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Signaling of high mobility group box 1 (HMGB1) through toll-like receptor 4 in macrophages requires CD14 - PubMed

  • ️Tue Jan 01 2013

Signaling of high mobility group box 1 (HMGB1) through toll-like receptor 4 in macrophages requires CD14

Sodam Kim et al. Mol Med. 2013.

Abstract

High mobility group box 1 (HMGB1) is a DNA-binding protein that possesses cytokinelike, proinflammatory properties when released extracellularly in the C23-C45 disulfide form. HMGB1 also plays a key role as a mediator of acute and chronic inflammation in models of sterile injury. Although HMGB1 interacts with multiple pattern recognition receptors (PRRs), many of its effects in injury models occur through an interaction with toll-like receptor 4 (TLR4). HMGB1 interacts directly with the TLR4/myeloid differentiation protein 2 (MD2) complex, although the nature of this interaction remains unclear. We demonstrate that optimal HMGB1-dependent TLR4 activation in vitro requires the coreceptor CD14. TLR4 and MD2 are recruited into CD14-containing lipid rafts of RAW264.7 macrophages after stimulation with HMGB1, and TLR4 interacts closely with the lipid raft protein GM1. Furthermore, we show that HMGB1 stimulates tumor necrosis factor (TNF)-α release in WT but not in TLR4(-/-), CD14(-/-), TIR domain-containing adapter-inducing interferon-β (TRIF)(-/-) or myeloid differentiation primary response protein 88 (MyD88)(-/-) macrophages. HMGB1 induces the release of monocyte chemotactic protein 1 (MCP-1), interferon gamma-induced protein 10 (IP-10) and macrophage inflammatory protein 1α (MIP-1α) in a TLR4- and CD14-dependent manner. Thus, efficient recognition of HMGB1 by the TLR4/MD2 complex requires CD14.

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Figures

Figure 1
Figure 1

HMGB1-induced TNF-α release is TLR4 dependent. (A) Supernatants from RAW264.7 cells treated with vehicle, LPS (0.1, 0.5 or 1 ng/mL) or HMGB1 (0.5, 1, 2, 5, or 10 μg/mL) with or without polymyxin B (PMB) (10 μg/mL) for 18 h were assessed for TNF-α production by using ELISA. (B) Supernatants of peritoneal macrophages from WT or TLR4−/−mice treated with LPS (1 ng/mL) and HMGB1 (2, 5 or 10 μg/mL) for 18 h were assessed for TNF-α by using ELISA. Data are representative of three independent experiments in triplicate (mean ± SEM, #P < 0.05 between designated, *P < 0.05 versus control).

Figure 2
Figure 2

HMGB1-dependent TLR4 activation requires MD2 and CD14 in vitro. (A) HEK293 cells expressing components of the TLR4 signaling complex were stimulated with 5 or 10 μg/mL HMGB1, 100 ng/mL LPS or 10 ng/mL TNF-α. NF-κB activity is expressed as a fold-increase in the inducible form of luciferase. (B) HEK/TLR4 and HEK/TLR4/MD2 cell lines were transfected with AdCD14 and similarly stimulated with LPS (10 ng/mL) or HMGB1 (1 μg/mL) treatment as above, and NF-κB activation was measured (mean ± SEM, *P < 0.05 versus HEK/WT in [A] and versus HEK/TLR4 + AdCD14 in [B]). Results shown are representative of at least three separate repeats in triplicate.

Figure 3
Figure 3

HMGB1-induced TNF-α and chemokine release are TLR4/CD14 dependent. (A) Peritoneal macrophages from WT, TLR4−/− and CD14−/− mice were treated with LPS (10, 100 or 1,000 ng/mL), HMGB1 (5 or 50 μg/mL) or Pam3CSK4 (1 μg/mL) in Opti-MEM for 18 h. TNF-α was then measured in supernatants by ELISA. (B) MCP-1, IP-10 and MIP-1α in supernatants of WT, TLR4−/− or CD14−/− macrophages stimulated with LPS (10 ng/mL) or HMGB1 (5 μg/mL) for 24 h. Data are representative of three independent experiments performed in triplicate (mean ± SEM, *P < 0.05 versus negative control).

Figure 4
Figure 4

HMGB1 stimulates clustering of TLR4 in the lipid raft. RAW264.7 macrophages were treated with 10 ng/mL LPS or 1 μg/mL HMGB1 for 60 min, and lysates were subjected to sucrose gradient fractionation. (A) Lysates were then drawn off in 12 separate fractions and run on SDS-PAGE before immunoblot (IB) with flotillin-1 to identify raft-enriched fractions. Fractions were then probed for TLR4. (B) RAW264.7 cells were treated with 1 μg/mL HMGB1 for 0, 15, 60 or 90 min and fixed. Immune fluorescence microscopy was performed for TLR4 (red) and the lipid raft protein GM1 (green), and colocalization was assessed (yellow). Images are representative of at least three independent experiments.

Figure 5
Figure 5

HMGB1 promotes localization of TLR4 coreceptors within the lipid raft. RAW264.7 macrophages were treated with 1 μg/mL HMGB1 for 60 min, and lysates were subjected to sucrose gradient fractionation. Lysates were then drawn off in 12 separate fractions and run on SDS-PAGE before immunoblot (IB) with flotillin-1 to identify raft-enriched fractions. Fractions were then probed for MD2 or CD14. Images shown are representative of at least three independent experiments.

Figure 6
Figure 6

HMGB1-induced TNF-α release depends on MyD88/TRIF. Peritoneal macrophages from WT, TLR4−/−, CD14−/− TRIF−/− and MyD88−/− mice were treated with HMGB1 (5 μg/mL) or LPS (10 ng/mL) in Opti-MEM for 18 h. Supernatants were then tested for TNF-α by ELISA. We found that the TNF-α release induced by HMGB1 and LPS is TRIF (A) and MyD88 (B) dependent. Data are representative of three independent experiments in triplicate (mean ± SEM).

Figure 7
Figure 7

HMGB1-induced MAP kinase activation depends on TLR4/CD14. Peritoneal macrophages from WT, TLR4−/− and CD14−/− mice were treated with HMGB1 (5 μg/mL) or LPS (10 ng/mL) in Opti-MEM for the time points up to 4 h. pp38 (A), pERK (B) and pJNK (C) activation were measured by imaging cytometry. Data are representative of three independent experiments in triplicate (mean ± SEM, *P < 0.05 versus WT at the same time point).

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