Interleukin 6 (il 6)

Considering linkages between inflammation and depressed mood in HF,8, 9, 26 we used commercial assays to assess the inflammatory biomarkers interleukin‐6 (IL‐6) and tumor necrosis factor‐R1 (TNF‐R1) (MSD, Rockville, Maryland). We also assessed C‐reactive protein (MSD, Rockville, Maryland) and cardiac biomarkers B‐type natriuretic peptide (BNP) immunoassay (Bayer, Tarrytown, New York) and soluble ST2 (sST2) (R&D Systems, Inc., Minneapolis, Minnesota). Whole blood was preserved with EDTA acid. Following centrifugation the plasma was stored at −80°C until assay. Intra‐ and inter‐assay coefficients were <7%).

The following biomarkers were measured to assess inflammatory profiles in study participants: C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-a), interleukin-10 (IL-10), monocyte chemoattractant protein 1 (MCP-1), chemerin, omentin, leptin, total adiponectin, high molecular weight (HMW) adiponectin, and fetuin-A. Non-HMW adiponectin was estimated based on the difference between total and HMW adiponectin. Venous blood samples were immediately centrifuged and frozen at −80 °C until analysis. CRP concentrations were determined by a highly sensitive immunoturbidimetric assay using ABX Pentra 400 reagents on an ABX Pentra 400 (Horiba ABX, Montpellier, France). Commercially available ELISA kits were used for the measurements of serum leptin (R&D Systems, Minneapolis, MI, USA), total adiponectin, chemerin, omentin, fetuin-A (all from Biovendor, Germany), and high molecular weight adiponectin (Merck Millipore KGaA, Darmstadt, Germany) and U-Plex assay was used to measure IL-6, TNF-a, MCP-1, and IL-10 (MSD, Rockville, ML, USA). Table S4 provides the detection limits of the kits for the measured biomarkers.

Hippocampal tissue was dissected from whole brain and frozen at −80 °C before homogenization. Tissue was homogenized in RIPA buffer (Thermo Scientific, Waltham, MA, USA) and PIC (HALT; Thermo Scientific, Waltham, MA, USA) using a tissue homogenizer (Bullet Blender; Next Advance, Troy, NY, USA). Protein concentration was determined using an infrared spectrometer (Direct Detect; Millipore, Burlington, MA, USA). A total of 50 μg of protein per sample (n = 7–13) was used for the inflammatory marker assay following manufacturer’s protocol (V-PLEX Proinflammatory Panel 1 (Mouse); IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, KC/GRO(CXCL1), IL-10, IL-12p70, and TNFα; MSD, Rockville, MD, USA).