Tnf α

Colon segments of approximatively 2 cm were placed in Lysing Matrix Tubes (MP Biomedicals) containing 1.4 mm-diameter ceramic spheres and 1 mL of RIPA buffer (1% Igepa, 0.5% deoxycholic acid and 0.1% SDS in Tris buffered saline solution 1×; pH 7.4) with protease inhibitors (Roche Diagnostics). The tubes were shaken at 6 m/s two times for 20 s each in a FastPrep (MP Biomedicals). After centrifugation at 10,000×g for 4 min (4 °C), the supernatants were recovered and protein concentrations were assessed using a Coomassie protein assay kit (Thermo Scientific) according to the manufacturer’s instructions. All supernatants were normalized at 1 mg/mL of protein with PBS and stored at − 80 °C until processing. ELISAs were performed on the supernatants to quantify the following mouse cytokines according to the manufacturer’s instructions: IL-10, IL-17A, TNFα and IFNγ (Mabtech, Nacka Strand, Sweden); IL-6, IL-1β and TGFβ (R&D Systems, Minneapolis, MN, USA).

ELISA was performed on purified phage preparations of different concentrations (10⁹, 10⁷, and 10⁵ PFU/well) using commercially available kits IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α (Mabtech AB, Stockholm, Sweden) according to the manufacturer’s instructions. Optical density (OD) was determined at 405 nm on a micro-plate reader (Molecular Devices Corp, Sunnyvale, CA, USA). Data were analyzed in the SoftMax Pro 5.2 rev C software package (Molecular Devices Corp, Sunnyvale, CA, USA).

Cytokine levels of BAL fluid were determined by ELISA, according to manufacturer’s instructions. Aliquots of BAL fluid were added to individual ELISA plate wells (Nunc MaxiSorp plates; Thermal Fisher Scientific). Turbo-TMB-ELISA Substrate (Thermal Fisher Scientific was used for detection. ELISA kits were purchased from R&D Systems (CCL2 [DY439], IL-1β [DY421], TNF-α [DY410], IL-12p70 [DY419], GM-CSF [DY415], M-CSF [DY416], IFN-γ [DY485], IL-17 [DY421] or IL-10 (MABTECH 3432). ELISA kit for norepinephrine was obtained from G-Biosciences. Corticosterone ELISA kit was obtained from ENZO. Protein concentrations of BAL fluid were determined by Bradford assay (BioRAD).

Primary human monocyte-derived macrophages were plated in 96 well plates at concentration of 3x10⁴ cell/well. Next day cells were pretreated with 1 μM of the TLR4 inhibitor TAK-242 (Invivogen, San Diego, CA) for 2 h followed by treatment with 20 ng/ml ProTα variants or controls: ultra-pure LPS (10 ng/ml) or TLR3 ligand PolyI:C (50 μg/ml) (Invivogen, San Diego, CA). Cells were then either infected with VSV envelope-pseudotyped HIV-1 (MOI: 0.01) expressing the luciferase reporter gene or left uninfected. Supernatants collected at 6 h and 48 h post-infection were analyzed by ELISA for the presence of IFN-β (VeriKine-HS^TM kit from PBL,Piscataway, NJ; sensitivity of the assay is 1.2 pg/ml), and TNF-α and IL-6 (Mabtech, Cincinnati, OH; assay sensitivity for both IL-6 and TNF-α is 13 pg/ml). The anti-HIV-1 activity was assessed by measuring in parallel the firefly luciferase expression in the cell lysates. Luciferase values were normalized relative to the corresponding sample’s protein concentration.