Lipoxin A4 Attenuates Constitutive and TGF-β1–Dependent Profibrotic Activity in Human Lung Myofibroblasts

Idiopathic pulmonary fibrosis (IPF) is a common, progressive, and invariably lethal interstitial lung disease with no effective therapy. The key cell driving the development of fibrosis is the myofibroblast. Lipoxin A4 (LXA4) is an anti-inflammatory lipid, important in the resolution of inflammation, and it has potential antifibrotic activity. However, the effects of LXA4 on primary human lung myofibroblasts (HLMFs) have not previously been investigated. Therefore, the aim of this study was to examine the effects of LXA4 on TGF-β1–dependent responses in IPF- and nonfibrotic control (NFC)–derived HLMFs. HLMFs were isolated from IPF and NFC patients and grown in vitro. The effects of LXA4 on HLMF proliferation, collagen secretion, α-smooth muscle actin (αSMA) expression, and Smad2/3 activation were examined constitutively and following TGF-β1 stimulation. The LXA4 receptor (ALXR) was expressed in both NFC- and IPF-derived HLMFs. LXA4 (10−10 and 10−8 mol) reduced constitutive αSMA expression, actin stress fiber formation, contraction, and nuclear Smad2/3, indicating regression from a myofibroblast to fibroblast phenotype. LXA4 also significantly inhibited FBS-dependent proliferation and TGF-β1–dependent collagen secretion, αSMA expression, and Smad2/3 nuclear translocation in IPF-derived HLMFs. LXA4 did not inhibit Smad2/3 phosphorylation. In summary, LXA4 attenuated profibrotic HLMF activity and promoted HLMF regression to a quiescent fibroblast phenotype. LXA4 or its stable analogs delivered by aerosol may offer a novel approach to the treatment of IPF.

I diopathic pulmonary fibrosis (IPF) is a progressive disease with a median survival of only 3 years (1,2). Patients present with breathlessness and disabling cough, progressing to respiratory failure and a distressful death. The cause is not known, but alveolar cell injury coupled with fibroblast/myofibroblast proliferation and activation are critical in the pathophysiology (3,4). There is little effective treatment, and there is therefore an urgent unmet clinical need for novel modulators of lung fibrosis and tissue remodeling.
The key cell driving the development of fibrosis is the myofibroblast (5,6). Myofibroblasts are intermediate in phenotype between fibroblasts and smooth muscle cells, expressing a-smooth muscle actin (aSMA) and exhibiting contractile activity, but they are also the principle cell responsible for the synthesis and deposition of the fibrotic matrix in IPF (7). The increased numbers of myofibroblasts found within IPF lungs occurs in part through the differenti-ation of resident fibroblasts (8); this involves reorganization of the actin cytoskeleton, increased expression of aSMA, and incorporation of actin stress fibers (9), a process regulated by the TGF-b1/ Smad pathway (10,11). Furthermore, myofibroblasts derived from IPF lungs demonstrate enhanced proliferation (12), migration (13), collagen production (14), aSMA expression (15),and actin stress fiber formation (15). The myofibroblast is therefore a highly attractive target for the treatment of IPF. As myofibroblasts rarely persist in healthy lungs, their differentiation is considered a key event in the pathogenesis of IPF, and it is likely that a therapy capable of reversing this phenotypic change would be efficacious.
A family of lipid mediators known as lipoxins, resolvins, protectins, and maresins (collectively called resolving mediators for convenience) (16)(17)(18) are important for the resolution of inflammation and generated soon after a tissue insult. Lipoxins are generated from membrane arachidonic acid through biochemical synthesis involving the enzymes 5-and 15-lipoxygenase (5-LOX and 15-LOX) (18). Several molecules are generated through transcellular synthesis with 15-LOX active in one cell such as an epithelial cell and 5-LOX active in a second cell such as an inflammatory leukocyte. In addition, aspirin-triggered forms of these molecules exist (epi-lipoxins and aspirin-triggered resolvins) in which acetylated cyclooxygenase-2 generates the initial metabolite, which is then modified further by 5-LOX. Several molecules can also be formed endogenously in the absence of aspirin, possibly via a cytochrome P450-dependent pathway (17), and 5-LOX-independent pathways for the production of protectins and maresins also exist with unicellular synthesis evident (18).
A key feature of these molecules is that they promote resolution of inflammation at low nanomole concentrations, but are not innately immunosuppressive as they also activate antibacterial mechanisms (17,19). Lipoxin A 4 (LXA 4 ) also has antifibrotic activity in a number of model systems. For example, it inhibits platelet-derived growth factor-dependent TGF-b1 production and profibrotic gene expression by renal mesangial cells (20), inhibits mesangial cell proliferation (21), and experimental renal fibrosis (22). LXA 4 also inhibits epithelial mesenchymal transition in renal epithelial cells (23), whereas knockout of the 12/15-LOX pathway prevents experimental dermal fibrosis (24). With respect to lung fibrosis, LXA 4 inhibited connective tissue growth factor-dependent proliferation of a human lung fibroblast cell line (25), whereas a stable epi-LXA 4 analog reduced bleomycin-induced pulmonary fibrosis in mice (26).
The effects of LXA 4 on the function of healthy and IPF-derived primary human lung myofibroblast (HLMF) function are unknown. We hypothesized that LXA 4 inhibits constitutive HLMF profibrotic responses and TGF-b1-driven profibrotic activity through the Smad signaling pathway. We therefore investigated the effects of LXA 4 on constitutive and TGF-b1-dependent HLMF Smad 2/3 activity, gene transcription, and profibrotic HLMF processes such as contraction, collagen secretion, proliferation, and differentiation.

