Leukotriene B4 (LTB4) and its functional receptor BLT1 are closely involved in tissue inflammation by primarily mediating leukocyte recruitment and activation. Elevated LTB4 was reported in patients with lung fibrosis; however, the role of the LTB4/BLT1 axis in lung fibrosis remains unknown. In this study, we demonstrated that BLT1−/− mice exhibited significantly attenuated bleomycin (BLM)-induced lung fibrosis. Interestingly, BLT1 blockade with its specific antagonist U75302 in the acute injury phase (days 0–10 after BLM treatment) significantly attenuated lung fibrosis, which was accompanied by significant decreases in early infiltrating neutrophils and later infiltrating CD4+ T cells and the production of TGF-β, IL-13, and IL-17A. In contrast, BLT1 blockade in the fibrotic phase (days 10–21 after BLM treatment) had no effect on lung fibrosis and TGF-β production, although it significantly decreased CD4+ T cell infiltration. Furthermore, depletion of neutrophils or CD4+ T cells had no effect on BLM-induced lung fibrosis, suggesting the independence of profibrotic activity of the LTB4/BLT1 axis on BLT1-dependent lung recruitment of these two leukocytes. Finally, although BLT1 blockade had no effect on the recruitment and phenotype of macrophages in BLM-induced lung fibrosis, the LTB4/BLT1 axis could promote TGF-β production by macrophages stimulated with BLM or supernatants from BLM-exposed airway epithelial cells in an autocrine manner, which further induced collagen secretion by lung fibroblasts. Collectively, our study demonstrates that the LTB4/BLT1 axis plays a critical role in acute injury phase to promote BLM-induced lung fibrosis, and it suggests that early interruption of the LTB4/BLT1 axis in some inflammatory diseases could prevent the later development of tissue fibrosis.

Lung fibrosis is the endpoint of a wide range of respiratory diseases. Environmental irritants, drugs, and chronic lung infection contribute to the progression of lung fibrosis, characterized by the accumulation of myofibroblasts and the excess deposition of collagen and other extracellular matrix proteins (1). Patients that develop the various forms of pulmonary fibrosis are refractory to treatment and have a high level of mortality (2). Idiopathic pulmonary fibrosis (IPF) is the most severe form of lung fibrosis with a median survival of ∼3 y after diagnosis (2). Thus, better understanding of the molecular and cellular mechanisms of lung fibrosis is needed to develop new therapeutic strategies.

Leukotrienes (LTs) that are generated from eicosanoid metabolites of arachidonic acid through 5-lipoxygenase (5-LO) pathways have been implicated in lung fibrosis (3). LTs fall into two classes, the cysteinyl LTs (cysLTs: LTC4, LTD4, and LTE4) and LTB4, generated by the critical enzymes LTC4 synthase and LTA4 hydrolase (LTA4H), respectively (4). Exaggerated levels of LTs have been reported in bronchoalveolar lavage fluid (BALF) or lungs of mouse models of lung fibrosis and patients with IPF (57). Mice deficient in 5-LO or LTC4 synthase had less severe lung fibrosis following bleomycin (BLM) treatment (5, 8). Administration of inhibitors of 5-LO or cysLT receptor 1 provided protection against BLM-induced lung fibrosis (9). These studies underscore the importance of cysLTs in the development of lung fibrosis. However, despite higher increased LTB4 levels than cysLTs observed in animal or patients with lung fibrosis (7, 10), little is known about whether LTB4 and its receptor play a causal role in the development of lung fibrosis.

LTB4 is generated from innate immune cells in response to a variety of stimuli (4), and it plays most of its functions by interacting with its high-affinity receptor BLT1 that is predominantly expressed on various immune cells, including neutrophils, macrophages, eosinophils, and effector T cells (11). The LTB4/BLT1 axis has been well documented to actively participate in a variety of inflammatory diseases by recruiting and activating various inflammatory cells (1214). For example, BLT1-dependent neutrophil recruitment is important for the development of collagen-induced arthritis and BLT1-dependent CD4+ T cell recruitment in allergic asthma (15, 16). Our previous studies demonstrated that BLT1-mediated early recruitment of neutrophils, which act as a primary source of LTB4, is essential for skin infiltration of allergic-specific T cells, contributing to allergic skin inflammation (17, 18). Accumulating evidence suggests that lung fibrosis is often linked to a strong inflammatory response, at least in the early phase (19). During the progression of lung fibrosis, different types of immune cells could be found in the foci, including neutrophils and T cells (20, 21), both of which are implicated in the development of lung fibrosis. BALF neutrophils were suggested to be an important predictor of early mortality in IPF patients (22). A recent study demonstrated that neutrophil elastase could promote myofibroblast differentiation in asbestos-induced lung fibrosis (23). IL-13 and IL-17A are two profibrotic cytokines derived from Th2 and Th17 cells, respectively, that have been suggested to be the important drivers of tissue fibrosis (24, 25). However, it is unclear whether the LTB4/BLT1 axis could contribute to the development of lung fibrosis through its ability to mediate the recruitment or activation of inflammatory immune cells.

In this study, we used BLT1−/− mice or BLT1 antagonist to investigate the role of the LTB4/BLT1 axis in a mouse model of BLM-induced lung fibrosis. We found that BLT1−/− mice or early BLT1 blockade attenuated lung fibrosis accompanied by decreased lung infiltration of neutrophils and CD4+ T cells; however, depletion of neutrophils or CD4+ T cells had no effect on lung fibrosis. We further demonstrated that the LTB4/BLT1 axis could promote TGF-β secretion by macrophages in an autocrine way, which promoted collagen deposition by primary mouse lung fibroblasts.

Female wild-type (WT) C57BL/6 mice were purchased from the Shanghai Experimental Center, Chinese Academy of Science (Shanghai, China). BLT1−/− mice on a C57BL/6 background were purchased from The Jackson Laboratory. All mice used were sex matched at 6–8 wk of age (20–22 g) and housed in the animal facility of Fudan University under specific pathogen-free barrier conditions. All mice received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals.

