Differential effects of Nintedanib and Pirfenidone on lung alveolar epithelial cell function in ex vivo murine and human lung tissue cultures of pulmonary fibrosis

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease. Repetitive injury and reprogramming of the lung epithelium are thought to be critical drivers of disease progression, contributing to fibroblast activation, extracellular matrix remodeling, and subsequently loss of lung architecture and function. To date, Pirfenidone and Nintedanib are the only approved drugs known to decelerate disease progression, however, if and how these drugs affect lung epithelial cell function, remains largely unexplored.

Methods: We treated murine and human 3D ex vivo lung tissue cultures (3D-LTCs; generated from precision cut lung slices (PCLS)) as well as primary murine alveolar epithelial type II (pmATII) cells with Pirfenidone or Nintedanib. Murine 3D-LTCs or pmATII cells were derived from the bleomycin model of fibrosis. Early fibrotic changes were induced in human 3D-LTCs by a mixture of profibrotic factors. Epithelial and mesenchymal cell function was determined by qPCR, Western blotting, Immunofluorescent staining, and ELISA.

Results: Low μM concentrations of Nintedanib (1 μM) and mM concentrations of Pirfenidone (2.5 mM) reduced fibrotic gene expression including Collagen 1a1 and Fibronectin in murine and human 3D-LTCs as well as pmATII cells. Notably, Nintedanib stabilized expression of distal lung epithelial cell markers, especially Surfactant Protein C in pmATII cells as well as in murine and human 3D-LTCs.

Conclusions: Pirfenidone and Nintedanib exhibit distinct effects on murine and human epithelial cells, which might contribute to their anti-fibrotic action. Human 3D-LTCs represent a valuable tool to assess anti-fibrotic mechanisms of potential drugs for the treatment of IPF patients.

Keywords: ATII; Epithelial cells; IPF; Lung disease; Nintedanib; PCLS; Pirfenidone; ex vivo.

Conflict of interest statement

Ethics approval

Human tissue

Human tissue has been obtained from the Comprehensive Pneumology Center cohort of the BioArchive CPC-M at the University Hospital Grosshadern of the Ludwig Maximilian University (Munich, Germany) and by the Asklepios Biobank of Lung Diseases (Gauting, Germany). Participants provided written informed consent to participate in this study, in accordance with approval by the local ethics committee of the LMU, Germany (Project 333-10, 455-12).

Animal experiment

All mouse experiments were performed in accordance with the guidelines of the ethics committee of the Helmholtz Zentrum Munich (Germany) and approved by the regional council of Upper Bavaria Germany (Project 55.2-1-54-2532-88-12).

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

Effect of ex vivo treatment with Pirfenidone and Nintedanib on the fibrotic phenotype of 3D-LTCs. a Representative immunofluorescence analysis of Collagen I, α-SMA and E-Cadherin in control (PBS) and fibrotic (Bleo) 3D-LTCs after 48 h in culture. Scale bar represents 50 μm. b Gene expression analysis by qPCR of fibrotic marker Fn1 and Col1a1 in control and fibrotic 3D-LTCs after 48 h in culture. ΔCp relative to Hprt is presented as mean ± SEM, n = 7. Means were compared using Wilcoxon matched pairs test. c Collagen I secretion of control and fibrotic 3D-LTCs was determined by WB and normalized to supernatant volume. n = 6. Means were compared using Mann-Whitney test. d WISP1 secretion of control and fibrotic 3D-LTCs was measured by ELISA. n = 7. Significance was assessed using Wilcoxon matched pairs test. e, f Fibrotic 3D-LTCs were cultured for 48 h in the presence of anti-fibrotic drugs (e) Nintedanib (0.1 μM, 1 μM, 10 μM) (f) and Pirfenidone (100 μM, 500 μM, 2.5 mM). Gene expression analysis by qPCR of fibrotic marker Fn1 and Col1a1. Log fold change is presented as mean ± SEM, n = 5–7. Means were compared to respective DMSO control using one-sample t-tests in comparison to a hypothetical value of 0. g Collagen I secretion of fibrotic 3D-LTCs treated with Nintedanib (1 μM) and Pirfenidone (500 μM) for 48 h was determined by WB and normalized to supernatant volume. n = 5. Significance was assessed using Wilcoxon matched pairs test. Significance: *p < 0.05, **p < 0.01

Fig. 2

Effect of ex vivo treatment with Pirfenidone and Nintedanib on lung epithelial cell marker in fibrotic 3D-LTCs. a-c Fibrotic 3D-LTCs were cultured for 48 h in the presence of anti-fibrotic drugs Nintedanib (1 μM) and Pirfenidone (500 μM). a proSP-C expression was assessed by Western blot. β-Actin was used as loading control. Quantification of proSP-C Western blot, n = 6. Data was normalized to β-Actin. b, c SP-C and WISP1 secretion of 3D-LTCs was determined by ELISA. n = 4–7. Significance was assessed using Wilcoxon matched pairs test. Significance: *p < 0.05

Fig. 3

Effect of in vitro treatment with Pirfenidone and Nintedanib on primary mouse (pm)ATII cells. a, b At day 14 after Bleomycin instillation, mice were sacrificed and control (PBS) and fibrotic (Bleo) pmATII cells were harvested. The pmATII cells were cultured in the presence of Nintedanib (1 μM) and Pirfenidone (500 μM) for 48 h. a Gene expression analysis by qPCR of fibrotic marker Fn1 in pmATII cells. ΔCp relative to Hprt is presented as mean ± SEM, n = 3. Means were compared using repeated-measures one-way ANOVA followed by Newmann-Keuls post test. b Gene expression analysis by qPCR of epithelial cell markers Sftpc, Nkx2.1, T1α, Hopx. ΔCp relative to Hprt is presented as mean ± SEM, n = 3. Means were compared using repeated-measures one-way ANOVA followed by Newmann-Keuls post test. Significance: *p < 0.05, **p < 0.01, ***p < 0.001 (DMSO vs Pirfenidone/Nintedanib). Significance: #p < 0.05, ##p < 0.01, ###p < 0.001 (PBS vs Bleo)

Fig. 4

Ex vivo treatment with Nintedanib stimulates alveolar epithelial marker expression in the human 3D-LTCs model of early pulmonary fibrosis. a Schematic of treatment with fibrotic cocktail (FC) or control cocktail (CC) and Nintedanib (Nint) and downstream analysis. 3D-LTCs were generated and treated with FC or CC for 48 h before FC or CC treatment was replenished and Nintedanib or control treatment was added. Treatment was stopped after 120 h and downstream experiments were performed. b Representative Immunofluorescence of punches treated with CC or FC for 120 h and stained for Fibronectin. Scale bars represent 1 mm. c-f Punches were treated with CC/FC and Nintedanib (1 μM) as indicated in (a). c Metabolic activity of punches 120 h after treatment with CC/FC and co-treatment with Nintedanib. N = 3. Significance was assessed by two-way ANOVA followed by Sidak’s multiple comparisons test. d Gene expression analysis by qPCR of epithelial cell marker SFTPC, NKX2.1, CDH-1, ZO-1. Log fold change is presented as mean ± SEM, N = 3. Means were compared to respective DMSO control using one-sample t-tests in comparison to a hypothetical value of 0. e Representative Immunofluorescence of punches treated with FC and Nintedanib for proSP-C. Scale bars represent 140um. f SP-C secretion of punches 120 h after treatment with CC/FC and co-treatment with Nintedanib was measured by ELISA. Values shown are normalized to CC treatment. Significance was assessed using Wilcoxon matched pairs test. N = 6. Significance: *p < 0.05