MP improved lung function and ameliorated tissue injury

We detected lung function at 6, 18 and 36 h in ALI model with or without MP treatment. Compared to the LPS group, MP significantly increased the peak expiratory flow (PEF) (Fig. 1a), maximal mid-expiratory flow (MMF) (Fig. 1b) and forced expiratory volume 0.2 (FEV_0.2)/forced vital capacity (FVC) (Fig. 1d) at 18 and 36 h. At 18 h after ALI, H&E staining showed interstitial edema, interalveolar septal thickening and intra alveolar and interstitial patchy hemorrhage in the LPS group (Fig. 2a). After MP treatment, the tissue injury was significantly attenuated (Fig. 2b). As for pulmonary edema evaluation, lung weight and dry ratio (W/D) was employed. The mice received LPS injection showed higher W/D than that in the MP group (Fig. 2c). In addition, MP treatment significantly improved acid–base disturbance, increased PaO₂ and reduced PaCO₂ (Fig. 2d).

MP ameliorated inflammation in vivo

To determine the inflammation level, we detected typical pro-inflammatory cytokines and chemokines in BAFL at 18 h after ALI. The expression of TNF-α, IL-2, IL-6, IL-12p40, CCL4 and CCL5 decreased in the MP-treated group. However, MP treatment increased the level of IL-4, IL-10, IL-13 and TGF-β (Fig. 3). These results suggested that MP inhibited the pro-inflammatory response, and reconstituted inflammatory microenvironment.

MP regulated the ratio of macrophage subsets in vivo

Next, we investigated the ratio of M1/M2 in the lung by flow cytometry (Fig. 4a). At 18 h after LPS injection, F4/80⁺iNOS⁺ M1 was significantly increased. Cmaf is an appropriate marker of M2 [15], so we gated Cmaf from F4/80⁺ cells. In the LPS group, F4/80⁺Cmaf⁺ M2 was decreased. However, MP treatment reduced M1 and increased M2 in the lung. No difference was observed between the control and MP treatment groups at 6 and 36 h (Fig. 4b), suggesting MP administration restored the balance of macrophage subsets at the middle stage after ALI.

MP promoted M2 differentiation in vitro

Since MP increased the M2 percentage in vivo, we then further investigated whether MP directly regulates macrophage differentiation in vitro. It has been demonstrated that adoptively transfer M2a or M2c into LPS-induced ALI mice reduced ALI [11]. We, therefore, detected the M2a and M2c percentage with different dose of MP (Fig. 5a). In the presence of IL-4 and IL-13, MP increased the CD206⁺ M2a percentage in a dose-dependent way (Fig. 5b). After stimulation by IL-10 and TGF-β, the percentage of CD163⁺ M2c was also increased in the MP-treated group. The trend presented in a dose-dependent way (Fig. 5b). This result indicated that MP promoted M2a and M2c differentiation.

M2c induced more Tregs than M2a

Tregs play an important role in inflammation resolution in ALI [16, 17]. We assumed M2 induce Tregs and ameliorated inflammation. Therefore, these induced-M2a and M2c were isolated by flow cytometry and co-cultured with CD4⁺ naïve T cells under Tregs induction environment. We detected CD4⁺CD25⁺Foxp3⁺ Tregs and CD4⁺IL-17a⁺RORγt⁺ Th17 (Fig. 6a). As expected, the pro-inflammatory M1 induced much more percentage of Th17. Compared to M0 and M1, both M2a and M2c significantly induced Tregs and inhibited Th17. Intriguingly, M2c induced more Tregs than M2a (Fig. 6b). To clarify the impact on Tregs immunosuppression function by M2, we further isolated Tregs that were induced by M2c. Compared to the normal Tregs, the expression of IL-10 and TGF-β in supernatant has no difference in M2c-induced Tregs (Fig. 6c). At last, mix lymphocyte reaction was performed to compare the immunosuppression function between the two groups of Tregs. The result further confirmed that M2c did not influence on Tregs immunosuppression function although increased their number (Fig. 6d).

M2c induced Tregs through IL-10 and TGF-β secretion

To elucidate the mechanism that M2c induces Tregs, we added anti-IL-10 and/or anti-TGF-β antibodies in the co-culture system (Fig. 7a). Either anti-IL-10 or anti-TGF-β antibody significantly reversed Tregs induction. In the mixed antibodies group, the number of Tregs is the lowest (Fig. 7b). The result indicated that IL-10 or TGF-β pathway is the key mechanism that involved in the induction of Tregs by M2c.

Animals

All animal use procedures were approved by the Committee on the Ethics of Animal Experiments of Zhongshan Hospital, Fudan University. This investigation was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Six weeks old specific pathogen-free male BALB/c mice (16–20 g) were maintained under specific pathogen-free conditions in the facilities of the animal center. The mice were kept in a temperature controlled room with 12 h dark/light cycles, and allowed food and water ad libitum. Animals underwent an acclimatization period of at least 14 days before use in our study.

ALI model

A total of 35 BALB/c mice were randomly divided into three main groups, control group (n = 5), LPS (Sigma, St. Louis, MO, USA) group (n = 15), MP (Pfizer, New York, NY, USA) treatment group (n = 15). For LPS and MP groups, the mice were subdivided into six subgroups (n = 5). Mice were sacrificed at 6, 18 and 36 h. ALI was induced by LPS via intravenous injection as previously described [28–32]. The mice in control group were administrated the sterile saline instead. Ten minutes after LPS injection, MP was given by intraperitoneal injection at 5 mg/kg in a single dose.

