Original article CED

Clinical and Experimental Dermatology

Effect of methotrexate monotherapy on T-cell subsets in the

peripheral circulation in psoriasis
M. Priyadarssini,1 L. Chandrashekar2 and M. Rajappa1
Departments of 1Biochemistry and 2Dermatology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry,India

doi:10.1111/ced.13795

Summary Background. Psoriasis is a T-helper (Th)1/Th17-mediated chronic inflammatory

disease. There is an increased population of Th cells in skin lesions and peripheral
circulation of patients with psoriasis. Systemic methotrexate (MTX) is an effective
treatment for moderate to severe psoriasis; however, its effect on different T-cell subsets
is not yet clear.
Aim. To study the effect of MTX monotherapy on the psoriatic T-cell profile in the
peripheral circulation of patients with psoriasis.
Methods. This was a follow-up study involving 50 patients with moderate to se-
vere psoriasis treated with systemic MTX for 12 weeks. Blood samples (5 mL) were
collected from participants, from which PBMCs were isolated, and T-cell phenotyping
was performed by flow cytometry.
Results. Following 12 weeks of MTX treatment, there was an increase in the per-
centages of Th2/Treg cells, and a relative decrease in the percentages of Th1/Th17
cells, along with a significant reduction in the median Psoriasis Area and Severity
Index (PASI).
Conclusion. MTX helps in the restoration of the immune balance by decreasing
the numbers of Th1 and Th17 cells and increasing the numbers of Th2 and Treg
cells, thus resulting in a significant reduction in disease severity. MTX converts a
proinflammatory T-cell phenotype to a protective anti-inflammatory phenotype, thus
significantly suppressing the inflammation in psoriasis.

Introduction and the cutaneous lesions of patients with psoriasis.2,3

Psoriasis is an immune disease of the skin, characterized
by an inflammatory infiltrate and excessive proliferation
of keratinocytes. Although the aetiopathogenesis of dis-
ease is not completely understood, the disease is known
to involve an immune dysregulation that initiates a cas-
cade of proinflammatory cytokines.1 This eventually
culminates in a predominant T-helper (Th)1 cell
response with defective Th2 function in the circulation
Correspondence: Dr Medha Rajappa, Department of Biochemistry,
Jawaharlal Institute of Postgraduate Medical Education and Research,
Puducherry, India
E-mail: linkmedha@gmail.com
Conflict of interest: the authors declare that they have no conflicts of
interest.
Accepted for publication 17 June 2018
Recent studies suggest that Th17 cells produce inter-
leukin (IL)-17, which orchestrates inflammation in such
conditions.4,5 Quantitative and qualitative defects in
Foxp3+ regulatory T cells (Tregs) fails to suppress T-cell
activation in psoriasis, thus contributing to defective T-
cell regulation.6–8 Thus, changes in the numbers and
functions of different subsets of T cells might have a
notable role in the disease pathogenesis. In our earlier
report,9 we assessed the Th/Treg cell pattern in the
peripheral circulation of 189 patients with psoriasis and
found a dysregulated T-cell immunophenotype, with an
elevation in Th1/Th17 cells and a relative depression in
Th2/Treg cells in patients with psoriasis compared with
healthy controls (HCs).
Despite considerable advances in treatment, psoriasis
remains a disease without complete cure. The concept

ª 2018 British Association of Dermatologists Clinical and Experimental Dermatology 1

