Complement activation by CpG in a human whole blood loop system: mechanisms and immunomodulatory effects

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• Phosphorothioate oligodeoxynucleotides (CpG) are known immune stimulants.
• Murine studies suggest combined TLR and complement activation yields different results than individual events.
• The interaction between CpG and complement in human blood requires further elucidation.

Purpose of the Study:

• To investigate the immune stimulatory effects of CpG 2006 in human blood.
• To determine the role of complement activation in CpG's effects.
• To elucidate the mechanism of complement activation by CpG.

Main Methods:

• Human blood loop system with selective C3 inhibition.
• Detection of complement activators (IgM, properdin) on CpG.
• Quartz crystal microbalance with dissipation monitoring for convertase activity.
• Assessment of CpG 2006 uptake into monocytes.

Main Results:

• Complement inhibition counteracted both backbone and sequence-specific CpG effects.
• Inhibition of complement reduced CD40, CD83 expression, and IL-6/TNF production.
• CpG-induced complement activation involved classical or alternative pathways.
• Alternative pathway convertase formation on CpG was observed, initiating complement activation.
• C3 fragment involvement in CpG internalization was indicated by suppressed monocyte uptake.

Conclusions:

• Complement activation significantly modulates CpG's immune stimulatory effects in human blood.
• The classical and alternative pathways are involved in CpG-induced complement activation.
• Complement fragments play a role in CpG uptake by monocytes.
• The interplay between complement and TLR9 signaling is critical and warrants further investigation.