Mesenchymal Stem Cells Induce Regulatory T-cell Population in Human SLE

Background: The mechanisms underlying peripheral disorders during systemic lupus erythematosus (SLE) were found to be shared with tolerance disorders and mediated by T-regulator (T-reg) cells. Mesenchymal stem cells (MSCs) may inhibit T-cell subset differentiation and induce the T-reg cell phenotype. However, the capacity of MSCs to promote functional T-reg cells in SLE patients remains unclear. Objectives: This study aimed to analyze the capacity of MSCs to induce the production of functional CD4+ CD25+ Foxp3+ T-reg cells, in vitro, under co-culture conditions with human SLE cells. Methods: This study used a preand post-test control group design. Peripheral blood mononuclear cells (PBMCs) were extracted from SLE patients at the Kariadi Hospital, and MSCs were derived from human umbilical cords (hUCs) The PBMC control group was treated with standard medium, and the treatment group was co-cultured with hUC-MSCs. After 24 hours of co-culture incubation, T-reg cells were removed from the PBMC pool, using magnetic-activated cell sorting (MACS), and the population was assessed using the trypan blue exclusion assay. Results: A significant increase in the population of T-reg cells was observed (P < 0.001) after 24 hours of co-culture incubation with hUC-MSCs. Conclusion: This study concluded that MSCs have the capacity to enhance the T-reg population in human SLE PBMCs. Keyword: MSCs, T-regs, SLE, T-cells, PBMCs Correspondence to: Dr. H. Agung Putra, MD, M.Si. Med., Chairman of Stem Cell and Cancer Research (SCCR) Laboratory, Faculty of Medicine, Sultan Agung Islamic University, Semarang, Jl. Raya Kaligawe KM. 4 Semarang, Central Java 50112 Phone.+628164251646, Fax. 0246594366, Email: dr.agungptr@gmail.com 1. Riyadh Ikhsan, Department of Dermatology and Venerology, Faculty of Medicine, Universitas Sumatera Utara (USU), Medan, Indonesia 2. Agung Putra, Stem Cell and Cancer Research (SCCR), Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang, Indonesia and Department of Postgraduate Biomedical Science, Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang, Indonesia and Department of Pathological Anatomy, Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang, Indonesia. 3. Delfitri Munir, Pusat Unggulan Iptek (PUI) Tissue Engineering, Faculty of Medicine, Universitas Sumatera Utara (USU), Medan, Indonesia 4. Dewi Masyithah Darlan, Department of Parasitology, Faculty of Medicine, Universitas Sumatera Utara (USU), Medan, Indonesia 5. Bantar Suntoko, Department of Internal Medicine, Faculty of Medicine, Diponegoro University (UNDIP), Semarang, Indonesia 6. Azizah Retno Kustiyah, Stem Cell and Cancer Research (SCCR), Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang, Indonesia and Department of Pediatric, Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang, Indonesia 7. Iffan Alif, Stem Cell and Cancer Research (SCCR), Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang, Indonesia. 8. Ardi Prasetio, Stem Cell and Cancer Research (SCCR), Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang, Indonesia. Bangladesh Journal of Medical Science Vol. 19 No. 04 October’20. Page : 743-748 DOI: https://doi.org/10.3329/bjms.v19i4.46635


Introduction
Systemic lupus erythematosus (SLE) is a chronic, autoimmune disease, in which the body's immune system produces excessive antibodies or other soluble molecules that trigger excessive inflammatory responses 1,2 . SLE patients continue to be at increased risk for premature mortality, according to a cohort study (1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014), indicating that mortality has not significantly improved among SLE patients, particularly among young adults 3 . These findings indicate that the development of new therapeutic agents is necessary to improve SLE management strategies. The mechanisms underlying peripheral disorders in SLE were found to be shared with those that underlie common tolerance disorders, which are mediated by CD4 + CD25 + Foxp3 + regulatory T (T-reg) cells. The disruption of T-reg cells may cause T-and B-cell hyperactivity, which reduces the function and number of T-reg cells 4

