Senescent fibroblasts secrete LTs as part of their SASP. We first investigated whether a class of biologically active signaling profibrotic lipids, namely the LT family, are part of the SASP. We interrogated the status of cytosolic phospholipase A2 (cPLA2), the major enzyme that releases arachidonic acid from the plasma membrane (28). cPLA2 is activated by phosphorylation on serine 505 by p38MAPK (29), which is activated in senescent cells (30). As expected, irradiated lung fibroblasts confirmed to be senescent by p21WAF1 expression (Figure 1A) showed activation (phosphorylation) of p38MAPK and cPLA2 (Figure 1B). Because p38MAPK signaling often culminates in increased transcription, we used quantitative PCR (qPCR) to measure the abundance of mRNAs encoding genes that specifically participate in eicosanoid synthesis. Compared with quiescent cells, senescent cells expressed higher levels of several of these genes (Figure 1C), including those encoding LT synthases, such as ALOX5 (5-LO), ALOX12, ALOX15, ALOX5AP, LTC4S, LTA4H, and ALOX15B (Figure 1C). To confirm the activation of the biosynthesis of LT in the senescent fibroblasts, increased levels of ALOX5 synthesis and activation as showed by a significant increased phosphorylated ALOX5 (6.5-fold increased) were measured in senescent cells compared with quiescent cells (Figure 1D). In addition, a significant 3-fold increase of cysteinyl LT level was measured in conditioned medium (CM) from senescent fibroblasts and significantly inhibited in the presence of the ALOX5 inhibitors BW-B70C (BW) or zileuton (Zil) (Figure 1E). Additionally, in agreement with our RNA data, lipid extracts from senescent cells showed elevated levels of LTB4 (Figure 1F). A time course revealed the complex dynamics that govern the expression of eicosanoid synthesis genes during the senescence response, as exemplified by the time course expression of LT synthases (ALOX5 and LTA4H) and PG synthases (COX2 or PTGS2 and PTGES). The LT expression was biphasic, showing a large increase 2 days after irradiation, followed by a decline and a smaller peak of expression 10–20 days following irradiation (Figure 1G). Interestingly, in the late phase of senescence (between 15–20 days following irradiation), PG biosynthesis enzymes, including COX2 (PTGS2), were increased (Figure 1H).

Figure 1 Senescent cells synthesize eicosanoids in a time-dependent manner. 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.

LT expression is a widespread component of the SASP. To determine whether the cell origin or the mode of senescence induction affected the LT biosynthesis activation, we also measured cysteinyl LT levels in human umbilical vein cells (HUVEC) and human liver carcinoma cells (HEPG2). We cultured the cells under standard conditions specific to each cell lines. A 3-fold and 2-fold increase in LT expression was measured in irradiated HUVEC and HEPG2 cell lines, respectively (Figure 2A).

Figure 2 Leukotriene secretion is a common feature of senescent cells. (A) HUVEC and HEPG2 cells were cultured in DMEM + 0.2% serum (quiescence, QUI) or irradiated (10 Gy, SEN[IR]). The secretion of leukotrienes was assessed measuring the level of cysteinyl leukotriene by ELISA in the conditioned medium 10 days after irradiation. (B) Level of cysteinyl leukotriene from IMR-90 fibroblasts in which senescence was promoted by diverse inducers (MiDAS, mitochondrial dysfunction-associated senescence; irradiation, SEN[IR]; Ras oncogene-induced, SEN [Ras]) and compared with the level from QUI cells. (C–E) IMR-90 were treated with 50 μg/mL bleomycin for 3 hours. Total RNA was isolated, reverse transcribed, and analyzed by qPCR. Signal was normalized to tubulin mRNA. Activation of eicosanoid biosynthesis was assessed by measuring the level of expression of genes encoding leukotriene (C) and prostaglandin (D) biosynthesis enzymes. Senescence was assessed by measurement of increased expression p16INK4a and p21WAF1 mRNA level (E). Data are presented as mean ± SEM of at least 3 replicates. Statistical analyses were performed using Student’s t test (A, C, D, and E) or 1-way ANOVA (B); *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

