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An insight into the role of magnesium in the immunomodulatory properties of mesenchymal stem cells
Accepted Manuscript An insight into the role of magnesium in the immunomodulatory properties of mesenchymal stem cells
Fabiana da Silva Lima, Amanda Batista da Rocha Romero, Araceli Hastreiter, Amanda Nogueira-Pedro, Edson Makiyama, Célia Colli, Ricardo Ambrósio Fock PII: DOI: Reference:
S0955-2863(17)30978-6 doi:10.1016/j.jnutbio.2018.02.006 JNB 7926
To appear in: Received date: Revised date: Accepted date:
7 November 2017 4 January 2018 5 February 2018
Please cite this article as: Fabiana da Silva Lima, Amanda Batista da Rocha Romero, Araceli Hastreiter, Amanda Nogueira-Pedro, Edson Makiyama, Célia Colli, Ricardo Ambrósio Fock , An insight into the role of magnesium in the immunomodulatory properties of mesenchymal stem cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jnb(2018), doi:10.1016/ j.jnutbio.2018.02.006
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ACCEPTED MANUSCRIPT An insight into the role of magnesium in the immunomodulatory properties of mesenchymal stem cells
Fabiana da Silva Lima1, Amanda Batista da Rocha Romero1, Araceli Hastreiter2,
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Amanda Nogueira-Pedro 2, Edson Makiyama 2, Célia Colli 1 , Ricardo Ambrósio
1
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Fock 1
Department of Food and Experimental Nutrition, Faculty of Pharmaceutical
Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical
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2
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Sciences, University of São Paulo, São Paulo, Brazil.
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Sciences, University of São Paulo, São Paulo, Brazil.
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* To whom correspondence should be addressed. Fock, Ricardo Ambrósio. Laboratory of Experimental Hematology, Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, University of São Paulo. Avenida Lineu Prestes, 580 - Bloco 17. São Paulo, SP, Brazil. 05508-900. Phone: +551130913639. email: [email protected]
Running Title: The role of magnesium in the immunomodulatory properties
ACCEPTED MANUSCRIPT Abstract Magnesium (Mg2+) is a mineral with the ability to influence cell proliferation and to modulate inflammatory/immune responses, due to its anti-inflammatory properties. In addition, mesenchymal stem cells (MSCs) modulate the function of all major immune cell populations. Knowing that, the current work aimed to investigate
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the effects of Mg2+ enrichment, and its influence on the immunomodulatory capacity of
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MSCs. Murine C3H/10T1/2 MSCs were cultivated in media with different
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concentrations of Mg2+ (0, 1, 3 and 5 mM), in order to evaluate the effects of Mg2+ on MSC immunomodulatory properties, cell proliferation rates, expression of NFB and
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STAT-3, production of IL-1, IL-6, TGF-, IL-10, PGE2 and NO, and TRPM7 expression. The results showed that TRPM7 is expressed in MSCs, but Mg2+, in the way
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that cells were cultivated, did not affect TRPM7 expression. Additionally, there was no difference in the intracellular concentration of Mg2+. Mg2+, especially at 5 mM, raised
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proliferation rates of MSCs, and modulated immune responses by decreasing levels of
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IL-1β and IL-6, and by increasing levels of IL-10 and PGE2 in cells stimulated with LPS or TNF-. In addition, MSCs cultured in 5 mM Mg2+ expressed lower levels of
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pNFB/NFB and higher levels of pSTAT-3/STAT-3. Furthermore, conditioned media from MSCs reduced lymphocyte and macrophage proliferation, but Mg2+ did not affect
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this parameter. In addition, conditioned media from MSCs cultured at 5 mM of Mg2+ modulated the production profile of cytokines, especially of IL-1 and IL-6 in macrophages. In conclusion, Mg2+ is able to modulate some immunoregulatory properties of MSCs.
Keywords: Magnesium, mesenchymal stem cells, macrophages and lymphocytes, cytokines, immunomodulation.
ACCEPTED MANUSCRIPT 1. Introduction Magnesium (Mg2+) is one of the most abundant minerals in the human body. Mg2+ participates as a cofactor for hundreds of enzymes, and is related to several metabolic and intracellular biochemical processes; beyond that, it is crucial for the synthesis of DNA and for the control of cellular proliferation [1,2].
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Experimental assays performed in vitro and in vivo using humans and animals
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have shown that Mg2+ is important in the modulation of inflammatory/immune
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responses, by affecting the function of several cells of the immune system [3–5], but little is known about the influence of this mineral on the mesenchymal stem cell (MSC).