Mice were anesthetized with sodium pentobarbital via i.p. injection and given 2 mg/kg BLM (Nippon Kayaku, Tokyo, Japan) in 40 μl of saline or saline alone via intratracheal (i.t.) injection. For BLT1 blockade in vivo, mice were i.t. administered 5 μg of U75302 (Cayman Chemical, Ann Arbor, MI) or vehicle (ethanol) as control every 3 d from day 0 to day 9 or from day 10 to day 21. For neutrophil depletion, mice were i.p. injected with anti-mouse Ly6G mAb (100 μg per mouse, clone RB6-8C5) or isotype Ab as control (all from Bio X Cell, West Lebanon, NH) every 2 d from day −1 to day 9. For CD4+ T cell depletion, mice were i.p. injected with anti-mouse CD4 mAb (200 μg per mouse, clone GK1.5) or isotype Ab as control (all from Bio X Cell) every 4 d from day −1 to day 21.

BALF was collected with 0.5 ml of cold PBS containing 0.5% FBS, and supernatants were collected for the TGF-β examination. Cells from all lavage were counted and differential cell counts were determined from stained cytospines using a Wright–Giemsa stain set (Sigma-Aldrich, St. Louis, MO). Infiltrating CD4+ T cells and CD8+ T cells and intracellular cytokine expression were examined by flow cytometric assay. The following anti-mouse Abs were used: CD4-FITC (clone GK1.5), CD8-Violet/PE-Cyanine7 (clone 53-6.7), IL-13–PE (clone eBio13A), and IL-17A–allophycocyanin (clone eBio17B7) (all from eBioscience, San Diego, CA). Samples were acquired by CyAn (Beckman Coulter) and analyzed by Summit 5.2 software (Beckman Coulter).

Lungs were removed, fixed in 10% buffered formalin, and embedded in paraffin. Lung sections were stained with H&E for histopathological analysis or with Masson’s trichrome for the evaluation of collagen content and distribution. The Ashcroft score for the evaluation of lung fibrosis was described previously (26). Immunohistochemical staining of α smooth muscle actin (α-SMA) was performed on paraffin-embedded lung sections. Briefly, after rehydration, sections were heated in the microwave for Ag retrieval and sequentially incubated with 3% H2O2 for blocking endogenous peroxidase activity, with 3% BSA (Sigma-Aldrich) for blocking nonspecific staining, with a rabbit polyclonal anti–α-SMA Ab (1:1000 dilution; Abcam, Cambridge, MA) at 4°C overnight in a humidified chamber. A ready-to-use peroxidase-conjugated anti-rabbit second Ab (GeneTech, Shanghai, China) was used. Then the lung sections were incubated and developed using diaminobenzidine solution (GeneTech) followed by the incubation of hematoxylin (Sangon Biotech, Shanghai, China) for 1 min. After wash, the lung sections were mounted with neutral balsam (CWBiotech, Beijing, China) and were examined under an XDS-1B inverted microscope (Leica, Wetzlar, Hessen, Germany).

Th frozen upper lobe of left lungs was digested by 0.1 mg/ml pepsin in acetic acid at room temperature overnight and then centrifuged at 12,000 rpm for 20 min. The content of collagen in lung was measured according to the instructions for a Sircol collagen assay kit (Biocolor, Carrickfergus, U.K.). Briefly, 50 μl of tissue-free supernatant of pepsin-digested lung and the standards were incubated with 1 ml of Sircol dye reagent for 30 min on a rotary table. After centrifugation and washing with 750 μl of ice-cold acid-salt wash reagent, the collagen-dye pellet was resuspended with 500 μl of alkali reagent and then samples were read at 555 nm by adding 200 μl of the collagen supernatant in alkali reagent into 96-well plates. The content of collagen in each sample was calculated according to the standard curve.

Lungs were homogenized in 1 ml of ice-cold PBS containing 1% proteinase inhibitor mixture (Sigma-Aldrich) and the tissue-free supernatants were collected. The concentrations of IL-13 and IL-17A in lung tissue homogenates were determined by ELISA (R&D Systems, Minneapolis, MN). TGF-β in BALF was activated by 0.1 M HCl for 10 min, and then 0.1 M NaOH was used to stop the reaction. The concentrations of activated TGF-β in BALF or culture supernatants were determined by ELISA (eBioscience).

The concentration of LTB4 in culture supernatants of BLM-stimulated macrophages was measured using LTB4 enzyme immunoassay kit (Cayman Chemical) according to the manufacturer’s instructions.

Total RNA was extracted from homogenized lung tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the provided instructions. cDNA was generated with a high-capacity cDNA reverse transcription kit (TaKaRa Bio, Shiga, Japan). Quantitative real-time PCR was performed with SYBR Green gene expression assay (TaKaRa Bio). Relative expression of the target gene was calculated using the comparative method for relative quantitation by normalization to the internal control β-actin. The primer sequences of all genes for PCR are as follows: β-actin, forward, 5′-CCAGCCTTCCTTCTTGGGTATG-3′, reverse, 5′-TGTGTTGGCATAGAGGTCTTTACG-3′; IL-1β, forward, 5′-ACCTGTCCTGTGTAATGAAAGACG-3′, reverse, 5′-TGGGTATTGCTTGGGATCCA-3′; CXCL1, forward, 5′-GATTCACCTCAAGAACATCCAG-3′, reverse, 5′-TGGGGACACCTTTTAGCATC-3′; CXCL2, forward, 5′-TTCCAGGTCAGTTAGCCTTG-3′, reverse, 5′-CAGACAGAAGTCATAGCCAC-3′; COL1A2, forward, 5′-GGACCCGTTGGCAAAGATG-3′, reverse, 5′-CACCAGGAGGACCAGGAG-3′; COL3A1, forward, 5′-GAGGAAACAGAGGTGAAAGAGG-3′, reverse, 5′-CAGCAATGGCAGCAGCAC-3′; α-SMA, forward, 5′-TCCTGACGCTGAAGTATCCGATA-3′, reverse, 5′-GGCCACACGAAGCTCGTTAT-3′.

Primary airway epithelial cells (AECs) were isolated from the lung of WT mice by dispase II digestion as previously described (27). Briefly, mice were anesthetized with sodium pentobarbital i.p. and perfused with 10 ml of sterile PBS via the postcava. Lungs lavage was performed with 1 ml of ice-cold PBS three times, and then the lungs were i.t. injected with 1 ml of dispase II and ligated. After a 30-min digestion, lungs were shredded with tweezers and filtered through 100- and 40-μm cell strainers. After centrifugation, the cell pellet was resuspended in DMEM/F12K (1:1) containing 10% FBS. Following incubation with anti-CD16/32 Ab and anti-CD45 Ab-coated dishes for 45 min (negative selection of leukocytes) and another 45 min for adherence (negative selection of fibroblasts), the nonadherent cells were cultured in six-well plates and stimulated with BLM (10 μg/ml) or saline as control for 24 h in 5% CO2 at 37°C. The culture supernatant was collected for the stimulation of macrophages in vitro.

