A molecular pattern of restrained inflammation and enhanced anti-parasite effector function upon <i>P. falciparum</i> re-exposure.

<p>(<b>A</b>) PBMCs were collected from 34 healthy children with blood smears negative for <i>P. falciparum</i> infection before the malaria season (HB) and 7 days after treatment of their first febrile malaria episode of the ensuing malaria season when malaria symptoms had resolved (d7). RNA was extracted from PBMCs immediately after thawing and hybridized onto Affymetrix GeneChip Human 1.0 ST arrays. RNA from all 68 PBMC samples was of sufficient quantity and quality for microarray analysis. Nine of 68 samples did not pass the microarray quality assessment and were removed from further analysis (see Supplemental <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004079#ppat-1004079-g001" target="_blank">Figure 1A</a>) such that 25 children with paired RNA samples at the healthy baseline and 7 days after malaria were analyzed. The heat map shows <i>ex vivo</i> RMA-normalized log₂ ratios (d7/HB) of differentially expressed genes (rows) for each child (columns). Genes are grouped and color-coded by function as indicated. (<b>B</b>) PBMCs analyzed by FACS for B cells (CD19⁺), T cells (CD3⁺), CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, and monocytes (CD14⁺) at the healthy baseline and after malaria. (n = 34 children; except CD14⁺ monocytes, n = 30). (<b>C</b>) Ratio of monocyte percentage (d7/HB) versus the ratio of the expression level of monocyte-derived pro-inflammatory cytokines and chemokines (d7/HB). Each point represents an individual subject (n = 21 children with paired samples). (<b>D</b>) RNA was extracted from PBMCs of the same 34 children after 18 h of <i>in vitro</i> stimulation with <i>P. falciparum</i>-infected red blood cell (iRBC) lysate. After stimulation with iRBC lysate, 22 of the 34 children had RNA samples from both time points of sufficient quantity and quality for microarray analysis and also passed the microarray quality assessment. The heat map shows RMA-normalized log₂ ratios (d7/HB) of differentially expressed genes (rows) for each child (columns) in response to <i>in vitro</i> iRBC lysate stimulation. Genes are grouped and color-coded by function as indicated. (<b>E</b>) q-RT-PCR confirmation of the microarray data. The data represent the results of one experiment with 6 genes (<i>IL1B</i>, <i>IL6</i>, <i>IL10</i>, <i>TGFB1</i>, <i>TLR2</i>, <i>CXCL5</i>) from 17 subjects at two time points (d7 and HB) from both the <i>ex vivo</i> unstimulated and <i>in vitro</i> iRBC-stimulated datasets. Each symbol represents a single gene at a given time point. PCR expression computed as antilog₂ –dCT. <i>n</i> = 497 XY pairs. (<b>F</b>) q-RT-PCR expression of genes encoding the pro-inflammatory cytokines IL1-β and IL-6 and the anti-inflammatory cytokine TGF-β in PBMCs of children (n = 17) collected at the healthy baseline (HB) and after resolution of febrile malaria (d7), either directly <i>ex vivo</i> (unstimulated) or after <i>in vitro</i> stimulation with iRBCs for 18 h. ns, not significant (<i>P</i>≥0.05), <i>P</i> values determined by the paired <i>t</i>test (B), Pearson's (C), Spearman's (E) or paired Wilcoxon rank sum test (F). Data are shown as the means ± s.d. (B) or means ± s.e.m. (F).