Quantitative genome-scale metabolic modeling of human CD4+ T cell differentiation reveals subset-specific regulation of glycosphingolipid pathways

T cell activation, proliferation, and differentiation involve metabolic reprogramming resulting from the interplay of genes, proteins, and metabolites. Here, we aim to understand the metabolic pathways involved in the activation and functional differentiation of human CD4+ T cell subsets (T helper [Th]1, Th2, Th17, and induced regulatory T [iTreg] cells). Here, we combine genome-scale metabolic modeling, gene expression data, and targeted and non-targeted lipidomics experiments, together with in vitro gene knockdown experiments, and show that human CD4+ T cells undergo specific metabolic changes during activation and functional differentiation. In addition, we confirm the importance of ceramide and glycosphingolipid biosynthesis pathways in Th17 differentiation and effector functions. Through in vitro gene knockdown experiments, we substantiate the requirement of serine palmitoyltransferase (SPT), a de novo sphingolipid pathway in the expression of proinflammatory cytokines (interleukin [IL]-17A and IL17F) by Th17 cells. Our findings provide a comprehensive resource for selective manipulation of CD4+ T cells under disease conditions characterized by an imbalance of Th17/natural Treg (nTreg) cells.</p

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells

Regulatory T (Treg) cells are critical in regulating the immune response. In vitro induced Treg (iTreg) cells have significant potential in clinical medicine. However, applying iTreg cells as therapeutics is complicated by the poor stability of human iTreg cells and their variable suppressive activity. Therefore, it is important to understand the molecular mechanisms of human iTreg cell specification. We identified hypermethylated in cancer 1 (HIC1) as a transcription factor upregulated early during the differentiation of human iTreg cells. Although FOXP3 expression was unaffected, HIC1 deficiency led to a considerable loss of suppression by iTreg cells with a concomitant increase in the expression of effector T cell associated genes. SNPs linked to several immune-mediated disorders were enriched around HIC1 binding sites, and in vitro binding assays indicated that these SNPs may alter the binding of HIC1. Our results suggest that HIC1 is an important contributor to iTreg cell development and function

Plant Hormone Salicylic Acid Produced by a Malaria Parasite Controls Host Immunity and Cerebral Malaria Outcome

<div><p>The apicomplexan parasite <i>Toxoplasma gondii</i> produces the plant hormone abscisic acid, but it is unclear if phytohormones are produced by the malaria parasite <i>Plasmodium</i> spp., the most important parasite of this phylum. Here, we report detection of salicylic acid, an immune-related phytohormone of land plants, in <i>P</i>. <i>berghei</i> ANKA and <i>T</i>. <i>gondii</i> cell lysates. However, addition of salicylic acid to <i>P</i>. <i>falciparum</i> and <i>T</i>. <i>gondii</i> culture had no effect. We transfected <i>P</i>. <i>falciparum</i> 3D7 with the <i>nahG</i> gene, which encodes a salicylic acid-degrading enzyme isolated from plant-infecting <i>Pseudomonas</i> sp., and established a salicylic acid-deficient mutant. The mutant had a significantly decreased concentration of parasite-synthesized prostaglandin E₂, which potentially modulates host immunity as an adaptive evolution of <i>Plasmodium</i> spp. To investigate the function of salicylic acid and prostaglandin E₂ on host immunity, we established <i>P</i>. <i>berghei</i> ANKA mutants expressing <i>nahG</i>. C57BL/6 mice infected with <i>nahG</i> transfectants developed enhanced cerebral malaria, as assessed by Evans blue leakage and brain histological observation. The <i>nahG</i>-transfectant also significantly increased the mortality rate of mice. Prostaglandin E₂ reduced the brain symptoms by induction of T helper-2 cytokines. As expected, T helper-1 cytokines including interferon-γ and interleukin-2 were significantly elevated by infection with the <i>nahG</i> transfectant. Thus, salicylic acid of <i>Plasmodium</i> spp. may be a new pathogenic factor of this threatening parasite and may modulate immune function via parasite-produced prostaglandin E₂.</p></div

Identification of salicylic acid from <i>Plasmodium berghei</i> ANKA.

<p><i>P</i>. <i>berghei</i> ANKA was purified from infected mice blood, and salicylic acid (SA) was extracted, and analyzed by LC-triple TOF mass spectrometry. (A) Structural formula of SA. (B) LC chromatogram of SA standard (control) and <i>P</i>. <i>berghei</i> ANKA sample. (C) Fragmentation analysis of peaks in (B) (colored in aqua). Collision energy was 20 eV.</p

Parasite SA influences the cerebral malaria outcome.

<p>(A-C) Histological observation of infected mouse cerebellums. (A) Cerebellum infected by <i>nahG</i>-expressing parasites. Note the sequestrated leukocytes in microvessels. The inset image shows a higher magnification of the boxed portion. Phagocytized hemozoin is observed (arrowhead). (B) Brain of a mouse infected with <i>gfp</i>-expressing parasites. Slight microbleeding was observed, but no sequestrated vessels were found. (C) Brain of an uninfected control. Sections were stained by hematoxylin and eosin. (D) Evans blue leakage analysis of the severity of cerebral malaria. Photographs of brains from mice infected with <i>nahG</i>- (left upper) and <i>gfp-</i> (left middle) expressing parasites and uninfected controls (left bottom), and quantification of dye leakage (right). Mice (n = 5) were sacrificed at 6 days post-infection. Solid line, p<0.01; dashed line, p<0.05. C57BL/6 mice at 6 days post-infection were used for all experiments. Bar: 50 μm.</p

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells

Regulatory T (Treg) cells are critical in regulating the immune response. In vitro induced Treg (iTreg) cells have significant potential in clinical medicine. However, applying iTreg cells as therapeutics is complicated by the poor stability of human iTreg cells and their variable suppressive activity. Therefore, it is important to understand the molecular mechanisms of human iTreg cell specification. We identified hypermethylated in cancer 1 (HIC1) as a transcription factor upregulated early during the differentiation of human iTreg cells. Although FOXP3 expression was unaffected, HIC1 deficiency led to a considerable loss of suppression by iTreg cells with a concomitant increase in the expression of effector T cell associated genes. SNPs linked to several immune-mediated disorders were enriched around HIC1 binding sites, and in vitro binding assays indicated that these SNPs may alter the binding of HIC1. Our results suggest that HIC1 is an important contributor to iTreg cell development and function. Ullah et al. find that HIC1 is induced during human iTreg cell differentiation. HIC1 binds to and regulates the expression of key genes during iTreg differentiation. Several autoimmune-disease-associated SNPs are enriched near HIC1 ChIP-seq peaks.Peer reviewe