Mesenchymal stem cells modulate IL-17 and IL-9 production induced by Th17-inducing cytokine conditions in autoimmune arthritis: an explorative analysis

Abstract

Background

The importance of proinflammatory T-cells and their cytokine production in patients with autoimmune arthritis has been widely described. Due to their immunomodulatory properties, mesenchymal stem cells (MSCs) have come into focus as a potential therapeutic concept. The aim of this study was to investigate the influence of MSCs on the phenotype, cytokine profile, and functionality of naive and non-naive CD4⁺ T-cells from healthy donors (HD) and patients with autoimmune arthritis under Th17-cytokine polarizing conditions in an explorative way using a transwell system prohibiting any cell–cell-contact.

Methods

Magnetically isolated naive and non-naive CD4⁺ T-cells were stimulated under Th17-polarizing proinflammatory cytokine conditions in presence and absence of bone marrow derived mesenchymal stromal cells (MSCs). After an incubation period of 6 days, the proportions of the T-cell subpopulations TEMRA (CD45RA⁺CD27⁻), memory (CD45RA⁻CD27⁺), effector (CD45RA⁻CD27⁻) and naive cells (CD45RA⁺CD27⁺) were determined. Quantitative immunofluorescence intensity was used as a measure for IL-9, IL-17 and IFN-γ production in each subpopulation.

Results

In isolated naive CD4⁺ T-cells from HD and patients, MSCs suppressed the differentiation of naive towards an effector phenotype while memory and naive cells showed higher percentages in culture with MSCs. In patients, MSCs significantly decreased the proportion of IL-9 and IL-17 producing effector T-cells. MSCs also reduced IFN-γ production in the naive and memory phenotype from HD.

Conclusions

The results of the study indicate significant immunomodulatory properties of MSCs, as under Th17-polarizing conditions MSCs are still able to control T-cell differentiation and proinflammatory cytokine production in both HD and patients with autoimmune arthritis.

Keywords
Mesenchymal stem cell; Arthritis; T lymphocytes

Introduction

Mesenchymal stem cells (MSCs) are multipotent adult stem (or stromal) cells with the capacity of self-renewal and differentiation potential towards several mesodermal linages such as osteogenic, chondrogenic and adipogenic differentiation (more citations) [¹]. The anti-inflammatory role and immune-modulating properties of MSCs have been shown in several studies and their potential is being tested and discussed already in clinical trials including indications for e.g. graft-versus-host disease and even COVID19 pneumonia [²–⁶] Specifically, inhibition of the effector T-cell activation, in both CD4⁺ and CD8⁺ T-cells, has been associated to an increase of proportions of regulatory T-cells (Treg). Moreover, reduction in the number of IL-17 and IFN-γ producing T-cells were observed in the presence of MSCs [1, 7]. A shift from Th1 to Th2 cells demonstrated by an increase in the levels of IL-4 has been observed upon treatment with MSCs [⁸]. Although, the mechanism how MSCs modulate T-cells has been still not elucidated, cell–cell contact and some soluble factors such as nitric oxide, IL-27, TGF-beta, human leukocyte antigen G, and prostaglandin E₂ may be the key factors in the process [4, 9–11]. Based on their potential regenerative and immunomodulatory properties, MSCs have been proposed as a possible therapeutic approach not only for the treatment of autoimmune-associated diseases [¹²–¹⁴] such as rheumatoid arthritis (RA) [¹⁵], systemic lupus erythematosus (SLE) [¹⁶], and ankylosing spondylitis (AS) [¹⁷], in graftversus-host-disease and COVID19 infection as discussed above, but also in tissue engineering [¹⁸].

Interleukin-9 (IL-9) helps to eliminate the pathogen in parasitic infections and promote the maintenance of a tolerant environment in skin transplants [⁸]. IL-9 is produced by mast cells [¹⁹] and Th2 cells [²⁰], whereas IL-9 may be also derived from natural killer cells (NK) [²¹], Th9 [²²], Th17 cells [²³], Tregs [²⁴] and CD8+ T-cells [²⁵]. In the presence of TGF-β, IL-9 may induce differentiation of naive CD4⁺ T-cells towards Th17 cells, while IL-9 also affects thymus-derived natural Tregs (nTregs) and enhances their suppressive function in vitro [²⁶]. However, IL-9 has been associated with allergies and autoimmunity [¹⁹, ²⁷]. IL-9 and Th9 cells are overexpressed in synovial tissue while proportion of Th9 cells are increased in peripheral blood from RA patients [²⁸]. Interestingly, some studies showed that MSCs decrease IL-9 production by reducing the percentage of pro-inflammatory T-cells and increased levels of Treg cells [²⁹]. However, if MSCs are co-cultured with CD4⁺ T-cells under Th17-polarizing conditions, they seem to have a pronounced immunosuppressive effect on Th1 and Th17 cells as already mentioned. After T-cell polarization has already been induced, MSCs appear to suppress Th1 differentiation, but stimulate Th17 differentiation. This indicates that there might be positive and negative effects on the T-cell modulation by therapeutically used MSCs depending on the existing inflammatory conditions [³⁰]. Other results confirm proinflammatory effects of MSCs on T-cells after cell–cell contact associated with an increased IL-17 production in CD4⁺CD45RO⁺ memory cells [³¹]. Ultimately, the immune response seems to be both promoted and inhibited by MSCs and seems to depend on the dynamics of the inflammation, the activation state of the immune system, current inflammatory cytokines and the effects of immunosuppressors [³²]. Nevertheless, there remain some significant inconsistencies and a general lack of information regarding a number of effects that MSCs have on T-cell subpopulations [¹].

Recently, in a study of Heim et al. we could show that Th9- and Th17- inducing cytokine conditions mimicking autoimmune inflammation in RA may have similar stimulatory effects regarding polarization of naive and non-naive T-cells into Th9 or Th17 cells [³³]. From our point of view, the influence on the polarization of naive and non-naive T-cells in Th9 and Th17 cells by MSC might be a possible therapeutic approach. Therefore, the aim of this study was to investigate the influence of MSCs on the phenotype, cytokine profile, and functionality of naive and non-naive CD4⁺ T-cells from HD and patients with autoimmune arthritis under Th17-cytokine polarizing conditions in an explorative way using a transwell system prohibiting any cell–cell-contact. This study provides insights for the potential prophylactic use of MSCs in the treatment of T-cell dysbalance found in autoimmune arthritis.

Materials and methods

Patients and healthy donors (HD)

Blood samples of 4 volunteers (37 ± 9 years) with autoimmune arthritis (2 patients with rheumatoid arthritis (RA), 1 patient with spondylarthritis (SA) and 1 patient with systemic lupus erythematosus (SLE) and significant polyarthritis and 4 ageand gender-matched healthy donors (HD) were taken at the Department of Internal Medicine II, Rheumatology and Clinical Immunology University Hospital Würzburg (Table 1). Patients meeting the ACR criteria for RA, SLICC criteria for SLE and ASDAS cri- teria for AS were included [³⁴–³⁶]. Blood samples from HD were randomly taken from immunologically healthy volunteers without history of autoimmune disease, malignancy, allergy, administration of blood products, monogenetic disorders, immunodeficiency or any infections in the last four weeks and gender matched to the study group.

