Anti citrullinated histone h3

Blood-derived human PMN were treated with vehicle or RvD1 100 nM as described above and added on top of HeLa cells at an E:T ratio of 10:1. After 18 h of co-incubation, PMN were harvested, counted and stained for FACS analysis, and supernatants were collected for western blotting of citrullinated histone H3 and for quantification of extracellular DNA release with the Qubit fluorometer (Life technologies). For apoptosis, 5 × 10⁵ cells were incubated for 15 min at room temperature with Annexin V-FITC (Merck KGaA), washed, incubated with 50 μg/ml Propidium Iodide (PI) and immediately processed. Samples were acquired using a FACSCanto II flow cytometer (Becton Dickinson), and data were analyzed with the FACS Diva software (BD Bioscience). For evaluation of neutrophil extracellular traps (NETs) formation, supernatants from an equal number of PMN (1 × 10⁶) were resolved by SDS-PAGE under reducing conditions and immunoblotted with the anti-citrullinated histone H3 (Abcam) as marker of NETtosis.

Murine neutrophils were isolated following harvest of bone marrow from 8-week-old naïve B6 or PD-L1 KO mice. In brief, skin, muscle and fat were removed from femurs and tibias. Bone marrow was then flushed using a 27g needle and RPMI (Gibco). Following isolation of pure neutrophils, cells were then treated with 250-500 nM phorbol-12-myristate-13-acetale (PMA, Sigma-Aldrich) for 4 hours and then spun at 480xg for 5 minutes. The pellet was discarded and supernatant containing NET chromatin was spun at 18,000xg for 10 minutes. Quantification of NETs was analyzed using a Nano Drop (Thermo Fisher Scientific). In-vivo NETs quantification was analyzed using myeloperoxidase (MPO) (Roche) associated with DNA ELISA (Roche). For the human studies, fresh peripheral blood obtained from healthy donors or patients was stained with anti-CD15 (clone W6D3, BioLegend), anti-CD16 (clone 3G8, BioLegend), anti-citrullinated histone H3 (Abcam), anti-PD-L1 (clone 29E.2A3, Biolegend) and specific secondary anti-antibodies (Thermo Fisher Scientific).

Whole lungs were homogenized in 4%CHAPS lysis buffer and mixed 1:1 in CHAPS-Urea buffer (9M urea, 4%CHAPS 10mM Tris) as previously described (30 (link)). Pelleted BAL cells were lysed in CHAPS urea buffer. Homogenates, lysates and BALF were mixed 3:1 in 4X laemmli buffer (Bio-rad) supplemented with 50mM DTT and boiled for five minutes. 20-60µg of protein or 5µl BALF was loaded on 10% SDS-PAGE gels (TGX Fastcast, Bio-rad) and run at 100V for ten minutes followed by 150V for an additional 45 minutes. Gels were then transferred onto PVDF membranes (Millipore) at 100V for one hour on ice. Blots were then blocked in 2.5% BSA-PBS for one hour at room temperature. Blots were incubated in primary antibody diluted in 2.5% BSA-PBS overnight at 4°C. Antibodies used include anti-NLRP3, anti-pSTAT3, anti-STAT3, anti-β-Actin (Cell signaling technologies), anti-Caspase-1 (Santa Cruz Biotechnology), anti-HMGB1 and anti-citrullinated-Histone H3 (Abcam) and anti-RAGE antibody (in house). Blots were then washed in PBS-T (0.1% Tween-20) 3X for 10 minutes. Blots were then incubated with HRP-conjugated secondary antibodies diluted in 2.5% non-fat milk-PBS for 1h at room temperature. Blots were again washed in PBS-T 3X for 10 minutes. Blots were incubated with Pierce ECL substrate (Thermo Scientific) and were imaged on a LI-COR Odyssey Fc by using LI-COR Image Studio software (LI-COR Biosciences).

The slicing preparation process is described above. After dewaxing, antigen retrieval was performed using citrate buffer and permeabilized with 0.1% Triton X-100 for 10 min, then specimens were blocked with goat serum. The sections were incubated with primary antibodies, specifically anti-citrullinated-histone H3 (1:100; Abcam; ab5103) and Anti-Myeloperoxidase antibody (1:200; Abcam; ab208670), followed by detection with the samples were incubated with Alexa Fluor-594-coupled (1:500, Invitrogen; A-11012) and/or Alexa Fluor-488-coupled (1:500, Invitrogen; A-11034) secondary antibodies overnight at 4°C. Coverslips were mounted with a VectaShield medium containing DAPI to detect DNA. The sections were imaged using a Zeiss inverted fluorescence microscope (AXI0; Zeiss) that was equipped with a Zensoftware or using a laser-scanning confocal microscope (SP8; Leica) with a 20×water immersion objective. Images were analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Inc. Rockville, MD, USA).