Research ArticleCELL BIOLOGY

Mesenchymal growth hormone receptor deficiency leads to failure of alveolar progenitor cell function and severe pulmonary fibrosis

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Science Advances  09 Jun 2021:
Vol. 7, no. 24, eabg6005
DOI: 10.1126/sciadv.abg6005
  • Fig. 1 Decreased expression of GHR in fibrotic human lungs.

    (A) GHR gene expression in lung tissues of healthy and individuals with ILD or IPF (Ctrl, n = 136; ILD, n = 255; IPF, n = 68) in LGRC cohort. (B and C) Representative images of immunofluorescence (B) and quantitative analysis (C) for the protein levels of GHR in lung tissues of healthy and IPF individuals. (D and E) RNAscope analysis (D) and quantitative analysis (E) for the RNA levels of GHR in lung tissues of healthy and IPF individuals. (F) Representative confocal microscopy GHR immunostaining for IPF lung tissues shows decreased GHR protein expression in severe fibrotic area of IPF lung tissues. (G) Quantification of Ghr protein expression with DNA stain [4′,6-diamidino-2-phenylindole (DAPI), blue] from staining in (F). (H) Pearson correlation coefficients of FEV1 and FVC before bronchodilator (bd) and DLCO as a function of GHR gene expression in the lungs of patients with IPF. R and P values, as well as the numbers of patients analyzed, are indicated in the figures. Scale bars, 100 μm (D), 200 μm (B), and 500 μm (F). ****P < 0.0001 by unpaired two-tailed Student’s t test, Bars represent means ± SEM.

  • Fig. 2 Most of the mesenchymal cells, and very few ATIIs, are GHR positive in normal lung but not in IPF fibrotic area.

    (A) Representative confocal microscopy images of GHR and COL1A1 coimmunostaining for healthy and IPF human lung tissues. (B) Quantification of GHR and COL1A1 coexpression by colocalization calculated from staining in (A). (C) GHR and HTII-280 coimmunostaining reveals that a small portion of HTII-280+ATII cells are GHR positive in normal human lungs and no GHR+HTII-280+ cells in fibrotic human lungs. (D) Quantification of GHR and HTII-280 coexpression by flow cytometry or by colocalization calculated from staining in (C). (E) Rare CD31+GHR+ endothelial cells were found in normal lungs but not IPF lungs. (F) Quantification of GHR and CD31 coexpression by colocalization calculated from staining in (E). Arrows show the coexpressing cells. Scale bars, 100 μm (A to C, lower power images) and 10 μm (A to C, higher power images). Bars represent means ± SEM, n = 7 biological replicates. ****P < 0.0001 by unpaired two-tailed Student’s t test.

  • Fig. 3 Mouse lung mesenchymal expression of Ghr.

    (A) Single-cell transcriptomic sequencing of wild-type (WT) mouse lung total single cells. Ghr gene expression is mainly in Col1a1+ cell cluster. (B) CAMC (mesenchymal cells defined by Col1a1 and Acta2) were reclustered and plotted for Ghr and Col1a1 coexpression. Around 88.35% of the total Ghr+ cells are Col1a1+. (C) Ghr expression in ATII cells by single-cell RNA-seq analysis. About 6.59% Sftpc+ ATII cells are Ghr+. (D) ATII expression of Ghr and Sftpc. (E) Top up-regulated genes for Ghr-positive mesenchymal cells compared with Ghr-negative mesenchymal cells. (F) Fluorescence-activated cell sorting (FACS) analysis of Ghr on cell surface of cultured passage 3 primary lung mesenchymal cells (Epcam+Cd31/45+ cells were excluded) from normal and bleomycin-treated mice (7, 14, and 21 days after bleomycin administration).

  • Fig. 4 Ghr-deficient mesenchymal cells are less supportive for epithelial progenitor cell colony formation.

