shanghai Experimental Center of the Chinese Science Academy (Shanghai, China). Young mice (10–16 weeks) and aged mice (20–24 months) were used. All mice were maintained under specific-pathogen-free and controlled conditions (22 °C, 55% humidity, and a 12-h day/night rhythm), in accordance with the Guide for the Care and Use of Laboratory Animals granted by University of Science and Technology of China.

Isolation of lung mononuclear cells (MNCs)

As previously described [42], MNCs were isolated from the lungs via density gradient centrifugation using 40% and 70% Percoll solution (Gibco BRL, Grand Island, NY, USA).

Purification of alveolar macrophages

Isolated lung MNCs were stained with fluorescein isothiocyanate (FITC)-conjugated anti-F4/80 (Clone BM8, eBioscience, San Diego, CA, USA), phycoerythrin (PE)-conjugated anti-CD11c (Clone N418, eBioscience, San Diego, CA, USA) and allophycocyanin/cyanine7 (APC-Cy7)-conjugated anti-CD45 (Clone 104, Biolegend, San Diego, CA, USA). Subsequently, alveolar macrophages (CD45+ F4/80+ CD11c+) were sorted using a FACS Aria II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The purity of the separated cells was > 95%.

mRNA sequencing

Total RNA was extracted from the purified alveolar macrophages (CD45+ F4/80+ CD11c+) using a miRNeasy Mini Kit (QIAGEN, GmBH, Germany). The mRNA sequencing was described in the supplemental materials and methods. Differentially expressed genes (DEGs) were analyzed by Gene Ontology (GO) and KEGG pathway analysis as described in the Supplemental Materials and Methods.

Single-cell RNA sequencing and analysis

Single-cell barcode scRNA-seq libraries were generated for purified alveolar macrophages (CD45+ F4/80+ CD11c+) using Chromium Single Cell 3′ Library (V2) (10 × Genomics, Pleasanton, CA, USA). A HiSeq X Ten system (Illumina) was used to sequence sc-RNA libraries. Data were mapped to the mouse genome mm10 using Cell Ranger 2.1.1 (10 × Genomics). For further analysis, raw data were converted to a Seurat object using Seurat R v2.3.4. [43] or a CellDataSet object using monocle R 2.10.0 [44].

Flow cytometry analysis

As previously described [45], for the surface phenotype assays, 1 × 106 cells were blocked with 10 μL rat serum for 30 min at 4 °C and then stained with the indicated antibody for 30 min at 4 °C in the dark. For the intracellular cytokine assay, the cells were stimulated with PMA (Sigma, St Louis, MO, USA), monensin (Sigma, St Louis, MO, USA) and ionomycin (Calbiochem, San Diego, CA, USA) for 4 h. The cells were labeled for surface markers, fixed, permeabilized, and then labeled with the indicated intracellular antibody for 30 min at 4 °C in the dark. All data were acquired using a FACS Aria II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo software version 10.0 (Treestar, Ashland, OR, USA). The monoclonal antibodies (mAb) used for FACS are shown in Supplemental Table 1.

Quantitative real-time polymerase chain reaction (PCR)

Total RNA was extracted from purified alveolar macrophages (CD45+ F4/80+ CD11c+) using a miRNeasy Mini Kit (QIAGEN, Duesseldorf, Germany). Total RNA was extracted from the lung tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The process was performed as described in the Supplemental Materials and Methods. Gene expression levels were quantified using the ΔΔCt method. Information on gene-specific primers is shown in Supplemental Table 2.

Stimulation of alveolar macrophages in vitro

Purified alveolar macrophages (1 × 105 cells/well) were stimulated with 300 ng/mL UGPR1 (LS-G56865-1, LifeSpan, Hamilton, OH, USA) in a total volume of 200 µL (DMEM supplemented with 10% fetal bovine serum) for 48 h. CCL6 in the culture supernatants was detected by an ELISA kit (EMCCL6, Thermo Scientific, Frederick, MD, USA). The anti-mMARCO antibody (Clone ED31, GeneTex, Alton Pkwy Irvine, CA, USA) was used to block the interaction at a concentration of 20 μg/mL in vitro. Immunoglobulin (Ig)G (clone HRPN, BioXcell, West Lebanon, NH, USA) was used as the control.

