RevIeWS

Methotrexate and its mechanisms

of action in inflammatory arthritis
Bruce N. Cronstein   1 ✉ and Thomas M. Aune   2
Abstract | Despite the introduction of numerous biologic agents for the treatment of rheumatoid
arthritis (RA) and other forms of inflammatory arthritis, low-​dose methotrexate therapy remains
the gold standard in RA therapy. Methotrexate is generally the first-​line drug for the treatment
of RA , psoriatic arthritis and other forms of inflammatory arthritis, and it enhances the effect of
most biologic agents in RA. Understanding the mechanism of action of methotrexate could be
instructive in the appropriate use of the drug and in the design of new regimens for the treatment
of RA. Although methotrexate is one of the first examples of intelligent drug design, multiple
mechanisms potentially contribute to the anti-​inflammatory actions of methotrexate, including
the inhibition of purine and pyrimidine synthesis, transmethylation reactions, translocation of
nuclear factor-​κB (NF-​κB) to the nucleus, signalling via the Janus kinase (JAK)–signal transducer
and activator of transcription (STAT) pathway and nitric oxide production, as well as the promotion
of adenosine release and expression of certain long non-​coding RNAs.

1
The New York University
School of Medicine, New York,
NY, USA.
2
Vanderbilt University
Medical Center, Nashville,
TN, USA.
✉e-​mail: bruce.cronstein@
nyumc.org
https://doi.org/10.1038/
s41584-020-0373-9
Since its introduction and widespread adoption in
the 1980s for the treatment of rheumatoid arthritis
(RA), low-​dose methotrexate therapy has considerably
changed the approach to therapy as well as the outcome
of therapy for RA1. On seeing a rheumatologist, most
patients with RA are initially given low-​dose metho­
trexate therapy, and many of the patients require no
additional therapies2. Even for patients who have had
an inadequate response to methotrexate and are treated
with a biologic agent, methotrexate therapy is generally
continued because of the seemingly additive effects of
biologic drugs and methotrexate and because metho-
trexate diminishes resistance to many biologic drugs1.
Moreover, in patients with RA, low-​dose methotrexate
therapy, with or without concomitant biologic therapy,
is associated with a lower rate of large joint replacement
than therapy with biologic agents alone, supporting
the hypothesis that low-​dose methotrexate therapy is
chondroprotective3–5.
The use and effectiveness of methotrexate in the
treat­ment of rheumatic disease will probably be further
enhanced by a better understanding of the pharmacol-
ogy and anti-​inflammatory mechanisms of action of this
drug. Indeed, emerging data suggest that appropriate
biomarkers might be identified that can determine who
will benefit most from methotrexate treatment. Some of
these biomarkers might reflect the mechanism of action
of methotrexate. In this Review, we discuss the pharma-
cology and metabolic and cellular mechanisms by which
methotrexate suppresses inflammation. We also examine
the mechanisms by which methotrexate induces some of
its well-​known toxic effects. A better understanding of
the mechanisms of action of methotrexate could lead to
more appropriate treatment regimens. The mechanisms
discussed in this Review are general and, although these
mechanisms are best documented in RA, they could
apply to other forms of inflammatory arthritis.
Pharmacology of methotrexate
Whether administered orally or parenterally at a low
dose, methotrexate has a relatively short half-​life of
approximately 6 hours and is undetectable in serum
after 18 hours6. The bioavailability of orally adminis-
tered methotrexate is highly variable owing to the lim-
ited capacity of the gut to absorb methotrexate, and the
maximal absorption of a single oral dose is <25 mg (ref.7).
Methotrexate is excreted both as methotrexate and as
7-hydroxymethotrexate (the major metabolite of metho­
trexate) in the urine. In the 1980s, methotrexate was first
reported as a ‘pro-drug’ that accumulates in tissue in a
polyglutamated form8,9. It is now known that metho­
trexate monoglutamate (the native form of the drug)
undergoes serial polyglutamation within cells, with vary­
ing numbers of glutamic acid additions, and metho-
trexate polyglutamates can be detected in the tissues for
weeks8 (Fig. 1). Polyglutamated methotrexate is the active
form of the drug, and the potency of poly­glutamated
methotrexate as an inhibitor of a number of enzymes
differs from the potency of the native compound. The
enzyme inhibited most potently by methotrexate poly­
glutamates is 5-aminoimidazole-4-carboxamide ribo­
nucleotide (AICAR) transformylase (ATIC), which