Bone marrow cells (2 × 105 cells/ml) from WT mice were culture in RPMI 1640 containing M-CSF (20 ng/ml). The adherent cells were mature bone marrow–derived macrophages (BMDM), reaching 80–90% confluence on day 6. After removing the nonadherent cells, BMDM were stimulated with BLM (10 μg/ml), the supernatant of BLM-exposed AECs, or LTB4 for 24 h, respectively. In some experiments, BMDM were preincubated with bestatin (20 μg/ml), the LTA4H inhibitor, for 30 min and then stimulated with BLM. The cell-free supernatant was collected for TGF-β measurement or for the stimulation of primary lung fibroblasts in vitro.

Lungs were removed from mice, and all the trachea and bronchi were removed. Primary lung fibroblasts were isolated from the lung of WT mice by culturing the lung explant in vitro. After culture and passage, the lung fibroblasts were seeded in six-well plates. After starvation in low serum medium (DMEM containing 1% FBS) for 24 h, lung fibroblasts were stimulated with conditioned medium from BMDM for 24 h in vitro. The secretion of collagen by lung fibroblasts in culture supernatant was examined following the instruction of Sircol collagen assay kit (Biocolor). In some experiments, after low serum medium starvation, primary lung fibroblasts were stimulated with LTB4 (100 nM) and vehicle (ethanol) for 24 h.

For Western blotting, BMDM (5 × 105 cells/ml) from WT mice were stimulated with BLM for 1 h with or without preincubation with bestatin in 12-well plates. In some experiments, BMDM were stimulated with 0.03 nM LTB4 for 1 h. The proteins were extracted form BMDM following the standard protocol. The following primary Abs was used: p-Akt1 Ab (Ser473; 1:2000), total Akt1 (1:1000), GAPDH (1:2000) (all from Cell Signaling Technology, Beverly, MA). After incubation with HRP-conjugated secondary anti-rabbit Abs (1:5000; Cell Signaling Technology), membranes were developed with the ECL detection system (Pierce/Thermo Fisher Scientific, Waltham, MA).

Statistical analysis was performed with Prism 5.0 (GraphPad Software). The two-tailed Student t test was used for unpaired data. A p value <0.05 was considered statistically significant.

We first attempted to evaluate the role of BLT1 in lung fibrosis by subjecting BLT1−/− mice to BLM-induced lung fibrosis. We i.t. administered BLM into mice on day 0 and then sacrificed them on days 7 and 21, when acute lung injury and lung fibrosis reached their peaks, respectively (Fig. 1A). We evaluated the extent of lung fibrosis histologically by Masson’s trichrome staining and biochemically by measuring lung collagen contents using a Sircol collagen assay kit. Masson’s trichrome staining of lung sections from WT mice showed that increased collagen deposition could be detected on day 7, which became much more severe on day 21 following BLM treatment (Fig. 1B). In contrast, BLT1−/− mice had obviously decreased collagen deposition (Fig. 1B). We further performed histopathological evaluation of lung fibrosis using an established Ashcroft scoring method (26). BLT1−/− mice exhibited significantly decreased scores of fibrotic lesions on day 21 compared with similarly treated WT mice (Fig. 1C). This was also confirmed by a Sircol collagen assay of collagen contents in lung tissue homogenates (Fig. 1D). Consistently, significantly decreased gene expression of COL3A1 and COL1A2 was found in lungs of BLT1−/− mice compared with those of WT controls on day 21 (Fig. 1E). The production of TGF-β, an important fibrotic cytokine, was also significantly reduced in BALF of BLT1−/− mice (Fig. 1F), which is consistent with impaired lung fibrosis. The expression of α-SMA is the hallmark of myofibroblasts that contributes to collagen deposition (28). Immunohistochemistry staining of lung sections with anti–α-SMA Ab demonstrated markedly more intense α-SMA+ cells in WT mice than in BLT1−/− mice on day 21 (Fig. 1G), suggesting impaired myofibroblast differentiation in BLT1−/− mice. Collectively, these results demonstrate that BLT1 mediates BLM-induced lung fibrosis.

FIGURE 1.

Attenuated lung fibrosis in BLT1−/− mice following BLM treatment. (A) Protocol for BLM-induced lung fibrosis and the time points when the tissue samples were collected. (B) Representative photomicrographs of lung sections stained with Masson’s trichrome. Original magnification, ×100. (C) Ashcroft scoring of lung fibrosis. (D) Collagen contents in lungs were measured by Sircol collagen assay. (E) Gene expression of COL3A1 and COL1A2 in the lung was examined by quantitative real-time PCR. (F) Protein levels of TGF-β in BALF were determined by ELISA. (G) α-SMA+ myofibroblasts were examined by immunohistochemistry staining with anti–α-SMA Ab in lung sections. Original magnification, ×200. Columns and error bars represent mean ± SEM. Similar results were obtained from two independent experiments. n = 5–7 per group. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 1.

Attenuated lung fibrosis in BLT1−/− mice following BLM treatment. (A) Protocol for BLM-induced lung fibrosis and the time points when the tissue samples were collected. (B) Representative photomicrographs of lung sections stained with Masson’s trichrome. Original magnification, ×100. (C) Ashcroft scoring of lung fibrosis. (D) Collagen contents in lungs were measured by Sircol collagen assay. (E) Gene expression of COL3A1 and COL1A2 in the lung was examined by quantitative real-time PCR. (F) Protein levels of TGF-β in BALF were determined by ELISA. (G) α-SMA+ myofibroblasts were examined by immunohistochemistry staining with anti–α-SMA Ab in lung sections. Original magnification, ×200. Columns and error bars represent mean ± SEM. Similar results were obtained from two independent experiments. n = 5–7 per group. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