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MSCs are known to have an immunomodulatory role, with participation in the regulation of cells from adaptive and innate immune responses [6,7]. In addition, MSCs
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have diverse immune regulatory features, especially linked to their ability to act in immunosuppressing the function of immune cell populations, thus affecting immune
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responses [6–8]. The immunomodulatory properties of MSCs depend on several factors,
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such as the production and secretion of soluble factors, especially cytokines and chemokines [9]. In recent decades, the relationship between MSCs and Mg2+, and its
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implications for skeletal tissue, has been investigated [10–12]. However, the ability of Mg2+ to modulate the immunomodulatory properties of MSCs is still unknown, as is the
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impact of Mg2+ and MSC interactions with other immune cells. Based on the assumption that Mg2+ and MSCs have important roles in the
modulation of inflammatory/immune responses, the current work aimed to evaluate if the effects of Mg2+ supplementation in vitro could influence some immunomodulatory properties of MSCs.
ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1 Mesenchymal cell line and treatments C3H/10T1/2 MSCs were obtained from the American Type Culture Collection (Rockville, MD), and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing a low glucose concentration (Vitrocell, São Paulo, Brazil),
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supplemented with 10% fetal calf serum (Vitrocell), 1% penicillin/streptomycin (Sigma
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Aldrich, Missouri, USA) and 2 mM of L-glutamine (Sigma). C3H/10T1/2 cells were incubated and grown at 37 °C in a 5% CO2 humidified incubator. Cells were harvested
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and plated in six-well culture plates at 5 × 105 cells per mL, and cultivated for 24 hours
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in DMEM without Mg2+ or in the presence of 1, 3 or 5 mM of Mg2+ [13] and as source of magnesium was used magnesium sulfate (MgSO4) [14]. After this period, cells were
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stimulated with 1.25 g/mL of lipopolysaccharide (LPS – Gram-negative bacterial coat component, Sigma, # L2880), which elicits a variety of immune responses and is a
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highly potent trigger of cytokine secretion. In addition, cells were also stimulated for 2
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h with 10 ng/mL of TNF- (R&D Systems, Abingdon, UK, # 410MT), a proinflammatory cytokine that acts directly in the inflammatory response. The dosage
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of LPS and TNF- were previously standardized in our laboratory and this concentration had the potential to amplify the inflammatory response and activate the
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innate immune response without resulting in high percentage of cell death [15-17]. MSC supernatants were collected to measure production of cytokines, prostaglandin E2 (PGE2) and nitric oxide (NO), or for conditioned culture with macrophages or lymphocytes as described below.
2.2 Expression of TRPM7
ACCEPTED MANUSCRIPT The expression of TRPM7 was determined by flow cytometry with antiTRPM7
(Abcam,
Cambridge,
UK,
#
ab109438),
and
checked
by
an
immunofluorescence technique (Abcam, # ab7007). Samples were incubated with 1 µg of antibody/106 cells/mL, incubated for 20 min at 25C, and sheltered from light. After this period, the samples were centrifuged at 400 × g for 3 min, the supernatant was
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discarded and the cell pellet was washed twice with phosphate-buffered saline (PBS;
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Sigma). Cells were resuspended in 500 L of PBS, and acquisition was performed in a
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FACSCanto II flow cytometer (Becton & Dickinson, California, USA). Fluorescence measurements were obtained with at least 1 × 104 cells. The data was analyzed using the
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software package FlowJo 7.6® (TreeStar, Oregon, USA). To determine the threshold of fluorochromes, we performed FMO controls for each fluorochrome. In addition,
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samples were verified by fluorescence microscopy, and representative images were
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obtained.
2.3 Intracellular and extracellular magnesium The concentrations of intracellular and extracellular Mg2+ in C3H/10T1/2
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MSCs were determined by atomic absorption spectroscopy (AAnalyst 100, Perkin
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Elmer, Connecticut, USA) [18, 19]. For the intracellular magnesium dosage the cells were lysed by addition of 250 L of demineralized water, while extracellular magnesium of samples of culture medium were first digested in nitric acid (1 : 1; v/v). In each case, an aliquot of 5% lanthanum (III) oxide solution was added to the sample to yield a final concentration of lanthanum equivalent to 0.1% (w/v). Appropriate calibration curves were constructed using magnesium chloride (Titrisol, Merck, Darmstadt, Germany). Accuracy of the employed methods was verified using Seronorm (SeronormTM Trace Elements Serum L-1 and Urine Blank, Sero, Billingstad, Norway)
ACCEPTED MANUSCRIPT certified standard trace elements, and secondary standards for the samples that were digested.
2.4 MSC viability and apoptosis
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C3H/10T1/2 cells (5 × 105) were cultured with or without Mg2+, as described previously, and stimulated for 24 h with 1.25 µg/mL of LPS or with 10 ng/mL of TNF-
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. Then, the cells were harvested in PBS (Sigma) and centrifuged; the pellet was
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resuspended with 50 L of annexin buffer, and incubated with 3 L of annexin-V
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(Becton & Dickinson, # BD 556419) and 5 L of propidium iodide (PI; Becton & Dickinson, # BD 556463) for 20 min, protected from light. After incubation, cells were
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centrifuged and resuspended in 200 L of annexin buffer for data acquisition using a FACSCanto II flow cytometer. At least 1 × 104 cells were acquired. Data analysis was
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performed using FlowJo 7.6® software (Tree Star).
