through CD163, also binds HMGB1. Haptoglobin-HMGB1 complexes elicit the production of antiinflammatory enzymes (heme oxygenase-1) and cytokines (e.g., IL-10) in WT but not in CD163-deficient macrophages. Genetic disruption of haptoglobin or CD163 expression significantly enhances mortality rates in standardized models of intra-abdominal sepsis in mice. Administration of haptoglobin to WT and to haptoglobin gene-deficient animals confers significant protection. These findings reveal a mechanism for haptoglobin modulation of the inflammatory action of HMGB1, with significant implications for developing experimental strategies targeting HMGB1-dependent inflammatory diseases. alog 3536), REDExtract-N-Amp Tissue PCR kit (catalog XNATS), Triton X-114, imidazole, monoclonal anti-human haptoglobin antibodies (catalog H6395), nonimmune mouse IgG, Drabkin’s reagents (catalog D5941), human M-CSF, DAPI, and dexamethasone (catalog D2915) were purchased from Sigma-Aldrich. TACS MTT Cell proliferation assay kit was from Trevigen. Trizol reagent was from Invitrogen. The Rever-tAid First Strand cDNA Synthesis Kit was from Fermentas. SYBR Premix Ex Taq II was from Takara Bio Inc. Thioglycollate medium was purchased from BD Biosciences. Primers for quantitative PCR (qPCR), trypsin-EDTA, and carbenicillin were from Invitrogen. Protein A/G agarose and isopropyl-D-thiogalacto-pyranoside (IPTG) were purchased from Pierce. NHS-activated Sepharose 4 fast flow beads were obtained from GE Healthcare (catalog 17-0906-01). E . coli strain DH5 α was from Novagen. Recombinant human CD163 protein, CD163 expression plasmid (SC117495, NM_004244.3), and MegaTran 1.0 transfection reagent were purchased from Origene. Antibodies for human HO-1 (catalog ab52946) and CD163 (cata-log MCA1853) were from Abcam and Serotec, respectively. Anti–human CD163-PE antibody was from BioLegend. FITC antibody labeling kit was from Thermo Scientific. Dynasore was from Tocris Bioscience. Preparations of recombinant HMGB1, redox-modified HMGB1, and HMGB1 mAbs . Recombinant HMGB1 was expressed in E . manufacturer’s immunoblotting The HMGB1 were calculated with reference to standard curves generated with purified HMGB1. Cytokine expression profile from primary human macrophages was determined by human cytokine array C1 (Ray-biotech) according to the manufacturer’s instructions. Twenty-two cytokines or chemokines were determined simultaneously. Serum hemoglobin levels were determined using Drabkin’s reagents (44) following the manufacturer’s instructions (Sigma-Aldrich). The amounts of hemoglobin in the samples were calculated with reference to standard curves generated with purified mouse hemoglobin. Haptoglobin levels were measured by quantitative ELISA kits following the manufacturer’s instructions (Genway Biotech Inc.) or by Western blot. sis of FITC-HMGB1 g/ml) plus of and we observed the optimal binding is 1:1 molar ratio for HMGB1 to haptoglobin. plasmon resonance analysis resonance analysis haptoglobin was conducted using the BIAcore 3000 or T200 instrument previously For HMGB1-haptoglobin binding analyses, purified haptoglobin (1 μ M, was immobilized onto the sensor chip. To evaluate binding, a series of concentrations of analytes (HMGB1 were then passed over the sensor chip. The association of analyte and ligand was recorded respectively by surface plasmon resonance. Results were analyzed using the software BIAeval 3.2 (BIAcore Inc.). For the binding studies of CD163 to the complexes of haptoglobin and HMGB1, human CD163 protein was coated on the sensor chip. Increasing concentrations of the complexes of HMGB1 and haptoglobin formed at 1:1 molar ratio were flowed over the sensor chip; analyses were performed in a similar manner as described above. amino acid isoforms haptoglobin the amide resonance of a residue with the carbonyl carbon of the same residue, as well as that of the preceding residues) experiments were used to sequentially assign the backbone NH via the carboxyl group. CBCACONH and HNCACB experiments were used to assign the backbone NH via the backbone C α and side-chain C β groups. 15 N- and/or 13 C-labeled recombinant proteins were expressed in minimal media and purified by ion-exchange and size-exclusion chromatography. NMR samples were prepared at 10–100 μ M concentrations in PBS with a final concentration of 10% D 2 O (deuterium oxide, 2 H 2 O). Samples were placed in standard 5-mm NMR tubes. Chemical shift perturbation studies were carried out using either a Bruker Avance III 600 or 800 MHz NMR system equipped with triple (TCI) resonance cryoprobes at 25 o C (298K). All data were processed using Topspin 3.1 (Bruker) and analyzed using CCPN (collaborative computing project for NMR) analysis. The redox status of cysteines on HMGB1 was confirmed by liquid chromatography tandem mass spectrometry as previously described (14). CD163 shRNA lentiviral transduction of human macrophages . Ficoll gradient–separated human monocytes were differentiated for 7 days in complete DMEM medium containing 10% heat-inactivated human serum, 2 mM glutamine, 100 U/ml penicillin, 100 μ g/ml streptomycin, and 2 ng/ml M-CSF in 24-well Primaria tissue culture plates at 5 × 10 5 cells/well. After screening of 5 human CD163 (NM_004244) Mis-sion shRNA lentiviral clones using FACS analysis, the clone that showed the best knockdown efficiency (TRCN0000421748) was selected. The selected clone was transduced into human macrophages by spinoc-ulation according to the manufacturer’s instructions (Sigma-Aldrich). anti-HMGB1 the linked haptoglobin beads (1.5 ml for at a rate of 1 ml/min. The filter were extensively with After with PBS 5 times, 2× Laemmli was added to the beads to elute bound proteins. Western blot was performed on bead eluate using anti-HMGB1 antibodies.