Histological examination

For histological examination, mouse lung samples or human lung samples from nonsmoking patients with bullous lung disease were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections of 4 μm thickness were stained with anti-mUGRP1 antibody (Clone 381,707, R&D, Abingdon, UK), anti-hUGRP1 antibody (Clone EPR11463, Abcam, Cambridge, UK), or anti-hMARCO antibody (NBP2-39,004, Novus Biologicals, Littleton, CO, USA) for IHC. The DAB Peroxidase Substrate Kit (PV-6000, ZSGB-BIOTECH Co., Ltd, Beijing, China). The sections were photographed using an Olympus IX73 microscope (Olympus, Tokyo, Japan). For immunofluescence analysis, sections of 4 μm thickness were stained with anti-mCCL6 antibody (Clone EPR23475-105, Abcam, Cambridge, UK), anti-mMARCO antibody (Clone EPR22944-64, Abcam, Cambridge, UK), and Four Color Multiplex Fluorescent Immunostaining Kit (Anti rabbit, abs50028, Absin, Shanghai, China) were used. The sections were photographed using Leica TCS SP5 confocal microscope (Leica, Wetzlar, Germany).

Western blotting

Western blotting was used to detect the protein expression levels of CCL6 and UGRP1 in the lung tissues of the aged mice compared with the young mice. The anti-CCL6 antibody (Clone 262,016, R&D, Abingdon, UK), anti-UGRP1 antibody (Clone 381,707, R&D, Abingdon, UK) and anti-β-actin antibody (Clone EPR21242, Abcam, Cambridge, UK) were used. The details were shown in the Supplemental Materials and Methods.

Mouse pulmonary fibrosis model

Bleomycin (BLM) (Nippon Kayaku Co., Ltd, Takasaki-shi, Japan) was used to induce pulmonary fibrosis in mice [46]. Histochemical analysis was performed by Masson Trichrome staining to indicate the fibrosis. Ashcroft scores were used to indicate the degree of fibrosis [47]. The hydroxyproline in lung tissue was detected by using hydroxyproline microplate assay kit (abs580066, Absin, Shanghai, China). The mRNA expression levels of Col1a1, Timp1 and α-SMA in lung tissue were detected by using real-time PCR. There were six mice in each group.

Antibody blockade and neutralization

The anti-mCCL6 mAb (Clone 262,016, R&D, Abingdon, UK) or anti-mMARCO mAb (Clone ED31, GeneTex, Alton Pkwy Irvine, CA, USA) was injected i.p. into the aged mice (100 μg/mouse in 100 μL of PBS) 7 days before bleomycin (Nippon Kayaku Co., Ltd, Takasaki-shi, Japan) challenge, and additional injections were performed every 7 days. Control mice were administrated equal amounts of control antibody Rat IgG2b (clone LTF-2; BioXcell, West Lebanon, NH, USA) or Rat IgG1 (clone HRPN, BioXcell, West Lebanon, NH, USA) respectively.

UGRP1 protein treatment

Recombinant murine UGPR1 (LS-G56865-1, LifeSpan, Hamilton, OH, USA) was injected i.p. into young mice (15 μg/mouse in 100 μL of PBS) 7 days before bleomycin (Nippon Kayaku Co., Ltd, Takasaki-shi, Japan) challenge, and additional injections were performed every 7 days. Control mice were administrated 100 μL PBS solution.

Depletion of alveolar macrophages

Clodronate liposomes (Liposoma, Amsterdam, NL) were administered intranasally (i.n.) into the recipient mouse (50 μL/mouse, once every 3 days for 28 days) to deplete alveolar macrophages in the bleomycin-treated mice, 7 days before bleomycin treatment. Control liposomes (Liposoma, Amsterdam, NL) were used for the control mice.

Statistical analysis

All data are shown as the mean ± standard error of the mean (SEM). Differences between individual data were analyzed using Student’s t-test, and two-way analysis of variance (ANOVA) when appropriate. Additional comparisons of proportions were made using the chi-squared test. Pearson’s test was performed for the correlation analysis. A p value < 0.05 was considered statistically significant.

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