Development of a whole blood stimulation assay to assess TLR-dependent innate immune responses

• Published in: Journal of allergy and clinical immunology 2004-02, Vol.113 (2), p.S50-S50
• Main Authors: Thurm, C.W., Manna, S., Halsey, J.F.
• Format: Article
• Language: English
• ISSN: 0091-6749; 1097-6825
• DOI: 10.1016/j.jaci.2003.12.140

Recent analysis of Toll-like receptors (TLRs) suggest that ex vivo WB stimulation with bacterial antigens such as CpG DNA and LPS may be useful in assessing TLR-mediated signaling pathways. We report a simple assay for assessing innate immune responses involving the stimulation of whole blood with various TLR-ligands followed by measurement of inflammatory cytokines in plasma. Whole blood samples from normal individuals were incubated for 4 or 24 hours with TLR2, TLR4 or TLR9 ligands (Zymosan A, LPS or CpG DNA, respectively). Non-CpG DNA and RPMI medium served as controls. Harvested plasma was analyzed for IL-1β, IL-6 and TNF-α content using the Luminex LMAP technology. Robust inflammatory cytokine concentrations were detected in response to LPS or Zymosan A after 4 hours of stimulation, whereas stimulation with CpG DNA required 24 hours for similar cytokine responses. Optimal concentrations of TLR-ligands were established for a 24 hour stimulation assay. IL-6 responses to each of the TLR-ligands were more robust than IL-1β or TNF-α. LPS-stimulated IL-6 concentrations ranged from 41.90-83.30 ng/ml, whereas CpG DNA−stimulated and Zymosan A −stimulated concentrations ranged from 3.0-59.18 ng/ml and 0.53-32.82 ng/ml, respectively. Clinical assessment of innate immune responsiveness through TLR-ligand stimulation of whole blood and subsequent measurement of inflammatory cytokines is feasible and may provide information in investigational studies involving immunotoxicity, immunomodulatory drugs and evaluating patients with recurrent infections.