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Identification of CD163 as an antiinflammatory receptor for HMGB1-haptoglobin complexes
Huan Yang, … , Ulf Andersson, Kevin J. Tracey
Huan Yang, … , Ulf Andersson, Kevin J. Tracey
Published May 19, 2016
Citation Information: JCI Insight. 2016;1(7):e85375. https://doi.org/10.1172/jci.insight.85375.
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Research Article Hepatology Immunology

Identification of CD163 as an antiinflammatory receptor for HMGB1-haptoglobin complexes

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Abstract

Secreted by activated cells or passively released by damaged cells, extracellular HMGB1 is a prototypical damage-associated molecular pattern (DAMP) inflammatory mediator. During the course of developing extracorporeal approaches to treating injury and infection, we inadvertently discovered that haptoglobin, the acute phase protein that binds extracellular hemoglobin and targets cellular uptake 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.

Authors

Huan Yang, Haichao Wang, Yaakov A. Levine, Manoj K. Gunasekaran, Yongjun Wang, Meghan Addorisio, Shu Zhu, Wei Li, Jianhua Li, Dominique P.V. de Kleijn, Peder S. Olofsson, H. Shaw Warren, Mingzhu He, Yousef Al-Abed, Jesse Roth, Daniel J. Antoine, Sangeeta S. Chavan, Ulf Andersson, Kevin J. Tracey

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Figure 1

Haptoglobin (Hp) binds HMGB1.

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Haptoglobin (Hp) binds HMGB1.
(A) Rats were subjected to CLP to induce s...
(A) Rats were subjected to CLP to induce sepsis and subsequently connected to an extracorporeal pump to filter blood through human haptoglobin–conjugated (Hp) or empty beads for 1 hour. Proteins bound to Hp or empty beads were eluted and subjected to Western blot analysis using anti-HMGB1 antibodies. Data are representative from 3 repeats. (B) Human Hp and HMGB1 was mixed and applied to beads coupled with anti–human Hp antibody or control (empty beads). Proteins bound to the beads were analyzed by Western blot probed with anti-HMGB1 antibodies. Data are representative from 5 experiments. (C) Human Hp was immobilized on the sensor chip, and binding to HMGB1 was assessed. The apparent Kd is ~64 nM. Data are presented as response units over time (Sec) and representative of 3 experiments. (D) Human Hp–coupled beads were incubated with various HMGB1 isoforms. Elutes were analyzed by Western blot using anti-HMGB1 mAbs. Data are representative from 6 repeats. (E) Upper: Schematics of human HMGB1 structure. Middle/lower: nuclear magnetic resonance (NMR) analysis of disulfide (middle) or sulfonyl (lower) Δ30-HMGB1 in complex with human Hp. Chemical shift perturbation of disulfide or sulfonyl Δ30-HMGB1 in complex with Hp is plotted as a function of residue number of HMGB1. Perturbations in chemical shift above 0.1 are considered a significant interaction (46). The majority of interacting amino acids are located within HMGB1 Box A. Data are representative from 3 repeats. (F) Upper: 15N-1H-heteronuclear single quantum coherence (HSQC) spectra for disulfide Δ30-HMGB1 in the presence or absence of Hp. HMGB1 residues (10 amino acids) showing significant chemical shift perturbations due to Hp binding are labeled. Middle: Representative 15N-HSQC spectra of disulfide Δ30-HMGB1 free (black) and in complex with haptoglobin (red); residues showing chemical shift changes upon complex formation are circled. Lower: 15N-HSQC spectra of sulfonyl Δ30-HMGB1 free (black) and in complex with Hp (red). Residues showing chemical shift changes seen with disulfide Δ30-HMGB1 upon complex formation with Hp are circled for comparison. Nonoverlapping black and red resonance spectra indicate a significant chemical shift and, hence, an interaction between proteins via that amino acid. (G) Serum samples (10 μl) from healthy individuals or sepsis patients were incubated with beads conjugated to anti–human Hp mAbs or empty (20 μl drained beads). Eluates were probed with anti-HMGB1 (upper) and anti–human Hp (lower) antibodies using Western blot analysis. Data are representative from 5 samples each.

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