Accumulating evidence from both clinical and animal studies shows that inflammatory responses precede and concur with collagen deposition (1). We therefore explored whether BLT1 deficiency influenced the lung inflammation following BLM treatment. To this end, we evaluated inflammatory responses by examining H&E staining of lung tissues and the number and composition of infiltrating leukocytes in BALF. Analysis of H&E-stained lung sections demonstrated greatly decreased inflammatory infiltrates in BLT1−/− mice compared with WT mice at both time points following BLM treatment (Fig. 2A). Consistently, significantly decreased total cell numbers were found in BALF of BLT1−/− mice compared with WT controls (Fig. 2B, 2C). Differential counts of BALF infiltrating leukocytes showed that neutrophils were dominant on day 7 (Fig. 2B), whereas neutrophils were very few, and, instead, macrophages and lymphocytes were the dominant cell types on day 21 (Fig. 2C), suggesting different types of inflammatory responses during the acute injury phase and the fibrotic phase. BLT1−/− mice had significantly decreased numbers of neutrophils but similar numbers of macrophages and lymphocytes compared with WT mice on day 7, and significantly decreased lymphocytes, but similar macrophages, on day 21 as compared with WT controls (Fig. 2B, 2C). Significantly increased gene expression of several proinflammatory cytokines related to neutrophil recruitment and activation, including IL-1β, CXCL1, and CXCL2, was found on day 7, but not on day 21 (Fig. 2D, data not shown). Significantly decreased gene expression of these cytokines was detected in lungs of BLM-treated BLT1−/− mice on day 7 compared with similarly treated WT mice (Fig. 2D). Further flow cytometric analysis of BALF cells demonstrated significantly decreased numbers of CD4+ T cells and CD8+ T cells in BLT1−/− mice on day 21 (Fig. 2E). IL-13 and IL-17A, two well-known profibrotic cytokines mainly produced by effector CD4+ T cells, were reported to be involved in BLM-induced lung fibrosis (24, 25). BLM-treated BLT1−/− mice had significantly reduced production of IL-13 and IL-17A in lung tissue homogenates compared with similarly treated WT mice on day 21 (Fig. 2F). The numbers of IL-13+CD4+ T cells and IL-17+CD4+ T cells were also significantly reduced in BALF of BLM-treated BLT1−/− mice (Supplemental Fig. 1A, 1B), whereas no signals for IL-13 and IL-17A were detected in CD8+ T cells by intracellular staining of BALF cells (Supplemental Fig. 1A). We also examined the gene expression of BLT2, the low-affinity receptor of LTB4, in lungs of WT and BLT1−/− mice. In contrast to greatly increased BLT1 gene expression in lungs of BLM-treated WT mice (Supplemental Fig. 2A), BLM treatment greatly decreased BLT2 gene expression in lungs of both WT and BLT1−/− mice (Supplemental Fig. 2B). Importantly, there was no difference between WT and BLT1−/− mice regardless of BLM treatment, indicating that BLT1 deficiency has no effect on BLT2 expression (Supplemental Fig. 2B). Collectively, these results demonstrate that BLT1 mediates neutrophil-dominated lung inflammation in the acute injury phase and lymphocyte-dominated lung inflammation in the fibrotic phase of BLM-induced lung fibrosis.

FIGURE 2.

Attenuated lung inflammation in BLT1−/− mice following BLM treatment. (A) Representative photomicrographs of lung sections stained with H&E. Original magnification, ×100. (B and C) Differential cell counts in BALF on day 7 (B) and day 21 (C) following BLM treatment. (D) Gene expression of IL-1β, CXCL1, and CXCL2 in the lung. (E) Representative flow cytometric analysis of the percentages of CD4+ and CD8+ T cells and the absolute cell numbers calculated based on the numbers of total cells in BALF. (F) Protein levels of IL-13 and IL-17A in the lung tissue homogenates. Columns and error bars represent mean ± SEM. n = 5–7 per group. Similar results were obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

Attenuated lung inflammation in BLT1−/− mice following BLM treatment. (A) Representative photomicrographs of lung sections stained with H&E. Original magnification, ×100. (B and C) Differential cell counts in BALF on day 7 (B) and day 21 (C) following BLM treatment. (D) Gene expression of IL-1β, CXCL1, and CXCL2 in the lung. (E) Representative flow cytometric analysis of the percentages of CD4+ and CD8+ T cells and the absolute cell numbers calculated based on the numbers of total cells in BALF. (F) Protein levels of IL-13 and IL-17A in the lung tissue homogenates. Columns and error bars represent mean ± SEM. n = 5–7 per group. Similar results were obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

Our results above suggest that lack of BLT1 attenuates both lung fibrosis and lung inflammation. To elucidate the role of BLT1-mediated lung inflammation in the development of lung fibrosis, we first investigated whether interfering with BLT1-mediated neutrophil-dominated early lung inflammation influenced the lung fibrosis and the accompanied T cell–dominated lung inflammation in the fibrotic phase. To this end, we i.t. administered a specific BLT1 antagonist, U75302, every 3 d from day 0 to day 9 after i.t. instillation of BLM, and then analyzed the extent of lung fibrosis and inflammation on day 21 (Fig. 3A). U75302 treatment greatly attenuated BLM-induced lung fibrosis on day 21, as evidenced by greatly decreased collagen deposition in the lungs assessed by Masson’s trichrome staining, Ashcroft scoring, and Sircol collagen assay (Fig. 3B–D), as well as significantly decreased gene expression of COL3A1 and COL1A2 (Fig. 3E). Consistently, U75302 treatment also caused significant decreases in TGF-β production in BALF and α-SMA+ cells in the lungs following BLM treatment (Fig. 3F, 3G). Furthermore, U75302 treatment during the acute injury phase significantly reduced the numbers of lymphocytes but not macrophages in BALF on day 21 (Fig. 3H). Consistently, the numbers of CD4+ T cells and CD8+ T cells were significantly reduced in BALF of mice treated with U75302 compared with vehicle controls (Fig. 3I). U75302 treatment also caused significantly decreased production of IL-13 and IL-17A in lung tissue homogenates (Fig. 3J).

FIGURE 3.