We induced senescence in IMR-90 fibroblasts following different modalities: mitochondria dysfunctional associated senescence (31), exposing them to a relatively high dose (10 Gy) of ionizing radiation (γRA [γ-irradiation]), oncogene induced (Ras), and oxidative stress (bleomycin). Each of these modalities was able to promote the secretion of LTs either evaluated by the measurement of cysteinyl LT in the CM (Figure 2B) or the activation of the expression of enzymes involved in the biosynthesis of LT (Figure 2C). Interestingly, these senescent cells also expressed enzymes related to the PG pathway (Figure 2D). Bleomycin-induced senescence was confirmed by the increased expression of p16Ink4a and p21Waf1 mRNA (Figure 2E). Thus, the secretion of LT appears to be a feature of the SASP, independently of the cell type or the modality of senescence induction.

Senescent fibroblasts promote fibrosis by secreting LTs as part of their SASP. Since the enzymatic machinery leading to the biosynthesis of LTs and PGs is activated and cysteinyl LT is released in senescent fibroblasts, we sought to test whether the secreted LT and PGs had a functional effect on naive fibroblasts. We generated CM from control (day 0) or senescent cells, 2 days (2-day CM) or 20 days (20-day CM) after irradiation, to determine whether the secreted LTs and PGs from senescent cells could alter fibrotic responses. Prior to collecting the CM, the senescent cells were treated with NS-398 to inhibit PG biosynthesis or Zil to prevent LT biosynthesis. Naive nonsenescent IMR-90 fibroblasts were then treated with the CMs, and the activation of COL1A2 and αSMA gene expression was assessed as surrogates for the evaluation of fibrotic response (Figure 3A). The upregulation of COL1A2 mRNA level measured after treatment with 2-day CM was prevented with 2-day CM from the senescent cells treated with Zil (2-day + Zil CM). Further, upregulation of COL1A2 mRNA was lower using 20-day CM, consistent with declined expression of LT in the 20-day postirradiation SASP. Treatment of the naive fibroblasts with 2-day or 20-day CM + NS-398 (2-day + NS-398 or 20-day + NS-398 CM) did not affect the upregulation of COL1A2 mRNA expression (Figure 3B). Similarly, the expression of α–smooth muscle actin (αSMA, Acta2), a marker of myofibroblast differentiation, was upregulated in the presence of the 2-day CM and significantly reduced after treatment with 20-day CM. The downregulation of αSMA mRNA expression was abrogated if the naive fibroblasts were treated with 20-day + NS-398 CM, suggesting that the decline of αSMA expression was, in part, controlled by the released of PGs by the senescent cells (Figure 3C). However, αSMA mRNA expression was not affected by CMs + Zil, suggesting that the upregulation of αSMA mRNA was independent of LT expression. Of note, TGF-β mRNA levels were not upregulated in irradiated senescence cells, and treatment with LT inhibitors did not change its expression (see below). It is, therefore, unlikely that the profibrotic effects are related to the presence of TGF-β in the SASP.

Figure 3 Leukotriene released by senescent cells induces profibrotic responses from naive fibroblasts in vitro. (A) Irradiated human lung fibroblasts (IMR-90) were treated with the ALOX5 inhibitor zileuton (Zi, 50 μM) or the PTGS inhibitor NS-398 (1 μg/mL) for 24 hours prior to the conditioned medium (CM) collection. CM was collected 2 and 20 days after radiation and applied to nonsenescent IMR-90 fibroblasts for 48 hours. (B and C) Total RNA was isolated, reverse transcribed, and analyzed by quantitative PCR. Signal was normalized to tubulin mRNA. The profibrotic responses were assessed by the expression of COL1A2 (B) and α-SMA mRNA (C). Data are presented as mean ± SEM of at least 3 replicates. Statistical analyses were performed using 1-way ANOVA. Asterisk represents the statistical differences calculated by time point. Statistical differences calculated within the same time point group (2 or 20 days); *P ≤ 0.05; **P ≤ 0.01.

