Folate and Inflammation – links between folate and features of inflammatory conditions

Abstract

Folate serves as a cofactor for one-carbon (1C) transfer reactions. These reactions are involved in the synthesis of DNA nucleotides, the amino acid methionine, and in the regulation of homocysteine (Hcy) levels. Emerging evidence suggests that these reactions have roles in the development and maintenance of inflammatory responses, with optimal folate availability having key importance in preventing endothelial dysfunction and DNA instability. Low folate levels are commonly observed in chronic inflammatory diseases, indicating that inadequate folate may be involved in the pathogenesis of inflammatory conditions or that chronic inflammation increases folate requirements. These findings highlight folate interventions as a potential treatment in inflammatory disorders. However, current understanding of folate and its influence on inflammatory phenotypes is limited. Evidence indicates that the relationship between folate and inflammation is dependent on several factors, including the timing of intervention, dosage, and interaction with environment and genes. These factors require further investigation before recommendations for folate intake can be made for the prevention and treatment of inflammation. This review outlines the emerging role of folate in inflammation and key factors that may influence this relationship.

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Journal of Nutrition & Intermediary Metabolism
journal homepage: www.elsevier.com/locate/jnim
Folate and Inflammation –links between folate and features of inflammatory
conditions
Patrice Jones
a,b,∗
, Mark Lucock
a
, Christopher J. Scarlett
a
, Martin Veysey
a,c
, Emma L. Beckett
a,b
a
School of Environmental & Life Sciences, University of Newcastle, Ourimbah, NSW, Australia
b
Public Health Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia
c
Hull York Medical School, University of Hull, Hull, UK
HIGHLIGHTS
•Optimal folate levels may prevent endothelial dysfunction in inflammatory diseases.
•Folate may also alter inflammatory responses via DNA methylation and synthesis processes.
•The link between folate and inflammation varies based on several factors, such as timing of intervention.
•Further studies are needed before making folate intake recommendations around inflammation.
ARTICLE INFO
Keywords:
Folate
Inflammation
Endothelial dysfunction
DNA methylation
DNA synthesis
ABSTRACT
Folate serves as a cofactor for one-carbon (1C) transfer reactions. These reactions are involved in the synthesis of
DNA nucleotides, the amino acid methionine, and in the regulation of homocysteine (Hcy) levels. Emerging
evidence suggests that these reactions have roles in the development and maintenance of inflammatory re-
sponses, with optimal folate availability having key importance in preventing endothelial dysfunction and DNA
instability. Low folate levels are commonly observed in chronic inflammatory diseases, indicating that in-
adequate folate may be involved in the pathogenesis of inflammatory conditions or that chronic inflammation
increases folate requirements. These findings highlight folate interventions as a potential treatment in in-
flammatory disorders. However, current understanding of folate and its influence on inflammatory phenotypes is
limited. Evidence indicates that the relationship between folate and inflammation is dependent on several
factors, including the timing of intervention, dosage, and interaction with environment and genes. These factors
require further investigation before recommendations for folate intake can be made for the prevention and
treatment of inflammation. This review outlines the emerging role of folate in inflammation and key factors that
may influence this relationship.
1. Introduction
Folate is a B vitamin derived from natural sources, such as green
leafy vegetables, or from fortified foods or supplements. Folate-related
roles within the human body are reliant on reduced folate compounds
(i.e., tetrahydrofolates (THF)) holding and donating one-carbon (IC)
units for 1C transfer reactions. These reactions are required in the
synthesis of DNA nucleotides and the amino acid methionine, and the
subsequent regulation of homocysteine (Hcy) levels [1].
Folate-dependent 1C reactions have been extensively studied in the
context of cardiovascular disease and cancers, with increased risk of
these diseases in those with inadequate folate status [1]. In addition to
these well-established roles, interest in folate as a contributory factor in
inflammation is emerging. This interest follows studies demonstrating
cardiovascular-related outcomes and chronic inflammatory diseases
share characteristics which may relate to folate status, such as en-
dothelial dysfunction [2,3]. Endothelial dysfunction is defined as an
impairment in vasomotor tone regulation via imbalanced levels of va-
sodilator and vasoconstrictors, which results in the endothelium
shifting towards a pro-inflammatory state [4,5]. Folate may prevent
endothelium dysfunction by maintaining levels of Hcy and vasodilator,
nitric oxide (NO) [4–6]. In addition, a link between chronic in-
flammatory conditions such as inflammatory bowel diseases and ele-
vated risk of carcinomas is well-established, and likely involves the role
https://doi.org/10.1016/j.jnim.2019.100104
Received 29 June 2019; Received in revised form 31 October 2019; Accepted 6 November 2019
∗
Corresponding author. School of Environmental & Life Sciences, University of Newcastle, Ourimbah, NSW, Australia.
