Methylenetetrahydrofolate reductase (MTHFR) deficiency enhances resistance against cytomegalovirus infection

Abstract

Folates provide one-carbon units for nucleotide synthesis and methylation reactions. A common polymorphism in the MTHFR gene (677C → T) results in reduced enzymatic activity, and is associated with an increased risk for neural tube defects and cardiovascular disease. The high prevalence of this polymorphism suggests that it may have experienced a selective advantage under environmental pressure, possibly an infectious agent. To test the hypothesis that methylenetetrahydrofolate reductase (MTHFR) genotype influences the outcome of infectious disease, we examined the response of Mthfr-deficient mice against mouse cytomegalovirus (MCMV) infection. Acute MCMV infection of Mthfr−/− mice resulted in early control of cytokine secretion, decreased viral titer and preservation of spleen immune cells, in contrast to Mthfr wild-type littermates. The phenotype was abolished in MTHFR transgenic mice carrying an extra copy of the gene. Infection of primary fibroblasts with MCMV showed a decrease in viral replication and in the number of productively infected cells in Mthfr+/− fibroblasts compared with wild-type cells. These results indicate that Mthfr deficiency protects against MCMV infection in vivo and in vitro, suggesting that human genetic variants may provide an advantage in the host response against certain pathogens.

Introduction

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism that generates essential precursors for DNA and protein synthesis. MTHFR synthesises the primary circulating form of folate, 5-methyltetrahydrofolate, which is required for the methylation of homocysteine to form methionine.1 (Supplementary Figure 1) MTHFR gene variation is common in humans with changes ranging from complete loss-of-function mutations to silent mutations2, 3, 4 It should be noted that certain MTHFR variants with reduced function persist at high frequency in some human populations;5, 6 the most common and widely studied in this group is the 677C → T mutation. The 677 variant encodes a thermolabile form of MTHFR with reduced activity and leads to an elevation of plasma homocysteine.7, 8 The variant is recognised as the first genetic risk factor for neural tube defects9 and also increases the risk of coronary heart disease.10 The high allele frequency of this MTHFR variant suggests that it may be of some benefit to the host.

Deleterious alleles commonly persist in the human population under pathogen selection, as exemplified by the maintenance of mutant hemoglobin alleles responsible for sickle cell anemia and other hemoglobinopathies in regions endemic for malaria.11 Recently, the 677C → T variant was reported to be associated with the presence of anti-HBV antibodies and reduced HBV-DNA levels in West Africa.12 However, no study has directly examined the influence of MTHFR expression on resistance to pathogens. Animal models exhibiting different levels of MTHFR expression13, 14 are informative tools to address such questions. We have previously reported that mice with two targeted null mutations in the gene (Mthfr−/−) and mice with a single null mutation (Mthfr+/−) exhibit similar pathological changes to humans with severe and mild-MTHFR deficiency, respectively.13 In contrast, MTHFR-transgenic mice with increased levels of MTHFR do not present overt abnormalities, but studies with methotrexate have shown increased sensitivity to the drug due to MTHFR's role in regulating folate distributions between nucleotide synthesis and methylation.14 This mouse model of MTHFR-related folate metabolism deficiency may help to untangle the relationship between folate metabolism and viral infections.

Cytomegaloviruses (CMVs) are thought to have co-evolved with their respective hosts ever since the radiation of mammals.15 Thus, more than 50% of the human population is infected with CMV. Most infections are asymptomatic, but they can lead to pneumonia, retinitis and hepatitis, especially in immunocompromised individuals.16 MCMV is a natural mouse pathogen that mimics the effects of CMV in humans.17 CMVs interact intimately with genes involved in host folate metabolism because they lack the enzymes involved in DNA-precursor synthesis, including dihydrofolate reductase (DHFR) and thymidylate synthase (TS), which are essential for their replication.18, 19 MTHFR shares the same substrate as thymidylate synthase and requires precursors generated by dihydrofolate reductase for its function (Supplementary Figure 1), suggesting the possibility that MTHFR is also important for MCMV replication.

