Abstract
The high mobility group box 1 (HMGB1) is a well-known late mediator of sepsis, secreted by multiple stimuli, involving pathways, such as the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways, and reactive oxygen species (ROS) under inflammation. Sulfatide, in contrast, is a sphingolipid commonly found in myelin sheets with a disputed immunological role. We sought to determine the immunological characteristics of sulfatide in the periphery by analyzing the secretion of HMGB1 triggered by lipopolysaccharide (LPS) stimulation in Raw 264.7 cells. Suppression of HMGB1 secretion by inhibiting its cytosolic translocation was observed after pre-treatment with sulfatide before LPS stimulation. Further analysis of the downstream molecules of toll-like receptor (TLR) signaling revealed suppression of c-Jun N-terminal kinase (JNK) phosphorylation and p65 translocation. LPS-mediated ROS production was also decreased when sulfatide pre-treatment was provided, caused by the down-regulation of the phosphorylation of activators, such as IRAK4 and TBK1. Investigation of the upstream mechanism that encompasses all the aforementioned inhibitory characteristics unveiled the involvement of lipid rafts. In addition to the co-localization of biotinylated sulfatide and monosialotetrahexosylganglioside, a decrease in LPS-induced co-localization of TLR4 and lipid raft markers was observed when sulfatide treatment was given before LPS stimulation. Overall, sulfatide was found to exert its anti-inflammatory properties by hindering the co-localization of TLR4 and lipid rafts, nullifying the effect of LPS on TLR4 signaling. Similar effects of sulfatide were also confirmed in the LPS-mediated murine experimental sepsis model, showing decreased levels of serum HMGB1, increased survivability, and reduced pathological severity.
Highlights
45 years have passed since the high mobility group box 1 (HMGB1) protein, an abundant nuclear protein and a well-defined danger-associated molecular pattern (DAMP) molecule, was first purified [1]
Sulfatide exhibited an inhibitory effect in HMGB1 secretion without toxicity in a dose-dependent manner, as shown (Figure 1A, Supplementary Figure 1A)
Our experiments showed sulfatide reducing HMGB1 secretion and cytosolic translocation upon LPS stimulation
Summary
45 years have passed since the HMGB1 protein, an abundant nuclear protein and a well-defined danger-associated molecular pattern (DAMP) molecule, was first purified [1]. We intend to limit the scope to active secretion of HMGB1, triggered by inflammatory signals transduced by toll-like receptor (TLR)-related signaling. Our research concentrates on TLR4, a member of the TLR family, which recognizes lipopolysaccharides (LPS), and its mechanism of action regarding the active secretion of HMGB1. The LPS– LBP complex, bound to CD14, is transferred to the myeloid differentiation protein-2 (MD-2)–TLR4 complex [14]. This complex forms a dimer, completing its activation process. TLR4, which contains lipid-binding motifs, is attracted and can readily form a dimer within the lipid rafts, providing a platform on which the TLR4s can be within closer proximity [16]
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