Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Exp Med. Aug 20, 2017; 7(3): 84-96
Published online Aug 20, 2017. doi: 10.5493/wjem.v7.i3.84
Treg/Th17 cell balance and phytohaemagglutinin activation of T lymphocytes in peripheral blood of systemic sclerosis patients
Ekaterina Krasimirova, Tsvetelina Velikova, Ekaterina Ivanova-Todorova, Kalina Tumangelova-Yuzeir, Desislava Kalinova, Vladimira Boyadzhieva, Nikolay Stoilov, Tsvetelina Yoneva, Rasho Rashkov, Dobroslav Kyurkchiev
Ekaterina Krasimirova, Tsvetelina Velikova, Ekaterina Ivanova-Todorova, Kalina Tumangelova-Yuzeir, Dobroslav Kyurkchiev, Laboratory of Clinical Immunology, University Hospital “St. Ivan Rilski”, Department of Clinical Laboratory and Clinical Immunology, Medical University of Sofia, 1431 Sofia, Bulgaria
Desislava Kalinova, Vladimira Boyadzhieva, Nikolay Stoilov, Tsvetelina Yoneva, Rasho Rashkov, Clinic of Rheumatology, University Hospital “St. Ivan Rilski”, Department of Internal Medicine, Medical University of Sofia, 1431 Sofia, Bulgaria
Author contributions: All authors contributed equally to this work; Krasimirova E, Velikova T, Ivanova-Todorova E, Tumangelova-Yuzeir K and Kyurkchiev D performed the research; Kalinova D, Boyadzhieva V, Stoilov N, Yoneva T and Rashkov R contributed patient assessment and clinical materials; Krasimirova E and Velikova T analysed the data; Krasimirova E, Ivanova-Todorova E and Kyurkchiev D wrote the paper; Kyurkchiev D approved the final version of the article to be published.
Institutional review board statement: All peripheral blood samples were taken from patients and healthy control subjects after informed written consent and ethical permission was obtained for participation in this study. The study was reviewed and approved by the Institutional Review Board of University Hospital Saint Ivan Rilski, Sofia, Bulgaria.
Conflict-of-interest statement: Authors declare no conflict of interests for this article.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dobroslav Kyurkchiev, MD, PhD, DSc, Associate Professor, Laboratory of Clinical Immunology, University Hospital “St. Ivan Rilski”, Department of Clinical Laboratory and Clinical Immunology, Medical University of Sofia, Ivan Geshov Str. 15, 1431 Sofia, Bulgaria. dkyurkchiev@medfac.mu-sofia.bg
Telephone: +359-2-8524957
Received: January 26, 2017
Peer-review started: February 8, 2017
First decision: May 10, 2017
Revised: May 26, 2017
Accepted: June 30, 2017
Article in press: July 3, 2017
Published online: August 20, 2017
Abstract
AIM

To investigate T-cell activation, the percentage of peripheral T regulatory cells (Tregs), Th17 cells and the circulating cytokine profile in systemic sclerosis (SSc).

METHODS

We enrolled a total of 24 SSc patients and 16 healthy controls in the study and divided the patients as having diffuse cutaneous SSc (dcSSc, n = 13) or limited cutaneous SSc (lcSSc, n = 11). We performed a further subdivision of the patients regarding the stage of the disease - early, intermediate or late. Peripheral venous blood samples were collected from all subjects. We performed flow cytometric analysis of the activation capacity of T-lymphocytes upon stimulation with PHA-M and of the percentage of peripheral Tregs and Th17 cells in both patients and healthy controls. We used ELISA to quantitate serum levels of human interleukin (IL)-6, IL-10, tissue growth factor-β1 (TGF-β1), and IL-17A.