In this study, we examined MCMV replication in Mthfr-deficient and transgenic mice. We observed that Mthfr−/− mice control viral replication more efficiently than heterozygous or wild-type littermates. This improved response was associated with controlled systemic levels of type I and type II IFNs, as well as increased numbers of immune effectors such as NK cells and CD8+ T cells. Finally, we showed that the levels of viral DNA synthesis were substantially lower in Mthfr-deficient mouse embryonic fibroblast (MEF) cells than wild-type cells. Overall, low levels of Mthfr expression were associated with improved infection outcome suggesting a protective effect.

Results and discussion

We examined the effect of Mthfr genotype on the control of MCMV replication using Mthfr−/− mice on a C57BL/6 background. These mice were fed a synthetic amino acid-defined diet containing seven times less folate than the daily requirement for rodents. In preliminary experiments, mice received a virus inoculum of 5000 PFU per mouse, which did not show significant differences in viral titers in the spleen by day 3 post-infection (p.i.), the time of maximal viral growth (data not shown). In fact, C57BL/6 mice are naturally resistant to MCMV and clear the virus during the early phase of the infection.20 Resistance is mediated by direct interaction between the activating NK cell receptor Ly49H and a viral protein (m157) expressed by infected cells21, 22 Therefore, to overcome Ly49H-mediated protection, mice were infected with a five-fold higher dose of MCMV. At this dose, we observed a strong influence of the Mthfr genotype on the host response. By day 3 p.i., there was a stepwise decrease in viral load (in log10PFU) from 4.49±0.074 in Mthfr+/+ mice to 4.11±0.17 (P=0.0085) and 3.8±0.12 (P=0.0013) in Mthfr+/− and Mthfr−/− mice, respectively (Figure 1a). To further explore the effect of MTHFR levels on the response to MCMV, we evaluated the viral load in transgenic mice carrying a copy of the human Mthfr cDNA. As expected, MTHFR over-expression reversed the direction of the response seen in deficient mice. We observed a marginal, although not significant, increase in spleen viral titer of the MTHFR-transgenic mice compared with wild-type littermates (Figure 2a). Collectively, these results indicate that relatively lower MTHFR levels are associated with an enhanced control of acute MCMV infection.

Figure 1
figure1

Mthfr genotype influences the host response against mouse cytomegalovirus (MCMV) infection. (a) Replication of MCMV in the spleen of mice with different levels of methylenetetrahydrofolate reductase (MTHFR) expression. Mthfr−/− and MTHFR-transgenic (Tg) mice were generated as previously described13, 14 Groups of Mthfr−/−, Mthfr+/− and Mthfr+/+ littermates, or MTHFR-transgenic (Tg) and wild-type (wt) littermates were fed a low-folate diet for 1 month. Mice were infected by the intraperitoneal route with 2.5 × 104 PFU of salivary gland propagated MCMV.30 Viral titers were determined in the spleen at day 3 post-infection (p.i.) by plaque-forming assays.30 (b) Innate cytokine response against MCMV infection in Mthfr−/− mice. Mthfr-deficient mice and controls were infected as before, and cytokine levels were determined in serum at day 1.5 p.i. IFNα activity was measured using L929 cells in a standard microtiter protection assay against infection with mouse vesicular stomatitis virus.31 IFNγ levels were quantified by standard sandwich enzyme linked immunosorbent assay (ELISA). (c) Spleen cell analyses in Mthfr −/− mice after MCMV infection. Mthfr-deficient mice and controls were infected as before. To analyse the lymphoid populations at day 3 p.i., splenocytes were prepared and Fc receptors were blocked by incubating the cells with 5 ug ml−1 of 2.4G2 antibody before staining with the indicated antibodies, anti-CD4 (clone Gk1.5) and anti-CD8a (clone 53-6.7) antibodies were from eBioscience (San Diego, CA, USA) and the anti-NK1.1 (clone PK136) antibody was from BD Pharmingen (Mississauga, ON, Canada). Data were captured with a FACSCalibur flow cytometer and analysed using CellQuest (BD Biosciences, Mississauga, ON, Canada). Cell numbers of the indicated populations are expressed as mean±s.e.m. of three mice per group.

