Bilirubin Targeting WNK1 to Alleviate NLRP3-Mediated Neuroinflammation

Area of Science:

• Biochemistry
• Cell Biology
• Neuroscience

Background:

• Bilirubin is an endogenous metabolite with known anti-inflammatory properties.
• The precise molecular targets and signaling pathways of bilirubin's anti-inflammatory effects are not fully understood.

Purpose of the Study:

• To identify the direct molecular target of bilirubin.
• To elucidate the downstream signaling mechanisms responsible for bilirubin's anti-inflammatory actions.
• To explore bilirubin's role in regulating the NLRP3 inflammasome.

Main Methods:

• Quantitative phosphorylation proteomics
• Cellular Thermal Shift Assay (CETSA)
• Molecular docking
• MicroScale Thermophoresis (MST)
• Genetic manipulation of bilirubin synthesis and metabolism enzymes (Blvra, Ugt1a1)
• Lipopolysaccharide (LPS)-induced inflammasome activation assays in mouse hippocampus

Main Results:

• With-no-lysine (K) kinase 1 (WNK1) was identified as the primary binding target of bilirubin at physiological concentrations.
• Bilirubin binds to the kinase domain of WNK1, activating its kinase activity.
• Activation of WNK1 by bilirubin leads to increased intracellular chloride ion concentration via the SPAK/OSR1-KCC2 pathway in neurons.
• Modulation of endogenous bilirubin levels significantly affected lipopolysaccharide (LPS)-induced NLRP3 inflammasome activation in mouse hippocampus.
• Exogenous bilirubin supplementation suppressed LPS-induced NLRP3 inflammasome activation in a WNK1-dependent manner.

Conclusions:

• WNK1 is a direct molecular target of bilirubin, mediating its anti-inflammatory effects.
• Bilirubin's action on WNK1 influences neuronal chloride ion concentration and inflammasome activation.
• These findings clarify the molecular basis of bilirubin's anti-inflammatory properties and suggest WNK1 as a key player in its biological functions.