ResolvinD1 stimulates epithelial wound repair and inhibits TGF-β-induced EMT whilst reducing fibroproliferation and collagen production

Acute and chronic inflammatory lung diseases are often associated with epithelial cell injury/loss and fibroproliferative responses. ResolvinD1 (RvD1) is biosynthesized during the resolution phase of inflammatory response and exerts potent anti-inflammatory and promotes resolution of inflammatory lung diseases. The aim of this study was to investigate whether RvD1 exerts protective effects on alveolar epithelial cell function/differentiation and protects against fibroproliferative stimuli. Primary human alveolar type II cells were used to model the effects of RvD1 in vitro upon wound repair, proliferation, apoptosis, transdifferentiation, and epithelial–mesenchymal transition (EMT). Effects of RvD1 upon primary human lung fibroblast proliferation, collagen production, and myofibroblast differentiation were also examined. RvD1 promoted alveolar type II (ATII) cell wound repair and proliferation. RvD1 protected ATII cells against sFas-ligand/TNF-α-induced apoptosis and inhibition on cell proliferation and viability. RvD1 promoted ATII cells transdifferentiation. Moreover, we demonstrate that RvD1 inhibited EMT in response to TGF-β. Furthermore RvD1 inhibited human lung fibroblast proliferation, collagen production, and myofibroblast differentiation induced by both TGF-β and bronchoalveolar lavage fluid from acute respiratory distress syndrome (ARDS) patients. The effects of RvD1 were PI3-kinase dependent and mediated via the resolvin receptor. RvD1 seems to promote alveolar epithelial repair by stimulating ATII cells wound repair, proliferation, reducing apoptosis, and inhibiting TGF-β-induced EMT. While RvD1 reduced fibroproliferation, collagen production, and myofibroblast differentiation. Together, these results suggest a potential new therapeutic strategy for preventing and treating chronic diseases (such as idiopathic pulmonary fibrosis) as well as the fibroproliferative phase of ARDS by targeting RvD1 actions that emphasizes natural resolution signaling pathways.

with sterile saline to remove blood and debris from the surface. Place the tissue into a fresh Petri dish ready for trypsinisation. Instill the trypsin solution (10-15 ml/5cm 3 piece, Gibco 25300) into the lung tissue in exactly the same way as the saline lavage. Place the covered Petri dish into a 37 °C incubator for 15 min. Repeat the procedure twice more to give a total trypsinisation period for 45 min. Chop the tissue finely into 1-2 mm 3 in the presence of FCS (10ml/5cm 3 piece) and DNase I (250µ g/ml HBSS; Sigma DN25, HB9394). Shake the minced tissue suspension vigorously by hand for 5 min to enhance type II cell recovery. Filter the tissue suspension through a large gauge mesh (400-500 µ m) and then a 40 µ m cell filter (BD Biosciences) to remove undigested tissues and debris from the enzymatically-released epithelial cells, which pass through the filter. Centrifuge the filtrate, containing mostly single cells, at 300g at 12°C for 7 min. Suspend the cell pellets in 50-100 ml 50% DCCM-1 and 50% HBSS containing 100 µ g/ml DNase I. Plate the resuspended cell suspension into either T-75 or T-175 culture flasks and incubate at 37°C for 1.30 h to enable any contaminating macrophages to adhere. Remove the media containing the nonadherent type II cell-enriched cell population and centrifuge at 300g at 12°C for 7 min. Resuspend the cell pellet in 3 ml of red cell lysis buffer and incubate for 3 min. Add enough HBSS to make up the volume and centrifuge the filtrate at 300g at 12°C for 7 min.
Resuspend the cell pellet in a known small volume of (5 ml) 10% DCCM-1(Biological Industries Ltd. Kibbutz Beit-Haemek, Israel) and make up the volume 10 ml. Count the epithelial cells using as haemocytometer by phase contrast microscopy. Filter again if clumps were found. Prepare a cytospin for alkaline phospatase staining if required. Add 10% DCCM-1 so that the cells are 1 x 10 6 epithelial cells/ml and plate onto collagen-coated plates; 1 x 10 6 /well of a 6 well plate (for western blot studies), 0.5 x 10 6 /well of a 24 well plate(for PCR, wound repair and Flow Cytometry supplemented with 10% FCS (sigma) at 37 °C and 5% CO 2 . Cells were subcultured at 60-80% confluence using trypsin/EDTA.Cells were abtained from three separate donors, and all experiments were repeated in triplicate.