E-mail address: Patrice.Jones@uon.edu.au (P. Jones).
Journal of Nutrition & Intermediary Metabolism 18 (2019) 100104
Available online 09 November 2019
2352-3859/ © 2019 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).

of folate in regulating DNA stability via mechanisms related to DNA
methylation, synthesis and repair [7,8].
Observational evidence suggests a relationship exists between folate
levels and risk of inflammatory disease. Low serum folate and elevated
Hcy levels are commonly reported in patients with chronic in-
flammatory disorders, such as inflammatory bowel disease and rheu-
matoid arthritis [9,10]. However, results of in vivo and in vitro studies
examining folate supplementation in inflammation have been incon-
sistent. Interpretation of these studies is further complicated by several
factors that may alter the influence folate has in inflammation, such as
the timing of intervention, folate dosage, and interaction with en-
vironmental and genetic factors. These factors require further con-
sideration before recommendations for folate intake can be made for
the prevention and/or treatment of inflammation.
This review outlines emerging roles of folate in inflammation, en-
compassing regulation of endothelial dysfunction, via both Hcy-de-
pendent and Hcy-independent mechanisms, and the maintenance of
DNA stability through DNA methylation, synthesis and repair reactions.
Key factors that may modulate the activity of folate in these processes
are also discussed.
2. Regulation of Hcy
Hcy is a non-essential amino acid produced as an intermediate
product in the synthesis of essential amino acids methionine and cy-
steine. At elevated levels, Hcy can cause endothelial dysfunction, re-
sulting in a shift towards a pro-inflammatory state [4].
Folate may modulate Hcy-induced endothelial dysfunction through
its role as a major regulator of Hcy. Folate, in the form of 5-methylTHF,
is used in the remethylation of Hcy to methionine (Fig. 1). This process
involves the irreversible action of 5,10-methylenetetrahydrofolate re-
ductase (MTHFR) in reducing 5,10-methyleneTHF to 5-methylTHF, and
then B
12
-dependent methionine synthase utilising 5-methylTHF as a
methyl donor in the remethylation of homocysteine to methionine
[11,12].
Hcy-induced endothelial dysfunction has been extensively studied
in atherosclerosis but is increasingly considered in other inflammatory
disorders, such as diabetes (types 1 and 2) and inflammatory bowel
disease, which also features endothelium dysfunction [2,3]. The major
mechanism by which Hcy induces endothelial dysfunction is through
decreasing production of the vasodilator NO, which may occur via
several mechanisms [4,5,13–19].
NO is the major vasodilator within the endothelium and is produced
from L-arginine via endothelial nitric oxide synthase (eNOS). The ac-
tivity of eNOS may be hindered by actions of Hcy in increasing levels of
reactive oxygen species (ROS) and decreasing levels of eNOS cofactor,
tetrahydrobiopterin (BH4). Hcy can cause ROS production through
auto-oxidation of its sulfhydryl group [15] or through upregulating
ROS-generating NADPH oxidases [5,13,14]. Increased ROS production
leads to decreased bioavailability of BH4, which is readily oxidised to 7,
8-dihydrobiopterin (BH2) in the circulation [4,16]. Decreased BH4
bioavailability can further increase oxidative stress, where, in the ab-
sence of BH4, eNOS uncoupling occurs and eNOS produces the super-
oxide radical instead of nitric oxide [17].