Nature Reviews | Rheumatology

Reviews
Key points
• Methotrexate polyglutamates inhibit aminoimidazole-4-carboxamide ribonucleotide
(AICAR) transformylase (ATIC), leading to intracellular accumulation of AICAR ment of inflammatory disease, for which methotrexate is
and increased adenosine release; adenosine binds to cell surface receptors and
suppresses many inflammatory and immune reactions.
• Methotrexate inhibits dihydrofolate reductase, preventing the reduction of
dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), leading to nitric oxide synthase
uncoupling and increased sensitivity of T cells to apoptosis, thereby diminishing
immune responses.
• Methotrexate inhibits activation of nuclear factor-​κB (NF-​κB) by increasing both
adenosine release and activation of adenosine receptor A2a and by inhibiting the
reduction of BH2 to BH4.
• Methotrexate increases the expression of long intergenic non-​coding RNA p21
(lincRNA-​p21), which is a multifunction long non-​coding RNA that regulates, both
directly and indirectly, a variety of critical immune and inflammatory processes.
• By modulating cell-​specific signalling pathways, methotrexate inhibits important
pro-inflammatory properties of major cell lineages involved in rheumatoid arthritis
pathogenesis, including T cells, macrophages, endothelial cells and fibroblast-like
synoviocytes.
catalyses the last committed step in de novo purine
biosynthesis; methotrexate polyglutamate is >2,000
times more potent as an inhibitor of ATIC than metho-
trexate monoglutamate9. Methotrexate polyglutamates
also inhibit other folate-​dependent enzymes involved in
de novo pyrimidine and purine synthesis (Fig. 2a) as well
as the enzymes involved in transmethylation reactions
and polyamine synthesis (Fig. 2c).
Postulated mechanisms of action
Methotrexate was initially developed to inhibit the
de novo synthesis of purines and pyrimidines that is
required for DNA and RNA synthesis and the prolifer-
ation of many different types of malignant cells, as well
as non-​malignant cells (Box 1). Although this mech-
anism might contribute to the mechanism by which
methotrexate suppresses inflammation (Fig. 2a), other
mechanisms of action have been postulated, includ-
ing enhanced adenosine release (Fig. 2b), inhibition of
transmethylation reactions that are required for some
cellular functions (Fig. 2c), diminished accumulation of
polyamines (Fig. 2c) and nitric oxide synthase uncoupling
(Fig. 2d). Methotrexate regulates, both directly and indi-
rectly, the function of nearly every cell type involved in
inflammation, including neutrophils, monocytes, T cells,
B cells, endothelial cells and fibroblast-like synovio-
cytes (FLSs). In this section, we discuss the previously
described molecular mechanisms of action of metho-
trexate and, where relevant, discuss the effects of these
molecular mechanisms on the functions of various cell
types involved in inflammation.
Metabolic mechanisms
Inhibition of purine and pyrimidine synthesis.
Methotrexate targets critical folate-​dependent enzy-
matic steps in the de novo synthesis of purines and
pyrimidines, the building blocks for RNA and DNA.
Reductions in the number of circulating leukocytes
(neutrophils and lymphocytes), cells that require rapid
proliferation of precursor cells in the bone marrow,
typically occur in patients taking methotrexate as an
antiproliferative drug for the treatment of cancer10. By
contrast, reductions in peripheral leukocyte counts are
considered a toxic reaction to methotrexate in the treat-
administered at doses 100-fold to 1,000-fold lower than
the dose administered for malignancies. Administration
of folic acid or even folinic acid (except on the day that
methotrexate is administered) can prevent the toxic
effects of methotrexate in patients with rheumatic dis-
eases11–14. Although folinic acid supplementation has
been reported to lead to flare of RA in patients taking
methotrexate15,16, a meta-​analysis does not support this
association in the treatment of RA17. Given that metho­
trexate is effective even when concurrent folic acid or
folinic acid supplementation prevents reductions in
peripheral leukocyte counts, the anti-​inflammatory
effects of methotrexate are unlikely to require inhibition
of cellular proliferation.
Inhibition of transmethylation reactions. Polyamines,
such as spermine and spermidine, accumulate in syn-
ovial tissue and fluid18, mononuclear cells19 and urine
of patients with RA20. Monocytes can hydrolyse these
polyamines to ammonia and H2O2, which function as
cytotoxins that can injure the cells and tissues of the
joint19,21,22. Inhibition of dihydrofolate reductase (DHFR;
an enzyme that catalyses the reduction of dihydrofolate
to tetrahydrofolate) by methotrexate diminishes the
formation of the methyl donors tetrahydrofolate and
5-methyltetrahydrofolate and reduces the synthesis of
polyamines (Fig. 2c). Thus, one hypothesis is that inhibi-
tion of transmethylation reactions by methotrexate and
subsequent reduction in polyamine production results
in diminished downstream production of ammonia and
H2O2, thereby diminishing synovial injury. This hypoth-
esis was the basis for clinical testing of a direct trans-
methylation inhibitor, 3-deazaadenosine, as a therapy
for RA; however, although this drug effectively inhib-
ited transmethylation reactions in the cells of patients
taking the drug, it had no effect on the course of RA
(reviewed elsewhere23). This finding suggests that inhib­
ition of transmethylation reactions has a small part in
the overall anti-​inflammatory actions of methotrexate
therapy in inflammatory disease.
Adenosine release. As noted already, ATIC is potently
inhibited by methotrexate polyglutamates 9,24, and
AICAR accumulates intracellularly in the tissues of
methotrexate-​t reated mice25. AICAR inhibits AMP
deaminase and adenosine deaminase, leading to the
release of adenine nucleotides into the extracellular
space (Fig. 2b); adenine nucleotides are converted into
adenosine by the action of the cell surface enzymes
ectonucleoside triphosphate dephosphorylase 1 (also
known as CD39) and ecto-5′-nucleotidase (also known
as CD73). Adenosine is a potent stimulus for a family
of receptors (adenosine receptors A1a, A2a, A2b and A3),
all of which have potent inhibitory effects on nearly all
inflammatory cell types (reviewed elsewhere26 and sum-
marized in Box 2). The hypothesis that adenosine, via
these receptors, mediates the anti-​inflammatory effects
of methotrexate was supported by studies in mice in
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a O COOH
O COOH
NH2 N
H
O N N
H N N COOH
N
HN N COOH CH3
H H2N N N
H2N N N
Folic acid Methotrexate
O COOH

