intermediate cells  (Alomone Labs)

Journal: Nature Communications

Article Title: Autocrine TGF-β-positive feedback in profibrotic AT2-lineage cells plays a crucial role in non-inflammatory lung fibrogenesis

doi: 10.1038/s41467-023-40617-y

Figure Lengend Snippet: a Serial assessment of BLM-induced lung fibrosis model in vivo using micro-CT ( upper ), Sirius Red (SR) staining ( middle ; scale bar: 1 mm), and DsRed reporter ( lower ; scale bar: 200 μm). Triangles on micro-CT images indicate fibrosis areas. Right graphs are the quantifications. CT score in each timing is derived from three independent mice. Other data represent the mean ± SEM of results obtained from three independent mice in each timing. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to day 0. b Lung sections on days 0 and 2 stained for SFTPC, γH2AX (scale bar: 100 μm). Triangles indicate γH2AX, SFTPC-double positive cells. c The number of total cells in BALF ( upper ) and percentages of CD45 + cells in whole lung lysates ( lower ). Data represent the mean ± SEM of results obtained from three independent mice. ** P < 0.01 compared to day 0. d Summary scheme of BLM-induced lung fibrosis model in vivo. e Diagram of ex vivo lung-lobe culture and sections of the cultured lung tissue stained for SFTPC, γH2AX, and DsRed fluorescence (scale bar: 100 μm). Arrows indicate γH2AX-positive AT2 cells. f qRT-PCR of ex vivo cultured lungs. Data represent the mean ± SEM of the results obtained from three independent mice. ** P < 0.01. g Evaluation of CD45 + cells by FACS for ex vivo cultured lung on day 12. Data represent the mean ± SEM of the results obtained from three independent mice. *** P < 0.001 compared to day 0. h Diagram of alveolar organoid (AO) culture and in vitro BLM treatment, images of untreated AOs in bright-field and GFP fluorescence on day 8 (scale bar: 1 mm), and sections stained for SFTPC and RAGE (scale bar: 20 μm). i Sections of BLM-treated AOs stained for γH2AX (scale bar: 20 μm). j Western blotting of CTRL- and BLM-AOs for γH2AX and GAPDH and the quantification. The lanes were run on the same gel. Data represent the mean ± SEM of results obtained from three independent mice. All statistical analyses are evaluated by unpaired two-tailed Student’s t -test or one-way analysis of variance with Bonferroni correction, as appropriate.

Article Snippet: In human lung fibrosis and in vitro culture of human AT2 cells, alveolar-basal intermediates (ABI) (hereafter ABI cells) have been reported as a functionally homologous cell type of PATS-like cells owing to their transcriptome similarity , – .

Techniques: In Vivo, Micro-CT, Staining, Derivative Assay, Ex Vivo, Cell Culture, Fluorescence, Quantitative RT-PCR, In Vitro, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Autocrine TGF-β-positive feedback in profibrotic AT2-lineage cells plays a crucial role in non-inflammatory lung fibrogenesis