Hcy may impact NO production through several other routes,
however these are less well-characterised. Endothelial cells supple-
mented in vitro with Hcy (0.5–2.5 nM) showed dose-dependent de-
creases in the expression of L-arginine transporters [18], suggesting
that Hcy may also influence NO production via regulation of L-arginine
cell uptake. Hcy may also regulate eNOS activity by influencing levels
of eNOS inhibitor, asymmetric dimethyl arginine (ADMA) [19]. In an in
vitro study, elevated Hcy caused increased ADMA levels and decreased
NO production via suppression of ADMA inhibitor dimethylarginine
dimethylaminohydrolase [19]. Impaired NO production is the major
mechanism by which Hcy may cause endothelial dysfunction, with
further roles of elevated Hcy levels in decreasing hydrogen sulphide
production and antioxidant activity, and increasing lipid peroxidation
also influencing factors [4,5].
3. Hcy-independent regulation of NO & ROS production
Folate has actions in regulating NO & ROS production that are in-
dependent of roles in regulating Hcy levels. These actions are not tra-
ditional 1C transfer reactions but arise due to structural similarities
between folates and biopterin compounds (i.e., between BH4 and THF,
and BH2 and DHF) [6,20–23].
As outlined, eNOS cofactor BH4 is readily oxidised to BH2 in the
circulation. Folate-dependent dihydrofolate reductase (DHFR) has a
role in reducing BH2 back to BH4 via the BH4 salvage pathway (Fig. 2)
[20,21]. This pathway has importance in maintaining an adequate
BH4:BH2 ratio, with DHFR inhibition shown to reduce BH4 and in-
crease BH2 levels in vitro, leading to enzymatic uncoupling of eNOS and
increased ROS production [20,21].
Due to structural similarities between folates and biopterins, in the
same way, BH2 may interact with DHFR, folate compounds are found to
interact with eNOS [22]. 5-methylTHF is shown to bind to the active
site of eNOS [22], and mimic the BH4's action in inducing NO pro-
duction and reducing ROS production [22,24]. Through these me-
chanisms, folate supplementation (50 μM) has been found to increase
NO production in an in vitro study examining human pulmonary artery
endothelial cells under hypoxia [25]. This finding has been replicated
in human subjects, with local perfusion of folate (5 mM of 5-MTHF) into
the dermal space, and folate supplementation (6 mg/day) over 6 weeks
both shown to improve in NO-dependent vasodilation and endothelial
function in healthy older adults [25,26].
In addition to the roles of folate in regulating ROS production in-
directly via prevention of eNOS uncoupling, folate may reduce oxida-
tive stress by acting directly as an antioxidant [27,28]. Folic acid and
reduced folate forms (THF and 5-MTHF) have shown ROS scavenging
Fig. 1. Folate-dependent remethylation of Hcy to methionine.
The remethylation of Hcy involves the irreversible action of MTHFR in reducing
5,10-methyleneTHF to 5-methylTHF to be used by B
12
-dependent MTR in the
remethylation of Hcy to methionine. This process has dual importance in reg-
ulating Hcy and methionine levels. Hcy at elevated levels may cause endothelial
dysfunction while methionine is the substrate for SAM, a universal methyl
donor used in DNA methylation.
Abbreviations; THF; tetrahydrofolate, MTHFR; 5,10methyleneTHF reductase,
MTR; methionine synthase, Hcy; homocysteine, Met; methionine, SAM; S-ade-
nosyl methionine, MAT; adenosylmethionine synthetase, SAH; S-adenosyl hcy,
SAHH; S-adenosylhomocysteine hydrolase.
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P. Jones, et al. Journal of Nutrition & Intermediary Metabolism 18 (2019) 100104
2

activity in vitro [27,28]. However, whether this activity has biological
relevance remains under question, with it difficult to examine the direct
and indirect effects of folate on ROS production separately in in vivo
studies [6].
4. Maintaining DNA methylation
Methionine is produced through folate-dependent Hcy remethyla-
tion and is the substrate for S-adenosyl methionine (SAM), a universal
methyl donor used in methyl transferase processes such as DNA me-
thylation (Fig. 1)[12,29]. Through roles in influencing SAM levels,
folate may modulate the bidirectional relationship between DNA me-
thylation and inflammation, where hypomethylation of inflammation-
related genes may predispose inflammatory diseases [30–32], and in-
flammation may promote DNA methylation processes [33–35]. DNA
methylation changes are most dynamic during development and as
such, a consistent research interest has centred on the potential impact
dietary folate may have in utero on DNA methylation and future disease
risk [36]. Of importance to inflammation, are several animal studies
that demonstrate maternal folate intake may influence the risk of
obesity and colitis in offspring [37–39].