Figure 2
figure2

Mthfr genotype influences mouse cytomegalovirus (MCMV) infectivity of primary fibroblasts. Mouse embryonic fibroblast (MEF) cells of Mthfr+/+ and Mthfr+/− mice were prepared from day 14 embryos from Mthfr+/− intercrossed mice. Cells were grown from individual embryos and genotyped using Mthfr-specific primers, as described previously.13 (a) MEF were cultured in 12-well plates in RPMI 1640 folate-free media (Invitrogen, Burlington, ON, Canada) and infected in quadruplicate with wild-type (wt) MCMV at a multiplicity of infection (M.O.I.) of 1. DNA was extracted from the cells at indicated times using the DNeasy Tissue Kit (Qiagen, Mississauga, ON, Canada) to monitor MCMV genomes by quantitative PCR using SYBR Green Platinum Supermix-UDG (Invitrogen). IE1 amplicons were produced by PCR using IE1 specific forward and reverse primers (F:5′-TCAGCCATCAACTCTGCTACCAAC-3′, R:5′-GTGCTAGATTGTATCTGGTGCTCCTC-3′). The β-actin gene was used as an internal control (F: 5′-TGGAGAAAATCTGGCACCAC-3′, R:5′-AATGGTGATGACCTGGCCGT-3′) as described.28 Copy number was calculated from a β-actin standard (amplicon in pGEM-T) over a range of 101–108 copies. Results are expressed as the mean±s.e.m. of the ratio of copies of IE1 to β-actin. (b) MEF cells from Mthfr+/+ and Mthfr+/− mice infected with an MCMV variant expressing green fluorescent protein (GFP)29 at a M.O.I. of 0.4 for 48 h. MEF were stained with a PE-conjugated monoclonal antibody (mAb) against H-2Db to assess MHC-I expression. Histograms show the relative proportions of cells which are either: MHC-I positive and GFP negative (MHC-I+/MCMV); both MHC-I and GFP positive (MHC-I+/MCMV+); or MHC-I negative and GFP positive (MHC-I/MCMV+). GraphPad Prism software was used to conduct unpaired, two-tailed t-tests.

Uncontrolled MCMV replication is usually accompanied by massive production of pro-inflammatory cytokines, such as IFNα, IFNγ and interleukin-12, and loss of spleen cell populations, which together contribute to MCMV-mediated pathology23, 24 In fact, detrimental effects of high levels of IFNα during viral infection have been observed25, 26 Abundant levels of IFNα can compromise host immune responses by inhibiting the differentiation of dendritic cells or by leading to CD8+ T cell attrition25, 26 In contrast, MCMV-resistant mice showing low viral load present limited production of pro-inflammatory cytokines and increased specific sub-populations of spleen cells in comparison with susceptible mice24, 27 Thus, we monitored serum levels of IFNs at 1.5 days p.i., the peak of cytokine production and spleen cell numbers at 3 days p.i., the peak of cell loss. Mthfr−/− and Mthfr+/− mice had lower cytokine levels than their wild-type counterparts even though the differences did not reach significance (Figure 1b). In addition, Mthfr−/− mice had a significantly higher number of total spleen cells as well as CD4+T, CD8+T and NK cell populations in comparison with Mthfr+/− or wild-type mice (Figure 1c). Moreover, the proportion of CD8+T cells in Mthfr−/− was 11.5±1.8% and significantly higher than Mthfr+/− and wild-type mice, with values of 7.7±1.1 and 8.2±1.7% respectively (P=0.01). Thus, in terms of cytokine response and expansion of spleen cell populations, Mthfr deficiency is associated with a MCMV-resistant immuno-phenotype. The increased size of the NK-cell pool and/or higher numbers of T cells may provide potential mechanisms for the relative resistance of Mthfr−/− mice to viral infection, as reported for MCMV-resistant mouse strains.27 However, it remains to be determined if the enhanced immune response is a direct or indirect effect of Mthfr deficiency. It would be important to determine if Mthfr−/− immune cells proliferate more efficiently because of the increased availability of folate derivatives for nucleotide synthesis when MTHFR activity is compromised (see Figure 1). Alternatively, the increased resistance to infection could be due to changes in DNA methylation status of key genes, an increase in pro-inflammatory homocysteine or a decrease in critical metabolites that are essential for viral replication.

