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.2021 Nov 26:12:739186.
doi: 10.3389/fimmu.2021.739186. eCollection 2021.

Tetracyclines DiminishIn Vitro IFN-γ and IL-17-Producing Adaptive and Innate Immune Cells in Multiple Sclerosis

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Tetracyclines DiminishIn Vitro IFN-γ and IL-17-Producing Adaptive and Innate Immune Cells in Multiple Sclerosis

Despoina T Florou et al. Front Immunol..

Abstract

Introduction: Limited data from clinical trials in multiple sclerosis (MS) reported that minocycline, a widely used antibiotic belonging to the family of tetracyclines (TCs), exerts a beneficial short-lived clinical effect A similar anti-inflammatory effect of minocycline attributed to a deviation from Th1 to Th2 immune response has been reported in experimental models of MS. Whether such an immunomodulatory mechanism is operated in the human disease remains largely unknown.

Aim: To assess thein vitro immunomodulatory effect of tetracyclines, and in particular minocycline and doxycycline, in naïve and treated patients with MS.

Material and methods: Peripheral blood mononuclear cells from 45 individuals (35 MS patients, amongst which 15 naïve patients and 10 healthy controls, HCs) were cultured with minocycline or doxycycline and conventional stimulants (PMA/Ionomycin or IL-12/IL-18). IFN-γ and IL-17 producing T-, NK- and NKT cells were assessed by flow cytometry. The effect of TCs on cell viability and apoptosis was further assessed by flow cytometry with Annexin V staining.

Results: Both tetracyclines significantly decreased, in a dose dependent manner, IFN-γ production in NKT and CD4+ T lymphocytes from MS patients (naïve or treated) stimulated with IL-12/IL-18 but did not decrease IFN-γ producing CD8+ T cells from naive MS or treated RRMS patients. They also decreased IL-17+ T and NKT cells following PMA and Ionomycin-stimulation. Tetracyclines did not affect the viability of cell subsets.

Conclusion: Tetracyclines canin vitro suppress IFN-γ and IL-17- producing cells from MS patients, and this may explain their potential therapeutic effectin vivo.

Keywords: NKT cells; doxycycline; interferon-γ; interleukin-17; minocycline; multiple sclerosis; proinflammatory.

Copyright © 2021 Florou, Mavropoulos, Dardiotis, Tsimourtou, Siokas, Aloizou, Liaskos, Tsigalou, Katsiari, Sakkas, Hadjigeorgiou and Bogdanos.

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Conflict of interest statement

