Figure 5
Trace levels of TLR2 and TLR4 agonists and IVIG aggregates synergize to augment the innate immune response. (A,B) mRNA of IL6, IL1β and IL8 were measured by real-time PCR in PBMC stimulated with intact or aggregated IVIG (80 µg/mL) in the absence or presence of increasing concentrations of LPS (A;1 to 100 pg/mL) or Pam3Cys4 (B; 0.5 ng to 50 ng/mL). Results shown represent 2–3 independent experiments with 1–3 subjects each. Differences in gene expression were tested by two way ANOVA # < 0.05. Synergy testing: Significant interactions in the effect of TLR agonist and aggregation were tested using SAS®9.4 as described in the materials and methods section and are denoted with *, where * < 0.05, ** < 0.01, and *** < 0.001. (C) STAT1 phosphorylation and IRF7 expression were measured in whole cell lysates of PBMC stimulated with intact or aggregated IVIG in the presence or absence of LPS (100 pg/mL for 12 h) by Western blot. Results are representative of 2 independent experiments with healthy blood donors. (D) TLR2 and TLR4 mRNA expression in Human PBMC after stimulation with aggregated or unaggregated proteins of IVIG, for 24 h was analyzed by Nanostring. Data shown as fold change in expression over the mean expression in unstimulated cells. Unstimulated cells were used as negative controls (denoted as Medium).