Obesity is a low-grade inflammatory disorder and a further risk
factor for chronic inflammatory diseases [40]. A relationship is evident
between maternal weight gain and increased weight in children
[41,42], with these findings replicated in several murine models
[30–32]. Offspring of dams fed high fat obesogenic diets during preg-
nancy and lactation showed increased adiposity as adults. These off-
spring also possess increased insulin resistance and levels of pro-in-
flammatory cytokines [30,31,43], with this shown to increase
susceptibility to colitis in one model [43]. In a murine model, supple-
mentation of maternal diets with methyl donors (15 mg folic acid and
15 g choline/betaine/kg diet) ameliorated the effect of a high fat ma-
ternal diet on weight in offspring [37]. A further study in sheep found
the maternal restriction of methyl donors resulted in heavier adult
offspring that possessed altered immune responses and insulin re-
sistance [38]. In a murine model of colitis, the offspring of dams fed
methyl-donor deficient diets had more severe colitis and overexpression
of pro-inflammatory pathways [39]. These findings indicate that folate
status can influence the relationship between DNA methylation and
inflammatory phenotype.
Inflammation is shown to induce DNA methylation changes in a
multitude of in vitro and in vivo studies [44–50]. In response to in-
flammation, the expression of DNA methyltransferases (DNMTs), S-
adenosylmethionine synthetase, and S-adenosylhomocysteine
hydrolase (SAHH) involved in regulating DNA methylation and me-
thionine production is increased [33–35]. This results in substantial
increases in DNA methylation that may exacerbate inflammatory re-
sponses and promote tumorigenesis [44–50]. Increases in folate have
been shown to be associated with increases in DNMT expression in
human umbilical vein endothelial cells [51], with an increase in pro-
inflammatory mediators reported in an murine model of folate re-
striction [52]. These findings indicate inflammatory responses may be
enhanced in cases of folate deficiency [51,52]. In a murine model of
colitis, inhibition of cellular methylation via SAHH inhibition lead to
disease exacerbation, with intraperitoneal injections of folate acid
(50 mg/kg) ameliorating colitis severity via controlled DNA methyla-
tion promotion [34]. This finding demonstrates that both hypomethy-
lation and hypermethylation may cause pro-inflammatory effects, and
that folate is an important factor balancing these effects.
5. Maintaining DNA synthesis & repair
Folate has a multifaceted role in nucleotide synthesis, with actions
in synthesising the thymine precursor, thymidylate, and purines. In
thymidylate synthesis, cytosolic serine hydroxymethyl transferase
(SHMT), thymidylate synthase (TYMS) and DHFR use 5,10-
methyleneTHF in the methylation of deoxyuridine monophosphate to
form thymidylate monophosphate. Folate in the form of 10-formylTHF
is also utilised in the synthesis of purine ring structures (Fig. 3)[12].
Nucleotide production is increasingly important during prolonged in-
flammation as it allows for DNA repair and the proliferation of immune
cells.
Increases in ROS during inflammation can cause DNA mutations
which may further contribute to inflammation and can lead to tumor-
igenesis [53]. Folate-dependent thymidylate is particularly important in
DNA repair, with involved enzymes, SHMT, TYMS and DHFR, forming a
multienzyme-complex that translocates to the nucleus during times of
DNA synthesis and repair [54,55]. In a randomised clinical trial ex-
amining arsenic-exposed individuals (n = 450), supplementation with
either 0.4 or 0.8 mg/day of folic acid for 8 weeks resulted in a sig-
nificant decrease in oxidative DNA damage [56]. In comparison, in vitro
and in vivo depletion studies both show folate deficiency to exacerbate
DNA damage via reductions in thymine causing uracil to be mis-
incorporated in DNA, and DNA repair responses to be hindered
[57–60].