To evaluate the effect of Mthfr genotype on MCMV cell replication, viral genome synthesis was determined in MEFs. Owing to different growth kinetics of Mthfr−/− cells, only Mthfr+/− and Mthfr+/+ MEF were analysed after culturing in low-folate medium. Viral-DNA replication was followed by quantitative PCR amplification of the IE1 early gene at various times p.i.28 The absence of MCMV DNA at 0 h p.i. indicated that the input virus was not amplified. The IE1 per actin copy number ratios were 1.5-and 4-fold higher in wild-type compared with Mthfr+/− MEF cells at 24 h and 48 h p.i., respectively (Figure 2a). To confirm these results, we analysed the presence of MCMV infection in combination with MHC class I (MHC-I) expression in Mthfr+/+ and Mthfr+/− fibroblasts infected with a recombinant green fluorescent protein (GFP)-MCMV virus.29 This virus carries the green fluorescent protein gene under the control of the IE1 promoter; thus, productively infected cells are fluorescently labeled. As MCMV encodes a number of genes dedicated to interfere with surface expression of MHC-I,29 the cells were stained with an antibody directed against the MHC-I product H2-Db Hence we distinguished three populations (by FACS analysis at 24 h p.i.: 1) of highly infected cells, which showed no MHC-I expression and high green fluorescent protein expression; 2) transiently infected cells, which were doubly labeled, but showed low MHC-I and green fluorescent protein expression and 3) uninfected cells, which were MHC-I positive, but showed no green fluorescent protein staining (not shown). The proportion of transiently infected cells was similar in Mthfr+/− and Mthfr+/+ fibroblasts. In contrast, we found that Mthfr+/− fibroblasts presented a significantly higher proportion of MCMV-uninfected cells (P=0.02) and a significantly lower proportion of infected cells (P=0.007) when compared with Mthfr+/+ cells (Figure 2b). Taken together, the cell-based data suggest that reduced MTHFR expression modulates MCMV viral-DNA synthesis in low-folate medium. Under these conditions, the limited availability of key folate intermediates in Mthfr+/− cells may negatively effect the formation of precursors for viral DNA synthesis, DNA methylation or synthesis of viral proteins. Our results are consistent with those previously obtained by inhibition of thymidylate synthase and dihydrofolate reductase, two important enzymes in folate metabolism,18, 19 and further underscore the crucial role of the folate pathway during MCMV replication. However, it remains to be determined whether the differences observed in MEF cells are sufficient to explain the phenotype seen in Mthfr -deficient mice.

Our study supports the hypothesis that MTHFR deficiency limits MCMV replication in vivo and in vitro. Subsequent testing of human MTHFR-deficient cell lines may provide insight into the role of MTHFR in the control of human CMV replication. Genetic epidemiological studies aimed at examining possible associations between MTHFR genotypes and human CMV susceptibility may indicate a potential advantage for these mutations in human populations, in addition to revealing targets for decreasing the CMV burden in individuals with active infection.

Homozygosity for the 677 polymorphism occurs in 10−15% of many Caucasian populations.1, 2 Several genetic disorders in humans may have reached a high prevalence through resistance to infection, including hemoglobinopathies and cystic fibrosis. This study provides intriguing data towards our hypothesis that the 677 polymorphism may also have achieved its high prevalence through resistance to infection. Although CMV may not have been the pathogen that directly contributed to the positive selection, our results warrant the investigation of the influence of MTHFR levels on resistance to other viral, bacterial and parasitic infections.

