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Secretion of leukotrienes by senescent lung fibroblasts promotes pulmonary fibrosis
Christopher D. Wiley, … , Judith Campisi, Claude Jourdan Le Saux
Christopher D. Wiley, … , Judith Campisi, Claude Jourdan Le Saux
Published November 5, 2019
Citation Information: JCI Insight. 2019;4(24):e130056. https://doi.org/10.1172/jci.insight.130056.
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Research Article Cell biology Pulmonology

Secretion of leukotrienes by senescent lung fibroblasts promotes pulmonary fibrosis

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Abstract

Accumulation of senescent cells is associated with the progression of pulmonary fibrosis, but mechanisms accounting for this linkage are not well understood. To explore this issue, we investigated whether a class of biologically active profibrotic lipids, the leukotrienes (LT), is part of the senescence-associated secretory phenotype. The analysis of conditioned medium (CM), lipid extracts, and gene expression of LT biosynthesis enzymes revealed that senescent cells secreted LT, regardless of the origin of the cells or the modality of senescence induction. The synthesis of LT was biphasic and followed by antifibrotic prostaglandin (PG) secretion. The LT-rich CM of senescent lung fibroblasts (IMR-90) induced profibrotic signaling in naive fibroblasts, which were abrogated by inhibitors of ALOX5, the principal enzyme in LT biosynthesis. The bleomycin-induced expression of genes encoding LT and PG synthases, level of cysteinyl LT in the bronchoalveolar lavage, and overall fibrosis were reduced upon senescent cell removal either in a genetic mouse model or after senolytic treatment. Quantification of ALOX5+ cells in lung explants obtained from idiopathic pulmonary fibrosis (IPF) patients indicated that half of these cells were also senescent (p16Ink4a+). Unlike human fibroblasts from unused donor lungs made senescent by irradiation, senescent IPF fibroblasts secreted LTs but failed to synthesize PGs. This study demonstrates for the first time to our knowledge that senescent cells secrete functional LTs, significantly contributing to the LT pool known to cause or exacerbate IPF.

Authors

Christopher D. Wiley, Alexis N. Brumwell, Sonnet S. Davis, Julia R. Jackson, Alexis Valdovinos, Cheresa Calhoun, Fatouma Alimirah, Carlos A. Castellanos, Richard Ruan, Ying Wei, Harold A. Chapman, Arvind Ramanathan, Judith Campisi, Claude Jourdan Le Saux

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Figure 1

Senescent cells synthesize eicosanoids in a time-dependent manner.

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Senescent cells synthesize eicosanoids in a time-dependent manner.
Senes...
Senescence was induced in human lung fibroblasts (IMR-90) using irradiation (10 Gy). Total RNA was isolated from mock irradiated, quiescent (QUI, cell cultured in 0.2%serum/DMEM), and irradiated cells after 10 days of culture, reverse transcribed, and analyzed by quantitative PCR. Signal was normalized to tubulin mRNA. (A) Increased p21WAF1 mRNA level confirmed the induction of senescence in irradiated cells compared with mock irradiated ones. (B) Proteins were extracted from QUI and irradiated senescent (SEN[IR]) cells and analyzed by Western blot for cPLA2 (phosphorylated on serine 505 or total cPLA2), p38MAPK (phosphorylated on threonine 180 or total), and tubulin (control). (C) Panel of expression of genes encoding leukotriene synthesis enzymes. (D) Lysates from QUI and 10-days postirradiation senescent IMR-90 fibroblasts were blotted for ALOX5 (total and phosphorylated on serine 271). Quantification of Western blot bands were first normalized to β-actin, and activation of ALOX5 is reported as the ratio p-ALOX5/ALOX5. (E) After ionizing radiation, fibroblasts were treated with DMSO (vehicle) or the ALOX5 inhibitors zileuton (Zil, 50 μM) or BW-B70C (BW, 10 μM) for 10 consecutive days, and conditioned medium (CM) was collected. Levels of cysteinyl leukotriene secreted in CM was measured by ELISA. (F) Intracellular level of leukotriene B4 measured by ELISA. (G) Time course expression of ALOX5 and LTA4H mRNA. (H) Time course expression of PTGS2 or COX2 and PTGES mRNA. Data are presented as mean ± SEM of at least 3 replicates. Statistical analyses were performed using Student’s t test (A, C, and D), 1-way ANOVA (D), or individual 2-tailed unpaired Student’s t test (E and F). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

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