Materials and Methods
Human lung myofibroblast isolation, characterization, and culture Nonfibrotic control (NFC) HLMFs were derived from healthy areas of lung from patients undergoing lung resection for carcinoma at Glenfield Hospital, Leicester, U.K. No morphological evidence of disease was found in the tissue samples used for HLMF isolation. IPF HLMFs were derived from patients undergoing lung transplant at the University of Pittsburgh Medical Center and were shown to have usual interstitial pneumonia on histological examination. Myofibroblasts were grown, cultured, and characterized as previously described (27). All NFC patients gave informed written consent, and the study was approved by the National Research Ethics Service (references 07/ MRE08/42 and 10/H0402/12). Written informed consent was also obtained from all IPF subjects, with the protocol approved by the University of Pittsburgh Institutional Review Board.
All cultures demonstrated the typical elongated spindle-shaped fibroblast morphology. All cultures underwent further extensive characterization via flow cytometry and immunofluorescence. They were found to be predominantly a myofibroblast-rich population (99% expressing aSMA) as described previously by us and others (15,(27)(28)(29)(30). They expressed aSMA, CD90, fibroblast surface protein, and collagen type I. No contaminating cells were found. Immunostaining for macrophages (CD68), T cells (CD3), and progenitor cells (CD34) was negative. There was no evidence of cobblestoneshaped epithelial cells among the cultures. Results of this detailed characterization have been shown previously (27,30).

Flow cytometry
HLMFs were grown in T25 flasks and serum-starved for 24 h prior to experiments. The myofibroblasts were incubated for 24 h and either left unstimulated or stimulated with TGF-b1 (10 ng/ml) (R&D Systems, Oxford, U.K.).
To study the inhibitory effects of LXA 4 on aSMA expression, HLMFs were incubated in the presence of serum-free medium alone or 0.1% ethanol vehicle control or LXA 4 at 10 210 and 10 28 mol. Cells were detached using 0.1% trypsin/0.1% EDTA, washed, then fixed, and permeabilized in 4% paraformaldehyde plus 0.1% saponin (Sigma-Aldrich, Poole, Dorset, U.K.) for 20 min on ice. Myofibroblasts were labeled with either: FITC-conjugated mouse monoclonal anti-aSMA (Sigma-Aldrich) or isotype control FITCconjugated mouse IgG 2a ; secondary Abs labeled with FITC were applied if appropriate. Analysis was performed using single-color flow cytometry on an FACScan (BD Biosciences, Oxford, U.K.).

Immunofluorescence
HLMFs were grown on eight-well chamber slides and serum-starved for 24 h prior to the experiment. The cells were left unstimulated and incubated in the presence of 0.1% ethanol vehicle control or LXA 4 at 10 210 and 10 28 mol for 48 h. Cells were then immunostained as described previously (27) using FITC-conjugated mouse monoclonal anti-aSMA (F3777; 10 mg/ml, Sigma-Aldrich) and isotype control FITC-conjugated mouse IgG 2a (X0933, 10 mg/ml; DakoCytomation, Ely, U.K.). Cells were mounted with fluorescent mounting medium and coverslipped. Original images were captured on an epifluorescent microscope (Olympus BX50; Olympus UK, Southend-on-Sea, U.K.); grayscale intensity was examined using Cell F imaging software (Olympus UK). Matched exposures were used for isotype controls.
Actin stress fibers were calculated using a specialized macro on Image J (National Institutes of Health) designed by Dr. Kees Straatman, University of Leicester (15). The macro is capable of providing a quantitative, unbiased score of the number of stress fibers per individual cell by determining the fluctuations of grayscale intensity created by the aSMA staining within the stress fibers.

