Extensive alterations of blood metabolites in pediatric cerebral malaria.

Cerebral malaria (CM) presents as an encephalopathy and is due to infection with Plasmodium falciparum. Patients are comatose, often with fever, recurrent seizures and this condition is associated with a high mortality rate. The etiology of the coma and seizures are poorly understood. Circulating small molecules and lipids have bioactive functions and alterations in their concentrations have been implicated in seizure disorders and other forms of encephalopathy. We carried out a comprehensive analysis of blood metabolites during CM to explore a biochemical basis of this encephalopathy. A paired metabolomics analysis was performed on the plasma samples of Malawian children (n = 11) during CM and at convalescence thirty days later, to identify differentially abundant molecules associated with CM. We also report plasma molecules associated with CM mortality (n = 4) compared to survival (n = 19). Plasma metabolites were identified through ultra high performance liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry to maximize compound detection and accuracy and then compared to a library for identification. We detected a total of 432 small molecules in the plasma and 247 metabolites were significantly differentially abundant between CM and convalescence (p 1.2). These results highlight the broad changes in blood chemistry during CM. We have identified metabolites that may impact central nervous system physiology and disease outcomes and can be further explored for their mechanistic roles into the pathophysiology of CM

STING-Licensed Macrophages Prime Type I IFN Production by Plasmacytoid Dendritic Cells in the Bone Marrow during Severe <i>Plasmodium yoelii</i> Malaria

<div><p>Malaria remains a global health burden causing significant morbidity, yet the mechanisms underlying disease outcomes and protection are poorly understood. Herein, we analyzed the peripheral blood of a unique cohort of Malawian children with severe malaria, and performed a comprehensive overview of blood leukocytes and inflammatory mediators present in these patients. We reveal robust immune cell activation, notably of CD14⁺ inflammatory monocytes, NK cells and plasmacytoid dendritic cells (pDCs) that is associated with very high inflammation. Using the <i>Plasmodium yoelii 17X YM</i> surrogate mouse model of lethal malaria, we report a comparable pattern of immune cell activation and inflammation and found that type I IFN represents a key checkpoint for disease outcomes. Compared to wild type mice, mice lacking the type I interferon (IFN) receptor exhibited a significant decrease in immune cell activation and inflammatory response, ultimately surviving the infection. We demonstrate that pDCs were the major producers of systemic type I IFN in the bone marrow and the blood of infected mice, via TLR7/MyD88-mediated recognition of <i>Plasmodium</i> parasites. This robust type I IFN production required priming of pDCs by CD169⁺ macrophages undergoing activation upon STING-mediated sensing of parasites in the bone marrow. pDCs and macrophages displayed prolonged interactions in this compartment in infected mice as visualized by intravital microscopy. Altogether our findings describe a novel mechanism of pDC activation <i>in vivo</i> and precise stepwise cell/cell interactions taking place during severe malaria that contribute to immune cell activation and inflammation, and subsequent disease outcomes.</p></div

CD169⁺ macrophages control early pDC activation in the bone marrow of infected mice.

<p>(<b>A</b>) DT-treated WT and <i>Cd169-Dtr</i>^<i>+/-</i> <i>Ifnb-Yfp</i>^<i>+/+</i>reporter mice (n = 7–14 mice/genotype) were inoculated i.v. with 2x10⁵ <i>Py 17X YM</i> iRBCs and blood and bone marrow cells were stained with the lineage markers CD11b, BST2 and Siglec-H and frequencies of YFP⁺ pDCs is shown. In (<b>B</b>), levels of IFNα in the blood and bone marrow of these mice were quantified and (<b>C</b>) shows the activation profiles of Ly6C⁺ monocytes, NK cells and pDCs using indicated markers (n = 3–7 mice/condition). (<b>D</b>) WT or <i>Cd169-Dtr</i>^<i>+/-</i> recipient mice (n = 7 mice/chimera) reconstituted with bone-marrow cells from WT <i>Ifnb-Yfp</i>^<i>+/+</i> mice were DT-treated and frequencies of YFP⁺ pDCs in the bone marrow was determined 1.5 days post <i>Py</i> infection. (<b>E</b>) Frequencies of YFP⁺ pDCs in the bone marrow of indicated <i>Py</i>-infected WT or <i>Sting</i>^<i>Gt/Gt</i> reciprocal chimeras n = 4 mice/chimera). Experiments were replicated 2–4 times. P-values are indicated when applicable.</p

High inflammation and immune cell activation in the blood of severe malaria patients and in a surrogate mouse model.