doi: 10.1038/s41467-023-40617-y

Figure Lengend Snippet: a Heatmap visualization of mRNA expression of SASP factors evaluated by qRT-PCR using normal AOs from Sftpc CreERT2 ; Rosa26 mTmG mice and p53-cKO AOs from Sftpc CreERT2 ; Trp53 flox/flox ; Rosa26 mTmG mice. AOs were treated with 100 μM BLM for 24 h, and harvested on culture day 9. Data were obtained from three independent mice. * P < 0.05, ** P < 0.01, *** P < 0.001 compared between BLM-treated normal AOs and BLM-treated p53-cKO AOs (unpaired, two-tailed Student’s t test). b Representative images of lung sections for in situ hybridization and quantification using Sftpc CreERT2 ; Rosa26 mTmG mice on days 0 and 7 after BLM injection in vivo. Yellow triangles indicate mRNA-dot-positive GFP + AT2-lineage cells. Data represent the mean ± SEM of results obtained from three independent mice. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired, two-tailed Student’s t test). (scale bar: 100 μm). c FACS panels with quantification of mean fluorescence intensity (MFI) for integrin αVβ6 using GFP + AT2-lineage cells (days 0 and 7, in vivo). Data represent the mean ± SEM of results obtained from three independent mice. *** P < 0.001 compared to day 0 (unpaired, two-tailed Student’s t test). d Experimental set-up, representative images of co-cultured AOs and myofibroblasts (scale bar: 1 mm), and quantification of the relative DsRed + area around the AO-containing gel using normal AOs treated with BLM (100 μM, 24 h) and subsequently with a neutralizing antibody against integrin αVβ6 (100 μg mL −1 , 72 h). Data represent the mean ± SEM of results obtained from three independent mice. * P < 0.05 (unpaired, two-tailed Student’s t test). e Diagram of the experiments and comparison of mRNA expression levels by qRT-PCR for primary lung fibroblasts. Lung fibroblasts were isolated from wild-type mice ( upper panel ) or tamoxifen-treated transgenic mice ( lower panel ): Pdgfra CreERT2 ; Tgfbr2 flox/flox or Pdgfra CreERT2 ; Tgfbr2 flox/+ . These fibroblasts were treated with culture supernatants from normal AOs that were pre-treated with BLM (100 μM, 24 h) and then cultured for the next 72 h. Data represent the mean ± SEM of results obtained from three independent mice. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni correction).

Article Snippet: In human lung fibrosis and in vitro culture of human AT2 cells, alveolar-basal intermediates (ABI) (hereafter ABI cells) have been reported as a functionally homologous cell type of PATS-like cells owing to their transcriptome similarity , – .

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, In Situ Hybridization, Injection, In Vivo, Fluorescence, Cell Culture, Comparison, Isolation, Transgenic Assay

Journal: Nature Communications

Article Title: Autocrine TGF-β-positive feedback in profibrotic AT2-lineage cells plays a crucial role in non-inflammatory lung fibrogenesis

doi: 10.1038/s41467-023-40617-y

Figure Lengend Snippet: a Representative images of immunostaining (scale bar: 100 μm) and quantification. Lung sections from BLM-treated Sftpc CreERT2 ; Rosa26 mTmG mice were stained with anti-GFP, anti-phosphorylated SMAD (p-SMAD) 2&3, and anti-αSMA antibodies. Triangles indicate p-SMAD2&3 + GFP + AT2-lineage cells. Data represent the mean ± SEM of results obtained from three independent mice. ** P < 0.01 compared to day 0 (unpaired, two-tailed Student’s t test). b Experimental set-up, representative images of protein expression of AOs via western blotting for total/phosphorylated SMAD2/3, and quantification. The samples were derived from the same experiment and gels/blots were processed in parallel. Data represent the mean ± SEM of results obtained from three independent mice. * P < 0.05 (unpaired, two-tailed Student’s t test). The lanes were run on the same gel. c Representative images of the lung sections for SR staining (scale bar: 1 mm), lung mCT, and lung sections stained with anti-GFP and anti-αSMA antibodies (scale bar: 100 μm). The Sftpc CreERT2 ; Rosa26 mTmG mice (CTRL) and Sftpc CreERT2 ; Tgfbr2 flox/flox ; Rosa26 mTmG mice (TR2-cKO) were treated with BLM in vivo. d Quantification of SR staining (day 21, n = 6), mCT (day 21, n = 6), αSMA + area (day 7, n = 3), and hydroxyproline in the left lungs (day 21, n = 6). Data represent the mean ± SEM of results obtained from three or six independent mice. * P < 0.05, ** P < 0.01 (unpaired, two-tailed Student’s t-test). e Representative images of co-cultured (myo)fibroblasts using TR2-cKO AO treated with BLM (100 μM, 24 h) (scale bar: 1 mm) and quantification of the relative DsRed + area around the AO-containing gel. Data represent the mean ± SEM of results obtained from three independent mice. * P < 0.05 (one-way analysis of variance with Bonferroni correction). f Representative images of co-cultured (myo)fibroblasts using normal AO treated with mixed TGF-β1/β2/β3 (5 pg mL −1 each, 24 h) (scale bar: 1 mm) and quantification of the relative DsRed + area around the empty or AO-containing gel. Data represent the mean ± SEM of results obtained from three independent mice. ** P < 0.01 (one-way analysis of variance with Bonferroni correction).