Effect of methotrexate on psoriatic T-cell profile  M. Priyadarssini et al.
of the ‘psoriatic march’ suggests a sequence of events
involved in the progression of inflammation from skin
to systemic involvement, eventually leading to oxida-
tive stress, endothelial dysfunction, altered glucose
metabolism and insulin resistance, and thus
atherosclerosis and coronary artery disease.10 Studies
related to various treatments such as phototherapy
and etanercept, and their effect on the psoriatic T-cell
population have been reported previously.11,12 Furu-
hashi et al. reported that there was elevation in Treg
cell counts and function in patients with psoriasis
treated with phototherapy.12 Methotrexate (MTX) is a
drug that exerts its anti-inflammatory actions by
decreasing recruitment of inflammatory cells and
increasing apoptosis of activated T cells in the skin of
patients with psoriasis.13,14 However, there is a dearth
of literature regarding the effect of MTX monotherapy
on the phenotypic pattern of T-cell subsets in the
peripheral circulation of patients with psoriasis. Hence,
we undertook this study to evaluate the effect of MTX
monotherapy on T-cell phenotype in the circulation in
psoriasis.
Methods
The study was approved by the JIPMER Institute
Ethics Committee (Human Studies) (project number
JIP/IEC/SC/2014/8/612), and was performed accord-
ing to the WMA Declaration of Helsinki ethical princi-
ples for medical research involving human subjects.
Written informed consent was obtained from all study
participants.
Participants
This was a hospital-based cohort study at Jawaharlal
Institute of Postgraduate Medical Education and
Research, Pondicherry, India. Of the 189 patients with
psoriasis recruited for our previous study,9 50 patients
aged 18–60 years with moderate to severe psoriasis [Pso-
riasis Area and Severity Index (PASI) > 10] diagnosed
according to the International Psoriasis Council Consen-
sus Classification of Psoriasis15 were enrolled in the
study. In addition, 50 healthy volunteers were recruited
for the HC group. Exclusion criteria included inflamma-
tion or infection, cancers, diabetes mellitus, coronary
artery disease, hypertension, and use of systemic or topi-
cal drugs in the 3 months prior to study enrolment.
Patient characteristics such as disease severity
(indicated by PASI16), disease duration, comorbidi-
ties, therapeutic history and family history were
noted.
2 Clinical and Experimental Dermatology
Methotrexate therapy
Patients with psoriasis were treated with systemic
MTX, which was administered orally in the form of a
tablet at a dose of 7.5 mg per week. The dose of MTX
was gradually increased to 10 mg per week after
4 weeks and then to 15 mg per week after 8 weeks.
The patients were followed up at 12 weeks, for assess-
ment of the response to therapy. At the end of
12 weeks, 32 patients had received 15 mg per week,
13 patients had received 12.5 mg per week and 5
patients had received a dose of 10 mg per week of
MTX.
Investigations
To assess T-cell populations, 5 mL of venous blood
was collected from each participant into anticoagula-
tion tubes, and peripheral blood mononuclear cells
(PBMCs) were isolated. Regular biochemical investiga-
tions such as glucose, lipid profile, and renal and liver
function tests were also performed.
Flow cytometry analysis
Density gradient centrifugation (Ficoll-Histopaque;
Sigma-Aldrich, St Louis, MO, USA) was used to sepa-
rate PBMCs from anticoagulated venous blood. A
four-colour cytometer (FACS Calibur; Becton Dickin-
son, San Diego, USA) was used to analyse the isolated
PBMCs based on their immunofluorescence. Surface
markers on PBMCs were analysed using antibodies
conjugated to different fluorochromes: fluorescein
isothiocyanate (FITC), phycoerythrin (PE), peridinin
chlorophyll protein (PerCP), allophycocyanin (APC)
and Alexa Fluor 647. In total, 30 000 lymphocytes
were acquired for surface marker analysis of PBMCs.
Levels of Th1, Th2, Th17 and Treg cells were anal-
ysed with a commercial kit (Human T-helper/Treg
Phenotyping Kit (BD Biosciences, San Diego, CA,
USA), following the manufacturer’s technical protocol
(Fig. 1).
Statistical analysis
SPSS software (v20; IBM, Armonk, NY, USA) was
used to analyse the data. Descriptive statistics were
used to analyse the baseline characteristics of the
patients. The normality of continuous data (age of
onset of psoriasis, disease duration, T-cell counts)
was assessed by Kolmogorov–Smirnov test. Normally
distributed data were expressed as mean  SD and
ª 2018 British Association of Dermatologists
 Effect of methotrexate on psoriatic T-cell profile  M. Priyadarssini et al.

