Macropinocytosis inhibition attenuates pro-fibrotic responses in lung fibroblasts and pulmonary fibrosis models.

Figures

• 

  Figure 1. Macropinocytosis is elevated in IPF-derived lung fibroblasts and its inhibition regulates profibrotic responses. (A and B) Control and IPF-derived lung fibroblasts were conditioned in low-serum media for 24 hours, as described in Methods. 24 hours later, culture media was changed, and cells were incubated with FITC-dextran (70 kDa, 0.5 mg/mL) for 1 hour. Then, cells were washed 3 times with PBS. Macropinocytosis was measured by fluorescent microscopy (A) (scale bar: 25 μm) and flow cytometry (B) to measure fluorescence intensity of FITC-dextran in cells (n = 5 for A and n = 8 for B in each condition). (C and D) Cells were treated with vehicle (Veh) or EIPA (12.5 μM) for 24 hours. Then, cells were harvested and subjected to Western blot (C) and qRT-PCR (D) to measure collagen 1 and α-SMA expression, as described in Supplemental Methods and Supplemental Table 1 (n = 5 each condition). (E and F) Control HLFs were treated with EIPA (12.5 μM) with or without TGF-β1 (10 ng/mL) for 24 hours. Then, cells were harvested and subjected to Western blot (E) and qRT-PCR (F), as described in Methods (n = 4 each condition). (G) Control HLFs were mixed with collagen 1 solution, as described in…

• 

  Figure 2. Macropinocytosis inhibition ameliorates mTORC1 signaling by regulating AA uptake in lung fibroblasts. (A) Control HLFs were treated with EIPA (12.5 μM) with or without TGF-β1 (10 ng/mL) for 24 hours. Then, p-P70S6K and p-S6 were measured by Western blot, as described in Methods (n = 4 each condition). (B and C) Cells were treated with EIPA (12.5 μM) with or without TGF-β1 (10 ng/mL) for 4 hours in AA-free and AA-supplemented conditions, as described in Methods. Then, cells were harvested to measure p-S6 by Western blot (B) and L-AA assay (C), as described in Methods (n = 4 or 6). (D) Cells were lentivirally transfected with scramble (Scr) or shTSC1, as described in Methods. Then, cells were treated with EIPA (12.5 μM) with or without TGF-β1 (10 ng/mL) for 24 hours, and cells were harvested, and COL1, α-SMA, and p-S6 were measured by Western blot (n = 4 each condition). Data are shown as the mean ± SEM. *P < 0.05, vs. Veh or Veh + AA free; †P < 0.05, vs. TGF-β1 + Veh or Veh + AA; §P < 0.05, vs. TGF-β1 + Veh + AA; #P < 0.05, vs. Scr + TGF-β1 + EIPA; significant comparisons by 1-way ANOVA.

• 

  Figure 3. Macropinocytosis inhibition regulates MEOX1 expression in activated lung fibroblasts. (A) Volcano plot representing treatment of MRC5 HLFs with and without EIPA in the presence of TGFB. Overlaid in yellow is a set of TGFβ1-induced genes from a separate RNA sequencing experiment in MRC5 HLFs treated with TGF-β1 (10 ng/mL) or vehicle. MRC-5 cells were treated with EIPA (12.5 μM) with or without TGF-β1 (10 ng/mL) for 24 hours. Then, RNA was isolated and subjected to bulk RNA sequencing, as described in Methods (n = 3). (B and C) Control HLFs were treated with EIPA (12.5 μM) and Rapalink-1 (10 nM) with or without TGF-β1 (10 ng/mL) for 24 hours. Then, MEOX1 was measured at mRNA and protein levels by qRT-PCR (B) and Western blot (C), respectively (n = 4 each condition). (D) Control HLFs were lentivirally transfected with Scr or shTSC1, as described in Methods. Then, cells were treated with vehicle (Veh) or EIPA (12.5 μM) for 24 hours. Then, cells were harvested and subjected to Western blot to measure MEOX1 expression. (E) Control HLFs were lentivirally transfected with empty vector (EV) or pLenti-MEOX1-DDK, as described in Methods. Then, cells were treated with TGF-β1 (10 ng/mL) for…