Measurement of lung mechanics

Each mouse was weighed, anesthetized by an intraperitoneal injection of pentobarbital sodium (40 mg/kg). Tracheostomy was performed and the trachea was cannulated with an 18-gauge intravenous catheter. After the tracheostomy, mice were placed in a body plethysmograph and connected to a computer-controlled ventilator. Lung mechanics were analyzed by the system of AniRes2005 (Bestlab High-Tech Co., Ltd, Beijing, China).

Lung wet/dry weight ratio

The severity of pulmonary edema was assessed by the wet to dry ratio (W/D ratio). The right lower lungs weighed and then dehydrated at 60 °C for 72 h in an oven.

Tissue injury score and arterial blood gas analysis

The left lower lung from each mouse was fixed in 10% formalin, embedded in paraffin, cut into 5 mm sections, stained with H&E. Lung injury score was measured by a blinded pathologist with a 0–4 point scale according to combined assessments of inflammatory cell infiltration in the airspace or vessel wall, alveolar congestion, hemorrhage, alveolar wall thickness and hyaline membrane formation. A score of 0 represented no damage; l represented mild damage; 2 represented moderate damage; 3 represented severe damage and 4 represented very severe histological changes. We collected blood from the abdominal aorta and performed arterial blood gas analysis.

Cytokines expression in bronchoalveolar lavage fluid

Bronchoalveolar lavage fluid (BALF) was collected at 18 h after LPS injection by cannulating the upper part of the trachea, by lavage three times with 1.0 ml PBS (pH 7.2). The fluid recovery rate was more than 90%. Lavaged sample from each mouse was kept on ice. BALF was centrifuged at 700g for 5 min at 4 °C. The BALF supernatant was collected after centrifugation (for 4 min at 4000 rpm) and stored at −80 °C before cytokine assay. TNF-α, IL-2, IL-4, IL-6, IL-10, IL-12 (p40), IL-13, TGF-β, CCL4 and CCL5 were measured by ELISA (R&D Systems, Minneapolis, MN, USA) as previously described [33].

Flow cytometry

The single cell suspension of lung was prepared as previously described [34]. Thereafter all single cells were resuspended in staining buffer (BD Bioscience, San Diego, CA, USA). The following monoclonal antibodies were added into the single-cell suspension according to manufacturers’ protocols: F4/80, CD163, CD206, Cmaf (eBioscience, San Jose, CA, USA). For Th17 and Treg detection, cells were stained with surface markers, fixed and permeabilized with fixation buffer [Dulbecco’s phosphate-buffered saline (pH 7.4) with 4% w/v paraformaldehyde (0.22 µm pore-filtered)]/permeabilization buffer [PBS, 1% fetal calf serum, 0.1% sodium azide, 0.1% saponin (0.2 µm pore-filtered; pH7.4-7.6); eBioscience] and then stained with RORγt and Foxp3 antibodies (eBioscience).

Cytokines in cell culture supernatant

The supernatant was collected and stored at −80 °C until further analysis. The level of IL-10 and TGF-β was measured using Mouse IL-10 and TGF-β ELISA Kit (Abcam, Cambridge, UK) in accordance with the manufacturer’s protocol respectively as previously described [35].

Macrophage culture and differentiation

BALB/c mice splenocytes were harvested and triturated with sterile syringes, and the resulting cell suspension was filtered through 40 mm nylon mesh and was incubated at 37 °C for 40 min, and then the culture supernatant that contained floating cells (T cells, natural killer cells, and dendritic cells) was discarded. The adherent spleen-derived macrophages were rinsed three times in RPMI-1640 medium and cultured for 48 h with the normal medium to be M0 (resting macrophages), with IL-4/IL-13 (10 ng/ml each) to become M2a and with IL-10/TGF-β (10 ng/ml each) to become M2c macrophages. IL-4, IL-13, IL-10, and TGF-β were purchased from Invitrogen (Mount Waverly, Australia).

Mixed lymphocyte reaction

Mononuclear cells (MNCs) of spleens from mice were obtained by Ficoll density gradient centrifugation. CD4⁺ T cells were magnetically purified from MNCs according to the manufacturer’s recommendations (Miltenyi Biotec, Auburn, CA, USA). The purity of sorted cells in this study was consistently more than 95%. Tregs with or without M2c induction were lethally irradiated (30 Gy). The Tregs were then cultured in graded doses with CD4⁺ T cells (3 × 10⁵ cells/well) in RPMI 1640 medium for 5 days. [³H]Thymidine (1 μCi/well, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, China) was added 18 h before the end of the culture period. The cells were then harvested onto glass fiber mats for the measurement of [³H]thymidine incorporation.

Statistical analysis

Results are expressed as the mean ± standard deviation (S.D.). Statistical analysis was performed using the Student’s t test (between two groups) and one-way ANOVA (among three or more groups) by SPSS 19.0 software (SPSS, Inc., Armonk, NY, USA). The Scheffe test was used for post hoc analysis. P < 0.05 was recognized as statistically significant.