<i>P. falciparum</i>-inducible IL-10 is mainly produced by CD4⁺CD25⁺Foxp3⁻ T cells that co-produce IFNγ and TNF.

<p>(<b>A</b>) PBMCs from the healthy baseline (HB), 7 days after malaria (d7), and at the healthy baseline at the end of the subsequent dry-season (HB′) were stimulated for 18 h with iRBC lysate and assayed for the production of IL-10, IFNγ and TNF by intra-cellular FACS. Results are shown as the ratio of live CD3⁺ CD4⁺ antigen-experienced cells (CD45RO⁺ CD27⁺, CD45RO⁺ CD27⁻, and CD45RO⁻ CD27⁻) producing IL-10, IFN-γ or TNF in response to stimulation with iRBC lysate vs. uninfected RBC (uRBC) lysate (n = 16, 13 paired samples). (<b>B</b>) Overlay of IL-10-producing cells (red) among all live cells (gray) in a CD3 vs. CD4 dot plot (top) (n = 14), and IL-10-producing CD4⁺ T cells (red) with all CD4⁺ T cells (gray) in CD25 vs. FoxP3 dot plot (bottom) (n = 9; representative subject shown). (<b>C</b>) Using SPICE analysis, cytokine-producing CD4⁺ T cells were divided into 7 distinct subpopulations producing any combination of IL-10, IFNγ and TNF (n = 16). (<b>D</b>) Pie chart representation of the combination of cytokines produced by CD4⁺ T cells after iRBC stimulation for 3 representative donors 7 days after malaria (d7). The black arcs indicate the IL-10-producing CD4⁺ T cells. (<b>E</b>) Representative FACS plots of live CD3⁺ CD4⁺ antigen-experienced cells producing IL-10, IFNγ and TNF after iRBC stimulation of PBMCs collected at the healthy baseline (HB), 7 days after malaria (d7) and at the healthy baseline at the end of the subsequent dry-season (HB′). (<b>F</b>) CD4⁺ T cells were isolated from PBMCs which had been collected from children 7 days after malaria and were then stimulated for 18 h with iRBC or uRBC lysate in the absence (CD4⁺T d7) or presence of non-CD4⁺T cells isolated from PBMCs of the same individuals collected at either the healthy baseline (CD4⁺T d7 + nonCD4⁺T HB) or 7 days after malaria (CD4⁺T d7 + nonCD4⁺T d7) (n = 8 paired samples). (<b>G</b>) PBMCs collected from children 7 days after malaria were stimulated for 18 h with iRBC lysate and assayed for the production of IL-10 in the presence (αMHC-II) or absence (isotype) of antibodies specific for HLA-DR, -DQ and -DP (n = 8). ns, not significant (<i>P</i>≥0.05), <i>P</i> values determined by a linear mixed model for repeated measures ANOVA with Tukey HSD post hoc tests (A) and permutation re-sampling tests (F, G). Data are shown as the means ± s.d.</p

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).</p

Proposed model by which children remain asymptomatic and control parasitemia upon <i>P. falciparum</i> re-exposure.

<p>In children without prior or recent malaria exposure, <i>P. falciparum</i> infection induces a robust pro-inflammatory cytokine and chemokine response (e.g. IL-1β, IL-6, IL-8) whereas effector mechanisms that mediate parasite clearance (phagocytosis, phagolysosome activation, antigen presentation, T cell co-stimulation and IFN-<b>γ</b> production by CD4⁺ T cells) are not readily inducible, leaving children susceptible to fever and other systemic symptoms of malaria as well as poorly controlled parasite replication. In contrast, febrile malaria induces an exposure-dependent regulatory state (shown here) whereby re-exposure to <i>P. falciparum</i> results in reduced production of pro-inflammatory cytokines and chemokines and enhanced expression of regulatory cytokines (e.g. IL-10 production by CD4⁺ T cells) and pathways involved in phagocytosis-mediated clearance of infected red blood cells and activation of adaptive immunity, thus enabling children to remain asymptomatic and control parasite replication in the face of ongoing <i>P. falciparum</i> exposure. In addition, <i>P. falciparum</i>-specific IgG levels are low in children who have not been recently exposed to malaria, but transiently increase in response to <i>P. falciparum</i> infection <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004079#ppat.1004079-Weiss1" target="_blank">[44]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004079#ppat.1004079-Crompton2" target="_blank">[45]</a>, further enhancing exposure-dependent parasite clearance through opsonization and phagocytosis of infected erythrocytes. Arrows indicate the direction of expression observed in this study of molecules at the mRNA and/or protein levels induced by <i>P. falciparum</i> re-exposure after febrile malaria relative to responses induced by <i>P. falciparum</i> exposure at the healthy baseline. Molecules are color-coded by biological function.</p