Table 1
Patient characteristics

	Patient 1	Patient 2	Patient 3	Patient 4
Age (y)	60.8	34.4	23.7	29.8
Sex (m/f )	f	f	f	m
Diagnosis	Seropositive RA	Seropositive RA	SLE polyarthritis	AS
Disease duration (y)	31	15	5	7
DAS28	3.50	3.35	–	–
BASDAI	–	–	–	2.3
BASMI	–	–	–	2
SLEDAI	–	–	12	–
ESR (1 h)	30 mm	4 mm	54 mm	4 mm
CRP (mg/dl)	0.25	0.01	0.04	0.31
Leukocytes (n*1000/µl)	9.9	6.7	6.4	12.8
Remission (yes/no)	No	No	No	Yes
Methotrexate	10 mg 1x/week	–	–	–
Leflunomide	20 mg 1–0-0	–	–	–
Prednisolone	40 mg 1–0-0	7.5 mg 1–0–0	40 mg 1–0–0	–
Infliximab	–	–	–	400 mg 1 × /8 weeks
Mycophenolate mofetil	–	–	500 mg 2–0–2	–
Etanercept	–	50 mg 1x/week	–	–

• Table shows age in years (y); sex—male/female (m/f); Diagnosis (RA = rheumatoid arthritis, SLE = systemic lupus erythematosus, AS = ankylosing spondylitis); Years since initial diagnosis; Disease Activity Score 28 (DAS28); Bath Ankylosing Spondylitis Disease Activity Index (BASDAI); Bath Ankylosing Spondylitis Metrology Index (BASMI); Systemic Lupus Erythematosus Disease Activity Index (SLEDAI); Erythrocyte sedimentation rate (ESR) after one hour (1 h); C-reactive protein (CRP) in mg/ dl; Leukocytes in n*1000/µl; Medication at the time of blood sample collection (MTX = methotrexate, LEF = leflunomide, PRE = prednisolone, ETA = etanercept, MYC = mycophenolate mofetil, INF = infliximab); Remission yes/no (patient in remission phase yes or no). A DAS28 < 2.6 was defined as remission for patients 1 and 2. Patient 3 showed an increased disease activity corresponding to a total of 12 points in the SLEDAI. In patient 4, a remission of the underlying disease could be assumed on the basis of low numerical values in the BASDAI and BASMI

The study was approved by the local ethics committee of the Medical Faculty of the University Hospital of Würzburg (No. 123/14) and performed in accordance with the Declaration of Helsinki 1967 and its later amendments and the Medical Research Involving Human Subjects Act (WMO).

Exclusion criteria were malignancies, congenital anomalies, syndromes, immunodeficiencies, clinically relevant infections in the last eight weeks and vaccinations in the last four weeks. Written informed consent was given from patients, HD and MSC donors.

Mesenchymal stem cells (MSC)

MSCs were harvested from spongy bone of the acetabulum during orthopedic hip surgery for the treatment of osteoarthritis of 2 otherwise HD and expanded in vitro [³⁷]. MSCs of both donors were in passage 1 at the beginning of the co-culture. Patients with malignancies, femoral head necrosis, femoral head trauma, congenital anomalies, syndromes, immune defects, clinically relevant infections in the last eight weeks and autoimmune inflammatory rheumatic diseases were excluded from the study (Table 2).

Table 2
MSC healthy donor characteristics

	MSC 1	MSC 2
Age (y)	48.9	72.2
Sex (m/f )	m	m
Diagnosis	Coxarthrosis	Coxarthrosis
Origin MSC	Acetabulum	Acetabulum
CRP (mg/dl)	1.6	0.2
Leukocytes (n*1000/µl)	7.7	6.5
Dabigatran	110 mg 0–0–2–0	–
Pantoprazol	20 mg 1–0–0–0	–
Paracetamol	500 mg 2–2–2–0	–
Diclofenac	75 mg 1–0–1–0	–
Levetiracetam	–	500 mg 1–0-1–0
Lorazepam	–	1 mg 0–0–1–0
Simvastatin	–	10 mg 0–1–0–0
Tromcardin® forte	–	0–2–0–0
Perindopril	–	4 mg 0–0–1/2–0

• Table shows age in years (y); sex—male/female (m/f); diagnosis; origin of MSCs; c-reactive protein (CRP) in mg/dl; leukocytes in n*1000/µl; medication (in mg) at the time of harvesting MSCs

Naive and non‑naive cell sorting

To isolate naive and non-naive CD4⁺ T-cells, two magnetic-bead-based isolation kits were combined. By a first step, CD4⁺ T-cells were isolated, in a second step, naive and non-naive cells were separated from the CD4⁺ T-cell fraction.

Step 1 (Isolation of CD4⁺T-cells): CD4⁺ cells were isolated from peripheral blood mononuclear cells (PBMCs) from HD and patients using the CD4⁺ T Cell Isolation Kit human.

Step 2 (Isolation of Naive and Non-Naive CD4⁺ T-cells): Naive (depleted) and non-naive CD4⁺ (retained) fractions were obtained using the Naive CD4⁺ T Cell Isolation Kit II human.

Both isolation steps were performed according to the manufacturers’ instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). A purity > 90% of the positive fractions was evaluated by flow cytometry. Non-CD4⁺ T-cells were irradiated (30 Gy) and used as autologous antigen presenting cells (feeder cells).

Co‑culture MSC and T‑cells

For co-culturing MSCs and T-cell subpopulations, adherent MSCs were removed from the cell culture surfaces by using trypsin/EDTA (1x) solution (PAA Laboratories GmbH, Pasching, Austria) following treatment with cell culture media containing 10% FCS to stop the reaction. To examine the effect of the soluble factors in the modulation of the T-cell response by MSCs, naive or non-naive CD4⁺ T-cells (2.5 × 10⁵ cells/well) together with feeder cells (5 × 10⁴) were seeded in the upper part of the Transwell system (Greiner Bio-one GmbH, Frickenhausen, Germany), while MSCs (0.25 × 10⁵ cells/well) were seeded on the bottom. Wells with direct cell-to-cell contact between MSCs and T-cells were not included in the study. For Th17-inducing cytokine conditions, wells were stimulated under a Th17 cytokine cocktail consisting of IL-1β (10 ng/mL), IL-6 (20 ng/mL), TGF-β (5 ng/ mL), IL-23 (100 ng/mL), anti-CD3/anti-CD28 (1 μg/mL) for 6 days at 37 °C. Wells containing no MSCs were used as control.

Flow cytometry analysis

The cytokine production profile on CD4⁺ naive and non-naive T-cells treated in the presence or absence of MSCs was determined upon stimulation with phorbol 12-myristate 13-acetate (PMA) (15 ng/ml), ionomycin (1 µg/ml) and brefeldin A (5 μg/ml) for 3 h at 37 °C. After incubation, expression of CD45RA and CD27 on naive and non-naive cells were determined by using monoclonal antibodies labeled with brilliant violet 421 (BV421, BioLegend, San Diego, USA) and CD27, labeled with phycoerythrin-cynanine7 (PE-Cy7, BioLegend, San Diego, USA) respectively. According to the expression of CD45RA and CD27, subpopulations were characterized as terminal effector memory cell reexpressing RA (TEMRA) (CD45RA⁺CD27−), memory (CD45RA−CD27⁺), effector (CD45RA−CD27−) and naive cells (CD45RA⁺CD27⁺) [³⁸].

Intracellular cytokine production of IL-9 and IL-17 was determined after treatment with fixation and permeabilization buffer (Biolegend, San Diego, USA) using IL-9 (PE, BD Biosciences, Franklin Lakes, USA) and IL-17 (Alexa Fluor 700, BioLegend, San Diego, USA) monoclonal fluorescent labeled antibodies. Surface marker and cytokine production was evaluated by flow cytometry using FACSCanto II, BD. Data analysis was performed using FACSDiva software V6 (BD, San Jose, CA). Absolute counts of T-cell subpopulations were calculated from total lymphocyte counts in cell cultures and from gated events by flow cytometric analysis.

Statistics

Statistical evaluation was performed non-parametrically using the Mann–Whitney U test. For calculation of the data sets IBM SPSS Statistics Version 22 (Armonk, New York, USA) was used. A p ≤ 0.05 was considered statistically significant.

Results

MSCs preserved the naive and memory T‑cell phenotype on CD4⁺ T‑cells

Distribution of the naive (CD45RA⁺CD27⁺), memory (CD45RA⁺CD27⁻), effector (CD45RA⁻CD27⁻), and TEMRA (CD45RA⁺CD27⁻) subpopulation after stimulation with a Th17 cytokine cocktail in the presence or absence of MSCs was evaluated in both CD4⁺ naive and non-naive isolated T-cells from patients and HD. The proportions of naive and memory cells in the naive fraction in both, patients and HD, in the presence of MSCs was higher compared to treated cells in the absence of MSCs (Fig. 1, Table 3). After culture of previously naive CD4⁺ T cells the proportion of effectors was significantly reduced in the presence of MSCs. Similar profile was observed in the non-naive isolated cells. In this fraction MSCs prevented the differentiation of memory into effector cells as well (Fig. 1, Table 4).