.
A previous study has reported that a decrease in circulating T-reg cells may promote the appearance of autoreactive T-and B-cells, the loss of homeostasis, and immune system failure, which is associated with SLE occurrence 5 . Another study reported that the enhancement of T-reg cell functions may have useful effects for human SLE 6 , and an SLE treatment that is currently being developed is the transplantation of mesenchymal stem cells (MSCs) 7 . MSCs are nonhematopoietic, plastic-adherent, multipotent, and fibroblastic-like cells that may express several surface markers, such as CD90, CD105, CD44, CD73, and lack the expression others, such as CD79, CD19, CD14, CD11b, CD45, CD34, and human leukocyte antigen (HLA)-DR 8 . MSCs can be differentiated into several cell and tissue types, including chondrocytes, osteoblasts, adipocytes, neural cells, and several types of immune cells 9 . MSCs can also inhibit the proliferation and expansion of T-and B-lymphocytes, dendritic cells, and natural killer (NK) cells. These immunosuppressive capacities have been supported by the identification of several specific mechanisms, such as the activation and expression of inducible nitric oxide synthase (iNOS) and indoleamine 2,3-dioxygenase (IDO) and the enhancement of suppressive cytokines 10 .
Several T-cell regions are known to play roles in inflammatory regulation 11,12 . A previous study revealed that MSCs may inhibit the differentiation of Th17 cells and induce the T-reg phenotype 13 . Moreover, other studies have reported that MSCs may enhance the expansion of T-reg cells through the induction of CD4 + T-cells, mediated by prostaglandin E 2 (PGE 2 ) and transforming growth factor β1 (TGF-β1) 14,15 . These findings indicated that MSCs may exert anti-inflammatory effects, even under inflammatory conditions. However, the capacity of MSCs to promote functional T-reg cells in SLE patients remains unclear. The aim of our study was to analyze the capacity of MSCs to induce the production of functional CD4 + CD25 + Foxp3 + T-reg cells, in vitro, under co-culture conditions with human SLE cells.

Research design
This study used a pre-and post-test control group design. This study was conducted at the Stem Cell and Cancer Research (SCCR) Laboratory, Faculty of Medicine, Sultan Agung Islamic University, Semarang, Indonesia, from September-October 2018. This research used peripheral blood mononuclear cells (PBMCs), which were extracted from SLE patients at the Kariadi Hospital after obtaining informed consent, and MSCs derived from human umbilical cords (hUCs). The control group comprised PBMCs treated with standard medium, and the treatment group comprised PBMCs cocultured with hUC-MSCs.

MSC Isolation
This study was approved and was performed in accordance with the guidelines established by the Committee Ethics Institutional Review Board of the Medical Faculty, Sultan Agung Islamic University, Semarang, Indonesia. MSCs were isolated and separated from cord blood obtained from donors, who provided informed consent. The isolation and expansion of MSCs were performed as described previously 20 . Briefly, cords were cut into smaller pieces and transferred into a T25 culture flask (Corning, Tewksbury, MA, USA) containing Dulbecco's modified Eagle medium (DMEM, Sigma-Aldrich, Louis St, MO) and augmented with 10% fetal bovine serum (FBS, Gibco™ Invitrogen, NY, USA) and 1% penicillin (100 U/mL)/streptomycin (100 µg/mL) (Gibco™ Invitrogen, NY, USA). Cultured cells then were incubated at 37°C and 5% CO 2 . The cell medium was renewed every 3 days, and cells were passaged after reaching 80% confluence (approximately every 14 days). hUC-MSCs from passages 4-6 were employed for this study.

Isolation of PBMCs and co-culture with MSCs
Human PBMCs, from healthy volunteers who provided informed consent, were separated using Ficoll-Paque (Sigma-Aldrich, Louis St, MO) density-gradient centrifugation, in a 15-mL conical tube. PBMCs were cultured and expanded in 2 ml of advanced RPMI 1640 culture medium (Gibco™ Invitrogen, NY, USA), supplemented with 10% FBS, 100 U/ml penicillin and streptomycin, and 2 mM glutamine, and incubated at 37°C, in a humidified atmosphere containing 5% CO 2 . For the treatment group, PBMCs were co-cultured with MSCs in a T25 flask, in RPMI supplemented with 1% penicillin and streptomycin and 10% FBS, at a 1:40 ratio of MSCs:PBMCs, for 24 hours. For the control group, isolated PBMCs (1×10 7 cells/flask) were cultured in a T25 flask, with the standard medium, for 24 hours.

T-reg cell sorting and counting
After co-culture incubation, T-reg cells were isolated from the PBMC pool, using the CD4 + CD20 + Regulatory T-Cell Isolation Kit (Miltenyi Biotec, Germany). CD4 + and CD25 + T-cells were removed from the pool by positive and negative selection, respectively. After incubation with antibodies, CD4 + CD25 + T-reg cells were sorted, using magneticactivated cell sorting (MACS) (Miltenyi Biotec, Germany), according to the manufacturer's instructions. The viability and population of T-reg cells were then analyzed using the trypan blue exclusion assay and an automated cell counter.