Treatment with U75302, a specific BLT1 antagonist, during the acute injury phase impairs BLM-induced lung fibrosis. (A) Protocol for the administration of U75302 or vehicle (ethanol) as control in BLM-induced lung fibrosis and the time point when the samples were collected. (B) Representative photomicrographs of lung sections stained with Masson’s trichrome. Original magnification, ×100. (C) Ashcroft scoring of lung fibrosis. (D) Collagen contents in lungs. (E) Gene expression of COL3A1 and COL1A2 in the lung. (F) Protein levels of TGF-β in BALF. (G) α-SMA+ myofibroblasts were examined by immunohistochemistry staining in lung sections. Original magnification, ×200. (H) Differential cell counts in BALF. (I) Cell numbers of CD4+ and CD8+ T cells in BALF. (J) Protein levels of IL-13 and IL-17A in the lung tissue homogenates. Columns and error bars represent mean ± SEM. n = 4–5 per group. Similar results were obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

Treatment with U75302, a specific BLT1 antagonist, during the acute injury phase impairs BLM-induced lung fibrosis. (A) Protocol for the administration of U75302 or vehicle (ethanol) as control in BLM-induced lung fibrosis and the time point when the samples were collected. (B) Representative photomicrographs of lung sections stained with Masson’s trichrome. Original magnification, ×100. (C) Ashcroft scoring of lung fibrosis. (D) Collagen contents in lungs. (E) Gene expression of COL3A1 and COL1A2 in the lung. (F) Protein levels of TGF-β in BALF. (G) α-SMA+ myofibroblasts were examined by immunohistochemistry staining in lung sections. Original magnification, ×200. (H) Differential cell counts in BALF. (I) Cell numbers of CD4+ and CD8+ T cells in BALF. (J) Protein levels of IL-13 and IL-17A in the lung tissue homogenates. Columns and error bars represent mean ± SEM. n = 4–5 per group. Similar results were obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

We next investigated whether interfering with BLT1-mediated T cell infiltration in the fibrotic phase influenced the lung fibrosis by i.t. administering U75302 every 3 d from day 10 to 21 after BLM instillation when neutrophil-rich inflammatory responses subside and active fibrogenesis occurs. The extent of lung fibrosis and inflammation was analyzed on day 21 (Fig. 4A). U75302 treatment during the fibrotic phase failed to attenuate lung fibrosis, as evidenced by comparable collagen deposition in the lungs assessed by Masson’s trichrome staining, Ashcroft scoring, and Sircol collagen assay (Fig. 4B–D), as well as comparable gene expression of COL3A1 and COL1A2 (Fig. 4E). Consistently, U75302 treatment had no effect on TGF-β production in BALF and α-SMA+ cells in the lungs following BLM treatment (Fig. 4F, 4G). Interestingly, in contrast to unchanged lung fibrosis, U75302 treatment during the fibrotic phase significantly reduced the number of lymphocytes, but not macrophages, in BALF (Fig. 4H). Consistently, the numbers of CD4+ T cells and CD8+ T cells were significantly reduced in BALF of mice treated with U75302 compared with vehicle controls (Fig. 4I). However, U75302 treatment had no effect on the production of IL-13 and IL-17A in lung tissue homogenates (Fig. 4J). Collectively, these results suggest that early, but not late, BLT1 blockade is critical for BLM-induced lung fibrosis.

FIGURE 4.

U75302 treatment, during the fibrotic phase, has an effect on BLM-induced lung fibrosis. (A) Protocol for the administration of U75302 or vehicle (ethanol) as control in BLM-induced lung fibrosis and the time point when the samples were collected. (B) Representative photomicrographs of lung sections stained with Masson’s trichrome. Original magnification, ×100. (C) Ashcroft scoring of lung fibrosis. (D) Collagen contents in lungs. (E) Gene expression of COL3A1 and COL1A2 in the lung. (F) Protein levels of TGF-β in BALF. (G) α-SMA+ myofibroblasts were examined by immunohistochemistry staining in lung sections. Original magnification, ×200. (H) Differential cell counts in BALF. (I) Cell numbers of CD4+ and CD8+ T cells in BALF. (J) Protein levels of IL-13 and IL-17A in the lung tissue homogenates. Columns and error bars represent mean ± SEM. n = 4–5 per group. Similar results were obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4.

U75302 treatment, during the fibrotic phase, has an effect on BLM-induced lung fibrosis. (A) Protocol for the administration of U75302 or vehicle (ethanol) as control in BLM-induced lung fibrosis and the time point when the samples were collected. (B) Representative photomicrographs of lung sections stained with Masson’s trichrome. Original magnification, ×100. (C) Ashcroft scoring of lung fibrosis. (D) Collagen contents in lungs. (E) Gene expression of COL3A1 and COL1A2 in the lung. (F) Protein levels of TGF-β in BALF. (G) α-SMA+ myofibroblasts were examined by immunohistochemistry staining in lung sections. Original magnification, ×200. (H) Differential cell counts in BALF. (I) Cell numbers of CD4+ and CD8+ T cells in BALF. (J) Protein levels of IL-13 and IL-17A in the lung tissue homogenates. Columns and error bars represent mean ± SEM. n = 4–5 per group. Similar results were obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

The above findings suggest that BLT1-mediated neutrophil-dominated acute lung inflammation seems to be important for the development of lung fibrosis and the accompanied T cell–dominated inflammation. We therefore investigated whether early infiltrating neutrophils contributed to BLM-induced lung fibrosis. To this end, we depleted neutrophils by i.p. injecting anti-Ly6G mAb to WT mice starting 1 d before BLM instillation every 2 d until day 9, and then analyzed the extent of lung fibrosis on day 21 (Fig. 5A). The isotype Ab was used as control. Neutrophils were efficiently depleted after the anti-Ly6G mAb treatment (Fig. 5B). Depletion of neutrophils had no effect on lung fibrosis, as evidenced by comparable collagen deposition in the lungs assessed by Masson’s trichrome staining, Ashcroft scoring, and Sircol collagen assay (Fig. 5C–E), as well as comparable gene expression of COL3A1 and COL1A2 (Fig. 5F). Consistently, depletion of neutrophils had no effect on TGF-β production in BALF and α-SMA+ cells in the lungs following BLM treatment (Fig. 5G, 5H). Additionally, significantly decreased numbers of CD8+ T cells, but not CD4+ T cells, were detected in BALF of neutrophil-depleted mice (Fig. 5I). Interestingly, significantly increased IL-17A production, but comparable IL-13 production, was detected in lung tissue homogenates of neutrophil-depleted mice compared with those of control mice (Fig. 5J).

FIGURE 5.

