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

Riekert, Maximilian; Almanzar, Giovanni; Schmalzing, Marc; Schütze, Norbert; Jakob, Franz; Prelog, Martina

doi:10.1186/s42358-023-00317-z

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Sumário

Research • Adv. rheumatol. 63 • 2023 • https://doi.org/10.1186/s42358-023-00317-z copy

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

Authorship SCIMAGO INSTITUTIONS RANKINGS

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

	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	–	–

	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

	HD		Pat
	MSCs	No MSCs	MSCs	No MSCs
TEMRA
CD45RA⁺CD27⁻
%	0.5 ± 0.3 (0.6; 0.2–0.8)^a a Differences between Effector T-cells in HD with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)	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 a Differences between Effector T-cells in HD with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)	2.9 ± 2.2 (2.6; 0.6–5.9)	19.3 ± 12.2 (14.9; 10.5–37.1)^b b Differences between Effector T-cells in Pat with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)
Absolute count	540 ± 480	6090 ± 2910	870 ± 660	5790 ± 3660
	(420; 150–1200)	(6540; 2460–8790)	(780; 180–1770)	(4470; 3150–11,130)

	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)

	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 a Differences between IFNγ-production of the naive phenotype in HD with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)	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 b Differences between IFNγ-production of the memory phenotype in HD with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)	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 c Differences between IL-17-production of the effector phenotype in Pat with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)
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 d Differences between IL-9-production of the effector phenotype in Pat with MSCs vs. without MSCs: p = 0.029 (Mann–Whitney-U-Test)
Absolute count	60 ± 30	1050 ± 1110	30 ± 30	810 ± 1020
	(60; 0–90)	(780; 30–2640)	(30; 0–30)	(2640; 60–2280)

	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)

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[1] *Correspondence: Maximilian Riekert max.riekert@web.de