Fig 2: Mesenchymal Ghr-deficient mice are more susceptible to pulmonary fibrosis.(A) A schematic diagram depicting the administration of bleomycin into the lungs of Ghr?BMC 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 Ghr?BMC and littermate control mice. (C) Hydroxyproline content in the lungs of Ghr?BMC and littermate control mice harvested before and 21 days after belomycin injection (Ctrl, n = 3; Ghr?BMC, n = 3; Ctrl + Bleo, n = 19; Ghr?BMC + Bleo, n = 15). (D) Cxcl1, Cxcl2, Cxcl5, Cxcl10, Cxcl11, and Cxcl12 protein secretion in BALF of Ghr?BMC 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 Ghr?BMC and littermate control mouse lungs 21 days after bleomycin injury (Ctrl, n = 19; Ghr?BMC, 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 3: 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 Ghr?BMC 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 Ghr?BMC mouse lungs (Ctrl, n = 18; Ghr?BMC + Ctrl vesicle, n = 16; Ghr?BMC + Ghr vesicle, n = 18). (F) Cxcl1, Cxcl2, Cxcl5, Cxcl10, Cxcl11, Cxcl12, and Cxcl16 protein secretion in BALF of Ghr?BMC 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 Ghr?BMC 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.

Fig 4: 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 5: 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.