Conflict of interest

The authors declare no conflict of interest.

References

  1. 1

    Stanger O . Physiology of folic acid in health and disease. Curr Drug Metab 2002; 3: 211–223.

  2. 2

    Goyette P, Christensen B, Rosenblatt DS, Rozen R . Severe and mild mutations in cis for the methylenetetrahydrofolate reductase (MTHFR) gene, and description of five novel mutations in MTHFR. Am J Hum Genet 1996; 59: 1268–1275.

  3. 3

    Goyette P, Frosst P, Rosenblatt DS, Rozen R . Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency. Am J Hum Genet 1995; 56: 1052–1059.

  4. 4

    Goyette P, Sumner JS, Milos R, Duncan AM, Rosenblatt DS, Matthews RG et al. Human methylenetetrahydrofolate reductase: Isolation of cDNA, mapping and mutation identification. Nat Genet 1994; 7: 195–200.

  5. 5

    Gueant-Rodriguez RM, Gueant JL, Debard R, Thirion S, Hong LX, Bronowicki JP et al. Prevalence of methylenetetrahydrofolate reductase 677 T and 1298C alleles and folate status: A comparative study in Mexican, West African, and European populations. Am J Clin Nutr 2006; 83: 701–707.

  6. 6

    Schneider JA, Rees DC, Liu YT, Clegg JB . Worldwide distribution of a common methylenetetrahydrofolate reductase mutation. Am J Hum Genet 1998; 62: 1258–1260.

  7. 7

    Weisberg I, Tran P, Christensen B, Sibani S, Rozen R . A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol Genet Metab 1998; 64: 169–172.

  8. 8

    Guenther BD, Sheppard CA, Tran P, Rozen R, Matthews RG, Ludwig ML . The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia. Nat Struct Biol 1999; 6: 359–365.

  9. 9

    van der Put NM, van den Heuvel LP, Steegers-Theunissen RP, Trijbels FJ, Eskes TK, Mariman EC et al. Decreased methylene tetrahydrofolate reductase activity due to the 677C-->T mutation in families with spina bifida offspring. J Mol Med 1996; 74: 691–694.

  10. 10

    Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG et al. A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995; 10: 111–113.

  11. 11

    Pouniotis DS, Proudfoot O, Minigo G, Hanley JL, Plebanski M . Malaria parasite interactions with the human host. J Postgrad Med 2004; 50: 30–34.

  12. 12

    Bronowicki JP, Abdelmouttaleb I, Peyrin-Biroulet L, Venard V, Khiri H, Chabi N et al. Methylenetetrahydrofolate reductase 677T allele protects against persistent HBV infection in West Africa. J Hepatol 2008; 48: 532–539.

  13. 13

    Chen Z, Karaplis AC, Ackerman SL, Pogribny IP, Melnyk S, Lussier-Cacan S et al. Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition. Hum Mol Genet 2001; 10: 433–443.

  14. 14

    Celtikci B, Leclerc D, Lawrance AK, Deng L, Friedman HC, Krupenko NI et al. Altered expression of methylenetetrahydrofolate reductase modifies response to methotrexate in mice. Pharmacogenet Genomics 2008; 18: 577–589.

  15. 15

    McGeoch DJ, Rixon FJ, Davison AJ . Topics in herpesvirus genomics and evolution. Virus Res 2006; 117: 90–104.

  16. 16

    Vancikova Z, Dvorak P . Cytomegalovirus infection in immunocompetent and immunocompromised individuals--a review. Curr Drug Targets Immune Endocr Metabol Disord 2001; 1: 179–187.

  17. 17

    Reddehase MJ, Podlech J, Grzimek NK . Mouse models of cytomegalovirus latency: Overview. J Clin Virol 2002; 25 (Suppl 2): S23–S36.

  18. 18

    Lembo D, Gribaudo G, Cavallo R, Riera L, Angeretti A, Hertel L et al. Human cytomegalovirus stimulates cellular dihydrofolate reductase activity in quiescent cells. Intervirology 1999; 42: 30–36.

