CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation

Area of Science:

• Immunology
• Cell Biology
• Microbiology

Background:

• Toll-like receptor 4 (TLR4) initiates innate immune responses to Gram-negative bacteria.
• TLR4 signaling involves two main pathways: Mal-MyD88 for pro-inflammatory cytokines and TRAM-TRIF for type I interferons.
• Understanding TLR4 and adaptor protein dynamics during lipopolysaccharide (LPS) stimulation is crucial for deciphering immune signaling.

Purpose of the Study:

• To investigate the dynamic behavior of TLR4 and TRAM during LPS stimulation.
• To elucidate the mechanisms of TLR4 and TRAM recruitment to cellular compartments.
• To understand the spatial and temporal regulation of TLR4-mediated immune signaling.

Main Methods:

• Utilized Total Internal Reflection Fluorescence (TIRF) microscopy to observe TLR4 and TRAM dynamics.
• Employed cell lines HEK293 and U373-CD14, a macrophage model.
• Investigated the roles of CD14, clathrin, and RAB11A in receptor complex formation and trafficking.

Main Results:

• LPS stimulation induced a CD14-dependent immobile fraction of TLR4 in the plasma membrane.
• TLR4 clustered into punctate structures with CD14/LPS and clathrin, indicating preparation for endocytosis.
• RAB11A mediated TRAM localization to endosomes, but CD14/LPS and TRAM were recruited independently.

Conclusions:

• TLR4 receptor complexes become laterally immobilized and prepared for endocytosis upon LPS stimulation.
• TRAM and CD14/LPS can be recruited to endosomes through distinct mechanisms.
• These findings provide insights into the spatiotemporal regulation of TLR4 signaling pathways.