NH2 N
H
N
N N COOH
H
H2N N N
Aminopterin

b Glutamyl
residues
O COOH FPGS O COOH

NH2 N NH2 N
H GGH H
N N OH
N N COOH N N
CH3 CH3 O
H2N N N H2N N N

Methotrexate (monoglutamate) Methotrexate polyglutamate

Fig. 1 | molecular structures of folic acid, aminopterin and methotrexate. a | Aminopterin and methotrexate (formerly
known as amethopterin) are structurally highly similar to folic acid. b | Within cells, methotrexate monoglutamate
undergoes serial polyglutamation to form methotrexate polyglutumates (methotrexateGlu(1–7)), the active form of
methotrexate. In this process, additional glutamate residues are added to the terminal glutamate moiety of methotrexate
by the action of the enzyme folylpolyglutamate synthase (FPGS). This reaction is reversible, and the peptidase γ-glutamyl
hydrolase (GGH) mediates the opposite reaction.

which methotrexate increased adenosine release into
inflamed tissue, and the anti-​inflammatory effects of
methotrexate were reversed by selective adenosine recep­
tor A2a antagonists25. In a subsequent study in rats with
adjuvant arthritis, non-​selective adenosine receptor
antagonists (theophylline and caffeine)27 reversed the
anti-​inflammatory effects of methotrexate. Methotrexate
does not inhibit inflammation in A2a-deficient mice,
A3-deficient mice or CD73-deficient mice, further con-
firming the hypothesis that adenosine release mediates
the anti-​inflammatory effects of methotrexate in animal
models28–30.
Demonstrating that adenosine has a role in medi-
ating the anti-​inflammatory effects of methotrexate in
patients with inflammatory diseases has been more
difficult. Measurement of adenosine concentrations
in biological fluids requires special care owing to the
rapid uptake of adenosine by cells and metabolism of
adenosine to inosine by adenosine deaminase present
in plasma. One study31 reported that methotrexate treat-
ment of patients with RA increases adenosine-​mediated
and dipyridamole-​mediated vasodilation (adenosine is
a potent vasodilator and dipyridamole functions as a
vasodilator by blocking adenosine uptake and thereby
increasing extracellular adenosine levels). This find-
ing confirms that methotrexate therapy does indeed
increase adenosine release in humans as the higher
levels of adenosine released in patients treated with
methotrexate would lead to increased adenosine levels
and adenosine-​mediated increases in vascular flow. In a
randomized prospective study, ingestion of caffeine,
a non-​selective adenosine receptor antagonist, reduced
the therapeutic response to methotrexate in patients
with RA32. By contrast, in a large retrospective study
of patients with RA receiving long-​term methotrexate
therapy, caffeine consumption was not associated with
poor responses to methotrexate; however, the patients
with a poor response to methotrexate early in the course
of disease were probably undercounted in this study33.
More recently, emerging evidence suggests that adeno-
sine production and release is one mechanism by which
regulatory T (Treg) cells diminish cellular immunity and
inflammation26 (leading to a growing number of stud-
ies of adenosine receptor antagonists in the treatment
of cancer34). Adenosine is produced by Treg cells via
CD39-mediated and CD73-mediated dephosphoryla-
tion of ATP, and some data indicate that low levels of
CD39 on Treg cells are associated with poor responses to
methotrexate in patients with RA35. In a parallel fashion,
methotrexate-​mediated increases in adenosine release
by B cells lead to diminished immunization against thera­
peutic monoclonal antibodies (such as anti-​TNF anti-
bodies) in a B-​cell activating factor (BAFF)-dependent
fashion and have been postulated to provide the basis
for methotrexate-​mediated suppression of antidrug anti-
bodies in patients receiving combined methotrexate and
anti-​TNF therapy36.
Nitric oxide synthase uncoupling. In addition to catalys-
ing the reduction of dihydrofolate to tetrahydrofolate,
DHFR also catalyses the reduction of dihydrobiopterin
(BH2) to tetrahydrobiopterin (BH4); methotrexate

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a Folinic
b
Methotrexate Folic acid Adenosine
acid release
RFC1 FRβ or PCFT Extracellular

Intracellular ATP ADP AMP Adenosine

Methotrexate Folic acid Folinic acid

CD39 CD73
FPGS GGH ATP ADP AMP Adenosine
DHF THF
MethotrexateGlu(1–7) DHFR
AMPDA ADA

5-CH3-THF 5,10-CH2-THF
MTHFR IMP Inosine
AICAR
MethotrexateGlu(1–7)
dTMP
ATIC
TYMS
dUMP
FAICAR ↑ AICAR

ATIC
Decreased purine
and pyrimidine
production FAICAR

d
MethotrexateGlu(1–7) Methotrexate/
methotrexateGlu(1–7)