Since TGF-β is the master regulator of fibrosis, we also treated the naive fibroblasts with TGF-β and the CMs. The level of COL1A2 mRNA expression was comparable when the naive fibroblasts were costimulated with 2-day CM or with 2-day CM + TGF-β, and inhibition of LT synthesis by Zil prevented the collagen expression induction using 2-day CM + TGF-β + Zil (Supplemental Figure 1B; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.130056DS1). The presence of TGF-β was required to maintain collagen expression when treated with 20-day CM (Supplemental Figure 1B). Treatment with TGF-β alone did not affect αSMA mRNA expression. Twenty-day CM was less effective at inducing αSMA mRNA expression than 2-day CM, but treating senescent cells with 20-day CM + 2-day CM + TGF-β + NS-398 prevented the downregulation of αSMA mRNA expression (Supplemental Figure 1C), consistent again with findings that PGs antagonize myofibroblast activation (32, 33).

Because LTs trigger cellular signaling through membrane-bound GPCRs, we then profiled known LT receptors in naive quiescent fibroblasts to determine which type was the most likely to trigger the profibrotic responses in naive fibroblasts. We first detected the mRNA expression of several LT receptors (Supplemental Figure 1A). Among all the ones we tested, the LT B4 receptors B1 and B2 (BLT1, BLT2), the GPCRs 99 and 17 (GPR99, GPR17), the adenosine diphosphate–reactive (ADP-reactive) purinergic (P2Y 12 ) receptor, and the cysteinyl LT receptors 1 and 2 (CYSLTR1, CYSLTR2) were expressed. This suggests that the naive fibroblasts were capable of responding to multiple members of the LT family. To investigate the requirement of any of the expressed receptors in mediating profibrotic effects induced by the SASP LTs, we used pharmacological antagonist LT receptors LY255283 (a selective, competitive BLT 2 receptor antagonist) and montelukast sodium (CysLT1 and GPR17 antagonist). We generated CM from control (day 0) or senescent cells, 10 days after irradiation, and treated naive fibroblasts for 48 hours with CM and LT receptor antagonists (Figure 4A). The activation of COL1A2 gene expression was assessed (Figure 4B). Treatment with vehicles alone didn’t affect the level of expression of COL1A2 gene, as expected. Decreased COL1A2 gene expression was only measured when the cells were treated with 10-day CM and montelukast sodium, in agreement with reports that montelukast antagonizes bleomycin-induced lung fibrosis in mice (34).

Figure 4 Pharmacological inhibition of the cysteinyl leukotrienes reduces the profibrotic effect of senescent cells on naive fibroblasts in vitro. (A) Schematic representation of conditioned media (CM) studies. The CM of senescent human lung fibroblasts (IMR-90) was collected 10 days after irradiation (10 Gy irradiation), LT antagonists LY255283 (LY, 50 nM), montelukast sodium (ML, 25 nM), or vehicle were added to the CM and applied to nonsenescent IMR-90 fibroblasts for 48 hours. Total RNA was isolated, reverse transcribed, and analyzed by quantitative PCR. Signal was normalized to β-actin mRNA. The inhibitory effect of LT antagonists was assessed by the expression of COL1A2 mRNA. (B) Relative levels of COL1A2 mRNA in cells treated with CM supplemented with either LY (left) or ML (right). Data are presented as mean ± SEM of at least 4 replicates. Statistical analyses were performed using unpaired Student’s t test; *P ≤ 0.05.

To assess whether senescence-associated LTs influence the expression of other SASP factors, senescent fibroblasts were continually treated with the ALOX5 inhibitors Zil or BW. After 10 days, as expected, the level of expression of proinflammatory factors known to be part of the SASP (IL-8, CXCL1, IL-6, IL-1b, IL-1a, CCL2, and VEGF) were upregulated in irradiated senescent cells compared with controls. Treatment with the ALOX5 inhibitors did not affect or moderately decreased the expression level of these genes, suggesting that the LTs secreted by senescent cells have little to no effect on the expression of other proinflammatory factors of the SASP (Figure 5A). Similarly, induction of profibrotic factors such as SERPINE-1, TIMP1, COL1A2, and FIBRONECTIN (FN1) mRNA were unchanged by the presence LT inhibitors (Figure 5B). Furthermore, mRNA expression of receptors for LTB4 (LTBR2) and cysteinyl LTs (CYSLTR2) was lost from senescent fibroblasts (Figure 5C), suggesting that LTs produced by senescent cells are most likely to have paracrine effects rather than autocrine ones.