Folate deficiency is shown to reduce proliferation in multiple cell
types by causing nucleotide imbalances and subsequent accumulation
of cells in the S phase [61–64]. Notably, changes in folate levels may
influence the proliferation of T cells [64], with aberrant T-cell pro-
liferation associated with autoimmune chronic inflammatory diseases
such as rheumatoid arthritis [65]. In cultured lymphocytes, folate de-
pletion reduced the proliferation of CD8
+
T lymphocytes and increased
the CD4
+
to CD8
+
T-cell ratio [64]. Supplementation of folate
(300 nM) to folate-depleted lymphocytes decreased the CD4
+
to CD8
+
ratio [64]. CD4
+
T (helper/inducer) and CD8
+
(cytotoxic/suppressor)
represent two T-cell subtypes, with an increased CD4
+
to CD8
+
ratio
indicative of increased immunodeficiency, and a hallmark of auto-
immune inflammatory diseases [65]. Further mouse model studies
support a role of folate in maintaining levels of CD4+Foxp3 regulatory
T cells (Tregs) [66,67], a regulatory CD4
+
subtype dysregulated in
chronic inflammatory diseases such as inflammatory bowel disease
[68]. Folate receptors have been found to be highly expressed in natural
Treg cells in mice, with blockage of these receptors resulting in de-
creased Treg levels [66,67].
6. Factors influencing inflammatory roles of folate
With emerging roles in maintaining inflammatory-related pathways,
the potential usefulness of folate supplementation in the prevention and
treatment of inflammation is being explored. However, data from
Fig. 2. The BH4 salvage pathway.
eNOS cofactor BH4 is readily oxidised to BH2 in circulation. In cases of de-
creased BH4 bioavailability, eNOS uncoupling occurs, and eNOS produces ROS
instead of NO. BH2 may be reduced back to BH4 via folate-dependent DHFR via
the BH4 salvage pathway. This pathway maintains an adequate BH4: BH2 ratio
to support NO production.
Abbreviations; eNOS; endothelial nitric oxide synthetase, BH4; tetra-
hydrobiopterin, BH2; 7, 8-dihydrobiopterin, DHFR; dihydrofolate reductase.
Figure created with BioRender.
P. Jones, et al. Journal of Nutrition & Intermediary Metabolism 18 (2019) 100104
3

animal and human studies examining the influence of folate supple-
mentation on inflammatory biomarkers is inconsistent and varies sig-
nificantly based on factors such as timing of intervention (i.e., pre-
ventative vs. treatment interventions), folate dose, levels of other
dietary factors, and genetic variance in folate enzymes.
Several human intervention studies demonstrate folate supple-
mentation effectively lowers Hcy levels but this has varied influence on
markers of endothelial dysfunction depending on time of intervention
[69–75]. Hcy-lowering had no effect on markers of endothelial dys-
function in studies examining healthy subjects or subjects with current
inflammatory conditions (2–5 mg mg/day of folic acid, 6 weeks–2
years) [69–72] but did improve endothelial function in subjects with
elevated Hcy (5–10 mg/day folic acid over 6–8 weeks) [74,75]. These
findings indicate folate supplementation is a more effective pre-
ventative measure rather than a potential treatment, particularly in
individuals with elevated Hcy at risk of future inflammatory conditions.
Folate interventions for prolonged periods or in advanced disease
stages may cause folate to promote inflammation by encouraging
aberrant methylation and cell proliferation activity. Intraperitoneal
injections of folate acid (50 mg/kg) ameliorated colitis severity in one
murine model of colitis [34] but in further murine models supple-
mentation of methyl donors to maternal diets (5 mg/kg folic acid, 5 g
betaine and 5.76 g choline/kg diet) was found to induce susceptibility
to colitis in offspring [76,77]. Supplementation of methyl donors to
maternal diets (15 mg folic acid and 15 g choline/betaine/kg diet) was
shown to combat the effect of a high fat maternal diets on weight gain
in offspring in one model [37], but the opposite effect was seen in cases
of excess folate supplementation (20 g folic acid/kg diet) [78]. This
suggests level of folate dose may also largely determine the influence
folate may have in inflammation.