DHFR
DHF THF

DHFR

Homocysteine BH2 BH4

5-CH3-THF 5,10-CH2-THF
NO NO
MTHFR
synthase synthase

SAH Methionine ROS production NO production

SAM 10-CH3-THF JNK activation

Polyamine synthesis Methyl donor AP1 activity

Fig. 2 | methotrexate regulates essential biochemical reactions.
a | Methotrexate is taken up by cells via reduced folate carrier 1 (RFC1),
where it is polyglutamated by folylpolyglutamate synthase (FPGS).
Methotrexate polyglutamates accumulate intracellularly and can
inhibit a number of enzymatic reactions, including those mediated by
dihydro­folate reductase (DHFR), methylenetetrahydrofolate reductase
(MTHFR), thymidylate synthase (TYMS) and 5-aminoimidazole-4-
carboxamide ribonucleotide (AICAR) transformylase (ATIC), leading to
diminished production of purines and pyrimidines. b | Methotrexate-​
mediated inhibition of ATIC leads to accumulation of intracellular AICAR ,
which ulti­mately leads to an increase in extracellular adenosine levels
owing to reduced activity of the enzymes AMP deaminase (AMPDA) and
adenine deaminase (ADA). c | Methotrexate inhibits the synthesis of
polyamines (owing to diminished concentrations of the methyl donor
5-methyltetra­hydrofolate (5-CH3-THF)). d | Methotrexate inhibition of
DHFR inhibits tetrahydrobiopterin (BH4) production, thereby diminish-
ing nitric oxide (NO) production and increasing the production of
reactive oxygen species (ROS) in a process known as ‘NO synthase
uncoupling’. Increased JUN N-​terminal kinase (JNK) activation promotes
activator protein 1 (AP1) activity and inhibits nuclear factor-κB activa-
tion (not shown). BH2, dihydrobiopterin; DHF, dihydrofolate; dTMP,
deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate;
FAICAR, formyl AICAR; FRβ, folate receptor-​β; GGH, γ-glutamyl hydro-
lase; IMP, inosine monophosphate; PCFT, proton-​coupled folate trans-
porter; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine;
THF, tetrahydrofolate.

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Long non-​coding RNAs

(lncRNAs). A newly discovered
class of RNAs that are
defined as RNAs of more than
200 base pairs in length, are
transcribed from genes but
not translated into proteins
due to the presence of multiple
translational stop codons
and, as RNAs, regulate the
expression of target genes and
proteins to carry out a vast
array of biologic functions.
Warburg effect
A shift from oxidative
phosphorylation to glycolysis
during cell activation.
inhibits the reduction of both dihydrofolate and BH2 at
similar concentrations (Fig. 2d). BH4 is a necessary cofac-
tor for all nitric oxide synthases (a family of enzymes that
catalyse the production of nitric oxide from l-​arginine).
In the absence of BH4, nitric oxide synthases produce
reactive oxygen species, such as hydrogen peroxide,
rather than nitric oxide, a process termed ‘nitric oxide
synthase uncoupling’37–39. Increased concentrations of
reactive oxygen species activate JUN N-​terminal kinases
(JNKs), leading to increased phosphorylation of the
transcription factor JUN and increased transcriptional
activity of the JUN–FOS heterodimer activator pro-
tein 1 (AP1). This transcription factor is a key regu­lator
of apoptosis as well as many other cellular processes.
AP1 activation results in the induction of genes encod-
ing proteins that induce cell cycle arrest and promote
sensitivity to apoptosis, including TP53, CDKN1A,
CDKN1B, CHEK2, BCL3 and HRK. HRK encodes a
protein, activator of apoptosis harakiri, that promotes
apoptosis by interacting with the apoptotic inhibitors
BCL-2 and BCL2L1, thus explaining the increase in sen-
sitivity of T cells to apoptosis in response to methotrexate
stimulation40,41. In T cells, methotrexate also inhibits
TNF-stimulated increases in nuclear factor-κB (NF-κB)
transcriptional activity, a major pro-​inflammatory signal­
ling pathway. Methotrexate-​mediated inhibition of NF-​κB
activation occurs through JNK-​dependent upregulation
of p53 via the pathway described above42.
LincRNA-​p21 expression. Emerging data indicate that
methotrexate regulates the expression of some long non-​
coding RNAs (lncRNAs; a general review of lncRNAs is
provided in ref.43). One such example, long intergenic
non-​coding RNA p21 (lincRNA-​p21)44, is encoded by
a gene adjacent to CDKN1A (encoding p21), hence its
name; lincRNA-​p21 is induced by p53 as part of the
DNA damage response. LincRNA-​p21 orchestrates
the p53-mediated apoptotic response by repressing
transcription of numerous genes encoding proteins that
inhibit apoptosis. As such, lincRNA-​p21 is required for
proper induction of apoptosis but does not seem to affect
regulation of the cell cycle, two of the central pathways
regulated by p53 in response to DNA damage or other
cellular stress45. LincRNA-​p21 is also induced by hypoxia
and promotes the expression of the transcription factor
HIF1α, an important mediator of the cellular response
to hypoxia that promotes the Warburg effect46. In the
cytoplasm, an additional function of lincRNA-​p21 is to
bind to select target mRNAs to inhibit their recruitment
to the ribosome and translation into proteins47. Thus,
lincRNA-​p21 is a multifunctional lncRNA regulating a
variety of critical biologic processes.
Circulating levels of both p53 and lincRNA-​p21 are
decreased in RA and are restored to levels in healthy
individuals during methotrexate therapy48. In T cells,
methotrexate induces the expression of lincRNA-​p21
(Fig. 3a). Induction of lincRNA-​p21 by metho­trexate
in T cells is not a result of nitric oxide synthase uncou-
pling or adenosine release and adenosine receptor
activation, but rather depends on activation of the
DNA damage sentinel DNA-​dependent protein kinase
(DNA-​P K). Exactly how methotrexate activates
DNA-​PK is not understood, but inhibition of DNA-​PK
clearly abrogates methotrexate-​mediated induction
of lincRNA-​p21 (ref.48). In T cells, lincRNA-​p21 inhib-
its NF-​κB activity but does not seem to target the
genes RELA and NFKB1, encoding key components of
NF-​κB, or disrupt intracellular signalling pathways that