    (A) Schematic of in vitro coculture colony formation assay for experiments in (B) to (D). (B) Scgb1a1-GFP+ (green fluorescent protein–positive) Club cell and Sftpc-GFP+ ATII organoids cocultured with mesenchymal cells isolated from Ghr−/− or control mouse lungs. The number and size of Scgb1a1-GFP+ Club cell and Sftpc-GFP+ ATII organoids were decreased when cocultured with Ghr−/− mesenchymal cells. (C) Expression of Scgb1a1, Sftpc, Pdpn, and Aqp5 in Scgb1a1+ Club cell organoids cocultured with lung mesenchymal cells isolated from Ghr−/− and control mice. (D) Expression of Sftpc, Pdpn, and Cdkn1a in Sftpc+ ATII cell organoids cocultured with lung mesenchymal cells isolated from Ghr−/− and control mice. (E) Pegvisomant (PEG) treatment for mesenchymal cells cocultured with ATII colonies. (F) FACS confirmation of Ghr expression on lung mesenchymal cells from Ghr−/− and control mice. (G) Ghr, Ghr TV1, Ghr TV2, Tbx4, Cemip, and Tgfbr3 expression in lung mesenchymal cells from Ghr−/− and control mice. (H) Has1, Has2, and Has3 expression in lung mesenchymal cells from Ghr−/− and control mice. (I) Chemokine gene (Cxcl1, Cxcl4, Cxcl5, Cxcl12, Cxcl14, and Cxcl16) expression in Ctrl and Ghr−/− mouse lung mesenchymal cells. (J) Il-6 gene expression in lung mesenchymal cells from Ghr−/− and control mice. Scale bars, 1 mm (B). n = 3, *P < 0.05, **P < 0.005, ***P < 0.001, and ****P < 0.0001 by unpaired two-tailed Student’s t test [B (panels 3 and 4) and J], one-way analysis of variance (ANOVA) (E), and two-way ANOVA [B (panel 2), C, D, G, and H], means ± SEM.

  • Fig. 5 GHR-enriched human lung mesenchymal cells promote ATII organoids associated with elevated CXC chemokine and cytokine expression.

    (A) Expression of GHR gene in GHR overexpressing (GHRTg) and control (Ctrl) human lung mesenchymal cells (n = 3 in each group). (B) Organoid culture of GhrLo and GhrHi ATIIs with Ctrl and GHRTg hLF (human lung fibroblasts). (C) CXC chemokine gene (CXCL1, CXCL2, CXCL3, CXCL4, CXCL6, CXCL8, and CXCL12) expression in GHRTg and Ctrl human lung mesenchymal cells (n = 3 in each group). (D) WNT5A gene expression in GHRTg and Ctrl human lung mesenchymal cells. (E) Bio-Plex Pro Human Chemokine analysis of secreted CXCL1, CXCL2, CXCL8, CXCL10, and CXCL12 levels in supernatants of cultured GHRTg and Ctrl human lung mesenchymal cells (Ctrl, n = 15; GHRTg, n = 13). (F) Enzyme-linked immunosorbent assay analysis of IGF-1 protein secretion in the supernatants of GHRTg and Ctrl human lung mesenchymal cells upon TNF-α and IL-6 stimulation (n = 4 in each group). (G) IL-6 cytokine secretion in supernatants of cultured GHRTg and Ctrl human lung mesenchymal cells (Ctrl, n = 15; GHRTg, n = 13). (H) CXCL1, CXCL2, CXCL3, CXCL8, CXCL16, and IL-6 gene expression in lung tissues of ILD (n = 255) and Ctrl (n = 136) individuals in the LGRC cohort. *P < 0.05, **P < 0.005, ***P < 0.001, and ****P < 0.0001 by unpaired two-tailed Student’s t test (A, D, E, G, and H) and two-way ANOVA (B and F), means ± SEM. ns, not significant.

  • Fig. 6 Mesenchymal Ghr-deficient mice are more susceptible to pulmonary fibrosis.

    (A) A schematic diagram depicting the administration of bleomycin into the lungs of GhrΔBMC and littermate control mice. Mouse lungs were harvested on day 21. (B) Quantitative RT-PCR analysis of Ghr mRNA expression (means ± SEM) in the lungs of GhrΔBMC and littermate control mice. (C) Hydroxyproline content in the lungs of GhrΔBMC and littermate control mice harvested before and 21 days after belomycin injection (Ctrl, n = 3; GhrΔBMC, n = 3; Ctrl + Bleo, n = 19; GhrΔBMC + Bleo, n = 15). (D) Cxcl1, Cxcl2, Cxcl5, Cxcl10, Cxcl11, and Cxcl12 protein secretion in BALF of GhrΔBMC and littermate control mice harvested on day 21 after bleomycin injury. (E and F) Representative images of hematoxylin and eosin (H&E) staining (E) and Masson’s trichrome staining (F) in GhrΔBMC and littermate control mouse lungs 21 days after bleomycin injury (Ctrl, n = 19; GhrΔBMC, n = 15). Ctrl, control. Scale bars, 10 μm (E and F, higher power images) and 100 μm (E and F, lower power images). *P < 0.05, ***P < 0.001, and ****P < 0.0001 by unpaired two-tailed Student’s t test (B) and two-way ANOVA (C), means ± SEM.

