Cortisol

Serum was isolated using serum-separating tubes (BD Biosciences). Quantitative detection of cortisol (Alpco, Salem, NH, USA) and dehydroepiandrosterone (DHEA; Alpco), was determined by means of ELISA according to manufacturer's instructions. Levels of the primary human anti-inflammatory glucocorticoid, cortisol, are often insufficient to evaluate the function of the hypothalamus-pituitary-adrenal (HPA) axis. Therefore, we also quantified the serum concentration of DHEA, a precursor of the sex hormones testosterone and estrogen and a glucocorticoid antagonist, in order to calculate the molar ratio of cortisol to DHEA [29 (link), 30 (link)]. In addition, serum levels of IL-6, IL-10, IL-12p70, TNF-α, MMP-9, and the inflammasome effector molecule caspase-1 (R&D, Minneapolis, MN, USA) were quantified using ELISA according to manufacturer's instructions. All kits were purchased from Meso Scale Discovery (MSD, Rockville, MD, USA), unless stated otherwise. For quantitative detection of the cytokines secreted following stimulation of peripheral blood, collected plasma samples were analyzed using the following commercially available ELISA kits: IL-1β, IL-6, IL-12p70, TNF-α, IFN-α (PBL InterferonSource, Piscataway, NJ, USA), caspase-1 (R&D), and MMP-9, according to manufacturer's instructions. All kits were purchased from eBioscience, unless stated otherwise.