Box 1 | Development of methotrexate

Methotrexate (formerly known as amethopterin) and its close analogue aminopterin were among the first products of
intelligent drug design. These drugs were developed by Yellapragada Subbarow, a biochemist at Harvard University,
for the treatment of cancers on the basis of their structural similarity to folic acid (Fig. 1) and their capacity to inhibit
folate-dependent enzymes. These drugs were first used, in high doses, to treat leukaemias and other forms of malignancy
in 1948, and aminopterin, in chemotherapeutic doses, was first used for the treatment of rheumatoid arthritis (RA) in 1951
(ref.100). Aminopterin was ultimately taken off of the market owing to manufacturing difficulties, leaving only methotrexate.
During the 1960s and 1970s, low-​dose methotrexate therapy was increasingly used to treat severe psoriasis, and its use was
extended to RA. In the mid-1980s, methotrexate was successfully tested in patients with RA in rigorous studies101–103 and
subsequently licensed by the FDA for the treatment of patients with RA in 1988 (ref.45). Since its initial approval, the use
of methotrexate has increased, as has the willingness of physicians to use higher, and more effective, doses of the drug.
More importantly, the recognition that oral bioavailability of the drug is quite limited led to the development and approval,
in 2013 and 2014, of two different parenteral (subcutaneous) formulations of the drug for use in the treatment of rheumatic
disease104,105.

Aminopterin and First report of Systematic reports Two different preparations of

amethopterin aminopterin use for of use of low-dose methotrexate for subcutaneous
(methotrexate) the treatment of methotrexate to use in the treatment of RA
synthesized psoriasis and RA treat RA licensed by the FDA

1947 1948 1951 1964 1985 1988 2013 2014

Aminopterin Aminopterin use Methotrexate

introduced for discontinued licensed for the
the treatment because of treatment of RA
of cancer manufacturing by the FDA
difficulties