Table 3
Phenotyping of naive CD4⁺ T-cells from healthy donors (HD) and patients (Pat) with/without MSCs

	HD		Pat
	MSCs	No MSCs	MSCs	No MSCs
TEMRA
CD45RA+CD27−
%	0.5 ± 0.3 (0.6; 0.2–0.8)a	1.8 ± 1.3 (1.8; 0.5–3.0)	2.0 ± 2.7 (0.7; 0.4–6.0)	2.9 ± 4.0 (1.2; 0.3–8.9)
Absolute count	150 ± 90	540 ± 390	600 ± 810	870 ± 1200
	(180; 60–240)	(540; 150–900)	(210; 120–1800)	(360; 90–2670)
Naive
CD45RA+CD27+
%	32.2 ± 7.8 (30.6; 25.8–41.9)	26.0 ± 3.8 (25.6; 21.8–31.1)	35.8 ± 22.8 (25.3; 22.8–69.9)	17.3 ± 6.5 (15.0; 12.4–26.9)
Absolute count	9660 ± 2340	7800 ± 1140	10,740 ± 6840	5190 ± 1950
	(9180; 7740–12,570)	(7680; 6540–9330)	(7590; 6840–20,970)	(4500; 3720–8070)
Memory
CD45RA+CD27+
%	65.5 ± 6.6 (66.6; 57.1–71.9)	52.1 ± 13.4 (51.5; 39.3–66.0)	59.3 ± 27.5 (72.4; 18.1–74.3)	60.5 ± 22.6 (69.8; 27.1–75.3)
Absolute count	19,650 ± 1980	15,630 ± 4020	17,790 ± 8250	18,150 ± 6780
	(19,980; 17,130–21,570)	(15,450; 11,790–19,800)	(21,720; 5430–22,290)	(20,940; 8130–22,590)
Effector
CD45RA−CD27−
%	1.8 ± 1.6 (1.4; 0.5–4.0)	20.3 ± 9.7 (21.8; 8.2–29.3)a	2.9 ± 2.2 (2.6; 0.6–5.9)	19.3 ± 12.2 (14.9; 10.5–37.1)b
Absolute count	540 ± 480	6090 ± 2910	870 ± 660	5790 ± 3660
	(420; 150–1200)	(6540; 2460–8790)	(780; 180–1770)	(4470; 3150–11,130)

• Phenotype switch of isolated naive CD4⁺ T-cells from healthy donors (HD) and patients (Pat) in presence/absence of MSCs after 6 days of Th17 stimulation. Average percentages and absolute count of the different phenotypes of all CD4⁺ T-cells are shown; Values are shown as follows: mean ± standard deviation (median; minimum–maximum)
• ^a
  Differences between Effector T-cells in HD with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)
• ^b
  Differences between Effector T-cells in Pat with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)
• There were no significant differences between other phenotypes in Pat or HD with MSCs vs. without MSCs (Mann–Whitney-U-Test)

Table 4
Phenotyping of non-naive CD4⁺ T cells from healthy donors (HD) and patients (Pat) with/without MSCs

	HD		Pat
	MSCs	No MSCs	MSCs	No MSCs
TEMRA
CD45RA+CD27−
%	2.0 ± 1.8 (1.4; 0.6–4.6)	2.7 ± 1.5 (2.8; 0.9–4.4)	4.7 ± 5.2 (2.7; 1.0–12.2)	2.5 ± 1.1 (2.3; 1.5–4.0)
Absolute count	600 ± 540	810 ± 450	1410 ± 1560	750 ± 330
	(420; 180–1380)	(840; 270–1320)	(810; 300–3660)	(690; 450–1200)
Naive
CD45RA+CD27+
%	6.5 ± 2.4 (6.1; 4.2–9.6)	9.5 ± 7.5 (8.3; 2.6–19.0)	14.3 ± 16.2 (8.3; 2.5–38.2)	6.6 ± 4.8 (7.1; 1.4–11.0)
Absolute count	1950 ± 720	2850 ± 2250	4290 ± 4860	1980 ± 1440
	(1830; 1260–2880)	(2850; 780–5700)	(2490; 750–11,460)	(2130; 420–3300)
Memory
CD45RA+CD27+
%	64.4 ± 29.5 (77.4; 20.3–82.6)	35.5 ± 15.9 (40.0; 12.6–49.5)	49.6 ± 28.9 (52.1; 16.3–77.9)	37.1 ± 21.9 (43.3; 6.8–54.9)
Absolute count	19,320 ± 8850	10,650 ± 4770	14,880 ± 8670	11,130 ± 6570
	(23,220; 6090–24,780)	(12,000; 3780–14,850)	(14,850; 4890–23,370)	(12,990; 2040–16,470)
Effector
CD45RA−CD27−
%	27.1 ± 29.3 (13.1; 11.4–71.0)	52.9 ± 20.6 (46.8; 36.4–81.6)	31.4 ± 30.6 (17.5; 13.6–77.1)	53.7 ± 25.4 (47.4; 32.4 – 87.8)
Absolute count	8130 ± 8790	15,870 ± 6180	9420 ± 9180	16,110 ± 7620
	(3930; 3420–21,300)	(14,040; 10,920–24,480)	(5250; 4080–23,130)	(16,110; 9720–26,340)

• Phenotype switch of isolated non-naive CD4⁺ T-cells from healthy donors (HD) and patients (Pat) in presence/absence of MSCs after 6 days of Th17 stimulation. Average percentages and absolute count of the different phenotypes of all CD4⁺ T-cells are shown; Values are shown as follows: mean ± standard deviation (median; minimum–maximum)
• There were no significant differences between the four phenotypes in Pat or HD with MSCs vs. without MSCs (Mann–Whitney-U-Test)

Fig. 1
Dot plots of different phenotypes (TEMRA, Naive, Memory and Effector T-cells) in the naive (A) and non-naive (B) CD4⁺ T-cell fraction after 6 days of Th17 stimulation in presence/absence (MSC+/MSC−) of MSCs; HD = healthy donors; Pat = patients; p-values are indicated (Mann–Whitney-U-Test)

Induction of IFN‑γ, IL‑17 and IL‑9 under Th17 polarizing conditions

Intracellular IFN-γ, IL-17, and IL-9 production was obtained in both, naive and non-naive CD4⁺ T-cells after stimulation under Th17-polarizing cytokine cocktail in vitro (Fig. 2). Cytokine production in the previously naive fraction was mainly carried out by memory and effector cells, whereas in the previously non-naive fraction effector cells mainly produced IFN-γ, IL-17, and IL-9. No significant differences were observed between patients and HD (Fig. 2). We also demonstrated that IL-9 can be induced by stimulation with Th17-specific stimuli, such as IL-1β, IL-6, TGF-β, IL-23, and anti-CD3/anti-CD28.

Fig. 2
Dot plots of IFNγ (A and B). IL-17 (C and D) and IL-9 (E and F) production of CD4⁺ T-cells in the shown phenotypes (TEMRA, Naive, Memory and Effector) in patients and healthy donors. based on isolated naive and non-Naive CD4⁺ T-cells after 6 days of Th17 stimulation. HD = healthy donors; Pat = patients; MSC − = without MSCs; MSC + = with MSCs; p-values are indicated (Mann–Whitney-U-Test)

MSCs reduced IFN‑γ, IL‑17, and IL‑9 cytokine production

The proportion of IL-9-, IL-17-, and IFN-γ producing cells was determined following intracellular staining in naive and non-naive isolated cells from patients and HD after treatment in the presence or absence of MSCs.

Figure 3 show representative flow cytometric plots for the cytokine production of naive (Fig. 3A–D) and non-naive (Fig. 3E–H) CD4⁺ T-cells of Patient 1 with RA (Table 1) after Th17 stimulation in presence and absence of MSCs. Both, patients with inflammatory arthritis and HD showed almost similar patterns of flow cytometric plots, albeit with different percentages of cytokine production.