Article Snippet: In human lung fibrosis and in vitro culture of human AT2 cells, alveolar-basal intermediates (ABI) (hereafter ABI cells) have been reported as a functionally homologous cell type of PATS-like cells owing to their transcriptome similarity , – .

Techniques: Immunostaining, Staining, Two Tailed Test, Expressing, Western Blot, Derivative Assay, In Vivo, Cell Culture

Journal: Nature Communications

Article Title: Autocrine TGF-β-positive feedback in profibrotic AT2-lineage cells plays a crucial role in non-inflammatory lung fibrogenesis

doi: 10.1038/s41467-023-40617-y

Figure Lengend Snippet: a Heatmap visualization of mRNA expression levels (for PATS-related and profibrotic genes) was performed by qRT-PCR using integrin αVβ6-positive or -negative GFP + AT2-lineage cells isolated from BLM-treated Sftpc CreERT2 ; Rosa26 mTmG mouse lungs (day 7, in vivo). Data were obtained from three independent mice. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired, two-tailed Student’s t test). b Heatmap visualization of mRNA expression levels (for PATS-related and profibrotic genes) was performed by qRT-PCR using normal AOs (from Sftpc CreERT2 ; Rosa26 mTmG mice), p53-cKO AOs (from Sftpc CreERT2 ; Trp53 flox/flox ; Rosa26 mTmG mice), and TR2-cKO AOs (from Sftpc CreERT2 ; Tgfbr2 flox/flox ; Rosa26 mTmG mice). AOs were treated with BLM (100 μM, 24 h), and sampling was performed on culture day 9. Data were obtained from three independent mice. * P < 0.05, ** P < 0.01, *** P < 0.001 compared between untreated CTRL normal AOs and BLM-treated normal AOs (unpaired, two-tailed Student’s t test). c Representative images of protein expression of TR2-cKO AOs treated with BLM (100 μM, 24 h) via western blotting for γH2AX, total p53, acetylated p53 (Lys379), p21, and GAPDH. The lanes were run in the same gel but were noncontiguous. The samples were derived from the same experiment and gels/blots were processed in parallel. The lower graph is the quantification. Data represent the mean ± SEM of results obtained from three independent mice analyzed using unpaired two-tailed Student’s t test (compared to each control). d Representative images of immunostaining (scale bar: 100 μm) and quantification. Lung sections from BLM-treated Sftpc CreERT2 ; Rosa26 mTmG mice (CTRL) and Sftpc CreERT2 ; Tgfbr2 flox/flox ; Rosa26 mTmG mice (TR2-cKO) were stained with anti-GFP and anti-p21 antibodies, respectively. Yellow triangles indicate p21 + GFP + AT2-lineage cells. Data represent the mean ± SEM of results obtained from three independent mice analyzed using unpaired two-tailed Student’s t test. e Comparison of mRNA expression levels of TGF-β-related genes evaluated by qRT-PCR using p53-cKO AOs (from Sftpc CreERT2 ; Trp53 flox/flox ; Rosa26 mTmG mice). AOs were treated with mixed TGF-β1/β2/β3 (5 ng·mL −1 each, 24 h), and sampling was performed on culture days 7 and 9. Data represent the mean ± SEM of results obtained from three independent mice. ** P < 0.01, *** P < 0.001 compared between untreated CTRL-AOs and TGF-β-treated AOs in each time point (unpaired, two-tailed Student’s t test).

Article Snippet: In human lung fibrosis and in vitro culture of human AT2 cells, alveolar-basal intermediates (ABI) (hereafter ABI cells) have been reported as a functionally homologous cell type of PATS-like cells owing to their transcriptome similarity , – .