<p>(<b>A</b>) Serum cytokine and chemokine levels in patients. Each point represents an individual patient, enrollment (blue), follow-up (black). (<b>B</b>) WT B6 mice were inoculated i.v. with 2x10⁵ <i>Py 17X YM</i> iRBCs. Kinetics of cytokine and chemokine levels in the blood (n = 3–15 mice/time point). (<b>C-E</b>) Frozen isolated human PBMCs from patients were thawed and stained with mAbs against cell-surface lineage markers for CD14⁺ monocytes (C), dendritic cells (myeloid or plasmacytoid) (D), or NK cells (E) including a viability stain, as well as indicated activation markers. Gating strategies and respective frequencies are shown. Scattered plots show the relative expression level (MFI) or percent of cell population expressing indicated marker for each individual patient at enrollment versus follow-up. (<b>F</b>) The proportion of blood Ly6C⁺ monocyte and NK cells expressing various surface markers in <i>Py</i>-infected WT mice at 1.5 and 4.5 days post infection (n = 3 = 14 mice/condition). Experiments were replicated 2–3 times. P-values are indicated when applicable.</p

Type I interferon enhances immune blood leukocyte activation and lethal outcomes in severe murine malaria.

<p>WT or <i>Ifnar1</i>^<i>-/-</i> B6 mice were inoculated i.v. with 2x10⁵ <i>Py 17X YM</i> iRBCs. (<b>A</b>) Survival and blood parasitemia of <i>Py</i>-infected mice over indicated times. (<b>B</b>) Serum and spleen cytokine and chemokine levels/contents 1.5 or 4.5 days post <i>Py</i>-infection (n = 3–8 mice). (<b>C</b>) 1.5 days after <i>Py</i> infection, blood cells were stained for the cell-surface lineage markers CD11b, CD3, CD19, Ly6C, Ly6G, NKp46, BST2, SiglecH and CD45. Frequencies of indicated cell subsets among all blood leukocytes (CD45⁺) in <i>Py</i>-infected WT compared to <i>Ifnar1</i>^<i>-/-</i> mice (upper bar graph, n = 6–10 mice) and either <i>Py</i>-infected or uninfected WT CD45.1⁺/<i>Ifnar1</i>^<i>-/-</i> (ratio 50:50) mixed bone marrow chimeras (lower bar graph, n = 7 mice) are shown. (<b>D</b>) The proportion of blood Ly6C⁺ monocyte and NK cells expressing various surface markers in <i>Py</i>-infected WT and <i>Ifnar1</i>^<i>-/-</i> mice (n = 6–10 mice) or WT CD45.1⁺/<i>Ifnar1</i>^<i>-/-</i> mixed chimeras (n = 7 mice) are reported. Experiments were replicated 2–3 times. P-values are indicated when applicable.</p

Mechanistic model summarizing findings.

<p>Mechanistic model summarizing findings.</p

Plasmacytoid dendritic cells produce immune-activating type I IFN via TLR7/MyD88 but not STING.

<p>(<b>A</b>) Frequencies of WT (CD45.1⁺) or KO (<i>Myd88</i>^<i>-/-</i>, <i>Tlr7</i>^<i>-/-</i>, <i>Sting</i>^<i>Gt/Gt</i>) YFP⁺ pDCs in the bone marrow of <i>Py</i>-infected WT/KO <i>Ifnb-Yfp</i>^<i>+/+</i> mixed chimeras (n = 4–6 mice/chimera). Bar graphs average all individual mice across 2 replicate experiments. (<b>B</b>) Frequency of YFP⁺ pDCs in the bone marrow of WT and <i>Tlr7</i>^-/- <i>Ifnb-Yfp</i>^<i>+/+</i> reporter mice 1.5 day post <i>Py</i> infection (n = 4 mice/genotype). Bar graphs summarize the frequencies of YFP⁺ pDCs in the blood and bone marrow. (<b>C</b>) IFNα levels in the blood and the bone marrow of <i>Py</i>-infected <i>Tlr7</i>^<i>-/-</i>, <i>Tlr9</i>^<i>-/-</i>, WT mice or uninfected mice (n = 4–8 mice/genotype). <b>(D)</b> <i>Bdca2-Dtr</i>^<i>+/-</i>/<i>Tlr7</i>^<i>-/-</i>, <i>Bdca2-Dtr</i>^<i>+/-</i>/<i>Tlr9</i>^<i>-/-</i>, <i>Bdca2-Dtr</i>^<i>+/-</i>/<i>Sting</i>^<i>Gt/Gt</i> or control WT/KO (ratios 50:50 or 30:70, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005975#ppat.1005975.s007" target="_blank">S5 Fig</a>) mixed bone marrow chimeras (n = 3–5 mice/chimera) were treated with DT prior <i>Py</i> infection, and activation profiles of blood Ly6C⁺ monocytes and NK cells in the blood using indicated markers were measured. Experiments were replicated 2–4 times. P-values are indicated when applicable.</p

Plasmacytoid dendritic cells produce immune-activating type I IFN in the bone marrow and the blood of <i>Py</i>-infected mice.