Figure 5 Inhibition of ALOX5 activity modulates the expression of proinflammatory SASP factors. Irradiated human lung fibroblasts (IMR-90) were treated with the ALOX5 inhibitor BW-B70C (BW, 10 μM) for 10 days. Total RNA was isolated, reverse transcribed, and analyzed by quantitative PCR. Signal was normalized to tubulin mRNA. (A and B) Heatmap representation of the level of expression mRNA levels of proinflammatory SASP components (A) and the level of expression mRNA levels of profibrotic SASP components (B). (C) Decreased mRNA expression of genes encoding LT receptors in senescent cells 10 days after irradiation. Data are presented as mean ± SEM of at least 3 replicates. Statistical analyses of A and B were performed using 2-way ANOVA, and significance was determined for C by 2-tailed Student’s t tests with Welch’s correction. **P ≤ 0.01; ****P ≤ 0.0001.

We also investigated whether senescence-associated LTs promoted senescence in naive fibroblasts by assessing p16 mRNA expression in these cells after 48 hours of treatment with CM collected from 10-day postirradiated fibroblasts (Supplemental Figure 1C). No change in expression was measured; therefore, our data would not support a paracrine effect for LT in the induction of senescence.

Taken together, these data support a model in which early LT synthesis by senescent cells stimulates collagen synthesis and fibrosis, but not senescence, by a paracrine mechanism, whereas the later PG synthesis by senescent cells antagonizes myofibroblast differentiation and collagen expression. In addition, among the senescence-associated LTs, our data indicate that naive fibroblasts could respond to any member of the LT family based on the expression of various receptors. However, based on treatment with antagonists, it is more likely that the cysteinyl LTC4 and LTD4 contribute to the profibrotic effect rather than the noncysteinyl LTB4.

Senescent cells release LTs in the lungs and contribute to fibrosis. To determine whether senescence-associated LT and PG biosynthesis occurs in vivo, and to be consistent with our in vitro data using lung fibroblasts, we asked whether senescent cells were responsible for LT-driven disorders in the lungs. LTs were previously shown to significantly contribute to fibrotic responses in the lungs (21, 35–37). Using the classical bleomycin-induced fibrosis model, WT C57BL/6J mice were subsequently treated with vehicle or ABT-263, a BCL2/BCL-XL/BCL-W inhibitor that selectively eliminates senescent cells in some tissues (38, 39). p16Ink4a and p21Waf1 RNA levels increased 14 days after bleomycin injury and were significantly reduced by ABT-263 (Figure 6A).

Figure 6 Senescence-associated leukotriene synthesis promotes pulmonary fibrosis. WT C57BL/6J mice received a single intratracheal injection of PBS (vehicle control) or bleomycin (Bleo, 1.9 U/kg). Mice received, from days 7–14,vehicle (Veh) or 50 mg/kg/day ABT-263 by gavage. (A) Level of p16INK4a and p21WAF1 mRNA levels normalized to tubulin mRNA. (B) Activation of cytosolic phospholipase A2 (cPLA2) was determined by calculating the ratio of the level of expression of phosphorylated cPLA2 to the level of expression of total cPLA2. Representative pictures of Western blot of lung lysates collected day 14 after the bleomycin injury for the expression of the phosphorylated form of cPLA2, cPLA2-S505P, and nonphosphorylated form cPLA2. n = 3 lysates per group. (C) Level of expression of mRNA of gene encoding enzymes of the leukotriene (ALOX5 and LTC4S) and the prostaglandins (PTGDS, PTGS2, and PTGES) biosynthesis pathways was measured by qPCR normalized to tubulin mRNA, in samples collected 14 days after bleomycin injury. Data are presented as dot plot graphs or heatmap. (D) Lipids were extracted from broncho-alveolar lavage fluid (BALF) collected 14 days after bleomycin injury and treatment with vehicle or ABT-263. BALF lipid content was analyzed for cysteinyl leukotrienes and for PGE2 by ELISA. (E) Representative pictures of Picrosirius red staining of lungs collected 21 days after bleomycin injury and treatment with vehicle or ABT-263. Original magnification, ×100. (F) Hydroxyproline levels obtained using the right lung lobes of mice, 21 days after bleomycin injury and treatment with vehicle or ABT-263. Unless stated otherwise, lung and BALF from at least 5 PBS-treated mice, 5 bleomycin + vehicle-treated mice, and 3 bleomycin + ABT-263 mice were analyzed. Statistical analyses were performed using 1-way ANOVA test. *P ≤ 0.05; **P ≤ 0.01.