Data analysis
All values are presented as the mean ± standard deviation (SD). All calculations were performed using SPSS 16.0 (IBM Corp., Armonk, NY, USA). Group comparisons were analyzed by paired Student's -tests, followed by Fisher's leastsignificant difference (LSD) post hoc test. A -value of < 0.05 was considered to be significant.

Ethical Clearance
All research activities were performed in accordance with and approved by the Health Research Ethical Committee Medical Faculty of Universitas Sumatera Utara (USU) Medan, under No. 564/TGL/KEPK FK USU-RSUP HAM/2018.

Isolation and Differentiation of hUC-MSCs
hUC-MSCs were isolated and cultured from UCs, based on their plastic attachment capacities, Figure 1. (a) hUC-MSCs were characterized by fibroblast-like appearance and spindle shape characteristics; (b) and calcium deposition appeared as a red color after Alizarin Red staining (10× magnification). under standard culture conditions for 4 passages. The cells were fibroblast-like, with spindle shape characteristics, became 80% confluent after 5-7 days in culture, and were regularly passaged (Figure 1a). Differentiation was examined by the administration of osteogenesis medium for 21 days. Calcium deposition was observed as a red color after immunodetection by Alizarin Red (Figure 1b).

MSCs enhance the T-reg population
We co-cultured the hUC-MSCs with PBMCs and examined directed autoimmunity. hUC-MSCs were co-cultured with PBMCs in the treatment group, and T-reg cell generation was compared with that for the control group after 24 hours of incubation. We found that hUC-MSCs significantly increased the T-reg cell population when co-cultured with PBMCs derived from SLE patients (P < 0.001). The number of T-reg cells among PBMCs after co-cultured with hUC-MSCs was 18.5 ± 1.84 × 10 5 cells, which was significantly higher than that for the control group (13.5 ± 1.274 × 10 5 cells; **, P < 0.001).
We co-cultured the hUC-MSCs with PBMCs and examined directed autoimmunity. hUC-MSCs were co-cultured with PBMCs in the treatment group, and T-reg cell generation was compared with that for the control group after 24 hours of incubation. We found that hUC-MSCs significantly increased the T-reg cell population when co-cultured with PBMCs derived from SLE patients (P < 0.001).

Discussion
The capacity of hUC-MSCs to modulate T-cell inflammatory conditions and proliferation may represent an alternative therapeutic route for clinical use. Several studies have shown that MSCs have the capacity to suppress inflammatory niches, depending on different mechanisms, including the capacity to generate and upregulate functional T-reg cells 16. Several studies have revealed that CD4 + CD25 + Foxp3 + cells, when co-cultured with hUC-MSCs, can inhibit alloantigen-activated T-cell proliferation 17 .
Another study reported that MSCs could induce the transformation of CD4 + T-cells into T-reg cells through cell contacts the contributions of PGE2 and TGF-β1 18 . MSCs may enhance the production of functional T-reg cells in human SLE patients. To prove this statement we co-cultured the hUC-MSCs with PBMCs and then sorted using MACS to find T-reg cell generation.
Our study showed that MSCs significantly increased the population of T-reg cells after 24 hours of coculture with human SLE PBMCs, at a ratio of 1:40 MSCs to PBMCs. (Figure 3 20 . This condition may enhance the expression of PGE 2 , which binds with the prostaglandin receptors EP 2 and EP 4 , activating the TIR-domain-containing adapterinducing interferon-β (TRIF)-TRIF-related adaptor molecule (TRAM)-mediated anti-inflammatory signaling pathway and resulting in the expression of several anti-inflammatory molecules, such as IL-10 and TGF-β, which may suppress the inflammatory niche 21 .
These regulated inflammatory conditions may lead to the generation of T-reg cells, induced by the expression of TGF-β. A previous study revealed that TGF-β could cause non-T-reg cells to transform into T-reg cells and inhibit effector T-cell development 22 .
Another study revealed that TGF-β plays a key role in signaling pathway regulation, inducing and preserving FoxP3 suppressor function and expression 23 .
These findings suggested that MSCs could induce T-reg cells, to modulate alloresponses into suppressive responses on effectors, resulting in the inhibition of the immune response to alloantigens. However, in our study, we did not explore TGF-β expression or the correlation between TGF-β and Foxp3 marker expression in T-reg cells.

Conclusion
Based on our study, we concluded that MSCs have the capacity to enhance the T-reg cell population in human SLE PBMCs.