Stimuli and Inhibitors
AT II cells and fibroblasts were treated with resolvinD 1 (Cayman Chemical Company,USA) at different concentrations. Inhibitors were used at the following concentrations according to manufacturers' instructions: LY294002, a PI3-kinase inhibitor (Calbiochem, Nottingham, UK) at 10 µ M; and the nonselective FPR antagonist, Boc-2 (N-t-Boc-Phe-Leu-Phe-Leu-Phe; GenScript USA Inc), at 10µ M. Inhibitors were added to cells 1hour prior to every treatment.

Bronchoalveolar Lavage Fluid Collection
BALF from ARDS patients is known to stimulate epithelial repair in the scratch wound assay in an IL-1 dependent fashion. 23 To test whether resolvin D1 could augment or synergise with this effect, the BALF from patients with ARDS were mixed 50:50 with appropriate culture media for each cell type as a positive control stimulus. We used BALF from patients enrolled into the BALTI-1 trial, demographics for whom have been published previously. 24 In Vitro Alveolar Epithelial Wound Repair Assay. Epithelial repair was determined using an in vitro epithelial wound repair assay as described before. 25 Briefly, primary human alveolar type II (AT II) cells were grown to confluent monolayers before wounding with a 1-mL pipette tip. Cells were serum starved for 24 hours before wounding. After

BRDU cell proliferation assay
4 X10 5 cells/ml (AT II cells) or 1.5 X10 5 cells/ml (HLF) were seeded into a 96 well culture dish. BrdU Label was added and cells were incubated with RvD1 or TGF-β (R&D Systems). After 24 hours culture, BrdU incorporation was assessed according to manufacturers' instructions (BRDU Cell Real time PCR was performed using total RNA from primary human alveolar type II (AT II) cells and fibroblats (RNeasy Mini Kit; Qiagen, Hilden, Germany), the cDNA synthesis kit (MBI Fermentas, St. Leon-Rot, Germany), RNA (1ug) was DNase treated at room temperature and reverse transcribed using superscript RTase and random primers, according to the manufacturer's protocol. mRNA expression was analyzed using Taqman primer/probe (Applied Biosystems) and multiplexed with 18S to account for total loading. Relative mRNA amounts were calculated using CT method 27 ∆CT =Ct target-Ct GAPDH, ∆∆CT =Ct treatment-Ct calibrator, where calibrator was the no-treatment group. Ct was then converted to fold change using the formula 2 -∆∆CT . Quantitative PCR was performed using commercially obtained primers. Details of PCR primers are showed in table 1. (Table S1):

Statistical Analysis
Data were normally distributed and analyzed by analysis of variance with Tukey's test for post hoc comparisons using Minitab 14.0 (Minitab, State College, PA). P value <0.05 was considered significant. Data are expressed as mean (SE).

Figure S1. Effect of RvD1 upon effects of soluble Fas-ligand and TNF-alpha on proliferation and cell viability
A: sFasL and TNF-α inhibited cellular proliferation compared with control media-treated cells. This effect was attenuated by 100 nM RvD1.
Experiments were performed using cells from 3 donors.

B: Cellular viability of AT II cells was reduced 24 hours after treatment
with 100 ng/mL sFasL or (and) 100 ng/mL TNF-α. Pre-treatment with RvD1 at 100nM significantly increased the viability of sFasL or (and) TNFα treated cells after 24 hours.