increase in the percentage of Th2 and Tregs in

patients compared with HCs at follow-up (Fig. 3). The
median percentages of Th2 and Tregs in the patients
after treatment were 85.20 and 85.66, compared with
78.66 and 92.45, respectively, in the HCs. Thus, the
increased percentages of Th2 and Treg cells in the
peripheral blood of patients with psoriasis after treat-
ment were close to healthy values.
We have illustrated this with the flow-cytometry
pattern of the T-cell phenotype in a patient with psori-
asis at baseline and follow-up (Fig. 4). The percentage
of Th1 cells dropped from 72.54% at baseline to
Figure 1 Mother flow cytometry plot depicting the lymphocytes, 0.44% at follow-up, while Th2 cells increased from
and the derived plot used to gate the CD4+ lymphocytes (R2), on 31.51% to 99.83%. Similarly, the percentage of Th17
which T-cell phenotyping was performed.
cells decreased from 87.69% at baseline to 3.06% at
follow-up, while Tregs increased dramatically, from
data without normal distribution were expressed as 9.74% to 87.58%.
median and interquartile range (IQR). Wilcoxon
signed rank test was used to compare the results
Changes in other parameters between follow-up and
before and after MTX therapy in patients, and corre-
baseline
lation analyses were performed using the Spearman
test. Analyses were carried out at the 5% level of At follow-up, the median PASI of patients with psoria-
significance and P < 0.05 was considered statistically sis at baseline was significantly reduced after MTX
significant. therapy (Table 2). Mean values of the liver enzymes
(aspartate aminotransferase and alanine aminotrans-
ferase) were not significantly different between base-
Results
line and at follow-up at 12 weeks.
Characteristics of the study population
Response to treatment
The baseline characteristics of the study population
are shown in Table 1. There were 50 patients with There were 11 patients who did not show adequate
psoriasis (35 men,15 women), and of these, 43 clinical response to MTX treatment (no reduction in
patients had chronic plaque psoriasis and 7 had pus- PASI) and were classified as nonresponders; however,
tular psoriasis. Median duration of psoriasis was of
6.00 years (IQR 3.00–10.00) and median PASI at
Table 1 Comparison of biochemical markers of cases at baseline
baseline was 18.12 (IQR 15.75–24.50). Various bio-
and the 12-week follow-up after methotrexate therapy.
chemical parameters of the patients were compared
between baseline and follow-up (Table 1). The base- Parameter Baseline (n = 50) 12 weeks (n = 50) P*
line characteristics and the baseline biochemical
Glucose, mg/dL 87.16  13.13 88.48  10.30 0.37
parameters were comparable between the patients and Urea, mg/dL 20.18  3.47 24.32  3.87 0.94
HCs. AST, IU/L 29.32  6.17 58.18  6.59 0.86
ALT, IU/aL 25.00  4.99 45.57  4.76 0.08
ALP, IU/L 72.78  21.24 89.84  18.15 0.11
Changes in T cells between follow-up and baseline GGT, IU/L 25.96  9.16 34.42  8.09 0.09
Sodium, IU/L 138.66  2.41 139.40  2.50 0.20
Figure 1 shows the gating of CD4+ lymphocytes (R2), Potassium, IU/L 4.12  0.42 4.19  0.45 0.59
from which T-cell phenotyping was performed. Flow Creatinine, mg/dL 0.90  0.13 0.89  0.15 0.49
cytometry analysis of the PBMCs at baseline showed Uric acid, mg/dL 4.91  1.04 4.77  0.94 0.40
an increase in the percentage of Th1 and Th17 cells Calcium, mg/dL 10.54  0.98 8.67  1.29 0.55
Phosphorus, mg/dL 4.06  0.27 5.17  0.26 0.91
and a decrease in the percentage of Th2 and Tregs in
patients compared with HCs at baseline (Fig. 2). How- ALP, alkaline phosphatise; ALT, alanine aminotransferase; AST,
ever, after treatment, there was a significant decrease aspartate aminotransferase; GGT, gamma-glutamyl transferase.
in the percentage of Th1 and Th17 cells and an Data are mean  SD. *Paired t-test.

ª 2018 British Association of Dermatologists Clinical and Experimental Dermatology 3

Effect of methotrexate on psoriatic T-cell profile  M. Priyadarssini et al.

Figure 2 Comparison of T-cell phenotype between patients and controls: (a) Th1 cells, (b) Th2 cells, (c) Th17 cells, (d) Treg cells.
Mann–Whitney U-test was used to analyse all these data.

Figure 3 Comparison of T-cell phenotype at baseline and at 12 weeks post methotrexate therapy: (a) Th1 cells, (b) Th2 cells, (c) Th17
cells, (d) Treg cells. Wilcoxon signed rank test was used to analyse all these data.

4 Clinical and Experimental Dermatology ª 2018 British Association of Dermatologists

 Effect of methotrexate on psoriatic T-cell profile  M. Priyadarssini et al.

Figure 4 Different T-cell phenotype in peripheral blood mononuclear cells (PBMCs) of a patient; flow cytometry analysis comparing the
percentages of different T-cell populations between baseline and follow-up.

the percentages of Th2 and Tregs were increased in 6 effects were not severe enough to necessitate drug
of these patients. PASI75 response rate was 78%. withdrawal.

Adverse effects Other correlations

Of the 50 patients, 9 experienced gastrointestinal
adverse effects such as nausea and vomiting during
the first few weeks of MTX treatment. However, these
Table 2 Comparison of Psoriasis Area and Severity Index at
baseline and at 12 weeks post-methotrexate therapy using Wil-
coxon signed rank test.
T-cell subsets in patients with psoriasis did not show
significant correlation with age. Both PASI and disease
duration showed a significant positive correlation with
percentages of Th1 and Th17 cells, and a significant
negative correlation with percentages of Th2 and
Tregs in patients with psoriasis at baseline (Table 3).