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Box 2 | effects of adenosine on inflammatory cells26
Neutrophils
• Inhibits oxidant generation
• Inhibits adhesion and recruitment
• Inhibits neutrophil extracellular trap formation
macrophages
• Increases M1 to M2 transformation
• Inhibits cytokine expression
• Inhibits osteoclast differentiation
T cells
• Inhibits T cell receptor-​triggered activation
• Inhibits activation-​induced cell death
• Inhibits FAS–FASL-mediated cell death
• Increases regulatory T (Treg) cell differentiation
• Mediates Treg cell-​mediated suppression of T cell
proliferation
endothelial cells
• Increases barrier integrity
• Inhibits oedema formation
Fibroblast-​like synoviocytes
• Inhibits metalloproteinase production
lead to NF-​κB activation. Rather, lincRNA-p21 binds to
RELA mRNA to inhibit its translation, thus reducing the
amount of NF-​κB available to mediate transcriptional
activation in response to external inflammatory stimuli,
such as TNF. Consistent with this notion, patients with
RA receiving methotrexate therapy have substantially
reduced levels of NF-​κB subunit p65 (encoded by RELA)
compared with patients with RA not receiving metho-
trexate therapy. In this way, induction of lincRNA-​p21
might also contribute to the efficacy of methotrexate
therapy in RA.
Inhibition of JAK–STAT signalling. IL-6 and other
stimuli activate a critical signalling system that leads
to phosphorylation of signal transducer and activator
of transcription (STAT) proteins by receptor-​associated
Janus kinases (JAKs), which stimulates production
of a variety of inflammatory signals49. On the basis of
high-​throughput screening data, methotrexate and its
analogue, aminopterin, were shown to inhibit signalling
via the JAK1–STAT3 and JAK2–STAT5 transcriptional
pathways in Drosophila cells and, subsequently, in human
macrophage cell lines50,51. The results suggested that the
inhibitory effects were folate independent as folinic
acid did not reverse methotrexate-​mediated inhibition
of STAT5 phosphorylation, and knockdown of various
folate-​dependent enzymes had no effect on the capacity
of methotrexate to inhibit STAT5 phosphorylation50,51.
It is unclear whether methotrexate treatment in these
cells directly inhibits JAK-​mediated phosphorylation of
STAT3 and STAT5 or whether other intracellular fac-
tors are involved as the effect of methotrexate was tested
only in whole cells. The authors of this report noted that
STAT phosphorylation was only moderately inhibited
in peripheral blood CD4+ T cells, B cells and monocytes
treated with methotrexate, suggesting that the effects of
methotrexate on JAK–STAT signalling might dampen
but not completely inhibit this inflammatory signalling
pathway in patients.
Inhibition of NF-​κB signalling. Activation and nuclear
translocation of NF-​κB has a central function in inflam-
mation and inflammatory changes in many tissues and
cell types. As noted already, adenosine has been postu-
lated to mediate many of the anti-​inflammatory actions
of methotrexate (Fig. 2b), including direct inhibition of
NF-​κB activation in a variety of cell types and tissues, such
as monocytes, macrophages and endothelial cells52–61.
In addition, as described earlier, methotrexate can also
inhibit NF-​κB activation by diminishing the expres­sion
of RELA (via the promotion of lincRNA-p21 expression)
in T cells and by promoting BH4 depletion and JNK
activation (Fig. 3a). A whole-​genome association study
identified a single-​nucleotide polymorphism in TNFAIP3
(encoding A20, a critical suppressor of NF-​κB activation)
that was associated with a favourable response to metho­
trexate in patients with inflammatory arthritis62. More
recently, researchers found that macrophages stimulated
with only granulocyte–macrophage colony-​stimulating
factor (GM-​CSF), but not other stimuli, responded
directly to methotrexate treatment through a thymidylate
synthase-​mediated and p53-mediated mechanism63,
and that GM-​CSF-stimulated macrophages upregu-
lated A20 in response to methotrexate64, rendering them
more ‘tolerant’. Thus, there are multiple mechanisms by
which methotrexate therapy suppresses the activation
of NF-​κB and downstream expression of inflammatory
mechanisms. Some of these mechanisms seem to be
limited to specific cell types (for example, lincRNA-​p21
and A20 induction in T cells and GM-CSF-stimulated
macrophages, respectively), whereas adenosine affects
activation of NF-​κB in multiple cell types.
Cellular mechanisms
Effects on T cells. T cells from patients with RA are resis­
tant to cell cycle checkpoint signals and apoptosis31,32.
The expression and function of genes that encode pro-
teins critical for cell cycle checkpoint programmes and
apoptosis are reduced in RA. The expression levels of
these genes are restored to normal or healthy control
levels in patients with RA receiving methotrexate ther-
apy40,41. Examples of such genes include TP53, CDKN1A,
CDKN1B and CHEK2. In T cells, these genes are directly
induced by methotrexate at submicromolar concentra-
tions, similar to the concentrations shown to achieve
a therapeutic benefit for patients with RA, thus supporting
the notion that checkpoint reprogramming by metho­
trexate might contribute to its therapeutic benefits.
Under these conditions, methotrexate does not directly
induce cell cycle arrest or apoptosis in T cells. Rather,
methotrexate markedly increases the sensitivity of T cells
to apoptosis (Fig. 3a). Thus, the known resistance of RA
T cells to apoptosis might be reversed by methotrexate
therapy.
In addition, the pro-​inflammatory transcription
factor NF-​κB is constitutively active in T cells from
patients with RA, and this chronic activity is corrected
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 Reviews

in patients with RA receiving methotrexate therapy65
(as discussed earlier) (Fig. 3a). Methotrexate-​mediated
inhibition of chronically active NF-​κB in RA T cells might
result from the ability of methotrexate to induce expres-
sion of p53, a transcription factor with anti-​inflammatory
properties66–79, or induction of lincRNA-​p21, both of
which reduce or inhibit NF-​κB activity.
Effects on fibroblast-​like synoviocytes. FLSs also partici­
pate in RA pathogenesis, and NF-​κB is highly acti­vated
in these cells in RA74,80. High NF-​κB activity in FLSs is
thought to contribute to their proliferation, angio­genesis
and additional inflammatory properties80. Methotrexate
inhi­bits NF-κB activity in FLSs. However, metho­trexate
does not inhibit NF-κB activity in FLSs through BH4
depletion and nitric oxide synthase uncoupl­ing. Rather,
methotrexate-stimulated release of adenosine and sub-
sequent activation of adenosine recep­tors seems to
inhibit the normally high basal levels of NF-​κB activity
in FLSs (Fig. 3b). The differences in responses to metho­
trexate between T cells and FLSs seem to be because
of differing levels of nitric oxide synthases, with T cells
having high levels of nitric oxide synthase activity, thus
enabling the nitric oxide synthase uncoupling pathway to
predominate, and FLSs having low levels of nitric oxide
synthase activity, enabling the adenosine–adenosine
receptor pathway to predominate. Thus, methotrex-
ate also inhi­bits NF-​κB activity in FLSs, which might
contribute to its therapeutic efficacy in RA.
Effects on monocytes. The effects of methotrexate have
also been examined in monocyte cell lines81. In con-
trast to T cells and FLSs, monocytes undergo apopto-
sis in response to methotrexate. Further, methotrexate
induces a dose-​dependent increase in expression of
pro-​inflammatory cytokines, including IL-1, TNF and
IL-6, in monocytic cell lines81. Induction of these pro-​
inflammatory cytokines by methotrexate seems to result
from inhibition of dihydrofolate reduction to tetra­
hydrofolate and not inhibition of BH2 reduction to BH4
and nitric oxide synthase ‘uncoupling’ or promotion of
adenosine receptor signalling (Fig. 3c). This effect might

