High Dietary Folate in Mice Alters Immune Response and Reduces Survival after Malarial Infection.

Malaria is a significant global health issue, with nearly 200 million cases in 2013 alone. Parasites obtain folate from the host or synthesize it de novo. Folate consumption has increased in many populations, prompting concerns regarding potential deleterious consequences of higher intake. The impact of high dietary folate on the host's immune function and response to malaria has not been examined. Our goal was to determine whether high dietary folate would affect response to malarial infection in a murine model of cerebral malaria. Mice were fed control diets (CD, recommended folate level for rodents) or folic acid-supplemented diets (FASD, 10x recommended level) for 5 weeks before infection with Plasmodium berghei ANKA. Survival, parasitemia, numbers of immune cells and other infection parameters were assessed. FASD mice had reduced survival (p<0.01, Cox proportional hazards) and higher parasitemia (p< 0.01, joint model of parasitemia and survival) compared with CD mice. FASD mice had lower numbers of splenocytes, total T cells, and lower numbers of specific T and NK cell sub-populations, compared with CD mice (p<0.05, linear mixed effects). Increased brain TNFα immunoreactive protein (p<0.01, t-test) and increased liver Abca1 mRNA (p<0.01, t-test), a modulator of TNFα, were observed in FASD mice; these variables correlated positively (rs = 0.63, p = 0.01). Bcl-xl/Bak mRNA was increased in liver of FASD mice (p<0.01, t-test), suggesting reduced apoptotic potential. We conclude that high dietary folate increases parasite replication, disturbs the immune response and reduces resistance to malaria in mice. These findings have relevance for malaria-endemic regions, when considering anti-folate anti-malarials, food fortification or vitamin supplementation programs

Differences in spleen immune cell populations at 7 dpi between CD and FASD mice.

<p>Values are means ± SEM. Two experiments are shown. FASD mice (grey; n = 8 (<i>Mthfr</i>^<i>+/+</i>) and n = 6 (<i>Mthfr</i>^<i>+/-</i>))had significantly decreased populations of (a) T cells (p = 0.01, linear mixed effects), (b) CD8⁺ T cells (p<0.01, linear mixed effects), (c) CD4⁺ T cells (p<0.05, linear mixed effects), (d) CCR4⁺ CD4⁺ T cells (p<0.05, linear mixed effects), (e) CCR4⁺ NK cells (p<0.01, linear mixed effects) and (f) IFNγ⁺ NK cells (p<0.05, linear mixed effects) compared with CD mice (black; n = 8 (<i>Mthfr</i>^<i>+/+</i>) and n = 8 (<i>Mthfr</i>^<i>+/+</i>)).</p

Survival of mice fed diets with variable folate content.

<p>(a) CD mice (n = 8) and CD+SST mice (n = 7) did not show differences in survival at 15 dpi (p = 1.0, log-rank test); MC mice (n = 7) had 100% mortality by 10 dpi. (b) Survival of CD^+/+ mice (n = 9), CD^+/- mice (n = 14), FASD^+/+ mice (n = 11), and FASD^+/- mice (n = 12) in two experiments combined. Mice fed CD had a greater chance of surviving infection than mice fed FASD (p<0.01, Cox proportional hazards). <i>Mthfr</i> genotype had no effect on chance of survival in mice fed CD or FASD (p = 0.45, Cox proportional hazards).</p

Parasitemia in CD and FASD mice.

<p>Parasitemia was measured every 48h from 6–20dpi. (a) Observed parasitemia trajectories from 5 randomly selected CD^<i>+/+</i> (n = 9), CD^<i>+/-</i> (n = 14), FASD^<i>+/+</i> (n = 11), and FASD^<i>+/-</i> (n = 12) mice from two combined experiments; selected trajectories are representative of the full data. (b) Statistical model showing estimated mean natural log of the parasitemia used to interpret parasitemia. The FASD mice had significantly higher overall levels of parasitemia than CD mice (p< 0.01, joint model of parasitemia and survival).</p

Primers for quantitative real-time RT-PCR.

<p>The table includes expected amplicon size, melting temperature (T_m) and reference source. Housekeeping genes (<i>Gapdh</i> and <i>Ywhaz</i>) were used to generate the normalization factor.</p><p>Primers for quantitative real-time RT-PCR.</p

Differences in TNFα immunoreactive protein in brain and in relative <i>Abca1</i> mRNA in liver between CD and FASD <i>Mthfr</i>^<i>+/+</i> mice at 7 dpi.

<p>Values are means ± SEM for A and B. (a) FASD mice (grey bar; 3 experiments combined; n = 10) had significantly higher levels of TNFα protein in brain compared with CD mice (black bar; 3 experiments combined; n = 7). Representative Western blot is shown below the graph. (b) Livers of FASD mice (grey bar; 3 experiments combined; n = 9) had significantly higher relative <i>Abca1</i> mRNA levels than livers of CD mice (black bar; 3 experiments combined; n = 9)). (c) Brain TNFα protein correlated with <i>Abca1</i> mRNA levels in liver of mice in both dietary groups (r_s = 0.63, Spearman correlation, p = 0.01; n = 16). **p<0.01, unpaired t-test.</p

Early Manifestations of Brain Aging in Mice Due to Low Dietary Folate and Mild MTHFR Deficiency

