Methylprednisolone

Recombinant Sh P28GST protein was expressed in Saccharomyces cerevisiae culture and purified under Good Manufacturing Practice conditions by Eurogentec S.A (Seraing, Belgium). Batches of P28GST (batchM-BIX-P03-225a) were conserved lyophilized in NH₄HCO₃ 10mM and 2.8% lactose. Control batches contained only NH₄HCO₃ and lactose. These preparations were re-suspended extemporaneously using NaCl 0.9% at the appropriate concentrations. Lyophilized GST from human placenta was purchased from Sigma-Aldrich and prepared according to the manufacturer’s instructions. For heat-inactivation, P28GST and GST from human placenta were incubated 15 minutes at 95°C. Quercetin was used as positive control (Sigma-Aldrich, Saint-Louis, Missouri). A commercial preparation of Methylprednisolone (Mylan, Canonsburg, Pennsylvania) was resuspended extemporaneously using 0.9% NaCl solution and administrated to mice by intraperitoneal injections at (2 mg/kg) at day 1 and day 2 after colitis induction. 2,4,6- Trinitrobenzenesulfonic acid (TNBS) (Sigma-Aldrich, Saint-Louis, Missouri) was used for colitis induction.

Monocytes immediately after adherence, or 7-day, 14-day, or 21-day culture macrophages were treated with 0.1 or 2 μg/mL LPS or 0.01, 1, 10 μM dexamethasone (Mylan, Saint Priest, France) for 24 h in the absence of HS unless otherwise stated. Results of time- and concentration-dependent experiments are presented in the supplementary data. Based on these results (S1 and S2 Figs), most of the treatments were done, unless otherwise specified, using 100 ng/mL LPS and 1μM dexamethasone. Methylprednisolone (Mylan 20 mg, Saint Priest France), another synthetic glucocorticosteroid was used on selected experiments performed on 7-day culture macrophages from 4 donors.
Monocytes were alternatively treated after washing with 1X PBS with 100 ng/mL IL1β (Cat No. 200-01B, Peprotech), 100 ng/mL IL6 (Cat No. 200–06, Peprotech) and 1μM dexamethasone (Mylan) for 24 h to compare with the results observed with the HepG2 cell line.
Following treatment, cell culture supernatants were collected and stored at -20°C for cytokine measurement by ELISA and cell lysates were used for RNA isolation, immunoprecipitation or western blot analysis.

The overall cross-circulation procedure is depicted in Supplemental Figure 1. Recipient pigs (n = 10) were anesthetized as described (19 (link)). Septotryl® (0.08 ml/kg) (Vetoquinol) was injected i.m. before catheterization. In the CST group, 20 mg/kg methylprednisolone (Mylan, Canonsburg, USA) was administered i.v. 1 h prior to the start of cross circulation. After a 25,000 U heparin bolus, the recipient pig was maintained on a continuous heparin infusion (100 U/Kg/h). The superior vena cava was cannulated in the recipient pig with a 20 F double lumen cannula as described (19 (link)). Carboxyfluorescein succinimidyl ester (CFSE, 25 mg, Sigma-Aldrich, Saint-Louis, MS, USA) was administered i.v. in the perfusing pig, diluted in 4 ml DMSO + 40 µl heparin, 30 min before the initiation of cross-circulation. Donor lungs were placed in dorsal position on XVIVO® chambers (XVIVO Perfusion) and the trachea was cannulated with a 7.5 mm diameter cuffed endotracheal tube (Mallinckrodt, Staines-upon-Thames, UK) and connected to a respirator. The vascular tubing was spliced to connect the recipient pig to the dedicated circuit, marking the start of cross-circulation. Sedation was kept for 10 h by permanent administration of 2 mg/kg/h propofol (Proposure®, Axience, Pantin, France) with 0.6% isofluorane and analgesia was achieved by i.v. administration of 0.2 mg/kg nalbuphine every 3 hours.