To assess whether the eicosanoid biosynthesis was affected by the senolytic treatment with ABT-263, we first measured the activation of cPLA2 by determining the level of expression of its phosphorylated form by Western blot. We measured a significant 1.5-fold expression increased in the bleomycin-injured mice that was reduced by ABT-263 (Figure 6B). Subsequently, the level of mRNA expression of enzymes, ALOX5, LTC4S, PTGS2, PTGDS, and PTGES, involved in eicosanoid biosynthesis was also measured. Their respective mRNA levels were increased in the lungs of bleomycin-injured mice, and elimination of senescent cells with ABT-263 lowered these levels to those of control animals (Figure 6C). Consequently, bleomycin increased the levels of both cysteinyl LTs and PG E2 in the bronchoalveolar lavage fluid (BALF) 21 days after injury, and ABT-263 reduced both levels (Figure 6D). The removal of senescent cells was also significantly associated with an attenuation of collagen content assessed by histological staining with Picrosirius red (Figure 6E) and measurement of hydroxyproline (Figure 6F) 21 days after injury. Furthermore, ABT-263 treatment attenuated the increase in collagen mRNA (Col3a1 and Col4a1, Supplemental Figure 2).

To confirm that the attenuation of cysteinyl LT levels in BAL after ABT-263 treatment was the result of deletion of p16Ink4a+ cells, we undertook 2 separated approaches. First, as one of the major cell populations secreting LTs, CD45+ cells were isolated from bleomycin-injured lungs 14 days after injection and were treated with ABT-263 for 48 hours. Our data indicate that the level of expression of LT or PG biosynthesis enzymes in CD45+ cells was not directly affected by ABT-263 treatment (Figure 7A). In addition, the percentage of CD45+ cells in the lungs 14 days after bleomycin injury were not affected by ABT-263 (Figure 7B). Second, p16Ink4a-3MR mice were injured with bleomycin and then treated with ganciclovir (GCV). p16Ink4a-3MR mice contain a transgene that permits the detection and selective killing of p16Ink4a+ senescent cells by administering GCV, an otherwise innocuous drug (10). Our data indicate that deletion of p16Ink4a+ cells resulted in reduced collagen deposition, as well as reduced level of cysteinyl LT in the BAL of the GCV-treated injured p16Ink4a-3MR to a similar level as the level after ABT-263 treatment (Figure 7, C–E).

Figure 7 Temporal changes in eicosanoid biosynthesis reveal pro- and antifibrotic activities of senescent cells. (A) Isolated CD45+ cells from 14-day bleomycin-injured lungs were treated with DMSO and 10 μM ABT-263 for 48 hours. Analysis of mRNA expression of LT and PG biosynthesis enzymes was performed by qPCR. (B) Analysis by flow cytometry of percent of CD45+ cells isolated from 14-day bleomycin-injured lungs treated ABT-263 or vehicle for 7 days. (C and D) p16Ink4a-3MR mice received a single intratracheal injection of PBS (vehicle control) or bleomycin (Bleo, 1.9U/kg). (C) Level of p16INK4a mRNA levels normalized to tubulin mRNA. (D) Hydroxyproline levels obtained using the right lung lobes of mice, 21 days after bleomycin injury. (E) Lipids were extracted from broncho-alveolar lavage fluid (BALF) collected 21 days after bleomycin injury. BALF lipid content was analyzed for cysteinyl leukotrienes by ELISA. (E) Whole lung tissues were collected 14, 21, and 42 days after PBS or bleomycin intratracheal injection for RNA extraction (7 mice per group) and analyzed by qPCR. (F) mRNA levels of p16INK4a (p16, green line) and collagen (COL1A2, orange line), normalized to tubulin mRNA. (G) mRNA levels of ALOX5 (gray line) and PTGDS (green line) normalized to tubulin mRNA. Statistical analyses were performed using Student’s t test (A), 1-way ANOVA (C, D, and E), or individual 2-tailed unpaired Student’s t test. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.001.

