Tnf α

ELISA assay was performed to quantify IL-1β, IL-10, IL-12, and TNF-α (BD Biosciences) from lung homogenate and culture supernatants. The minimum detectable concentrations are 62.5 pg.mL⁻¹ for IL-12 (BD Pharmingen) and 31.25 pg.mL⁻¹ for IL-1β and IL-10, respectively (BD Pharmingen), and 15.65 pg.mL⁻¹ for TNF-α (BD Pharmingen). All procedures were performed according to the manufacturer’s instructions.

Cells were incubated with surface markers against pre-conjugated antibodies CD45-APC (eBioscience, San Diego, CA) and CD86-FITC (BD bioscience, San Jose, CA) for 20 min. Cells were then permeabilized for intracellular staining with a fixation/permeabilization solution kit (eBioscience, San Diego, CA). Cells were then separated into two groups and incubated with markers CD206 (Abcam, Cambridge, MA), TNFα (BD bioscience, San Jose, CA), and IL-10 (BD bioscience, San Jose, CA), or TNFα (BD bioscience, San Jose, CA) and TMEM119 (Novus, Centennial, CO). Secondary antibodies PE (eBioscience, San Diego, CA) and PerCP (BD Bioscience, San Jose, CA) were used in both groups. Cells were then washed and analyzed using the Cytoflex (Beckman Coulter, Indianapolis, Indiana).

Before migration experiments, immune cells were isolated from 8-week-old Cort^+/+ mice. Macrophages and T cells were isolated as described in Additional file 1. Immune migration was evaluated in a BECs monolayer grown onto coated inserts of 24-well plates, as described above. 24 h before the assay, cells were activated with TNF-α (10 ng/ml, BD Pharmigen). On the day of the experiment, transwells inserts were transferred to a new 24-well culture plate containing 1% FBS medium in the bottom side supplemented with MCP-1 (50 μg/ml, Bionova) or IP-10 (50 μg/ml, Bionova) for macrophages and T cells, respectively. Immediately, immune cells (2 × 10⁵/transwell) were added to the upper side of the transwell in contact with BECs. When indicated, mouse cortistatin-29 (100 nM) was added to both sides of the transwell. After 24 h, migrated T cells were counted on the bottom side using a Neubauer chamber (VWR). Macrophages were identified at the bottom of the well after plate fixation and DAPI staining. As a positive control of migration, macrophages/T cells were incubated in a BECs-free coated transwell. The number of migrated cells was represented as the percentage of immune migrated cells vs the positive control.

Before migration experiments, immune cells were isolated from 8-week-old Cort^+/+ mice. Macrophages and T cells were isolated as described in Additional file 1. Immune migration was evaluated in a BECs monolayer grown onto coated inserts of 24-well plates, as described above. 24 h before the assay, cells were activated with TNF-α (10 ng/ml, BD Pharmigen). On the day of the experiment, transwells inserts were transferred to a new 24-well culture plate containing 1% FBS medium in the bottom side supplemented with MCP-1 (50 μg/ml, Bionova) or IP-10 (50 μg/ml, Bionova) for macrophages and T cells, respectively. Immediately, immune cells (2 × 10⁵/transwell) were added to the upper side of the transwell in contact with BECs. When indicated, mouse cortistatin-29 (100 nM) was added to both sides of the transwell. After 24 h, migrated T cells were counted on the bottom side using a Neubauer chamber (VWR). Macrophages were identified at the bottom of the well after plate fixation and DAPI staining. As a positive control of migration, macrophages/T cells were incubated in a BECs-free coated transwell. The number of migrated cells was represented as the percentage of immune migrated cells vs the positive control.

Surface and intracellular staining of freshly isolated PBMCs was performed using appropriate combinations of antibodies for detection of CD3, CD4, CD28, CD25, CD127, FoxP3, CTLA-4, IL-10, TNF-α, Th1-type cytokines IL-2 and IFN-γ, Th2-type cytokine IL-4 and Th17-type cytokine IL-17 (all Becton Dickinson, San Diego, CA, USA), and γH2AX (Cell Signaling, Danvers, MA, USA). Surface staining for 20 min was followed by permeabilization for 30 min and intracellular staining for 30 min according to a routine protocol. Golgi transport was inhibited by brefeldin A (10 ng/ml) or monensin (10 ng/ml) 4 h prior to cytokine staining. Appropriate isotype controls were used. Stained cells were analyzed on a FACS Canto II (Becton Dickinson). Data are analyzed with DIVA software and FlowJo.

Cytokine levels of supernatants were measured with a human enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer's instructions. The ELISA kit of IL-10 was manufactured by Pierce Biotechnology, Illinois, USA. The IL-6, TGF-β1, and IL-17 were purchased from R&D Systems, Inc., Minnesota, USA. The IL-12, TNF-α, and IL-1β were purchased from Becton Dickinson, CA, USA. Cytokine responses were defined as the difference in supernatant levels with and without LPS stimulation. Negative responses were set as 0 pg/mL. Changes in cytokine responses were defined as the difference in cytokine response on day 7 minus the cytokine response on day 1.