  19. 19

    Gribaudo G, Riera L, Lembo D, De Andrea M, Gariglio M, Rudge TL et al. Murine cytomegalovirus stimulates cellular thymidylate synthase gene expression in quiescent cells and requires the enzyme for replication. J Virol 2000; 74: 4979–4987.

  20. 20

    Krmpotic A, Bubic I, Polic B, Lucin P, Jonjic S . Pathogenesis of murine cytomegalovirus infection. Microbes Infect 2003; 5: 1263–1277.

  21. 21

    Smith HR, Heusel JW, Mehta IK, Kim S, Dorner BG, Naidenko OV et al. Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc Natl Acad Sci USA 2002; 99: 8826–8831.

  22. 22

    Arase H, Mocarski ES, Campbell AE, Hill AB, Lanier LL . Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science 2002; 296: 1323–1326.

  23. 23

    Bekiaris V, Timoshenko O, Hou TZ, Toellner K, Shakib S, Gaspal F et al. Ly49H+ NK cells migrate to and protect splenic white pulp stroma from murine cytomegalovirus infection. J Immunol 2008; 180: 6768–6776.

  24. 24

    Fodil-Cornu N, Lee SH, Belanger S, Makrigiannis AP, Biron CA, Buller RM et al. Ly49h-deficient C57BL/6 mice: A new mouse cytomegalovirus-susceptible model remains resistant to unrelated pathogens controlled by the NK gene complex. J Immunol 2008; 181: 6394–6405.

  25. 25

    Hahm B, Trifilo MJ, Zuniga EI, Oldstone MB . Viruses evade the immune system through type I interferon-mediated STAT2-dependent, but STAT1-independent, signaling. Immunity 2005; 22: 247–257.

  26. 26

    McNally JM, Zarozinski CC, Lin MY, Brehm MA, Chen HD, Welsh RM . Attrition of bystander CD8 T cells during virus-induced T-cell and interferon responses. J Virol 2001; 75: 5965–5976.

  27. 27

    Robbins SH, Bessou G, Cornillon A, Zucchini N, Rupp B, Ruzsics Z et al. Natural killer cells promote early CD8 T cell responses against cytomegalovirus. PLoS Pathog 2007; 3: e123.

  28. 28

    Wheat RL, Clark PY, Brown MG . Quantitative measurement of infectious murine cytomegalovirus genomes in real-time PCR. J Virol Methods 2003; 112: 107–113.

  29. 29

    Pinto AK, Hill AB . Viral interference with antigen presentation to CD8+ T cells: Lessons from cytomegalovirus. Viral Immunol 2005; 18: 434–444.

  30. 30

    Depatie C, Lee SH, Stafford A, Avner P, Belouchi A, Gros P et al. Sequence-ready BAC contig, physical, and transcriptional map of a 2-Mb region overlapping the mouse chromosome 6 host-resistance locus Cmv1. Genomics 2000; 66: 161–174.

  31. 31

    Ishikawa R, Biron CA . IFN induction and associated changes in splenic leukocyte distribution. J Immunol 1993; 150: 3713–3727.

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Acknowledgements

We are grateful to Nadia Prud’homme for technical assistance and Gregory Boivin for critical review of the text. This work was supported by the Canadian Institutes of Health Research MOP-7781 (SMV) and MOP-4232 (RR).

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Correspondence to S M Vidal.

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Supplementary Information accompanies the paper on Genes and Immunity website (http://www.nature.com/gene)

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Fodil-Cornu, N., Kozij, N., Wu, Q. et al. Methylenetetrahydrofolate reductase (MTHFR) deficiency enhances resistance against cytomegalovirus infection. Genes Immun 10, 662–666 (2009). https://doi.org/10.1038/gene.2009.50

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Keywords

  • MTHFR
  • folate metabolism
  • cytomegalovirus
  • mouse models
  • innate resistance

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