Folate is an important B vitamin required for methylation reactions, nucleotide and neurotransmitter synthesis, and maintenance of homocysteine at nontoxic levels. Its metabolism is tightly linked to that of choline, a precursor to acetylcholine and membrane phospholipids. Low folate intake and genetic variants in folate metabolism, such as the methylenetetrahydrofolate reductase (MTHFR) 677 C>T polymorphism, have been suggested to impact brain function and increase the risk for cognitive decline and late-onset Alzheimer’s disease. Our study aimed to assess the impact of genetic and nutritional disturbances in folate metabolism, and their potential interaction, on features of cognitive decline and brain biochemistry in a mouse model. Wild-type and Mthfr+/− mice, a model for the MTHFR 677 C>T polymorphism, were fed control or folate-deficient diets from weaning until 8 and 10 months of age. We observed short-term memory impairment measured by the novel object paradigm, altered transcriptional levels of synaptic markers and epigenetic enzymes, as well as impaired choline metabolism due to the Mthfr+/− genotype in cortex or hippocampus. We also detected changes in mRNA levels of Presenillin-1, neurotrophic factors, one-carbon metabolic and epigenetic enzymes, as well as reduced levels of S-adenosylmethionine and acetylcholine, due to the folate-deficient diet. These findings shed further insights into the mechanisms by which genetic and dietary folate metabolic disturbances increase the risk for cognitive decline and suggest that these mechanisms are distinct.This work was supported by the Canadian Institutes of Health Research (MOP-43232 to RR). RHB is the recipient of a Doctoral Award from the Fonds de Recherche du Québec-Santé. MCT is the recipient of a Predoctoral Fellowship from MINECO (FPU 2013) and Post-Doctoral Award from the Fonds de Recherche du Québec-Santé. The Research Institute is supported by a Center’s grant from the Fonds de Recherche du Québec-Santé

High dietary folate in pregnant mice leads to pseudo-MTHFR deficiency and altered methyl metabolism, with embryonic growth delay and short-term memory impairment in offspring

Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reactions. Severe MTHFR deficiency results in homocystinuria and neurologic impairment. Mild MTHFR deficiency (677C > T polymorphism) increases risk for complex traits, including neuropsychiatric disorders. Although low dietary folate impacts brain development, recent concerns have focused on high folate intake following food fortification and increased vitamin use. Our goal was to determine whether high dietary folate during pregnancy affects brain development in murine offspring. Female mice were placed on control diet (CD) or folic acid-supplemented diet (FASD) throughout mating, pregnancy and lactation. Three-week-old male pups were evaluated for motor and cognitive function. Tissues from E17.5 embryos, pups and dams were collected for choline/methyl metabolite measurements, immunoblotting or gene expression of relevant enzymes. Brains were examined for morphology of hippocampus and cortex. Pups of FASD mothers displayed short-term memory impairment, decreased hippocampal size and decreased thickness of the dentate gyrus. MTHFR protein levels were reduced in FASD pup livers, with lower concentrations of phosphocholine and glycerophosphocholine in liver and hippocampus, respectively. FASD pup brains showed evidence of altered acetylcholine availability and Dnmt3a mRNA was reduced in cortex and hippocampus. E17.5 embryos and placentas from FASD dams were smaller. MTHFR protein and mRNA were reduced in embryonic liver, with lower concentrations of choline, betaine and phosphocholine. Embryonic brain displayed altered development of cortical layers. In summary, high folate intake during pregnancy leads to pseudo-MTHFR deficiency, disturbed choline/methyl metabolism, embryonic growth delay and memory impairment in offspring. These findings highlight the unintended negative consequences of supplemental folic acid.Canadian Institutes of Health Research (MOP-43232 to R.R.); Medical Research Council (N003713 to N.G.); Doctoral Award from the Fonds de Recherche du Que´bec-Sante´ (to R.H.B.); Fonds de Recherche du Que´bec-Sante´ (grant to McGill University Health Centre). Funding to pay the Open Access publication charges for this article was provided by Medical Research Council (N003713 to NG).Peer reviewe

High dietary folate in pregnant mice leads to pseudo-MTHFR deficiency and altered methyl metabolism, with embryonic growth delay and short-term memory impairment in offspring

Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reactions. Severe MTHFR deficiency results in homocystinuria and neurologic impairment. Mild MTHFR deficiency (677C>T polymorphism) increases risk for complex traits, including neuropsychiatric disorders. Although low dietary folate impacts brain development, recent concerns have focused on high folate intake following food fortification and increased vitamin use. Our goal was to determine whether high dietary folate during pregnancy affects brain development in murine offspring. Female mice were placed on control diet (CD) or folic acid-supplemented diet (FASD) throughout mating, pregnancy and lactation. Three-weekold male pups were evaluated for motor and cognitive function. Tissues from E17.5 embryos, pups and dams were collected for choline/methyl metabolite measurements, immunoblotting or gene expression of relevant enzymes. Brains were examined for morphology of hippocampus and cortex. Pups of FASD mothers displayed short-termmemory impairment, decreased hippocampal size and decreased thickness of the dentate gyrus. MTHFR protein levels were reduced in FASD pup livers, with lower concentrations of phosphocholine and glycerophosphocholine in liver and hippocampus, respectively. FASD pup brains showed evidence of altered acetylcholine availability and Dnmt3a mRNA was reduced in cortex and hippocampus. E17.5 embryos and placentas from FASD dams were smaller. MTHFR protein and mRNA were reduced in embryonic liver, with lower concentrations of choline, betaine and phosphocholine. Embryonic brain displayed altered development of cortical layers. In summary, high folate intake during pregnancy leads to pseudo-MTHFR deficiency, disturbed choline/methyl metabolism, embryonic growth delay and memory impairment in offspring. These findings highlight the unintended negative consequences of supplemental folic acid