Timepoint PASI P* Discussion

Baseline (n = 50) 18.12 (15.75–24.5) < 0.001 Psoriasis is an immune-mediated systemic inflamma-
12 weeks (n = 50) 8.23 (5.75–14.5)
tory disease in which T lymphocytes trigger and main-
PASI, psoriasis area and severity index. Data are median (interquar- tain the inflammation. Our earlier report described
tile range). *Wilcoxon signed rank test. higher percentages of Th1 and Th17 cells and lower

ª 2018 British Association of Dermatologists Clinical and Experimental Dermatology 5

Effect of methotrexate on psoriatic T-cell profile  M. Priyadarssini et al.
Table 3 Correlation of Psoriasis Area and Severity Index and dis-
ease duration with T-cell phenotype of patients at baseline.
PASI Disease duration
Percentage of cells q P* q P
Th1 0.72 < 0.001 0.48 0.06
Th2 0.67 < 0.001 0.75 0.03
Th17 0.61 < 0.001 0.64 0.35
Tregs 0.68 < 0.001 0.82 < 0.001
PASI, psoriasis area and severity index; Th, T helper cells; Tregs,
regulatory T cells. *Spearman correlation.
percentages of Th2 and Treg cells in the psoriasis
group compared with the HC group.9 There was a pre-
dominance of Th1 and Th17 cell populations both in
the skin and circulation of patients with psoriasis,
which was further supported by the diminished anti-
inflammatory response mounted by Th2 and Treg
cells.9 MTX decreases recruitment of inflammatory
cells, increasing the apoptosis of activated T cells.
These effects are mediated by increased aminoimida-
zole 4-carboxamide ribonucleotide levels17 and
decreased thymidylate synthase activity,18 which
decrease the epithelial hyperplasia found in psoriasis
lesions.19 Despite these effects of MTX, its actions on
different T-cell subsets has not been studied to date.
Hence, we followed up 50 patients with moderate to
severe psoriasis after treatment with standard systemic
MTX for 12 weeks to assess the drug’s effects on T-cell
profile.
We observed that the percentages of Th1 and Th17
cells were decreased and those of Th2 and Treg cells
were increased after MTX therapy. Th2/Treg percent-
ages in patients with psoriasis after 12 weeks of MTX
therapy reached values near to those found in the HC
population. This may be due to the inhibitory effect of
increased IL-4 and IL-10 produced by Th2 cells on the
Th1-predominant pattern of inflammation.20 Addition-
ally, Th2 cells inhibit the interaction of IL-1 with its
receptor IL-1R,21 thus MTX decreases the numbers of
proinflammatory Th1 cells, which produce IL-2 and
interferon-c, with a concomitant increase in the anti-
inflammatory Th2 cells, which produce IL-4 and IL-
10.22 MTX has been shown to reduce serum levels of
IL-17 in various inflammatory diseases by inhibiting
mRNA expression of IL-17.23 It was also shown to
result in elevation in Tregs, probably by increasing
expression of transforming growth factor-b and thus
differentiation of Tregs, which in turn assisted in
attenuating the inflammation.24 This net anti-inflam-
matory effect of MTX makes it very effective in the
treatment of immune-mediated inflammatory diseases
6 Clinical and Experimental Dermatology
such as psoriasis. Our findings are in agreement with
those of Torres-Alvarez et al.,14 who reported a down-
regulation of inflammatory cell infiltrate in psoriatic
skin samples after MTX treatment.
MTX therapy also reduced PASI; median PASI at
baseline was 18, which was reduced to 8 after
12 weeks of MTX therapy. Thus, we found that MTX
restores the immune imbalance in psoriasis, causing
elevation in the Th2/Treg phenotype and depression
in the Th1/Th17 phenotype. This suggests that
appropriate treatment with MTX could convert the
proinflammatory T-cell phenotype to a protective anti-
inflammatory phenotype.
The percentages of Th and Treg cells in patients
with psoriasis did not correlate with patient age, but
did correlate with disease duration and PASI, suggest-
ing that disease duration and severity have synergistic
effects on inflammation.
Our study has some limitations. Other T-cell subsets,
such as Th22/Th9, which would have further aided in
establishing the inflammatory pattern, were not
assessed. Another limitation was that joint disease
improvement was not studied in this research. Owing
to resource constraints, we did not assess single
nucleotide polymorphisms or the molecular expression
of these T-cell cytokines which would have helped in
better comprehension of the genotype–phenotype cor-
relation of disease aetiology.
Conclusion
MTX converts the proinflammatory T-cell phenotype
to a protective anti-inflammatory phenotype, thus sig-
nificantly suppressing the inflammation in psoriasis,
and limiting the progression of disease and its associ-
ated cardiovascular comorbidity.
Acknowledgements
Funding by JIPMER (sanction number JIP/Res/Intra-
MD-MS/first/04/2014) to MR is gratefully acknowl-
edged.
What’s already known about this topic?
• Psoriasis is a T-helper (Th1/Th17)-mediated
chronic inflammatory disease.
• Systemic MTX is an effective treatment for
moderate to severe psoriasis.
ª 2018 British Association of Dermatologists
 Effect of methotrexate on psoriatic T-cell profile  M. Priyadarssini et al.

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ª 2018 British Association of Dermatologists Clinical and Experimental Dermatology 7