  • Fig. 7 Fibrotic lung mesenchymal cells retard epithelial regeneration and express less GHR.

    (A) Schematic of in vitro coculture human ATII colony formation assay for experiments in (B) and (C). (B) Representative images of ATII organoids cocultured with lung mesenchymal cells from healthy and IPF individuals. (C) Bar graph depicting the organoid-forming efficiency and colony area in (B). (D) GHR mRNA expression in secreted vesicles from cultured lung mesenchymal cells of healthy and IPF individuals (n = 7 in each group). (E) Schematic of in vitro cocultured mouse ATII colony formation assay for experiments in (F) to (H). (F) Representative images of ATII organoids cocultured with mesenchymal cells isolated from normal and bleomycin-treated mice (21 days after treatment), as well as the MLg mouse fibroblast cell line. (G) Organoid-forming efficiency of ATII cocultured with normal, bleo d21, and MLg mesenchymal cells. (H) Colony size of ATII cocultured with normal, bleo d21, and MLg mesenchymal cells. (I) Vesicular expression of Ghr from normal and bleomycin-treated day 21 mouse lung mesenchymal cells cultured in vitro. Scale bars, 1 mm (B and F). *P < 0.05 and ****P < 0.0001 by unpaired two-tailed Student’s t test (C, D, and I) and one-way ANOVA (G and H), means ± SEM.

  • Fig. 8 Ghr vesicles reduce pulmonary fibrosis in mesenchymal Ghr-deficient mice.

    (A) Immunostaining of red fluorescent protein, Scgb1a1, Sftpc, and DAPI in mouse lungs administrated with mCherry sequence containing Ghr vesicles. Yellow solid line, mCherry and Scgb1a1 or Sftpc colocalized cells; white dotted line, Scgb1a1- or Sftpc-negative cells. (B) Ghr mRNA expression in ATII cells sorted from mouse lungs treated with Ghr or control vesicles. (C) Ghr protein expression in ATII cells sorted from mouse lungs treated with Ghr or control vesicles. (D) A schematic diagram depicting the administration of Ghr or control vesicles into the lungs of GhrΔBMC mice after bleomycin treatment. Mouse lungs were harvested on day 21. (E) Significantly decreased hydroxyproline levels were detected in Ghr vesicles and control vesicle–administered GhrΔBMC mouse lungs (Ctrl, n = 18; GhrΔBMC + Ctrl vesicle, n = 16; GhrΔBMC + Ghr vesicle, n = 18). (F) Cxcl1, Cxcl2, Cxcl5, Cxcl10, Cxcl11, Cxcl12, and Cxcl16 protein secretion in BALF of GhrΔBMC mice with Ghr or control vesicle treatments. (G and H) Representative images of H&E staining (G) and trichrome staining (H) in Ghr or control vesicle–treated GhrΔBMC mouse lung 21 days after bleomycin injury. (I) A diagram illustrating how subepithelial mesenchymal cells support ATII cells through Ghr vesicle and Ghr-coordinated CXC chemokines in normal and fibrotic conditions in the lung. Scale bars, 10 μm (A), 100 μm (G and H, higher power), and 1 mm (G and H, lower power). *P < 0.05, **P < 0.005, ***P < 0.001, and ****P < 0.0001 by unpaired two-tailed Student’s t test (B and C) and one-way ANOVA (E), means ± SEM.

Supplementary Materials

  • Supplementary Materials

    Mesenchymal growth hormone receptor deficiency leads to failure of alveolar progenitor cell function and severe pulmonary fibrosis

    Ting Xie, Vrishika Kulur, Ningshan Liu, Nan Deng, Yizhou Wang, Simon Rowan, Changfu Yao, Guanling Huang, Xue Liu, Forough Taghavifar, Jiurong Liang, Cory Hogaboam, Barry Stripp, Peter Chen, Dianhua Jiang, Paul W. Noble

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