Fig. 3
Representative FACS plots for the cytokine production of naive (A–D) and non-naive (E–H) CD4⁺ T-cells of Patient 1 with RA (Table 1) after Th17 stimulation. Data in the upper right area of each FACS plot indicate the percentage of IL-17⁺, IL-9⁺, IL-17⁺/IL-9⁺ and IL-17⁻/IL-9⁻ CD4⁺ T-cells in the naive and non-naive fraction. A and E IL-17 and IL-9 production of naive/non-naive CD4⁺ T-cells without MSCs (MSC−); B and F IL-17 and IFNγ production of naive/non-naive CD4⁺ T-cells without MSCs (MSC−); C and G IL-17 and IL-9 production of naive/non-naive CD4⁺ T-cells with MSCs (MSC+); D and H IL-17 and IFNγ production of naive/non-naive CD4⁺ T-cells with MSCs (MSC+)

In general, non-naive isolated cells showed higher proportions of cytokine-producing cells following stimulation by Th17 polarizing conditions. Memory (CD45RA⁻CD27⁺) and especially effector (CD45RA⁻CD27⁻) CD4⁺ T-cells produced high percentages of cytokine in the absence of MSCs. However, in the presence of MSCs the cytokine production was impaired (Table 6). The proportion of IL-9-, IL-17-, and IFN-γ-producing CD4⁺ T-cells, especially in the memory and effector subpopulations, was reduced compare to the proportion of these cells in the absence of MSCs (Table 6). Although, the cytokine production in the previously naive fraction was lower compared to the counterpart (non-naive fraction), the same cytokine production retarding effect was observed in the presence of MSCs (Table 5). Cytokine contribution by naive (CD45RA⁺CD27⁺) and TEMRA (CD45RA⁺CD27⁻) cells appeared very low in both fractions. Nevertheless, a reduction in their contribution by IL-9, IL-17, and IFN-γ was determined in both but more pronounced in the non-naive fraction (Tables 5, 6). Comparing the influence of MSCs on cytokine production in patients and HD, it appears that IL-17 and IL-9, which are mainly produced by effector cells, are significantly reduced in patients by MSCs. Although not significant, this trend of reduced IL-9 (naive- and non- naive fraction: p = 0.114) and IL-17 (naive fraction p = 0.114 and non-naive fraction: p = 0.486) production in the presence of MSCs could also be observed in HD. In contrast, IFNγ production was significantly reduced in HD, mainly in the memory and naïve subpopulations (Fig. 2).

Table 5
Cytokine production of naive CD4⁺ T-cells from healthy donors (HD) and patients (Pat) with/without MSCs

	HD		Pat
	MSCs	No MSCs	MSCs	No MSCs
Cytokine production
% CD4+ T-cells Absolute count
IFNγ-production
TEMRA in %	0.0 ± 0.1 (0.0; 0.0–0.1)	0.1 ± 0.2 (0.1; 0.0–0.3)	0.2 ± 0.2 (0.2; 0.0–0.5)	0.3 ± 0.5; (0.0; 0.0–1.0)
Absolute count	0 ± 30	30 ± 60	60 ± 60	90 ± 150
	(0; 0–30)	(30; 0–90)	(60; 0–150)	(0; 0–300)
Naive in %	0.0 ± 0.1 (0.0; 0.0–0.1)	0.3 ± 0.1 (0.3; 0.1–0.4)a	0.3 ± 0.4 (0.3; 0.0–0.8)	0.6 ± 0.9 (0.2; 0.0–1.9)
Absolute count	0 ± 30	90 ± 30	90 ± 120	180 ± 270
	(0; 0–30)	(90; 30–120)	(90; 0–240)	(60; 0–570)
Memory in %	1.1 ± 0.9 (1.3; 0.0–1.9)	4.5 ± 1.6 (4.0; 3.3–6.8)b	1.6 ± 2.1 (1.1; 0.0–4.4)	3.3 ± 1.8 (3.0; 1.4–5.8)
Absolute count	330 ± 270	1350 ± 480	480 ± 630	990 ± 540
	(390; 0–570)	(1200; 990–2040)	(330; 0–1320)	(900; 420–1740)
Effector in %	0.3 ± 0.5 (0.1; 0.0–0.1)	2.6 ± 2.1 (2.3; 0.6–5.4)	0.1 ± 0.2 (0.1; 0.0–0.3)	2.2 ± 2.2 (1.7; 0.1–5.3)
Absolute count	90 ± 150	780 ± 630	30 ± 60	660 ± 660
	(30; 0–30)	(690; 180–1620)	(30; 0–90)	(510; 30–1590)
IL-17-production
TEMRA in %	0.0 ± 0.0 (0.0; 0.0–0.0)	0.0 ± 0.0 (0.0; 0.0–0.0)	0.0 ± 0.0 (0.0; 0.0–0.0)	0.0 ± 0.0 (0.0; 0.0–0.0)
Absolute count	0 ± 0	0 ± 0	0 ± 0	0 ± 0
	(0; 0–0)	(0; 0–0)	(0; 0–0)	(0; 0–0)
Naive in %	0.1 ± 0.1 (0.1; 0.0–0.3)	0.1 ± 0.1 (0.1; 0.0–0.3)	0.3 ± 0.4 (0.2; 0.0–0.8)	0.2 ± 0.2 (0.3; 0.0–0.3)
Absolute count	30 ± 30	30 ± 30	90 ± 120	60 ± 60
	(30; 0–90)	(30; 0–90)	(60; 0–240)	(90; 0–90)
Memory in %	0.4 ± 0.2 (0.4; 0.3–0.7)	0.3 ± 0.2 (0.4; 0.1–0.7)	0.4 ± 0.2 (0.5; 0.1–0.5)	0.8 ± 0.8 (0.6; 0.1–1.9)
Absolute count	120 ± 60	90 ± 60	120 ± 60	240 ± 240
	(120; 90–210)	(120; 30–210)	(150; 30–150)	(180; 30–570)
Effector in %	0.2 ± 0.1 (0.2; 0.0–0.3)	0.5 ± 0.2 (0.5; 0.2–0.7)	0.1 ± 0.1 (0.2; 0.0–0.2)	0.6 ± 0.2 (0.7; 0.4–0.8)c
Absolute count	60 ± 30	150 ± 60	30 ± 30	180 ± 60
	(60; 0–90)	(150; 60–210)	(60; 0–60)	(210; 120–240)
IL-9-production
TEMRA in %	0.1 ± 0.1 (0.1; 0.0–0.1)	0.2 ± 0.2 (0.2; 0.0–0.4)	0.3 ± 0.4 (0.1; 0.0–0.9)	0.5 ± 0.7 (0.2; 0.0–1.5)
Absolute count	30 ± 30	60 ± 60	90 ± 120	150 ± 210
	(30; 0–30)	(60; 0–120)	(30; 0–270)	(60; 0–450)
Naive in %	0.2 ± 0.2 (0.2; 0.1–0.5)	0.2 ± 0.1 (0.2; 0.1–0.3)	0.9 ± 1.4 (0.3; 0.1–3.0)	0.8 ± 0.9 (0.5; 0.1–2.0)
Absolute count	60 ± 60	60 ± 30	270 ± 420	240 ± 270
	(60; 30–150)	(60; 30–90)	(90; 30–900)	(150; 30–600)
Memory in %	0.5 ± 0.5 (0.3; 0.2–1.2)	1.6 ± 1.4 (1.1; 0.5–3.6)	0.7 ± 0.6 (0.5; 0.3–1.6)	1.9 ± 1.3 (1.8; 0.5–3.6)
Absolute count	150 ± 150	480 ± 420	210 ± 180	570 ± 390
	(90; 60–360)	(330; 150–1080)	(150; 90–480)	(480; 150–1080)
Effector in %	0.2 ± 0.1 (0.2; 0.0–0.3)	3.5 ± 3.7 (2.6; 0.1–8.8)	0.1 ± 0.1 (0.1; 0.0–0.1)	2.7 ± 3.4 (1.6; 0.2–7.6)d
Absolute count	60 ± 30	1050 ± 1110	30 ± 30	810 ± 1020
	(60; 0–90)	(780; 30–2640)	(30; 0–30)	(2640; 60–2280)