Collagen gel contraction assay
HLMFs were serum-starved for 24 h and then pretreated for 24 h with serumfree medium alone, 0.1% ethanol control, LXA 4 10 210 , or LXA 4 10 28 mol. Cells were detached and embedded in collagen gels as described previously (31). TGF-b1 was then added to appropriate wells to a final concentration of 10 ng/ml. Photographs were taken at 0 and 24 h. The surface area was measured at each time point using ImageJ software (National Institutes of Health; http://rsbweb.nih.gov/ij/).

Smad2/3 nuclear localization
HLMFs were grown on eight-well chamber slides and serum-starved for 24 h prior to the experiment. The cells were either unstimulated or stimulated with TGF-b1 (10 ng/ml) in the presence of serum-free medium alone, 0.1% ethanol control, LXA 4 10 210 mol, or LXA 4 10 28 mol. After 1 h, cells were immunostained using rabbit monoclonal anti-Smad2/3 (0.174 mg/ml; Cell Signaling Technology). Secondary Ab labeled with FITC (F0313; Dako-Cytomation) was applied and the cells counterstained with DAPI (Sigma-Aldrich). Cells were mounted with fluorescent mounting medium and coverslipped. Images were analyzed as above. The intensity of nuclear Smad2/3 staining was quantified by measuring the grayscale intensity of DAPI-positive nuclei to whole-cell staining.

Quantitative RT-PCR
HLMF RNA was isolated using the RNeasy Plus Kit (Qiagen, West Sussex, U.K.) according to the manufacturer's instructions. Primers were designed for aSMA (ACT2A): forward, 59-TTCAATGTCCCAGCCATGTA-39 and reverse, 59-GAAGGAATAGCCACGCTCAG, product size 222 bp from the National Center for Biotechnology Information Reference sequence NM_001141945.1; collagen type I (COL1A1) forward, 59-TTCTGCAA-CATGGAGACTGG and reverse, 59-CGCCATACTCGAACTGGAATC; and collagen type IV (COL4A1), forward, 59-GGACTACCTGGAACAA-AAGGG and reverse, 59-GCCAAGTATCTCACCTGGATCA, product size 240 bp from reference sequence NM_001845.4; b-actin primers were analyzed using gene-specific Quantitect Primer Assay primers (Qiagen), HS_ACTB_1_SG. All expression data were normalized to b-actin and corrected using the reference dye ROX. Gene expression was quantified by realtime PCR using the Brilliant SYBR Green QRT-PCR 1-Step Master Mix (Stratagene). PCR products were run on a 1.5% agarose gel to confirm the product amplified was the correct size, and each of the products was sequenced to confirm the specificity of the primers.

Collagen secretion assay
HLMFs were cultured in serum-free medium alone or 0.1% ethanol control and stimulated with TGF-b1 10 ng/ml in the presence of ethanol control, and LXA 4 at 10 210 or 10 28 mol for 24 h. Soluble collagen re-leased by HLMFs was quantified using the Sircol collagen assay (Biocolor, County Antrim, U.K.) according to the manufacturer's instructions (27,32,33).

Proliferation assay
HLMFs were seeded into six-well plates, and when 50%, confluent cells were serum starved for 24 h in serum-free medium. Cells were then stimulated with serum-free medium plus 0.1% ethanol, 10% FBS medium plus 0.1% ethanol, or 10% FBS plus LXA 4 at 10 210 and 10 28 mol. After 48 h, cells were mobilized with 0.1% trypsin/0.1% EDTA and counted using a standard hemocytometer. Cell viability was assessed by trypan blue exclusion. Results were counted by two blinded observers with high agreement (intraclass correlation of 0.969). All conditions were performed in duplicate.