a T cells b Fibroblast-like synoviocytes c Monocytes

Methotrexate Methotrexate Methotrexate

DHFR Unknown ATIC DHFR

BH2 BH4 mechanism AICAR FAICAR DHF TTF

DNA-PK
NOS uncoupling Adenosine release Unknown
NF-κB–dependent
mechanism

ROS production LincRNA-p21 Adenosine receptor binding

JNK NF-κB activity NF-κB activity Apoptosis IL-1 production IL-6 production

Apoptosis Increase in Anti-inflammatory Anti-inflammatory Pro-inflammatory

sensitivity expression of cell
cycle checkpoints

Anti-inflammatory

Fig. 3 | Cell-specific mechanisms of methotrexate in rheumatoid
arthritis. The actions of methotrexate in distinct cell lineages differ
depending on the metabolism of each particular cell type. a | In T cells,
methotrexate inhibits dihydrofolate reductase (DHFR)-mediated reduc-
tion of dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), leading to
nitric oxide synthase (NOS) uncoupling and increased reactive oxygen
species (ROS) production. ROS activate JUN N-​terminal kinase (JNK),
and activated JNK induces genes encoding proteins that regulate sen-
sitivity to apoptosis and cell cycle progression. In T cells, methotrexate
also activates DNA-​dependent protein kinase (DNA-​PK) by unknown
mechanisms, which leads to the induction of long intergenic non-​
coding RNA p21 (lincRNA-​p21). LincRNA-p21 inhibits the translation of
REL A mRNA , thus reducing levels of the pro-inflammatory transcrip-
tion factor nuclear factor-κB (NF-κB), inhi­biting inflammation. b | In
fibroblast-like synoviocytes, methotrexate also inhibits NF-κB activity
but this effect occurs via inhibition of 5-aminoimidazole-4-carboxamide
ribonucleotide (AICAR) transformylase (ATIC), increased adenosine
release and activation of adenosine receptors on the same cell, lead-
ing to inhibition of NF-​κB with subsequent anti-​inflammatory effects.
c | In monocytes, methotrexate can promote apoptosis and increase the
expression of pro-inflammatory cytokines via an unknown NF-κB-
dependent mechanism that might occur via inhibition of DHFR-mediated
reduction of dihydrofolate (DHF) to tetrahydrofolate (TTF). FAICAR ,
formyl AICAR.

Nature Reviews | Rheumatology

Reviews
Box 3 | Predictors of poor response to methotrexate
• High DAS28 (refs93,95) or low DAS28 (ref.94) methotrexate and has resulted in a reduced prevalence
(conflicting results)
• High Health Assessment Questionnaire (HAQ) score93,94
• High BMI93
• High score on Hospital Anxiety and Depression Scale94
• High tender joint count94
• Low erythrocyte folate level93
• Single-​nucleotide polymorphism in ABCB1 and trexate, being able to predict which patients are most
ABCC3 (ref.93)
• Rheumatoid factor negative94
• Current smoker95
• No alcohol consumption95
have clinical implications and might help explain some
of the adverse effects of methotrexate, including mucosi-
tis and pneumonitis, which could be mediated by these
pro-​inflammatory cytokines.
Adenosine also regulates monocyte function via
stimu­lation of adenosine receptors, and hence metho-
trexate might affect monocytes indirectly by promoting
adenosine release by other cells. Binding of adenosine
to adenosine receptor A1 on human peripheral blood
monocytes stimulates the formation of multinucleated
giant cells82. In addition, the binding of adenosine to
adenosine receptors A2a and A3 on monocytes inhib-
its the synthesis and release of TNF and IL-6 and pro-
motes the transition of inflammatory M1 monocytes to
anti-inflammatory M2 monocytes (reviewed elsewhere26).
Toxic effects of methotrexate
Clearly many of the toxic effects of long-​term low-​dose
methotrexate therapy result from the antifolate proper-
ties of the drug. These toxic effects include diminished
peripheral blood white cell counts, stomatitis and hair
loss and probably result from methotrexate-​mediated
inhibition of cellular proliferation. Many of these toxic
effects can be prevented by concomitant administration
of folic acid or folinic acid (except on the day of metho-
trexate administration) as methotrexate and folinic acid
compete for the same transporter for cellular uptake.
By contrast, a number of the toxic effects of methotrex-
ate probably result from methotrexate-​mediated adeno-
sine release. Among these adenosine-​related toxic effects
are the feeling of tiredness many patients experience on
the day of methotrexate administration (adenosine is
released in the brain and binds to adenosine receptors
in the central nervous system, promoting sleep83–85),
rheumatoid nodulosis82 and hepatic fibrosis86,87.
Therapeutic implications
Methotrexate remains the most commonly used drug
in the treatment of RA and other rheumatic diseases.
Because the bioavailability of orally administered metho­
trexate is limited, many patients can probably achieve
better responses to the drug by parenteral adminis­tration
of methotrexate or by dividing the dose of the drug over
the course of a day as opposed to taking a single daily
oral dose. The demonstration that folic acid administra-
tion can prevent many of the toxic effects associated with
methotrexate therapy without affecting therapeutic effi-
cacy has led to most clinicians prescribing folic acid with
of methotrexate-​related adverse effects12,13,88,89.
Currently nearly all patients with inflammatory arth­
ritis are given methotrexate first, in the absence of a con-
traindication, and if patients do not respond, then they
are prescribed another therapy, usually in combination
with methotrexate2. As not all patients respond to metho­
likely to respond would be useful to more effectively pre-
scribe the right treatment to patients to diminish disease
activity and to reduce unnecessary exposure to poten-
tially toxic drugs. Great hope was placed in identifying
genetic markers of metho­trexate response, including
some discussed in this Review (TNFAIP3), but, to date,
no factors or biomarkers have been clearly validated as
predictive. Both candidate gene studies and genome-​
wide association studies have identified a variety of
potential genetic variants associated with a favourable
response to methotrexate, although these associations do
not seem to be reproducible when tested in other popu-
lations90,91. More recently, a machine learning analysis of
transcriptomic data of whole blood from patients with
RA showed the potential predictive value for metho-
trexate response of the expression of genes involved in a
number of inflammatory pathways (most notably, genes
involved in the response to type I interferon); however,
the inclusion of all of the measured gene transcripts
in the model gave a better predictive value than the inclu-
sion of gene transcripts of the interferon pathway alone92.
Other studies have attempted to define clinical and labo-
ratory markers that are useful in predicting methotrexate
response (Box 3), although the markers identified do not
appear to be constant across these studies93–95, perhaps
because of the use of different end points.
Conclusions
Examination of the molecular basis for the therapeutic
activity of methotrexate shows that multiple pathways
are activated by methotrexate, all of which contribute to
the suppression of inflammation in RA. The underlying
biochemical mechanisms include inhibition of ATIC
(leading to increased release of adenosine, which sup-
presses inflammatory and immune functions via inter­
action with cell surface receptors); inhibition of DHFR and
diminished reduction of BH2 to BH4 (leading to uncou-
pling of nitric oxide synthases and increased production
of reactive oxygen species) and JNK activation; increased
expression of lincRNA-​p21, a lncRNA that diminishes
a variety of immune and inflammatory reactions; and
direct and indirect (adenosine-​mediated) inhibition
of signalling via NF-​κB and the JAK–STAT pathway,
critical promoters of inflammatory and immune reac-
tions. Many of the effects of methotrexate that have
been described seem to be cell-​type specific. In T cells,
dominant pathways include increased sensitivity to
apoptosis and inhibition of NF-​κB activity. Moreover,
the suppression of T cell activity by Treg cells via the
secretion of adenosine is promoted by methotrexate, and
adenosine itself promotes the differentiation of Treg cells
that can suppress inflammation. Many of these in vitro
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 Reviews

