Tumor necrosis factor (tnf)

Cell culture supernatants from human PHHs (n = 3–5), KCs (n = 3–5) and PBMCs (n = 3–5) were collected 24 h after treatment with the different concentrations (0.05, 0.5, 5 and 50 μM) of NAPs and TLR ligands Poly(I:C) [25 μg/ml], ssRNA40, [10 μg/ml] and ODN2216 [2 μM] (scheme shown in Fig. 1A). Cytokine secretion of IFNA4, IFNB1, IFNG, IFNL2, TNF, IL6 and IL10 were determined by ELISA according to the manufacturer’s instructions (IFNA4, IFNB1, IFNG, TNF, IL6, IL10 [R&D Systems, Wiesbaden, Germany]; IFNL2 [IL28A, RayBiotech Inc, Norcross, GA, USA]). Untreated controls represent basal cytokine secretion.

Hemodynamics, gas exchange (calorimetric O₂ uptake and CO₂ production), arterial blood gas tensions, acid-base status, glucose, lactate, creatinine, neutrophil gelatinase-associated lipocalin (NGAL), aspartate transaminase (AST), alanine transaminase (ALT), 8-Isoprostane, bilirubin, and troponin were determined as described previously (32 (link), 35 (link)–37 (link)). In brief, blood gas analysis, glucose, and lactate levels were measured using a standard blood gas analyzer (ABL 800 Flex, Radiometer GmbH, Krefeld, Germany). Creatinine (#KIT044, BioPorto, Hellerup, Denmark), AST (#AS 1204, Randox, Crumlin, Northern Ireland), ALT (#AL 1205, Randox), 8-Isoprostane (#516351, Cayman Chemical, Ann Arbor, USA), bilirubin (#BR 2361, Randox), and troponin (#2010-4-HSP, Life Diagnostics, West Chester, USA), tumor necrosis factor (TNF, #PTA00, R&D Systems, Minneapolis, USA), interleukin 6 (IL6, #P6000B, R&D Systems), interleukin 10 (IL10, #P1000, R&D Systems), and super oxide dismutase (SOD, #S311, Dojindo Molecular Technologies, Rockville, USA) were determined as recommended by the manufacturer.

Human primary fibroblasts were seeded at 1 × 10⁵ cells per well on coverslips placed in 12-well plates containing RPMI-1640 supplemented with antibiotics and 1% FBS. Before stimulations the cells were synchronized as described above. For cellular stimulation the media were replaced with RPMI-1640 containing antibiotics and 1% FBS, without or with 10 ng/ml TNF (R&D Systems). After 24 h cells were washed with PBS and fixed in 4% PFA for 15 min at RT. Fixed cells were permeabilized with 0.1% Triton-X in PBS for 10 min at RT, blocked with 1% BSA-PBS for 1 h at RT, after which slides were incubated with 4 μg/ml rabbit anti-human ARNTL2 IgG (Santa Cruz Biotechnology, Dallas, USA; cat# sc-98300 X) or 4 μg/ml non-immune rabbit IgG at 4°C overnight. Next day slides were incubated in 1:100 dilution of Alexa Fluor 568 labeled goat anti-rabbit IgG secondary antibody (Molecular Probes, Leiden, The Netherlands; cat# ab175471) for 1 h at RT, counterstained in 5 μg/ml DAPI and mounted.

FTY720 (C/N 10006292) and FTY720-P (C/N 10008639) was purchased from the Cayman Chemical Company (Ann Arbor, MI). TNF was from R&D Systems (Minneapolis, MN). Unless otherwise specified, all chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO).