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.
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.
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.
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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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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
- folate metabolism
- mouse models
- innate resistance
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