These data indicate that removal of senescent cells is associated with both reduced eicosanoid biosynthesis and collagen deposition, consistent with the hypothesis that the senescent cells are an underlying driver of bleomycin-induced pulmonary fibrosis due to the secretion of LTs as part of their SASP or by inducing LT expression in other cells via a paracrine effect.

An interesting feature of the bleomycin-induced pulmonary fibrosis mouse model is that it resolves over time (40, 41), suggesting that either senescent cells are eventually cleared or the fibrosis-promoting properties of senescent cells change over time. We therefore analyzed p16Ink4a and collagen (COL1A2) mRNA levels over a 42-day period following bleomycin-induced injury. Notably, although p16Ink4a expression increased over 14 days after injury, peaked 30 days after injury, and remained elevated through day 42 (Figure 7F, green line), collagen expression peaked at 14 days after injury but, thereafter, progressively declined (Figure 7F, orange line). These data suggest that the fibrosis-inducing properties of senescent cells change over time. Because we observed an early rise in LT synthases expression (Figure 1G), followed by a later rise in PG synthases expression (Figure 1H) in cultured cells, we tested whether a similar pattern developed in vivo. Indeed, bleomycin injury caused an early spike in whole lung ALOX5 expression (Figure 7G), followed by a progressive rise in the expression of PTGDS, which synthesizes PGD2 (Figure 7G), consistent with a switch in senescent cell phenotype from profibrotic to antifibrotic.

Senescent IPF fibroblasts synthesize LTs but fail to produce PGs. Previous reports indicate that fibroblasts from IPF patients fail to elevate PG synthesis (42) or fail to respond to PG treatment (43). To determine whether senescent IPF fibroblasts synthesize LTs and PGs in a similar fashion to senescent normal lung fibroblasts, we compared mRNA levels of ALOX5, PTGS2, PTGDS, and PTGES in control and radiation-induced senescent fibroblasts isolated from normal and IPF lungs. As expected at baseline, IPF lung fibroblasts expressed high levels of ALOX5 mRNA compared with normal lung fibroblasts. However, even though ALOX5 mRNA expression increased upon irradiation-induced senescence in both cell types, IPF fibroblasts still expressed significantly higher levels of ALOX5 mRNA (Figure 8A). Compared with nonirradiated cells, senescent normal lung fibroblasts increased expression of mRNA of the PG synthases PTGS2 and PTGD. This induction of PG biosynthesis enzyme expression is also measured in senescent IPF fibroblasts but to a much lesser extend (Figure 8A). These data suggest that IPF patients respond differently to senescence-inducing stimuli with regard to PG synthesis. Senescent cells, as defined as p16Ink4a+cells, ALOX5+ cells, and dual-positive cells, are detected in IPF lung in greater numbers than in normal lungs (Figure 8, B and C). Overall, the p16Ink4a+ cells represented an average 30% of total cells in the IPF lungs, of which 50% expressed ALOX5 (Figure 8C). Because several cell types, mainly type II epithelial cells and fibroblasts, have been previously reported to undergo senescence in IPF lungs (44), we costained ALOX5+ cells with marker for type II epithelial cells (surfactant protein C) and for fibroblasts (vimentin). Very few ALOX5+ cells, less than 3%, were detected in the SPC+ population (Figure 8, D and E). However, 23.3% ± 5.7% of ALOX5+ cells also costained for vimentin (Figure 8, D and E). Despite this staining, we did not detect the expression of ALOX5 in myofibroblast cells, as defined as α-SMA+ (data not shown), indicating that the producers of LTs are a distinct population of lung fibroblasts. Together, our experiments point to senescent lung fibroblasts as a key source of profibrotic LTs during lung fibrosis.