<p>(<b>A</b>) WT <i>Ifnb-Yfp</i>^<i>+/+</i> reporter mice (n>5) were inoculated i.v. with 2x10⁵ <i>Py 17X YM</i> iRBCs and blood and bone marrow cells were stained with the lineage markers CD11b, CD3, CD19, NK1.1, Ly6C, BST2 and Siglec-H. The phenotype of YFP⁺ cells is shown. (<b>B</b>) Frequencies of YFP⁺ pDCs (CD11b^loBST2⁺SiglecH⁺) in bone marrow, blood, spleen, and liver of <i>Py</i>-infected WT <i>Ifnb-Yfp</i>^<i>+/+</i> reporter mice (1.5 day, n = 3–10 mice). (<b>C</b>) Kinetics of YFP expression by pDCs in the bone marrow and blood of <i>Py</i>-infected WT <i>Ifnb-Yfp</i>^<i>+/+</i> reporter mice and absolute numbers of YFP⁺ pDCs in the indicated compartments (n = 3–11 mice/time point). (<b>D</b>) The bar graph shows the absolute number of pDCs in the bone marrow, blood, and spleen 1.5 days post infection (n = 7 mice). Kinetics of total pDC frequency among CD45⁺ cells in the bone marrow, blood, and spleen during the first 48 hours of <i>Py</i>-infection (n = 5–7 mice/time point). (<b>E</b>) Activation profiles (CD86, BST2, ICAM-1) of pDCs in the bone marrow of <i>Py</i>-infected (YFP⁺, YFP^neg) or uninfected (YFP^neg) <i>Ifnb-Yfp</i>^<i>+/+</i> reporter mice (n>5 mice/condition). (<b>F, G</b>) DT-treated <i>Bdca2-Dtr</i>^<i>+/-</i> or control WT B6 mice were inoculated with 2x10⁵ <i>Py 17X YM</i> iRBCs and 1.5 days later, levels of IFNα in the blood and bone-marrow (F, n = 7–13 mice), and activation profiles of Ly6C⁺ monocytes and NK cells using indicated markers were measured (G, n = 3–7 mice). (<b>H</b>) FACS histogram overlays of indicated chemokine receptor expression on pDCs from <i>Py</i>-infected versus naïve mice (n = 3 mice/condition). Experiments were replicated 2–4 times. P-values are indicated when applicable.</p

Production of systemic type I IFN during severe murine blood stage malaria requires both MyD88 and STING sensing pathways.

<p>WT, <i>Myd88</i>^<i>-/-</i> or <i>Sting</i>^<i>Gt/Gt</i> B6 mice crossed to <i>Ifnb-Yfp</i>^<i>+/+</i> reporter mice were inoculated i.v. with 2x10⁵ <i>Py 17X YM</i> iRBCs. 1.5 days later, levels of IFNα (<b>A,</b> n = 4–10 mice/genotype) and frequencies of YFP⁺ cells among pDCs, as well as absolute numbers (<b>B</b>), in the blood and bone marrow were determined (n = 3–15 mice/genotype). (<b>C</b>) Blood cells were stained for the cell-surface lineage markers CD11b, Ly6C, NKp46, BST2, Siglec-H, CD45, and indicated activation markers. Expression of activation markers on Ly6C⁺ monocytes, NK cells and pDCs in the blood of <i>Py</i>-infected compared to uninfected mice is shown (n = 3–8 mice/genotype). Experiments were replicated 2–3 times. P-values are indicated when applicable.</p

Hierarchical clustering of differentially abundant plasma metabolites during CM and convalescence in a paired analysis of eleven Malawian children.

<p>A heatmap of a heirarchical clustering of 247 differentially abundant (p < 0.05, FDR < 0.10, paired t test) plasma metabolites between CM and convalescence (one month later). Unsupervised hierchical clustering segregates the samples by clinical state. The asterisk notes one child with mild malaria diagnosed during the convalescent visit.</p