Neutrophil depletion has no effect on BLM-induced lung fibrosis. (A) Protocol for the administration of neutralizing Ly6G mAb or isotype Ab as control in BLM-induced lung fibrosis. (B) Percentages of neutrophils in blood were determined by flow cytometric analysis. (C) Representative photomicrographs of lung sections stained with Masson’s trichrome. Original magnification, ×100. (D) Ashcroft scoring of lung fibrosis. (E) Collagen contents in lungs. (F) Gene expression of COL3A1 and COL1A2 in the lung. (G) Protein levels of TGF-β in BALF. (H) α-SMA+ myofibroblasts were examined by immunohistochemistry staining in lung sections. Original magnification, ×200. (I) Cell numbers of CD4+ and CD8+ T cells in BALF. (J) The protein levels of IL-13 and IL-17A in the lung tissue homogenates. Columns and error bars represent mean ± SEM. n = 4–6 per group. Similar results were obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

Neutrophil depletion has no effect on BLM-induced lung fibrosis. (A) Protocol for the administration of neutralizing Ly6G mAb or isotype Ab as control in BLM-induced lung fibrosis. (B) Percentages of neutrophils in blood were determined by flow cytometric analysis. (C) Representative photomicrographs of lung sections stained with Masson’s trichrome. Original magnification, ×100. (D) Ashcroft scoring of lung fibrosis. (E) Collagen contents in lungs. (F) Gene expression of COL3A1 and COL1A2 in the lung. (G) Protein levels of TGF-β in BALF. (H) α-SMA+ myofibroblasts were examined by immunohistochemistry staining in lung sections. Original magnification, ×200. (I) Cell numbers of CD4+ and CD8+ T cells in BALF. (J) The protein levels of IL-13 and IL-17A in the lung tissue homogenates. Columns and error bars represent mean ± SEM. n = 4–6 per group. Similar results were obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

We next explored the role of CD4+ T cells in BLM-induced lung fibrosis by depleting CD4+ T cells using i.p. injection of anti-CD4 mAb to WT mice starting 1 d before BLM instillation every 4 d until day 18. The extent of lung fibrosis was analyzed on day 21 (Fig. 6A). There were no detectable CD4+ T cells, but slightly increased numbers of CD8+ T cells, in BALF of mice treated with anti-CD4 mAb compared with those treated with isotype Ab (Fig. 6B). Meanwhile, significantly decreased IL-13, but comparable IL-17A production, was detected in lung tissue homogenates of CD4+ T cell–depleted mice compared with those of controls (Fig. 6C). Depletion of CD4+ T cells did not affect lung fibrosis, as evidenced by comparable collagen deposition in the lungs assessed by Masson’s trichrome staining, Ashcroft scoring, and Sircol collagen assay (Fig. 6D–F), as well as comparable gene expression of COL3A1 and COL1A2 (Fig. 6G). Consistently, TGF-β production in BALF and α-SMA+ cells in the lungs were comparable in mice treated with neutralizing anti-CD4 mAb and isotype controls (Fig. 6H, 6I). Collectively, these results suggest that both neutrophils and CD4+ T cells are dispensable for BLM-induced lung fibrosis.

FIGURE 6.

CD4+ T cell depletion has no effect on BLM-induced lung fibrosis. (A) Protocol for the administration of neutralizing anti-CD4 mAb or isotype Ab as control in BLM-induced lung fibrosis and the time point when the samples were collected. (B) Cell numbers of CD4+ and CD8+ T cells in BALF. (C) Protein levels of IL-13 and IL-17A in the lung tissue homogenates. (D) Representative photomicrographs of lung sections stained with Masson’s trichrome. Original magnification, ×100. (E) Ashcroft scoring of lung fibrosis. (F) Collagen contents in lungs. (G) Gene expression of COL3A1 and COL1A2 in the lung. (H) Protein levels of TGF-β in BALF. (I) α-SMA+ myofibroblasts were examined by immunohistochemistry staining in lung sections. Original magnification, ×200. Columns and error bars represent mean ± SEM. n = 4–6 per group. Similar results were obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6.

CD4+ T cell depletion has no effect on BLM-induced lung fibrosis. (A) Protocol for the administration of neutralizing anti-CD4 mAb or isotype Ab as control in BLM-induced lung fibrosis and the time point when the samples were collected. (B) Cell numbers of CD4+ and CD8+ T cells in BALF. (C) Protein levels of IL-13 and IL-17A in the lung tissue homogenates. (D) Representative photomicrographs of lung sections stained with Masson’s trichrome. Original magnification, ×100. (E) Ashcroft scoring of lung fibrosis. (F) Collagen contents in lungs. (G) Gene expression of COL3A1 and COL1A2 in the lung. (H) Protein levels of TGF-β in BALF. (I) α-SMA+ myofibroblasts were examined by immunohistochemistry staining in lung sections. Original magnification, ×200. Columns and error bars represent mean ± SEM. n = 4–6 per group. Similar results were obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

We attempted to seek out the potential target cell on which the LTB4/BLT1 axis acted to influence BLM-induced lung fibrosis. Although we previously demonstrated that cysLTs have profibrotic activities by promoting the proliferation and collagen deposition by skin fibroblasts (29), we excluded the possibility that the LTB4/BLT1 axis enhanced collagen deposition by directly promoting fibroblast activation or epithelial–mesenchymal transition, as very little, if any, BLT1 mRNA and no BLT1 protein were detected in primary mouse fibroblasts and AECs (Supplemental Fig. 3A, 3B). Moreover, LTB4 stimulation did not change gene expression of α-SMA, COL3A1, and COL1A2 in primary mouse lung fibroblasts (Supplemental Fig. 3C). Macrophages that express BLT1 are also closely involved in tissue fibrosis by producing various profibrotic cytokines, including TGF-β (30). Recently, Larson-Casey et al. (31) demonstrated that macrophage-derived TGF-β is required for the progression of BLM-induced lung fibrosis. Although we did not observe the change in the number of macrophages, we noticed that BLT1 deficiency or early BLT1 blockade caused significantly decrease TGF-β levels (Figs. 1F, 3F), which was consistent with the attenuated lung fibrosis. We speculated that the LTB4/BLT1 axis might promote TGF-β production by macrophages, which initiated the lung fibrosis in the acute injury phase. BLM treatment could directly activate macrophages or cause the damage of AECs, which indirectly activate macrophages (32, 33). We thus stimulated BMDM from WT or BLT1−/− mice with BLM or supernatants from saline- or BLM-exposed AECs. Comparable TGF-β levels were found in the cultures of WT and BLT1−/− BMDM without stimulation (Fig. 7A, 7B). Stimulation with BLM or supernatants from BLM-exposed AECs caused significantly increased TGF-β production by WT BMDM, which was significantly reduced in BLT1−/− BMDM (Fig. 7A, 7B). Similar results were obtained when peritoneal macrophages were used (Supplemental Fig. 4). These results suggested that BLT1−/− macrophages have no intrinsic defect in TGF-β production, but produce less TGF-β in response to BLM stimulation. We next found that BLM stimulation significantly increased LTB4 production by BMDM (Fig. 7C), which is consistent with the previous findings that macrophages are also a cell source for LTB4 (4, 34, 35). Interestingly, pretreatment of bestatin, the LTA4H inhibitor, significantly reduced TGF-β production by BLM-treated BMDM (Fig. 7D). Together with significantly reduced TGF-β production in BLM-treated BLT1−/− BMDM, these data suggest that the LTB4/BLT1 axis could promote TGF-β production by macrophages in an autocrine way. Importantly, supernatants from BLM-stimulated BMDM induced significantly higher collagen secretion by primary mouse lung fibroblasts than did those from bestatin-pretreated BMDM (Fig. 7E).