RESULTS

We identified a decreased percentage of CD3+CD69+ cells in PHA-stimulated samples from SSc patients in comparison with healthy controls (13.35% ± 2.90% vs 37.03% ± 2.33%, P < 0.001). However, we did not establish a correlation between the down-regulated CD3+CD69+ cells and the clinical subset, nor regarding the stage of the disease. The activated CD4+CD25+ peripheral lymphocytes were represented in decreased percentage in patients when compared to controls (6.30% ± 0.68% vs 9.36% ± 1.08%, P = 0.016). Regarding the forms of the disease, dcSSc patients demonstrated lower frequency of CD4+CD25+ T cells against healthy subjects (5.95% ± 0.89% vs 9.36% ± 1.08%, P = 0.025). With regard to Th17 cells, our patients demonstrated increased percentage in comparison with controls (18.13% ± 1.55% vs 13.73% ± 1.21%, P = 0.031). We detected up-regulated Th17 cells within the lcSSc subset against controls (20.46% ± 2.41% vs 13.73% ± 1.21%, P = 0.025), nevertheless no difference was found between dcSSc and lcSSc patients. Flow cytometric analysis revealed an increased percentage of CD4+CD25-Foxp3+ in dcSSc patients compared to controls (10.94% ± 1.65% vs 6.88% ± 0.91, P = 0.032). Regarding the peripheral cytokine profile, we detected raised levels of IL-6 [2.10 (1.05-4.60) pg/mL vs 0.00 pg/mL, P < 0.001], TGF-β1 (19.94 ± 3.35 ng/mL vs 10.03 ± 2.25 ng/mL, P = 0.02), IL-10 (2.83 ± 0.44 pg/mL vs 0.68 ± 0.51 pg/mL, P = 0.008), and IL-17A [6.30 (2.50-15.60) pg/mL vs 0 (0.00-0.05) pg/mL, P < 0.001] in patients when compared to healthy controls. Furthermore, we found increased circulating IL-10, TGF-β, IL-6 and IL-17A in the lcSSc subset vs control subjects, as it follows: IL-10 (3.32 ± 0.59 pg/mL vs 0.68 ± 0.51 pg/mL, P = 0.003), TGF-β1 (22.82 ± 4.99 ng/mL vs 10.03 ± 2.25 ng/mL, P = 0.031), IL-6 [2.08 (1.51-4.69) pg/mL vs 0.00 pg/mL, P < 0.001], and IL-17A [14.50 (8.55-41.65) pg/mL vs 0.00 (0.00-0.05) pg/mL, P < 0.001]. Furthermore, circulating IL-17A was higher in lcSSc as opposed to dcSSc subset (31.99 ± 13.29 pg/mL vs 7.14 ± 3.01 pg/mL, P = 0.008). Within the dcSSc subset, raised levels of IL-17A and IL-6 were detected vs healthy controls: IL-17A [2.60 (0.45-9.80) pg/mL vs 0.00 (0.00-0.05) pg/mL, P < 0.001], IL-6 [2.80 (1.03-7.23) pg/mL vs 0.00 pg/mL, P < 0.001]. Regarding the stages of the disease, TGF-β1 serum levels were increased in early stage against late stage, independently from the SSc phenotype (30.03 ± 4.59 ng/mL vs 13.08 ± 4.50 ng/mL, P = 0.017).

CONCLUSION

It is likely that the altered percentage of Th17 and CD4+CD25-FoxP3+ cells along with the peripheral cytokine profile in patients with SSc may play a key role in the pathogenesis of the disease.

Keywords: Systemic sclerosis, T-cell activation, Th17, Tregs, CD4+CD25-Foxp3+ cells, Interleukin-17, Tissue growth factor-β, Interleukin-10, Interleukin-6

Core tip: Systemic sclerosis (SSc) is a devastating autoimmune disorder, which can be subclassified into limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc) based on the skin manifestations. One of the original contributions of our study has demonstrated a decreased capacity for PHA-induced peripheral T-cell activation in patients with SSc. For the first time, our research group has identified an up-regulated percentage of CD4+CD25-FoxP3+ cells in the dcSSc subset. Regarding the peripheral cytokine profile in SSc, the serum levels of interleukin (IL)-17A have been increased in lcSSc as opposed to the dcSSc subset. The rest of our data, concerning the elevated circulating IL-6, IL-10, and TGF-β in SSc patients, has confirmed literature-based results.