• Average percentage of cytokine producing positive cells and absolute count within the CD4⁺ T cell subpopulations of TEMRA, Naive, Memory and Effector cells based on naive CD4⁺ T-cells after Th17 stimulation over 6 days. HD = healthy donors. Pat = patients. Values are shown as follows: mean ± standard deviation (median; minimum–maximum)
• ^a
  Differences between IFNγ-production of the naive phenotype in HD with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)
• ^b
  Differences between IFNγ-production of the memory phenotype in HD with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)
• ^c
  Differences between IL-17-production of the effector phenotype in Pat with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)
• ^d
  Differences between IL-9-production of the effector phenotype in Pat with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)
• There were no significant differences between the cytokine production of other phenotypes in Pat or HD with MSCs vs. without MSCs (Mann–Whitney-U-Test)

Table 6
Cytokine production of non-naive CD4⁺ T-cells from healthy donors (HD) and patients (Pat) with/without MSCs

	HD		Pat
	MSCs	No MSCs	MSCs	No MSCs
Cytokine production
% CD4+ T-cells
Absolute count
IFNγ-production
TEMRA in %	0.6 ± 0.5 (0.5; 0.1–1.3)	0.5 ± 0.4 (0.3; 0.1–1.1)	1.1 ± 1.6 (0.4; 0.1–3.5)	0.6 ± 0.6; (0.4; 0.2–1.4)
Absolute count	180 ± 150	150 ± 120	330 ± 480	180 ± 180
	(150; 30–390)	(90; 30–90)	(180; 30–1050)	(120; 60–420)
Naive in %	0.7 ± 0.5 (0.7; 0.1–1.3)	0.5 ± 0.4 (0.5; 0.1–1.1)	2.2 ± 3.9 (0.4; 0.1–8.0)	0.8 ± 1.1 (0.4; 0.2–2.4)
Absolute count	210 ± 150	150 ± 120	660 ± 1170	240 ± 330
	(210; 30–390)	(150; 30–330)	(120; 30–2400)	(120; 60–720)
Memory in %	4.0 ± 3.3 (4.2; 0.1–7.7)	4.7 ± 1.5 (4.3; 3.5–6.9)	4.0 ± 4.2 (3.0; 0.2–9.6)	5.0 ± 4.7 (3.4; 1.2–11.8)
Absolute count	1200 ± 990	1410 ± 450	1200 ± 1260	1500 ± 1410
	(1260; 30–2310)	(1290; 1050–2070)	(900; 60–2880)	(1020; 360–3540)
Effector in %	6.0 ± 8.3 (2.3; 0.8–18.4)	11.2 ± 10.0 (6.8; 5.0–26.1)	3.8 ± 3.0 (3.9; 0.2–7.3)	9.2 ± 8.3 (7.1; 2.5–20.1)
Absolute count	1800 ± 2490	3360 ± 3000	1140 ± 900	2760 ± 2490
	(690; 240–5520)	(2040; 1500–7830)	(1170; 60–2190)	(2130; 750–6030)
IL-17-production
TEMRA In %	0.1 ± 0.2 (0.0; 0.0–0.3)	0.1 ± 0.1 (0.0; 0.0–0.2)	0.9 ± 1.7 (0.1; 0.0–3.5)	0.1 ± 0.1 (0.1; 0.0–0.3)
Absolute count	30 ± 60	30 ± 30	270 ± 510	30 ± 30
	(0; 0–90)	(0; 0–60)	(30; 0–1050)	(30; 0–90)
Naive in %	0.2 ± 0.1 (0.2; 0.0–0.3)	0.1 ± 0.1 (0.1; 0.0–0.2)	0.7 ± 1.3 (0.1; 0.0–0.1)	0.4 ± 0.5 (0.1; 0.1–1.1)
Absolute count	60 ± 30	30 ± 30	210 ± 390	120 ± 150
	(60; 0–90)	(30; 0–60)	(30; 0–30)	(30; 30–330)
Memory in %	2.6 ± 2.9 (1.8; 0.3–6.6)	1.2 ± 0.7 (1.0; 0.6–2.2)	0.9 ± 0.6 (0.9; 0.1–1.6)	1.3 ± 0.9 (1.0; 0.5–2.5)
Absolute count	780 ± 870	360 ± 210	270 ± 180	390 ± 270
	(540; 90–1980)	(300; 180–660)	(270; 30–480)	(300; 150–750)
Effector in %	3.6 ± 4.1 (2.5; 0.0–9.4)	6.7 ± 3.9 (7.2; 1.9–10.5)	3.9 ± 3.7 (3.4; 0.0–8.9)	8.3 ± 5.4 (7.8; 2.9–14.8)
Absolute count	1080 ± 1230	2010 ± 1170	1170 ± 1110	2490 ± 1620
	(750; 0–2820)	(2160; 570–3150)	(1020; 0–2670)	(2340; 870–4440)
IL-9-production
TEMRA in %	0.6 ± 0.5 (0.6; 0.1–1.1)	0.5 ± 0.4 (0.4; 0.1–1.1)	1.0 ± 1.4 (0.4; 0.1–3.1)	0.7 ± 0.5 (0.5; 0.3–1.4)
Absolute count	180 ± 150	150 ± 120	300 ± 420	210 ± 150
	(180; 30–330)	(120; 30–330)	(120; 30–930)	(150; 90–420)
Naive in %	0.7 ± 0.5 (0.8; 0.1–1.1)	0.5 ± 0.4 (0.4; 0.1–1.0)	1.2 ± 1.3 (0.9; 0.1–3.0)	0.9 ± 1.0 (0.5; 0.3–2.4)
Absolute count	210 ± 150	150 ± 120	360 ± 390	270 ± 300
	(240; 30–330)	(120; 30–300)	(270; 30–900)	(150; 90–720)
Memory in %	1.3 ± 0.8 (0.9; 0.8–2.5)	3.0 ± 1.6 (2.9; 1.3–4.8)	1.1 ± 0.9 (0.8; 0.4–2.5)	3.2 ± 3.0 (2.1; 1.1–7.6)
Absolute count	390 ± 240	900 ± 480	330 ± 270	960 ± 900
	(270; 240–750)	(870; 390–1440)	(240; 120–750)	(630; 330–2280)
Effector in %	3.6 ± 5.0 (1.4; 0.4–11.0)	15.5 ± 14.1 (11.1; 4.4–35.4)	2.1 ± 2.1 (1.2; 0.7–5.2)	14.3 ± 12.5 (12.0; 3.6–29.6)
Absolute count	1080 ± 1500	4650 ± 4230	630 ± 630	4290 ± 3750
	(420; 120–3300)	(3330; 1320–10,620)	(360; 210–1560)	(3600; 1080–8880)

• Average percentage of cytokine producing positive cells and absolute count within the CD4⁺ T-cell subpopulations of TEMRA, Naive, Memory and Effector cells based on non-naive CD4⁺ T-cells after Th17 stimulation over 6 days. HD = healthy donors. Pat = patients. Values are shown as follows: mean ± standard deviation (median; minimum–maximum); There were no significant differences between the cytokine production of the phenotypes in Pat or HD with MSCs versus without MSCs (Mann–Whitney-U-Test)

Discussion

The effect on the phenotype and cytokine production profile of naive and non-naive CD4⁺ T-cells from patients with T-cell mediated autoimmune arthritis and HD under stimulation with Th17-polarizing conditions in the presence or absence of MSCs was determined. We could show, that co-culture of MSC with previously naïve or non-naïve CD4⁺ T cells results in an abrogation of the differentiation into the effector T cell subtype and significantly reduced production of IFN-γ, IL-17 and IL-9 in both T-cell fractions derived from patients with autoimmune arthritis and from HD.

The differentiation from naive (CD45RA⁺CD27⁺) and memory (CD45RA⁻CD27⁺) cells into effector (CD45RA⁻CD27⁻) or TEMRA (CD45RA⁺CD27⁻) cells was impaired in the presence of MSCs. Our findings indicate that MSCs prevent the differentiation of naive and memory cells under pro-inflammatory conditions. Preservation of the naive phenotype in co-culture with MSCs has already been shown in other studies [²⁹, ³⁹, ⁴⁰]. Additionally, Batorov et al. showed that autologous MSCs can increase the recovery of lymphocytes in the naive stage after autologous hematopoietic stem cell transplantation in vivo and this process could be also observed in memory cells [⁴¹].