FIGURE 7.

Blockade of the LTB4/BLT1 axis inhibits TGF-β production by BLM-treated macrophages in vitro. (A and B) TGF-β concentrations in the supernatants of BMDM stimulated with BLM (10 μg/ml) or vehicle (saline) (A), or with supernatants of saline- and BLM-exposed AECs (B). (C) LTB4 levels in the supernatants of saline- or BLM-stimulated BMDM were measured by enzyme immunoassay. (D) TGF-β concentrations in the supernatants of BMDM stimulated with BLM alone or BLM following bestatin pretreatment (20 μg/ml). (E) Collagen contents in the culture of primary lung fibroblasts stimulated with the supernatants of BMDM treated with BLM alone or BLM following bestatin pretreatment (20 μg/ml). (F) TGF-β concentrations in the supernatants of BMDM stimulated with different dosage of LTB4. ***p < 0.001 versus medium group. (G and H) Representative Western blots showing p-Akt1 and total Akt1 (t-Akt1) in BMDM stimulated with LTB4 at 0.03 nM (G) or BLM alone or BLM following bestatin pretreatment (20 μg/ml) (H). Data are pooled from two or three independent experiments and expressed as mean ± SEM. *p < 0.05, ***p < 0.001.

FIGURE 7.

Blockade of the LTB4/BLT1 axis inhibits TGF-β production by BLM-treated macrophages in vitro. (A and B) TGF-β concentrations in the supernatants of BMDM stimulated with BLM (10 μg/ml) or vehicle (saline) (A), or with supernatants of saline- and BLM-exposed AECs (B). (C) LTB4 levels in the supernatants of saline- or BLM-stimulated BMDM were measured by enzyme immunoassay. (D) TGF-β concentrations in the supernatants of BMDM stimulated with BLM alone or BLM following bestatin pretreatment (20 μg/ml). (E) Collagen contents in the culture of primary lung fibroblasts stimulated with the supernatants of BMDM treated with BLM alone or BLM following bestatin pretreatment (20 μg/ml). (F) TGF-β concentrations in the supernatants of BMDM stimulated with different dosage of LTB4. ***p < 0.001 versus medium group. (G and H) Representative Western blots showing p-Akt1 and total Akt1 (t-Akt1) in BMDM stimulated with LTB4 at 0.03 nM (G) or BLM alone or BLM following bestatin pretreatment (20 μg/ml) (H). Data are pooled from two or three independent experiments and expressed as mean ± SEM. *p < 0.05, ***p < 0.001.

Close modal

Furthermore, we examined the direct effect of LTB4 on TGF-β production by BMDM by adding exogenous LTB4 at different concentrations. We demonstrated that a similar concentration (0.03 nM) as found in the culture of BLM-treated BMDM significantly enhanced TGF-β production (Fig. 7F). Interestingly, LTB4-induced TGF-β production by BMDM was dose-independent, as addition of exogenous LTB4 at higher concentrations (0.1, 1, 10, 100 nM) failed to further enhance TGF-β production by BMDM (Fig. 7F). These data suggest that LTB4 derived from macrophages is sufficient to amplify TGF-β production by macrophages in response to BLM stimulation. A recent study showed that Akt1 activation mediates TGF-β production by macrophages, leading to BLM-induced lung fibrosis (31). We therefore examined whether the LTB4/BLT1 axis could promote Akt1 activation by Western blotting. We found that addition of exogenous LTB4 at a low concentration (0.03 nM) was able to induce Akt1 activation (Fig. 7G). More importantly, pretreatment of bestatin greatly reduced Akt1 activation in BLM-treated BMDM (Fig. 7H). Taken together, these data suggest that the LTB4/BLT1 axis could enhance TGF-β production by BLM-treated macrophages in an autocrine way.

Although elevated LTB4 was found in BALF and lungs of IPF patients as well as mouse models of lung fibrosis (57), surprisingly, the precise role of the LTB4/BLT1 axis during the progression of lung fibrosis has yet been thoroughly investigated. In this study, we demonstrate that interruption of the LTB4/BLT1 axis during the acute injury phase, but not the fibrotic phase, attenuates BLM-induced lung fibrosis. Although interruption of the LTB4/BLT1 axis impaired lung infiltration of neutrophils and CD4+ T cells, the profibrotic ability of the LTB4/BLT1 axis appeared to be independent of these two types of immune cells, as depletion of neutrophils or CD4+ T cells had no effect on BLM-induced lung fibrosis. Furthermore, we demonstrate that the LTB4/BLT1 axis could increase TGF-β secretion by BLM-stimulated macrophages in an autocrine way, which may contribute to the development of lung fibrosis by promoting collagen deposition.

We first observed significantly attenuated BLM-induced lung fibrosis in BLT1−/− mice. The development of BLM-induced lung fibrosis consists of two phases, the acute injury phase characterized by abundant neutrophil infiltration and the fibrotic phase by fibrogenesis and T cell infiltration. Notably, the protection from lung fibrosis in BLT1−/− mice was associated with a great reduction in early neutrophil recruitment and later T cell recruitment to the lung, as well as the production of fibrotic cytokines, including TGF-β, IL-13, and IL-17A. We noted that BLT2 had much lower baseline levels than did BLT1 in lungs; moreover, BLM treatment comparably decreased BLT2 gene expression in lungs of WT and BLT1−/− mice, indicating that BLT1 deficiency has no effect on BLT2 expression. Together with a previous study showing that BLT1 blockade did not affect the efficiency of LTB4 binding to BLT2 (36), it is unlikely that BLT2 could compensate for BLT1 in our model. However, we could not exclude the possible role of BLT2 in lung fibrosis, as recent studies suggest that BLT2 is involved in lung diseases, including asthma and chronic obstructive pulmonary disease (37, 38).

