Figures & data
Figure 1. Expression of alveolar epithelial cell type 2 markers in iAT2 cells derived from human iPSC. Each specific protein was detected by western blotting. Normal human lung tissue was used as the positive control (Lane 1). iAT2 cells expressed both surfactant proteins B and C, confirming differentiation (Lane 2). LPCAT1, Lysophosphatidylcholine Acyltransferase 1; HTII-280, 280–300 kDa protein specific to apical surface of AT2 cells; SP-B, Surfactant Protein B; SP-C, Surfactant Protein C.
Figure 2. iAT2 cell single cell RNA sequencing. (a) Single cell RNA-seq revealed multiple clusters within the iAT2 cell population. (b) Volcano plot showing the six most prominently down (left) and up (right) regulated genes after cytokine exposure. As examples, Cellular communication network factor 1, SRY-box transcription factor 4, Early growth response 1, KLF transcription factor 6, Fibronectin 1 and Claudin 18 were markedly over expressed. Cell adhesion molecule 6, S100 calcium binding protein P, Ferritin heavy chain 1, Solute carrier family 34 member 2, Chitinase 3 like 2 and Cystatin B were markedly under expressed.
Figure 3. iAT2 cell single cell RNA sequencing. Heat map showing genes highly expressed in clusters. Surfactant proteins B and C were highly expressed in cluster 2, as was HOPX, consistent with the cells’ alveolar epithelial differentiation. Differential gene expression due to cytokines is shown qualitatively by the distribution of up and down regulated genes on the heat maps and by the prominently expressed genes listed in the text columns to the left of each heat map.
Figure 4. Expression of GHRH-R in lung tissues and iAT2 cells. Western blotting was done to confirm protein expression of the GHRH receptor in lung tissues and iAT2 cells. The full-length pituitary type GHRH-R (pGHRH-R) was predominantly expressed in normal lungs, while the truncated splice variant 1 (SV1) was expressed strongly in lungs from patients with IPF, as well as in cultured iAT2 cells, normal lung fibroblasts and fibroblasts isolated from patients with IPF.
Figure 5. Effect of GHRH and peptide analogs on proliferation of iAT2 cells. (a) GHRH and its agonist, MR-409, stimulated proliferation of iAT2 cells in CK + DCI medium, while GHRH antagonist MIA-602 significantly decreased proliferation of iAT2 cells, demonstrating a trophic role for the GHRH-R in iAT2 cells. (b) Growth inhibition, induced by cytokines (CK), was partially reversed by native GHRH peptide or its agonist, MR-409. These results indicate that GHRH-R activation protects iAT2 cells from growth inhibition by cytokines. (Data are means ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001 and ns, not significantly different; one-way ANOVA and Bonferroni’s correction.).
Figure 6. iAT2 cells express pro-inflammatory genes after incubation with LPS. iAT2 cells were incubated with LPS (500 ng/ml) alone or LPS plus 1 or 5 µM MIA-602 for 48 h. Quantitative RT-PCR was used to analyze the expression of IL-1β, TNF-α and TGF-β1 in the iAT2 cells. Compared to non-treated cells, iAT2 cells treated with LPS showed significantly enhanced expression of IL-1β and TNF-α (but not TGF-β1). Incubation with GHRH-R antagonist, MIA-602, at the same time significantly counteracted the effect of LPS. (Data are shown as medians with the upper/lower quartiles in the Box and Whisker plots, LPS vs LPS + MIA-602 ***, p < 0.001, ****, p < 0.0001; ns, not significantly different; One-way ANOVA and Bonferroni’s correction.).
Figure 7. iAT2 cells express mesenchymal genes after incubation with LPS. iAT2 cells were incubated with LPS (500 ng/mL) alone or LPS plus 1 or 5 µM MIA-602 for 48 h. RNA was isolated and used for RT-PCR to detect expression of ACTA2, CTGF, SERPINE1, FN1, COL1A1, TNC, WNT5A and MMP 7 genes by the iAT2 cells. In each case, LPS significantly enhanced expression of genes typical of mesenchymal differentiation, and GHRH-R antagonist, MIA-602, suppressed the LPS-induced mesenchymal gene expression. (Data are shown as medians with the upper/lower quartiles in the Box and Whisker plots, LPS vs LPS + MIA-602 ***, p < 0.001, ****, p < 0.0001; One-way ANOVA and Bonferroni’s correction.).
Figure 8. Cytokines stimulate iAT2 cell expression of pro-inflammatory mediators. iAT2 cells were incubated with cytokines for 48 h. Quantitative RT-PCR revealed significant increases in genes related to extracellular matrix (ECM) deposition and myofibroblast activation. The increase of pro-inflammatory cytokine expression suggested positive feedback, stimulating an inflammatory response by iAT2 cells. (Data are means ± SEM; ****, p < 0.0001; Unpaired student t-test.).
Figure 9. iAT2 cells treated with cytokines expressed mesenchymal proteins. iAT2 cells were treated with cytokine cocktail every other day for 2 wk. The loss of AT2 marker SP-B and expression of cytokeratin 5, N-cadherin, and vimentin were consistent with transdifferentiation toward a basal cell or myofibroblast phenotype and expression of mesenchymal markers. The band intensities, after being normalized to GAPDH, indicated a 6.9-fold decrease in SP-B and 1.2-fold increase in N-cadherin. The increases in cytokeratin 5 and vimentin were not calculated because they are barely detectable in the cells not treated with cytokines.