Techniques: Expressing, Quantitative RT-PCR, Isolation, In Vivo, Two Tailed Test, Sampling, Western Blot, Derivative Assay, Control, Immunostaining, Staining, Comparison

Journal: Nature Communications

Article Title: Autocrine TGF-β-positive feedback in profibrotic AT2-lineage cells plays a crucial role in non-inflammatory lung fibrogenesis

doi: 10.1038/s41467-023-40617-y

Figure Lengend Snippet: a Diagram of AO culture for the lung fibrosis model using human AT2 cells and primary lung fibroblasts from Acta2-DsRed mice. Human lung samples were collected from three subjects. b Representative images of sections of hAOs treated with BLM (100 μM, 24 h) on day 11 of culture stained with anti-SFTPC, anti-H2AX, anti-p53, and anti-p21 antibodies, and DAPI (scale bar: 20 μm). For p53 and p21 expressions, an identical sphere is stained. c Representative images of co-cultured (myo)fibroblasts treated with BLM (100 μM, 24 h) (scale bar: 1 mm) and quantification of the relative DsRed + area around the hAO-containing gel. Data represent the mean ± SEM of results obtained from three subjects. * P < 0.05 (unpaired, two-tailed Student’s t-test). d Comparison of mRNA expression levels evaluated by qRT-PCR in BLM-treated hAOs. Data represent the mean ± SEM of results obtained from three subjects. * P < 0.05, ** P < 0.01 (unpaired, two-tailed Student’s t test). e Diagram of AO culture for the lung fibrosis model using human AT2 cells and primary lung fibroblasts from Acta2-DsRed mice. The AOs were treated with a mixture of TGF-β1/β2/β3 (5 ng mL −1 each, 24 h). f Representative images of co-cultured (myo)fibroblasts using hAOs treated with mixed TGF-β1/β2/β3 (scale bar: 1 mm) and quantification of the relative DsRed + area around the hAO-containing gel. Data represent the mean ± SEM of results obtained from three subjects. ** P < 0.01 (unpaired, two-tailed Student’s t test). g Comparison of mRNA expression levels evaluated by qRT-PCR in TGF-β-treated hAOs. Data represent the mean ± SEM of the results obtained from three subjects. * P < 0.05, ** P < 0.01 (unpaired, two-tailed Student’s t test). h Reanalysis of single-cell RNA-seq data from lung samples isolated from patients with idiopathic pulmonary fibrosis. The interaction of TGF-β signaling between each cell type, including autocrine, was estimated using the CellChat algorithm.

Article Snippet: In human lung fibrosis and in vitro culture of human AT2 cells, alveolar-basal intermediates (ABI) (hereafter ABI cells) have been reported as a functionally homologous cell type of PATS-like cells owing to their transcriptome similarity , – .

Techniques: Staining, Cell Culture, Two Tailed Test, Comparison, Expressing, Quantitative RT-PCR, RNA Sequencing, Isolation

Journal: Nature Communications

Article Title: Autocrine TGF-β-positive feedback in profibrotic AT2-lineage cells plays a crucial role in non-inflammatory lung fibrogenesis

doi: 10.1038/s41467-023-40617-y

Figure Lengend Snippet: a DNA damage by BLM activates p53 signaling in AT2 cells to upregulate TGF-β-related profibrotic gene expression, which initiates a positive-feedback loop for TGF-β signaling within AT2 cells. This process directly induces fibroblast-to-myofibroblast differentiation even without immune cells. b Cascade schema in each setting (genotype/stimulation): wild-type/BLM; p53-null/BLM; Tgfbr2-null/BLM; p53-null/recombinant TGF-β.

Article Snippet: In human lung fibrosis and in vitro culture of human AT2 cells, alveolar-basal intermediates (ABI) (hereafter ABI cells) have been reported as a functionally homologous cell type of PATS-like cells owing to their transcriptome similarity , – .

Techniques: Gene Expression, Recombinant