A possible mechanism of regulation of MSCs on T-cells may be by enhancing the proportion of regulatory T-cells. MSCs promote the induction of Treg cells in vivo and in vitro [⁴², ⁴³]. Conversion of conventional T-cells into Treg is facilitated in the presence of MSCs [⁴⁴, ⁴⁵]. Liu et al. showed that MSCs could enhance CD8⁺CD28⁻Treg cell activity by inhibiting naive CD4⁺ T-cell activation and decreasing IFN-γ production in activated CD4⁺ T-cells [⁴⁶]. Consistent with the results of our experiments, intracellular IFN-γ production was significantly higher in “effector”-sorted cells from HD which were not co-cultered with MSC compared to MSC + cultures. Although no significant difference could be observed, the reduction in IFN-γ production in the presence of MSCs could also be shown in patients (Fig. 2). This might rise the impression that the immunomodulatory effect of MSCs is mitigated in patients. In addition, patients produced less cytokines than HD after stimulation. Reduced IFN-γ levels in the patient group might be explained by the substantial individual variations in the cytokinemediated immunological microenvironment at different stages of disease development and medication. Additionally, and in contrast to the suspected unilateral IFN-γ suppression by MSCs, recent studies indicate that IFN-γ is a key factor in the management of RA patients with MSCs [⁴⁷–⁴⁹]. He et al. showed that the combination of IFN-γ and MSC can increase the clinical effectiveness of MSC-based therapy. Hypothetically, therefore, it would not make sense to significantly decrease IFN-γ production by MSCs in patients with rheumatic diseases which is also reflected in the results of our study.

Present results demonstrate the fundamental role of IL-31/IL-33 axis in the development of chronic inflammatory immune-mediated diseases via stimulating pro-inflammatory cytokines, regulating cell proliferation and controlling tissue remodeling. In addition, it is assumed that inhibition of IL-33 increases the expansion of Tregs and myeloid-derived suppressor cells and thus causes inhibition of Th17 cells. Although the direct influence of MSCs on the IL-31/IL-33 axis was not examined in our study, there is evidence that IL-33 can modulate MSCs and thus most likely targeting Th1/Th17 axis [⁵⁰]. There is increasing evidence that IL-17 plays a major role in various steps in the development of RA, SLE and other autoimmune and chronic inflammatory diseases. Furthermore IL-17 is important for the elimination of extracellular and intracellular pathogens [⁵¹–⁵³].

High levels of IL-17 are accompanied by an increase in the number of Th17 cells. These cells are associated with dysfunction of Treg cells observed in diseases such as RA, systemic sclerosis (SSc), and inflammatory bowel diseases (IBD) and support osteoclast stimulation by osteoblastand osteocyte derived RANKL thereby promoting bone loss [⁵⁴–⁵⁸]. Our in vitro model demonstrated that the presence of MSCs reduced the number of IL-17 producing CD4⁺ T-cells in patients and in HD despite being in a Th17 polarizing microenvironment. So far unknown secondary changes in gene regulation in MSC exerted by such pro-inflammatory environments may also modulate this effect. It has been described in earlier work that TLR4 stimulation can trigger proinflammatory cascades in MSC, which comprise secretion of besides others IL1b, IL6, IL8 and finally wnt5A and ROR2 expression as first response mechanisms after injury and in the very early phase of osteogenic differentiation. The latter may be again relevant in the crosstalk between MSC and T-cells as non-canonical wnt-signaling has immunomodulatory properties [⁵⁹, ⁶⁰]. MSCs also promote polarization of monocytes/macrophages into anti-inflammatory type 2 phenotype increasing levels of IL-10 having as consequence reduction in the levels of TNFa and IL-17 [⁶¹, ⁶²] and is in accordance with our findings.

Interestingly, naive and non-naive CD4⁺ T-cells stimulated under Th17 conditions in our system induce production of high levels of IL-9. IL-9 is a cytokine produced by various cells including Th2, Th17, Treg, and Th9 cells and can be mainly produced by stimulation in the presence of IL-4 and TGFβ [⁸, ⁶³]. IL-9 is important in the regulation of the airway inflammation inducing proliferation of goblet cells and mucin producing cells [⁶⁴]. However, IL-9 has been related in some pathogenic processes of diseases such as lupus erythematosus, RA, and SSc [²⁸, ⁶⁵–⁶⁷]. Our results are in line with a recently published article of our group, showing the Th17-inducing cytokines are effective to induce IL-9 production and Th9-polarized cells [³³].

This study has several limitations. First, we included a small number of patients associated with different underlying rheumatic diseases, pathophysiological aspects, and therapeutical characteristics comprising different doses of corticosteroids and immunosuppressants. Due to small numbers, outliers may prominently contribute to the differences in results. Although our study is limited by the small case numbers, the highly explorative character of the experiments allow generation of the hypothesis that MSC are able to reduce the differentiation into cytokine-producing effector helper T cells and clearly showed reduction of pro-inflammatory cytokines, such as IL-17, IFN-γ and IL-9. The use of the two magnetic-bead-based isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany) for the cell isolation of naive and non-naive CD4⁺ cells with an average purity of > 90% leads to the assumption that the remaining 10% of the cell fractions might be contaminating cell subpopulations, which could falsely contribute to the investigated subpopulations and possibly to overall cytokine production after expansion in vitro. It should therefore be mentioned that the data gained from the non-naïve CD4⁺ T cell fraction may include cytokine-producing cells of the complementary fraction.

Applying a transwell system, we could prove that these effects were exclusively based on soluble factors and not on direct cell–cell-contacts between MSCs and sorted naïve or non-naïve T cells, respectively. Although we failed to measure soluble factors in the supernatants due to technical limitations, as we had to remove the fairly small amounts T cells by several centrifugation steps, our study showed that the presence of MSCs reduced the proportion of IL-9, IL-17, and IFN-γ producing cells, suggesting a modulatory effect of the MSCs on T-cells inhibiting the differentiation of naive and memory cells into cytokine producing effector T-cells even in proinflammatory conditions. In addition, however, it should be mentioned that the expression of individual cytokines by Th1, Th9 and Th17 for each Th lineage may ultimately be biased, as no other driving cytokines and their effect on Th cells, such as IL-12 on Th1 or IL-23 on Th17, have been addressed. Of note, intracellular cytokine production does not automatically mean cytokine release and provides a indirectly view on cytokine producing ability of defined Th subpopulations.

Conclusion

The distribution of different T-cell phenotypes in the course of autoimmune diseases seems to play an important role for targeted therapeutic approaches with the aim of reducing the proinflammatory cytokine production in order to control the immune response [⁶⁸]. Today, various known inhibitors like monoclonal antibodies against TNF-α (e.g. Adalimumab) and IL-6 (Tocilizumab) receptor, TNF-α soluble receptor, IL-1 receptor antagonist and analogous tools are available, targeting the IL-17 pathway [⁵³].

As a cell-based therapy, MSCs might represent a promising alternative for patients who do not respond to a conventional therapy with disease-modifying antirheumatic drugs or biologicals [²⁹]. Anyway, preventing the phenomenon of T-cell differentiation into effector cells or using MSCs as a prophylactic option in affected patients should be addressed in pre-clinical and clinical studies.

• Funding
  Open Access funding enabled and organized by Projekt DEAL. We acknowledge support for the Article Processing Charge from the DFG (German Research Foundation, 491454339).
• Declarations
  Ethics approval and consent to participate
  Ethical approval was obtained from the local ethics committee of the Medical Faculty of the University Hospital of Würzburg (No. 123/14). Informed consent was obtained from all subjects and/or their legal guardian(s). All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by a named institutional and/or licensing committee.
• Consent for publication
  Not applicable.
• Publisher's Note
  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Availability of data and materials

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Acknowledgements

The authors would like to thank the research group headed by Professor Norbert Schütze, Orthopedic Clinic, König-Ludwig-Haus, Würzburg, Germany for providing the mesenchymal stem cells.