Statistical analysis
Experiments from an individual donor were performed either in duplicate or triplicate, and a mean value was derived for each condition. Data distribution across donors was tested for normality using the Kolmogorov-Smirnov test. For parametric data, the one-way ANOVA or repeatedmeasures ANOVA for across-group comparisons was used followed by the appropriate multiple-comparison post hoc test; otherwise an unpaired or paired t test was used. For nonparametric data, the Friedman test was used for across group comparisons followed by the appropriate multiple-comparison post hoc test, or the Wilcoxon signed-rank test was used where there were paired groups. GraphPad Prism for windows (version 6; GraphPad Software, San Diego, CA) was used for these analyses. A p value , 0.05 was taken to assume statistical significance, and data are represented as mean (6 SEM) or median (interquartile ratio).
NFC-and IPF-derived HLMFs expressed ALXR (FPRL1) with a whole population shift in comparison with the control Ab (p = 0.0056, Mann-Whitney U test) (Fig. 1A). There was a trend for a higher expression in the IPF-derived myofibroblasts, although this was not statistically significant, and expression of ALXR did not change following 24 h of TGF-b1 stimulation (Fig. 1B). Thus, HLMFs express LXA 4 receptors.
Constitutive aSMA expression and actin stress fiber formation is inhibited by LXA 4 We and others have shown that fibroblasts obtained from human lung parenchyma express high quantities of aSMA and have the typical myofibroblast contractile phenotype (27,29,35,36). Furthermore, these HLMFs derived from IPF donors express higher numbers of actin stress fibers constitutively than cells from NFC donors (15). After 48 h of treatment with LXA 4 (10 210 and 10 28 mol), the amount of constitutive aSMA staining was dose dependently reduced ( Fig. 2A-C). Following treatment with LXA 4 cells were less stellate and became more spindle like, a similar morphology to inactivated fibroblasts (Fig. 2D). IPF-derived HLMFs again expressed more aSMA stress fibers at baseline in comparison with NFC-derived cells (p = 0.0177). LXA 4 reduced the number of actin stress fibers in both IPF-and NFC-derived cells and to a similar extent (Fig. 2E, 2F).

Constitutive HLMF contraction is inhibited by LXA 4
One of the main features of a myofibroblast is its ability to drive tissue repair and wound closure by reorganizing the ECM via contraction (37). We therefore investigated the effects of LXA 4 on constitutive HLMF contraction. HLMF contraction was examined over 24 h in the presence of LXA 4 at 10 210 and 10 28 mol using collagen gels (Fig. 3A). With both NFC-and IPF-derived HLMFs, basal contraction was dose dependently reduced by LXA 4 (p = 0.0009 and p = 0.0007, respectively, for LXA 4 at 10 28 mol) (Fig. 3B).

Constitutive HLMF Smad2/3 nuclear translocation is inhibited by LXA 4
It has previously been reported that aSMA expression and stress fiber formation in myofibroblasts is regulated in part by the TGF-b1/ Smad signaling pathway (10,11). As LXA 4 inhibited both consti- tutive aSMA expression and HLMF contraction, we examined whether LXA 4 disrupts constitutive Smad2/3 nuclear translocation using immunofluorescent staining. HLMFs had detectable Smad2/3 within the nucleus at baseline, and the IPF-derived HLMFs displayed a higher proportion of nuclear staining in comparison with the NFC-derived cells (p = 0.043), in keeping with previous work (15). This nuclear Smad2/3 immunostaining was reduced and to a similar extent in both IPF-and NFC-derived HLMFs in the presence of LXA 4 (10 28 mol) for 1 h (Fig. 3C, 3D).
TGF-b1-induced aSMA expression and contraction are inhibited by LXA 4
To study the effects of LXA 4 on TGF-b1-dependent HLMF contraction, HLMFs were cultured within collagen gels and their contraction monitored over 24 h. Pictures were taken at 0 and 24 h and the contraction measured using ImageJ software (National Institutes of Health); representative images are displayed in Fig. 4E. TGF-b1 stimulation increased HLMF contraction in comparison with vehicle control (0.1% ethanol) (p = 0.0026), with no differences in response between NFC-and IPF-derived cells (Fig. 4F). LXA 4 significantly reduced TGF-b1-dependent HLMF contraction at both 10 210 mol (p = 0.0057) and 10 28 mol (p = 0.0006). Thus, LXA 4 not only inhibits constitutive HLMF aSMA expression and contraction, but also the increased aSMA expression and contraction induced by the potent profibrotic mediator TGF-b1 (38,39).