effects also occur in patients with RA who are receiving
methotrexate therapy.
One hypothesis that arises from the various mecha­
nisms of action of methotrexate in RA is that metho-
trexate regulates responses to ‘danger signals’. RA is
considered to arise from an adaptive immune response
to self-​antigens, and in the search for such antigens,
citrullinated peptides, the levels of which are increased
in RA, have been implicated96,97. An adaptive immune
response to a foreign antigen is generally thought
to require both a danger signal, to activate the innate
immune response, and a foreign antigen, to stimulate
the adaptive immune response98,99. By altering the con-
centrations of adenosine, methotrexate alters responses
to these antigenic danger signals. Moreover, the cell cycle
checkpoints and DNA repair machinery responsible for
maintaining appropriate cellular function also inhibit
pro-​inflammatory pathways, and methotrexate regulates
these repair mechanisms. Therefore, we speculate that
the danger signal that initiates the innate immune
response in RA could also be an internal signal produced
by this defective repair machinery rather than an exter-
nal signal and might ultimately result in a continuous
cycle of chronic inflammation. Methotrexate directly tar-
gets this proposed internal danger signal, which might
contribute to its therapeutic effects in RA in addition
to the suppression of an overly exuberant immune and
inflammatory response.
Clearly, all of the mechanisms described to date will
contribute to the therapeutic efficacy of methotrexate
therapy in RA. Insights into non-​folate-dependent anti-​
inflammatory mechanisms of methotrexate have led to
a marked reduction in clinical toxicity of the drug and
thereby increased its use in the treatment of RA.
Published online xx xx xxxx

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Acknowledgements
The work of T.M.A. was supported by grants from the US
National Institutes of Health (R21AR063846, R42AI53948,
R01AI044924), the American College of Rheumatology
‘Within Our Reach’ grant programme (ACR124405), the
US National Center for Advancing Translation Sciences
(UL1TR000445) and the US National Science Foundation
Graduate Research Fellowship Program.
Author contributions
The authors contributed equally to all aspects of the article.
Competing interests
B.N.C. declares that he holds equity in Regenosine, a biotech
start-​up developing therapies for osteoarthritis, and CanFite
Biopharma, that he has received grant support from AstraZeneca
and Kairos Inc., and that he has a number of patents involving
adenosine receptors and hepatic fibrosis, wound healing, bone
regeneration and osteoarthritis. T.M.A. declares that he has no
competing interests.
Peer review information
Nature Reviews Rheumatology thanks W. Wei and the other,
anonymous, reviewer(s) for their contribution to the peer
review of this work.
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