As methyl donor supplementation is not a targeted approach, af-
fecting methylation of both anti-inflammatory and pro-inflammatory
genes, it is possible that prolonged or excessive exposure to methyl
donors or intervention at specificdisease stage may promote DNA
methylation patterns that contribute to inflammation [79]. Prolonged
or excessive exposure to folate during advanced diseases stages may
exacerbate inflammation by also encouraging aberrant cell prolifera-
tion of pro-inflammatory and cancerous cell types [80]. Notably,
methotrexate is an anti-folate medication with action in deterring
proliferation of pro-inflammatory cells by inhibition of the role of folate
in DNA synthesis. Methotrexate (MTX) is commonly prescribed in
rheumatoid arthritis and other inflammatory disorders [81]. However,
increased Hcy levels and risk of cardiovascular disease is commonly
reported in methotrexate users [82] with current consensus that users
should regularly use folate supplementation to offset these risks (re-
commended 5 mg folic acid following MTX treatments) [81].
The interpretation of current findings is made difficult by the co-
existence of several dietary and genetic factors that regulate folate or
folate-related processes. These factors are not always considered in
study designs but are important nutrient-nutrient and nutrient-gene
interactions that may determine study outcomes. In addition to folate,
adequate levels of vitamin B
12
and vitamin B
6
, and methyl donor cho-
line are required for Hcy-regulation, DNA methylation, and DNA
synthesis [12,83]. Consequently, results of interventions can vary de-
pending on whether folate supplementation was considered in isolation
or in combination with relevant B vitamins and methyl donors. The
occurrence of functional polymorphisms in folate-dependent enzymes
may also influence the actions of folate in inflammation. MTHFR-C677T
is the most extensively studied polymorphism in folate metabolism and
is associated with significant reductions in MTHFR activity and folate
status [84,85]. Results of in vivo and in vitro studies indicate MTHFR-
C677T as a major genetic determinant of Hcy levels [86–89], with this
variant also linked to changes in NO production [90], DNA methylation
[91,92] and DNA damage [93,94] through interaction with folate
status. Other functional polymorphisms in rate-limiting enzymes such
as TYMS and DHFR are associated with changes in folate levels [85]
and could theoretically alter inflammatory roles, but are yet to be ex-
amined. Considering the current available evidence, further con-
sideration of factors such as timing of intervention, folate dose and
interactions with dietary and genetic factors is needed in future studies
Fig. 3. Roles of folate in nucleotide synthesis.
Folate synthesises thymine precursor, thymidylate,
and purine ring structures. In thymidylate synth-
esis, SHMT generates 5,10methyleneTHF which is
then used by TYMS in methylation of dUMP to
dTMP. DHFR regenerates oxidised DHF back to
THF. Folate in the form of 10-formylTHF is utilised
in the synthesis of purines, where GAR and AICAR
transformylases transfer one carbon units from 10-
formylTHF to purine ring structures. MTHFD1 has
action in generating the different THF forms of
folate supporting these reactions, as well as reac-
tions in Hcy remethylation.
Abbreviations; THF; tetrahydrofolate, MTHFD1;
methylenetetrahydrofolate dehydrogenase 1, GAR
Glycinamide ribonucleotide; AICAR 5-aminoimi-
dazole-4-carboxamide ribonucleotide; SHMT;
serine hydroxymethyl transferase, DHFR; dihy-
drofolate reductase, TYMS; thymidylate synthase,
dUMP; deoxyuridine monophosphate, dTMP;
deoxythymidine monophosphate, Hcy; homo-
cysteine.
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P. Jones, et al. Journal of Nutrition & Intermediary Metabolism 18 (2019) 100104
4

to elucidate the roles of folate in inflammation.
7. Conclusion
There is a growing body of evidence supporting the role of optimal
folate availability in the development and maintenance of processes
linked to inflammation. Folate may prevent endothelial dysfunction by
Hcy-dependent and Hcy-independent regulation of NO levels.
Additionally, actions of folate in DNA methylation, repair and synthesis
processes may influence the inflammatory phenotype via epigenetic
changes and modulation of cell proliferation. However, much of our
current knowledge in how folate may influence inflammation is based
on findings from in vitro studies and murine models, which have not
been yet been translated into investigations of human cohorts. The
interpretation of current findings is further complicated by several
confounders that may alter the influence of folate, which need to be
further examined before folate recommendations around inflammation
can be made.
Author statement
Conception and design of review by PJ and EB. Drafting, revision,
and approval of final submission by all authors.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgements
Patrice Jones is supported by an Australian Government Research
Training Program scholarship and a Hunter Medical Research Institute
(HMRI) Greaves Family Scholarship. Emma Beckett is supported by a
NHMRC Early Career Fellowship.
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