Discussion
The myofibroblast is implicated as the key cell driving the progression of IPF through the synthesis of excess fibrotic extracellular matrix and tissue contraction. The myofibroblast is therefore an attractive target for novel antifibrotic therapies. In this study, we have demonstrated that HLMFs express LXA 4 receptors and that LXA 4 attenuates: 1) constitutive HLMF aSMA expression, stress fiber formation, contraction, and Smad2/3 nuclear localization; 2) TGF-b1-dependent increases in HLMF aSMA expression, contraction, collagen mRNA expression, collagen secretion, and Smad2/3 nuclear localization; and 3) serum-dependent HLMF proliferation.
Fibroblasts and myofibroblasts demonstrate marked functional and phenotypic heterogeneity between tissues and across species. As an example, there is a marked difference in the phenotype of fibroblasts grown from human airway and lung parenchyma, with those from lung spontaneously displaying a myofibroblast phenotype (29,35,36). When considering novel therapeutic targets, it is therefore important to study cells from the species, tissue compartment and disease of interest. Expression of the LXA 4 receptor ALXR (FPRL1) has been demonstrated previously in human synovial fibroblasts by RT-PCR (41), and these cells and rat fibroblast cell lines respond to LXA 4 implying functional receptor expression (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). In this study, we have demonstrated that ALXR protein is expressed on the surface of the majority of parenchymal HLMFs derived from both NFC and IPF tissue.
In a rat fibroblast cell line, LXA 4 at 10 29 mol attenuated TGF-b1dependent Smad2 phosphorylation (but not Smad3 phosphorylation) and MAPK activation, and inhibited TGF-b1-dependent gene transcription (22). We also found that LXA 4 inhibited TGF-b1dependent gene transcription, but in contrast to Börgeson et al. (22), we could not detect inhibition of Smad2/3 phosphorylation, although there was reduced TGF-b1-dependent nuclear Smad2/3 translocation. The means by which LXA 4 would prevent nuclear translocation of activated Smads without affecting phosphorylation is intriguing and will require further work to elucidate the mechanism. Nevertheless, the ability of LXA 4 to inhibit Smad nuclear localization is in keeping with its ability to inhibit TGF-b1-dependent gene transcription and the downstream production of collagen and aSMA in HLMFs.
Several phenotypic differences have been observed previously between control and IPF-derived HLMFs in culture, suggesting that there may be genetic and/or epigenetic changes that promote a profibrotic HLMF phenotype in patients who develop IPF. Interestingly, we found that LXA 4 not only reduced TGF-b1-dependent profibrotic responses, but promoted HLMF differentiation toward a fibroblast phenotype by reducing constitutive aSMA actin expression and actin stress fiber formation. This in turn is likely to explain the reduced constitutive HLMF contraction evident in collagen gels. This occurred in conjunction with a reduction in nuclear Smad2/3 immunostaining, which occurred within 1 h of LXA 4 exposure, suggesting there is a component of constitutive Smad2/3 signaling in HLMFs at rest. This ability of LXA 4 to promote dedifferentiation toward a fibroblast phenotype suggests that if there is a degree of constitutive profibrotic HLMF activity in IPF lungs due to genetic/ epigenetic factors, LXA 4 or a stable analog might be a particularly useful therapy.
In addition to the inhibition of TGF-b1-dependent stimulation, we found that LXA 4 also inhibited HLMF proliferation induced by serum, and this is in keeping with previous work showing that LXA 4, inhibited connective tissue growth factor-dependent proliferation of a human fibroblast cell line (25). Previous studies using LXA 4 in cell-culture systems suggest it is highly active in the nanomole range. Our data are consistent with this, with effects readily evident in HLMFs at 10 210 mol (0.1 nmol), although more consistent at 10 28 mol.
In summary, we have shown that LXA 4 inhibits many TGF-b1dependent profibrotic responses in healthy and IPF-derived HLMFs, which may result from the inhibition of Smad2/3 nuclear translocation. Furthermore, LXA 4 promotes HLMF dedifferentiation in the resting state, suggesting it may have the potential to reverse the fibrotic process. In support of this, and of particular relevance to this study, a stable epi-LXA4 analog markedly inhibited bleomycininduced pulmonary fibrosis in mice when administered either preventively or curatively, with reversal of fibrosis evident in the latter (26). This was associated with a reduction in the accumulation and differentiation of myofibroblasts in the lung parenchyma. Thus, there are consistent in vitro and in vivo data from primary HLMFs and the mouse bleomycin model, respectively, indicating that LXA 4 or its stable analogs may not only prevent the progression of lung fibrosis, but also potentially reverse it. This indicates that clinical trials of LXA 4 analogs should be considered in patients with IPF.

Disclosures
The authors have no financial conflicts of interest.