Methemoglobin is an endogenous toll-like receptor 4 ligand-relevance to subarachnoid hemorrhage

Area of Science:

• Neuroscience
• Immunology
• Biochemistry

Background:

• Neuroinflammation is a key factor in subarachnoid hemorrhage (SAH) complications.
• Toll-like receptor 4 (TLR4) signaling in microglia contributes to neuronal damage post-SAH.
• Methemoglobin (metHgb), heme, and hemin are potential endogenous TLR4 ligands from erythrocyte breakdown, but their roles are unclear due to purification challenges.

Purpose of the Study:

• To rigorously purify methemoglobin (metHgb) free from lipopolysaccharide (LPS) contamination.
• To investigate whether purified metHgb functions as a Toll-like receptor 4 (TLR4) ligand.
• To elucidate the role of metHgb in mediating neuroinflammation following subarachnoid hemorrhage (SAH).

Main Methods:

• A 5-step purification process was employed to obtain highly purified, LPS-free methemoglobin (metHgb).
• Fourier Transform Ion Cyclotron Resonance mass spectrometry and Limulus amebocyte lysate assay confirmed metHgb purity.
• In vitro studies used microglial and macrophage cell lines, while in vivo studies involved injecting purified metHgb into the rat subarachnoid space.

Main Results:

• Purified metHgb was confirmed as a Toll-like receptor 4 (TLR4) ligand at physiologically relevant concentrations.
• MetHgb induced time- and dose-dependent release of tumor necrosis factor α (TNFα) from immune cells, inhibited by TLR4 antagonists.
• Intracerebral injection of metHgb in rats activated microglia and upregulated TNFα, indicating in vivo neuroinflammatory effects.

Conclusions:

• Methemoglobin (metHgb) is a potent endogenous Toll-like receptor 4 (TLR4) ligand.
• MetHgb contributes to neuroinflammation following subarachnoid hemorrhage (SAH) by activating TLR4 signaling in microglia.
• Targeting metHgb-mediated TLR4 activation may offer a therapeutic strategy for SAH complications.