References

• ¹
  Duffy MM, Ritter T, Ceredig R, Griffin MD. Mesenchymal stem cell effects on T-cell effector pathways. Stem Cell Res Ther. 2011;2:34.
• ²
  Kavanagh H, Mahon BP. Allogeneic mesenchymal stem cells prevent allergic airway inflammation by inducing murine regulatory T cells. Allergy. 2011;66:523–31.
• ³
  Griffin MD, Ritter T, Mahon BP. Immunological aspects of allogeneic mesenchymal stem cell therapies. Hum Gene Ther. 2010;21:1641–55.
• ⁴
  Barry FP, Murphy JM, English K, Mahon BP. Immunogenicity of adult mesenchymal stem cells: lessons from the fetal allograft. Stem Cells Dev. 2005;14:252–65.
• ⁵
  Chen KG, Johnson KR, McKay RDG, Robey PG. Concise review: conceptualizing paralogous stem-cell niches and unfolding bone marrow progenitor cell identities. Stem Cells (Dayton, OH). 2018;36:11–21.
• ⁶
  Herrmann M, Jakob F. Bone Marrow Niches for skeletal progenitor cells and their inhabitants in health and disease. Curr Stem Cell Res Ther. 2019;14:305–19.
• ⁷
  Ghannam S, Pene J, Moquet-Torcy G, Jorgensen C, Yssel H. Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype. J Immunol (Baltimore, Md: 1950). 2010;185:302–12.
• ⁸
  Noelle RJ, Nowak EC. Cellular sources and immune functions of interleukin-9. Nat Rev Immunol. 2010;10:683–7.
• ⁹
  Caplan AI. Why are MSCs therapeutic? New data: new insight. J Pathol. 2009;217:318–24.
• ¹⁰
  Wang Q, Sun B, Wang D, et al. Murine bone marrow mesenchymal stem cells cause mature dendritic cells to promote T-cell tolerance. Scand J Immunol. 2008;68:607–15.
• ¹¹
  English K, Ryan JM, Tobin L, Murphy MJ, Barry FP, Mahon BP. Cell contact, prostaglandin E(2) and transforming growth factor beta 1 play non-redundant roles in human mesenchymal stem cell induction of CD4⁺CD25(High) forkhead box P3+ regulatory T cells. Clin Exp Immunol. 2009;156:149–60.
• ¹²
  Darlington PJ, Boivin MN, Renoux C, et al. Reciprocal Th1 and Th17 regulation by mesenchymal stem cells: implication for multiple sclerosis. Ann Neurol. 2010;68:540–5.
• ¹³
  Resnick IB, Barkats C, Shapira MY, et al. Treatment of severe steroid resistant acute GVHD with mesenchymal stromal cells (MSC). Am J Blood Res. 2013;3:225–38.
• ¹⁴
  Zappia E, Casazza S, Pedemonte E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood. 2005;106:1755–61.
• ¹⁵
  Gonzalez-Rey E, Gonzalez MA, Varela N, et al. Human adipose-derived mesenchymal stem cells reduce inflammatory and T cell responses and induce regulatory T cells in vitro in rheumatoid arthritis. Ann Rheum Dis. 2010;69:241–8.
• ¹⁶
  Liang J, Sun L. Mesenchymal stem cells transplantation for systemic lupus erythematosus. Int J Rheum Dis. 2015;18:164–71.
• ¹⁷
  Wang P, Li Y, Huang L, et al. Effects and safety of allogenic mesenchymal stem cell intravenous infusion in active ankylosing spondylitis patients who failed NSAIDs: a 20-week clinical trial. Cell Transplant. 2014;23:1293–303.
• ¹⁸
  Ng J, Bernhard J, Vunjak-Novakovic G. Mesenchymal stem cells for osteochondral tissue engineering. Methods Mol Biol (Clifton, NJ). 2016;1416:35–54.
• ¹⁹
  Hültner L, Kölsch S, Stassen M, et al. In activated mast cells, IL-1 upregulates the production of several Th2-related cytokines including IL-9. J Immunol (Baltimore, Md: 1950). 2000;164:5556–63.
• ²⁰
  Gessner A, Blum H, Röllinghoff M. Differential regulation of IL-9-expression after infection with Leishmania major in susceptible and resistant mice. Immunobiology. 1993;189:419–35.
• ²¹
  Monteiro M, Agua-Doce A, Almeida CF, Fonseca-Pereira D, VeigaFernandes H, Graca L. IL-9 expression by invariant NKT cells is not imprinted during thymic development. J Immunol (Baltimore, Md: 1950). 2015;195:3463–71.
• ²²
  Jones CP, Gregory LG, Causton B, Campbell GA, Lloyd CM. Activin A and TGF-β promote T(H)9 cell-mediated pulmonary allergic pathology. J Allergy Clin Immunol. 2012;129:1000-10.e3.
• ²³
  Nowak EC, Weaver CT, Turner H, et al. IL-9 as a mediator of Th17-driven inflammatory disease. J Exp Med. 2009;206:1653–60.
• ²⁴
  Eller K, Wolf D, Huber JM, et al. IL-9 production by regulatory T cells recruits mast cells that are essential for regulatory T cell-induced immune suppression. J Immunol (Baltimore, Md: 1950). 2011;186:83–91.
• ²⁵
  Visekruna A, Ritter J, Scholz T, et al. Tc9 cells, a new subset of CD8(+) T cells, support Th2-mediated airway inflammation. Eur J Immunol. 2013;43:606–18.
• ²⁶
  Elyaman W, Bradshaw EM, Uyttenhove C, et al. IL-9 induces differentiation of TH17 cells and enhances function of FoxP3+ natural regulatory T cells. Proc Natl Acad Sci USA. 2009;106:12885–90.
• ²⁷
  Abd-Elhalem SS, Haggag NZ, El-Shinnawy NA. Bone marrow mesenchymal stem cells suppress IL-9 in adjuvant-induced arthritis. Autoimmunity. 2018;51:25–34.
• ²⁸
  Ciccia F, Guggino G, Rizzo A, et al. Potential involvement of IL-9 and Th9 cells in the pathogenesis of rheumatoid arthritis. Rheumatology (Oxford). 2015;54:2264–72.
• ²⁹
  Pedrosa M, Gomes J, Laranjeira P, et al. Immunomodulatory effect of human bone marrow-derived mesenchymal stromal/stem cells on peripheral blood T cells from rheumatoid arthritis patients. J Tissue Eng Regen Med. 2020;14:16–28.
• ³⁰
  Carrion F, Nova E, Luz P, Apablaza F, Figueroa F. Opposing effect of mesenchymal stem cells on Th1 and Th17 cell polarization according to the state of CD4⁺ T cell activation. Immunol Lett. 2011;135:10–6.
• ³¹
  Hsu SC, Wang LT, Yao CL, et al. Mesenchymal stem cells promote neutrophil activation by inducing IL-17 production in CD4⁺ CD45RO+ T cells. Immunobiology. 2013;218:90–5.
• ³²
  Wang Y, Chen X, Cao W, Shi Y. Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications. Nat Immunol. 2014;15:1009–16.
• ³³
  Heim J, Almanzar G, Schmalzing M, Gernert M, Tony HP, Prelog M. Induction of IL-9 in peripheral lymphocytes of rheumatoid arthritis patients and healthy donors by Th17-inducing cytokine conditions. Front Immunol. 2021;12:668095.
• ³⁴
  Kay J, Upchurch KS. ACR/EULAR 2010 rheumatoid arthritis classification criteria. Rheumatology (Oxford). 2012;51(Suppl 6):vi5–9.
• ³⁵
  Magallares B, Lobo-Prat D, Castellví I, et al. Assessment of EULAR/ ACR-2019, SLICC-2012 and ACR-1997 classification criteria in SLE with longstanding disease. J Clin Med. 2021;10:2377.
• ³⁶
  van der Heijde D, Lie E, Kvien TK, et al. ASDAS, a highly discriminatory ASAS-endorsed disease activity score in patients with ankylosing spondylitis. Ann Rheum Dis. 2009;68:1811–8.
• ³⁷
