Pre-exposure of monocytes to P. falciparum dampens subsequent inflammatory responses to P. falciparum and LPS.

Monocytes of U.S. adults were incubated with medium, RBC or Pf-iRBC. At 24 hours, cells were analyzed by (A) cytokine gene expression Taqman arrays (n = 3 subjects), and supernatants were analyzed by a bead-multiplexed assay (n = 9 subjects) to quantify (D) TNF, (E) IL-6 and (F) IL-1β. In the same experiment, replicate monocytes were incubated with medium, RBC or Pf-iRBC, washed at 24 hours and incubated for 3 additional days in human serum to permit macrophage (Mf) differentiation. On day 5, the three populations of macrophages were analyzed by (B) cytokine gene expression arrays (n = 3 subjects). Finally, in the same experiment, replicate monocytes were incubated with medium, RBC or Pf-iRBC, washed at 24 hours, incubated for 3 additional days in human serum to permit Mf differentiation, and then co-cultured with Pf-iRBC or LPS for 24 hours. Cells were harvested for (C) cytokine gene expression arrays (n = 3 subjects), and supernatants were analyzed (n = 9 subjects) to quantify TNF, IL-6 and IL-1β induced by (G-I) Pf-iRBC or (L-N) LPS (n = 14 subjects). ΔCt values (mean ±SE) for (J) TNF and (K) IL-6 at the indicated timepoints for the 3 subjects shown in A-C. (A-C) Heatmaps were generated from ΔCt values, with lower ΔCt values corresponding to higher gene expression. ΔCt values were normalized to 18S rRNA expression. (D-I and L-N) Lines represent median values. Data were analyzed by the Wilcoxon test with Bonferroni adjustment, and significance levels between the groups are indicated by P values. (J and K) Two-way ANOVA was performed followed by Tukey’s multiple comparisons test. Significance level between conditions (Pf-iRBC stim vs. Control Medium and Pf-iRBC stim vs. RBC stim, respectively) are indicated by P values at each timepoint.