Author ED: Allergan, Novartis, Genesis, ELPEN, Bayer, Teva, Merck- Serono, Genzyme-Sanofi, Roche, UCB - speaker or chairman horonaria, advisory or travel grants, and clinical research-educational support grants. Author DB: AbbVie, Novartis, Genesis, ELPEN, Pfizer, Aenorasis, Menarini, Kopper, ITF Hellas, Roche, MSD, GSK, Hospital Line - speaker or chairman horonaria or paid investigator or clinical research and educational support grants. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Tetracycline-mediated inhibition of CD4+ IFNγ+ T cells. PBMCs from naïve MS patients (n=15), RRMS patients (n=20) and HCs (n=10) were seeded in cell culture plates (1 x 106 per well) and stimulated with IL-12 plus IL-18 (IL-12/IL-18), IL-12/IL-18 plus minocycline (MIN) and IL-12/IL-18 plus doxycycline (DOX) for 5h. Cells were collected, washed, surface stained with appropriate monoclonal antibodies an analyzed for intracellular IFN-γ production by flow cytometry (see also methods section). Individual cell subsets were sub-gated according to expression of CD3 and CD4 surface epitopes.(A) Flow cytometry dot-plots showing the frequency of CD4+IFNγ+ T cells in IL-12/IL-18, IL-12/IL-18/MIN and IL-12/IL-18/DOX treated cells from representative MS cases and HCs.(B) Box and whiskers graphical representation showing significant reduction in the percentages of IFN-γ-producing CD4+ T cells in the presence of tetracyclines in naïve MS, RRMS and HCs.
Figure 2
Figure 2
Tetracycline-mediated inhibition of CD8+ IFNγ+ T cells. PBMCs from naïve MS patients (n=15), RRMS patients (n=20) and HCs (n=10) were seeded in cell culture plates (1 x 106 per well) and stimulated with IL-12 plus IL-18 (IL-12/IL-18), IL-12/IL-18 plus minocycline (MIN) and IL-12/IL-18 plus doxycycline (DOX) for 5h. Cells were collected, washed, surface stained with appropriate monoclonal antibodies an analyzed for intracellular IFN-γ production by flow cytometry (see also methods section). Individual cell subsets were sub-gated according to expression of CD3 and CD8 surface epitopes.(A) Flow cytometry dot-plots showing the frequency of CD8+IFNγ+ T cells in IL-12/IL-18, IL-12/IL-18/MIN and IL-12/IL-18/DOX treated cells from representative MS cases and HCs.(B) Box and whiskers graphical representation showing significant reduction in the percentages of IFNγ-producing CD8+ T cells in the presence of tetracyclines in HCs but not naïve MS and RRMS patients.
Figure 3
Figure 3
Tetracycline-mediated inhibition of CD56+CD3+IFN-γ+ T cells. PBMCs from naïve MS patients (n=15), RRMS patients (n=20) and HCs (n=10) were seeded in cell culture plates (1 x 106 per well) and stimulated with IL-12 plus IL-18 (IL-12/IL-18), IL-12/IL-18 plus minocycline (MIN) and IL-12/IL-18 plus doxycycline (DOX) for 5h. Cells were collected, washed, surface stained with appropriate monoclonal antibodies an analyzed for intracellular IFN-γ production by flow cytometry (see also methods section). Individual cell subsets were sub-gated according to expression of CD3 and CD56 surface epitopes.(A) Flow cytometry dot-plots showing the frequency of IFN-γ+ NKT cells (gated CD56+CD3+ cells two-dimensionally depicted in CD3vs IFN-γ plots) in IL-12/IL-18, IL-12/IL-18/MIN and IL-12/IL-18/DOX treated cells from representative MS cases.(B) Box and whiskers graphical representation showing significant reduction in the percentages of IFN-γ-producing NKT cells in the presence of tetracyclines in naïve MS, RRMS and HCs.
Figure 4
Figure 4
Tetracyclines have no significant effect on IFN-γ+ NK cells. PBMCs from naïve MS patients (n=15), RRMS patients (n=20) and HCs (n=10) were seeded in cell culture plates (1 x 106 per well) and stimulated with IL-12 plus IL-18 (IL-12/IL-18), IL-12/IL-18 plus minocycline (MIN) and IL-12/IL-18 plus doxycycline (DOX) for 5h. Cells were collected, washed, surface stained with appropriate monoclonal antibodies an analyzed for intracellular IFN-γ production by flow cytometry (see also methods section). Individual cell subsets were sub-gated according to expression of CD3 and CD56 surface epitopes.(A) Flow cytometry dot-plots showing the frequency of CD56+CD3-IFNγ+ NK cells (gated CD56+CD3- cells two-dimensionally depicted in CD56vs IFN-γ plots) in IL-12/IL-18, IL-12/IL-18/MIN and IL-12/IL-18/DOX treated cells from representative MS cases.(B) Box and whiskers graphical representation show no significant reduction in the percentages of IFN-γ-producing NK cells in the presence of tetracyclines in either HCs or MS patients.
Figure 5
Figure 5
Tetracycline-mediated inhibition of CD4+CD3+IL-17A+ T cells and CD56+CD4+IL-17A+ T (NKT) cells. PBMCs from naïve MS patients (n=9), RRMS patients (n=9) and HCs (n=9) were seeded in cell culture plates (1 x 106 per well) and stimulated with PMA plus ionomycin (PMA/ION), PMA/ION plus minocycline (MIN) and PMA/ION plus doxycycline (DOX) for 5h. Cells were collected, washed, surface stained with appropriate monoclonal antibodies an analyzed for intracellular IL-17A production by flow cytometry (see also methods section).(A) Flow cytometry dot-plots showing the frequency of total CD4+IL-17A+ cells in PMA/IONO, PMA/IONO/MIN and PMA/IONO/DOX treated cells from a representative naïve MS case.(B) CD4+IL-17A+ cells were sub-divided according to expression of CD3 or CD56 surface epitopes. Box and whiskers graphical representation showing significant reduction in the percentages of IL-17A-producing T and NKT cells in the presence of tetracyclines in naïve MS patients, RRMS patients and HCs.
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