We further demonstrated that the act of the LTB4/BLT1 axis in the acute injury phase, but not in the fibrotic phase, is essential for BLM-induced lung fibrosis by using a specific BLT1 antagonist in different phase. Early BLT1 blockade during the acute injury phase (immediately after BLM administration until day 10) attenuated BLM-induced lung fibrosis to a similar extent to those observed in BLT1−/− mice, whereas BLT1 blockade during the fibrotic phase (days 10–21 after BLM administration) had no effect on lung fibrosis. Similarly to BLT1−/− mice, attenuated lung fibrosis caused by early BLT1 blockade was associated with a great reduction in early neutrophil recruitment (data not shown) and later T cell recruitment to the lung, as well as the production of fibrotic cytokines. In contrast, late BLT1 blockade had no effect on the production of TGF-β, IL-13, and IL-17A, although decreased recruitment of T cells was also observed.

Neutrophils were recently implicated in tissue fibrosis via several mechanisms (39, 40). We therefore hypothesized that BLT1-dependent neutrophil recruitment and/or activation may contribute to BLM-induced lung fibrosis and the accompanying T cell recruitment. However, neutrophil-depleted mice developed similar BLM-induced lung fibrosis to control mice, indicative of a dispensable role of neutrophils in BLM-induced lung fibrosis. Unexpectedly, neutrophil depletion caused significantly increased IL-17A production with no effect on CD4+ T cell recruitment and the production of TGF-β and IL-13 in the lungs (Supplemental Fig. 1C). Th2 and Th17 cells have long been considered to promote a variety of tissue fibrosis by secreting different cytokines, including IL-13 and IL-17A (25, 41). A previous study has shown that BLM-induced lung fibrosis is IL-17A–dependent but IL-13–independent (25). We found that CD4+ T cell–depleted mice developed similar BLM-induced lung accompanied by significantly decreased IL-13 but comparable TGF-β and IL-17A production when compared with control mice. These data suggest that CD4+ T cells are dispensable for BLM-induced lung fibrosis. There are two interesting findings: first, we found that CD4+ T cells are not the major cell source for IL-17A in BLM-induced lung fibrosis, as ∼70% of IL-17A+ cells are CD4 T cells (data not shown); second, neutrophil depletion greatly enhanced recruitment of both IL-17A+CD4+ T cells and IL-17A+CD4 cells (Supplemental Fig. 1C). It was reported that IL-17A produced by CD4+ and γδ T cells contributes to BLM-induced lung fibrosis (25, 42). These IL-17A+CD4 cells are very likely to be γδ T cells, which have been identified to be a major innate cell source for IL-17A under several inflammatory conditions (43, 44), but we still could not exclude type 3 innate lymphoid cells, which also produced IL-17A (45). However, we found that early BLT1 blockade had no effect on lung recruitment of IL-17A+CD4 cells (Supplemental Fig. 1D), suggesting that they are unlikely to participate in BLT1-mediated lung fibrosis. It would be worthy to identify these IL-17A+CD4 cells and investigate the relationship between neutrophils and IL-17A–producing cells in the future study.

In addition to neutrophils and T cells, macrophages participate in the development of tissue fibrosis, playing either profibrotic or antifibrotic activities, depending on different populations of macrophages in the different phase of tissue fibrosis (1). Although M1 and M2 macrophages have been long considered to be antifibrotic and profibrotic, respectively, it was recently reported that Arg1-expressing M2 macrophages are required for the suppression and resolution of hepatic fibrosis (46). We found that gene expression of M2 markers, including Arg1, Ym1, and Fizz1, was equal in lungs of WT and BLT1−/− mice following BLM treatment (data not shown), suggesting that the protection of lung fibrosis in BLT1−/− mice is not associated with macrophage phenotype. Interestingly, in a mouse model of hepatic fibrosis, macrophages have been suggested to promote fibrosis during the early inflammatory phase, but suppress fibrosis during the late remodeling phase (47). Therefore, it is very likely that macrophages are the major target cells of the LTB4/BLT1 axis in the acute injury phase to promote BLM-induced lung fibrosis. Macrophages are demonstrated to be the major cell source for TGF-β, one of the key drivers of fibrosis in BLM-induced lung fibrosis (30). Our in vitro results demonstrated that LTB4 produced by BLM-treated macrophages could enhance TGF-β production in a BLT1-dependent way, and inhibition of LTB4 synthesis reduced TGF-β production by BLM-treated macrophages, leading to less collagen deposition by primary mouse lung fibroblasts. Moreover, we found that LTB4 could promote the activation of Akt1 in macrophages, which was recently reported to be critical for TGF-β production by macrophages and BLM-induced lung fibrosis (31). Interestingly, the levels of LTB4 produced by BLM-stimulated macrophages are sufficient to promote TGF-β production by macrophages, which was not further enhanced by increasing the dose of exogenous LTB4, suggesting that LTB4 at low concentration is able to amplify TGF-β production by macrophages upon BLM stimulation, thereby leading to lung fibrosis. Thus, it is likely that LTB4 derived from macrophages could be sufficient to promote TGF-β production in an autocrine way, which further activates fibroblasts, whereas neutrophils, as a major source of LTB4, are important for providing LTB4 at higher levels to recruit more neutrophils and T cells to promote tissue inflammation (12, 15, 17, 18). This may explain, at least in part, why depletion of neutrophils did not affect BLM-induced lung fibrosis.

In summary, our study demonstrates that the LTB4/BLT1 axis plays a critical role in the acute injury phase to promote BLM-induced lung fibrosis, which is independent of BLT1-dependent recruitment of neutrophils and CD4+ T cells to the lung. Our results also have an important clinical implication that early interruption of the LTB4/BLT1 axis in the acute injury phase of some inflammatory diseases could prevent the later development of tissue fibrosis.

We thank Haiyan Chu (School of Life Science, Fudan University) for technical assistance with Sircol collagen assays.

This work was supported by National Natural Science Foundation of China Grant 813220437 (to R.H.), Shuguang Project 13SG03 (to R.H.), and by State Key Basic Research Program (973) Project 2015CB553404 (to R.H.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

AEC

airway epithelial cell

BALF

bronchoalveolar lavage fluid

BLM

bleomycin

BMDM

bone marrow–derived macrophage

cysLT

cysteinyl leukotriene

IPF

idiopathic pulmonary fibrosis

i.t.

intratracheal(ly)

5-LO

5-lipoxygenase

LT

leukotriene

LTA4H

leukotriene A4 hydrolase

LTB4

leukotriene B4

α-SMA

α smooth muscle actin

WT

wild-type.

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The authors have no financial conflicts of interest.

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