  Dotterweich J, Tower RJ, Brandl A, et al. The KISS1 receptor as an in vivo microenvironment imaging biomarker of multiple myeloma bone disease. PLoS ONE. 2016;11:e0155087.
• ³⁸
  Appay V, van Lier RA, Sallusto F, Roederer M. Phenotype and function of human T lymphocyte subsets: consensus and issues. Cytometry A. 2008;73:975–83.
• ³⁹
  Mareschi K, Castiglia S, Sanavio F, et al. Immunoregulatory effects on T lymphocytes by human mesenchymal stromal cells isolated from bone marrow, amniotic fluid, and placenta. Exp Hematol. 2016;44:138-50.e1.
• ⁴⁰
  Fujii S, Miura Y, Fujishiro A, et al. Graft-versus-host disease amelioration by human bone marrow mesenchymal stromal/stem cell-derived extracellular vesicles is associated with peripheral preservation of naive T cell populations. Stem Cells (Dayton, OH). 2018;36:434–45.
• ⁴¹
  Batorov EV, Shevela EY, Tikhonova MA, et al. Mesenchymal stromal cells improve early lymphocyte recovery and T cell reconstitution after autologous hematopoietic stem cell transplantation in patients with malignant lymphomas. Cell Immunol. 2015;297:80–6.
• ⁴²
  González MA, Gonzalez-Rey E, Rico L, Büscher D, Delgado M. Adiposederived mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory and autoimmune responses. Gastroenterology. 2009;136:978–89.
• ⁴³
  Ge W, Jiang J, Arp J, Liu W, Garcia B, Wang H. Regulatory T-cell generation and kidney allograft tolerance induced by mesenchymal stem cells associated with indoleamine 2,3-dioxygenase expression. Transplantation. 2010;90:1312–20.
• ⁴⁴
  Khosravi M, Karimi MH, Hossein Aghdaie M, Kalani M, Naserian S, Bidmeshkipour A. Mesenchymal stem cells can induce regulatory T cells via modulating miR-126a but not miR-10a. Gene. 2017;627:327–36.
• ⁴⁵
  Engela AU, Hoogduijn MJ, Boer K, et al. Human adipose-tissue derived mesenchymal stem cells induce functional de-novo regulatory T cells with methylated FOXP3 gene DNA. Clin Exp Immunol. 2013;173:343–54.
• ⁴⁶
  Liu Q, Zheng H, Chen X, et al. Human mesenchymal stromal cells enhance the immunomodulatory function of CD8(+)CD28(−) regulatory T cells. Cell Mol Immunol. 2015;12:708–18.
• ⁴⁷
  Yang Y, He X, Zhao R, et al. Serum IFN-γ levels predict the therapeutic effect of mesenchymal stem cell transplantation in active rheumatoid arthritis. J Transl Med. 2018;16:165.
• ⁴⁸
  Kato M. New insights into IFN-γ in rheumatoid arthritis: role in the era of JAK inhibitors. Immunol Med. 2020;43:72–8.
• ⁴⁹
  Polchert D, Sobinsky J, Douglas G, et al. IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft versus host disease. Eur J Immunol. 2008;38:1745–55.
• ⁵⁰
  Terraza C, Fuentes R, Pino-Lagos K. IFN-γ and IL-33 modulate mesenchymal stem cells function targeting Th1/Th17 axis in a murine skin transplantation model. Cytokine. 2018;111:317–24.
• ⁵¹
  Rudner XL, Happel KI, Young EA, Shellito JE. Interleukin-23 (IL-23)-IL-17 cytokine axis in murine Pneumocystis carinii infection. Infect Immun. 2007;75:3055–61.
• ⁵²
  Huang W, Na L, Fidel PL, Schwarzenberger P. Requirement of interleukin- 17A for systemic anti-Candida albicans host defense in mice. J Infect Dis. 2004;190:624–31.
• ⁵³
  Murdaca G, Colombo BM, Puppo F. The role of Th17 lymphocytes in the autoimmune and chronic inflammatory diseases. Intern Emerg Med. 2011;6:487–95.
• ⁵⁴
  Ugor E, Simon D, Almanzar G, et al. Increased proportions of functionally impaired regulatory T cell subsets in systemic sclerosis. Clin Immunol (Orlando, FL). 2017;184:54–62.
• ⁵⁵
  Almanzar G, Klein M, Schmalzing M, et al. Disease manifestation and inflammatory activity as modulators of Th17/Treg balance and RORC/ FoxP3 methylation in systemic sclerosis. Int Arch Allergy Immunol. 2016;171:141–54.
• ⁵⁶
  Fieldhouse KA, Ukaibe S, Crowley EL, Khanna R, O’Toole A, Gooderham MJ. Inflammatory bowel disease in patients with psoriasis treated with interleukin-17 inhibitors. Drugs Context. 2020. https://doi.org/10.7573/dic.2020-2-1
  » https://doi.org/10.7573/dic.2020-2-1
• ⁵⁷
  Pacifici R. The role of IL-17 and TH17 cells in the bone catabolic activity of PTH. Front Immunol. 2016;7:57.
• ⁵⁸
  Li JY, Yu M, Tyagi AM, et al. IL-17 receptor signaling in osteoblasts/osteocytes mediates PTH-induced bone loss and enhances osteocytic RANKL production. J Bone Miner Res. 2019;34:349–60.
• ⁵⁹
  Rauner M, Stein N, Winzer M, et al. WNT5A is induced by inflammatory mediators in bone marrow stromal cells and regulates cytokine and chemokine production. J Bone Miner Res. 2012;27:575–85.
• ⁶⁰
  Ebert R, Benisch P, Krug M, et al. Acute phase serum amyloid A induces proinflammatory cytokines and mineralization via toll-like receptor 4 in mesenchymal stem cells. Stem Cell Res. 2015;15:231–9.
• ⁶¹
  Deng Y, Zhang Y, Ye L, et al. Umbilical cord-derived mesenchymal stem cells instruct monocytes towards an IL10-producing phenotype by secreting IL6 and HGF. Sci Rep. 2016;6:37566.
• ⁶²
  Melief SM, Geutskens SB, Fibbe WE, Roelofs H. Multipotent stromal cells skew monocytes towards an anti-inflammatory interleukin-10-producing phenotype by production of interleukin-6. Haematologica. 2013;98:888–95.
• ⁶³
  Schmitt E, Germann T, Goedert S, et al. IL-9 production of naive CD4⁺ T cells depends on IL-2, is synergistically enhanced by a combination of TGF-beta and IL-4, and is inhibited by IFN-gamma. J Immunol (Baltimore, Md: 1950). 1994;153:3989–96.
• ⁶⁴
  Zhao P, Xiao X, Ghobrial RM, Li XC. IL-9 and Th9 cells: progress and challenges. Int Immunol. 2013;25:547–51.
• ⁶⁵
  Ouyang H, Shi Y, Liu Z, et al. Increased interleukin-9 and CD4⁺IL-9+ T cells in patients with systemic lupus erythematosus. Mol Med Rep. 2013;7:1031–7.
• ⁶⁶
  Dantas AT, Marques CD, da Rocha Junior LF, et al. Increased serum interleukin-9 levels in rheumatoid arthritis and systemic lupus erythematosus: pathogenic role or just an epiphenomenon? Dis Markers. 2015;2015:519638.
• ⁶⁷
  Yanaba K, Yoshizaki A, Asano Y, Kadono T, Sato S. Serum interleukin 9 levels are increased in patients with systemic sclerosis: association with lower frequency and severity of pulmonary fibrosis. J Rheumatol. 2011;38:2193–7.
• ⁶⁸
  Laranjeira P, Pedrosa M, Pedreiro S, et al. Effect of human bone marrow mesenchymal stromal cells on cytokine production by peripheral blood naive, memory, and effector T cells. Stem Cell Res Ther. 2015;6:3.

Publication Dates

• Publication in this collection
  25 Aug 2023
• Date of issue
  2023

History

• Received
  08 Dec 2022
• Accepted
  19 July 2023
• Published
  31 July 2023