• 

  Figure 4. Macropinocytosis inhibition attenuates pulmonary fibrosis in Bleo-injured mice. (A and B) Slc9a1fl/fl (WT) or Slc9a1fl/fl Col1a2Cre-ER(T)+/0 (Slc9a1 fCKO) mice were treated with tamoxifen to induce Slc9a1 knockout in fibroblasts, as described in Methods. Then, mice were exposed to Bleo-induced pulmonary fibrosis, as described in Methods. At day 21 after Bleo exposure, lungs were harvested, stained with Masson’s trichrome staining (scale bar: 1,000 μm) (A) and subjected to hyproxyproline assay (B) (n = 5 for sham groups, n = 10–11 for Bleo groups). (C–E) Mouse lung fibroblasts from WT and Slc9a1 fCKO mice were isolated, as described in Methods. Cells were stimulated with TGF-β1 (10 ng/mL) for 24 hours. Then, cells were subjected to Western blot or qRT-PCR, as described in Methods. (F and G) WT mice were exposed to Bleo to induce pulmonary fibrosis, as described in Methods. 10 days after Bleo exposure, mice were intraperitoneally treated with EIPA (10 mg/kg) or vehicle (Veh) every other day until the end of experiment. At day 21 after Bleo exposure, lungs were harvested, stained with Masson’s trichrome staining (n = 3–4) (scale bar: 1,000 μm). (F) and subjected to hyproxyproline…

• 

  Figure 5. EIPA inhibits profibrotic responses in IPF-derived PCLSs. IPF-derived PCLSs were treated with EIPA (12.5 μM), as described in Methods. (A) UMAP plot represents different mesenchymal cell populations identified in IPF PCLSs (n of 4 for DMSO and n of 3 for EIPA-treated PCLSs). (B) Volcano plot representing treatment of IPF PCLSs with and without EIPA, as described in A. Overlaid in yellow is a set of myofibroblast IPF-induced genes from a separate PCLS experiment comparing IPF + DMSO (n of 4) and control (n of 4) PCLS samples, which were reused from McKenna et al. (29). (C) Regulatory network plot for genes repressed in EIPA-treated IPF PCLSs compared with IPF alone. TFs with the largest and most significant footprints within the gene set are distributed toward the top right of the plot. (D) After treatment, PCLSs were lysed and subjected to total RNA isolation. mRNA levels of POSTN, IGFBP5, ACTA2, and COL1A1 were measured with qRT-PCR, as described in Methods (n = 5 for each condition). (E) After treatment, PCLSs were washed with PBS and fixed for COL1 staining, as described in Methods. The COL1 fluorescent intensity was measured with ImageJ software (NIH) (n = 5 for each…

• 

  Figure 6. Imipramine ameliorates profibrotic responses in lung fibroblasts, Bleo-injured mice, and PCLSs. (A) Control and IPF-derived lung fibroblasts were treated with imipramine (Imi, 10 μM). 24 hours later cells were harvested, and COL1 and α-SMA were measured with Western blot (n = 4 each condition). (B and C) Control HLFs were treated with Imi (10 μM) with or without TGF-β1 (10 ng/mL) for 24 hours. Then, cells were harvested and subjected to Western blot to measure COL1, α-SMA, p-S6, and MEOX1 (n = 4 for each condition). (D–F) WT mice were exposed to Bleo-induced pulmonary fibrosis, as described in Methods. 10 days after Bleo exposure, mice were intraperitoneally treated with Imi (10 mg/kg) or vehicle (Veh) every other day until the end of the experiment. At day 21 after Bleo exposure, lungs were harvested, stained with Masson’s trichrome staining (n = 3–4) (D), and subjected to hyproxyproline assay (n = 5 or 10) (E) or mRNA assessment (n = 5) (F). (G) IPF-derived PCLSs were treated with Imi (10 μM), as described in Methods. Then, cells were lysed and mRNA levels of POSTN, IGFBP5, COL1A1, and ACTA2 were measured by qRT-PCR (n = 5 each condition). *P < 0.05, vs. unstimulated or Control…