2.5 MSC cell cycle and proliferation
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To assess the effect of Mg2+ on MSC proliferation, cells were cultivated in fetal bovine serum (FBS)-starved media (DMEM with 1% penicillin/streptomycin only).
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After 16 h, 5 × 105 MSCs were cultured with or without Mg2+ as described previously, and stimulated for 24 h with 1.25 µg/mL of LPS or with 10 ng/mL of TNF-. Cells were harvested and fixed with 70% ethanol for 20 min on ice. After centrifugation, the pellet of the cells was resuspended with 4 mg/mL ribonuclease A (USBiological, Massachusetts, USA, # R2011) and 4 L of PI for 45 min at 37 °C, protected from light. Data acquisition and analysis by flow cytometry were performed as described above. Cell cycle status was assessed by quantifying the percentage of histogram regions
ACCEPTED MANUSCRIPT corresponding to G1/G0 and S/G2/M phases. The MSC proliferation rate was determined by MTT colorimetric assay using MTT-tetrazolium (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (Sigma, # M2128)) [20].
2.6 Cytokine, PGE2 and NO production by MSCs
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C3H/10T1/2 cells were cultured in different Mg2+ concentrations as previously
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described. After 24 h, cells were stimulated for 2 h with 1.25 µg/mL of LPS or with 10
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ng/mL of TNF-. C3H/10T1/2 cell supernatants were collected, and the production of IL-1, IL-6, transforming growth factor beta 1 (TGF-), IL-10, PGE2 and NO was
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determined. Cytokine production was assessed by enzyme-linked immunosorbent assay (ELISA) kits (Quantikine ELISA®, R&D Systems) and PGE2 by Prostaglandin E2
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ELISA (Cayman Chemical, Michigan, USA); NO concentrations were measured using a chemiluminescence analyzer (NOATM 280, Sievers, Colorado, USA), sampled in
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triplicate.
2.7 NFB and STAT-3 expression by MSCs
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Expression of NFB and STAT-3 was evaluated in C3H/10T1/2 cells by Western blot technique. C3H/10T1/2 cells were cultured as previously described; when
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confluent, cells were trypsinized and seeded into six-well plates at a density of 5 × 105 cells per mL of MSC medium, without magnesium (0 mM) or with 1, 3 or 5 mM of Mg2+, for a period of 24 h, and were then stimulated for 2 h with 1.25 µg/mL of LPS or with 10 ng/mL of TNF-. Subsequently, MSCs were lysed with RIPA® buffer (Pierce, Illinois, USA, #89900) containing protease and phosphatase inhibitors (0.5 mM PMSF, 50 mM NaF, 10 µg/mL leupeptin and 10 µg/mL aprotinin; Sigma). Protein quantification was performed based on the Bradford method, and a commercial kit
ACCEPTED MANUSCRIPT (BCATM protein assay kit®, Pierce) was used for this aim. Subsequently, sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%) was performed using 20 µg of protein sample, followed by a polyvinylidene fluoride membrane (PVDF®, Amersham Biosciences, Pennsylvania, USA) transfer. A molecular weight standard (Bio-Rad, Philadelphia, USA) was used to compare separated molecular weight fractions. Primary
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antibodies anti-NFB (C-20, # sc-372), -pNFB (Ser 311, # sc-33039), -STAT-3 (C-
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20, # sc-482) and -pSTAT-3 (Ser 727, # sc-8001-R) from Santa Cruz Biotechnology were diluted in TBS-Tween buffer, respectively to 1 : 1000, 10 : 1000, 4 : 1000 and
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4 : 1000, and incubated overnight. Finally, membranes were incubated for 1 h with anti-
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IgG rabbit biotin-conjugated secondary antibody (R&D Systems) diluted to 1 : 10000 in TBS-Tween buffer. Immunoreactive bands were visualized using the ECL detection
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system® (Amersham Biosciences), and images were captured using ImageQuantTM 400® version 1.0.0 (Amersham Biosciences, USA). For standardization and quantification,
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images were analyzed using ImageQuant TL® (Amersham Biosciences). Results were
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normalized to the intensity of β-actin (Sigma, St. Louis, USA, # A3854), which was
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diluted to 3 : 10000 in TBS-Tween buffer.
2.8 MSCs and their effects on macrophage lineage proliferation and cytokine
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production
Macrophages (mouse leukemic monocyte macrophage cell line Raw 264.7,
American Type Culture Collection (Rockville, MD)) were cultured in high glucose DMEM (Vitrocell) supplemented with 10% FBS (Vitrocell), 1% penicillin/streptomycin (Sigma, St. Louis, MO, USA, # P3032 and # S9137) and 2 mM L-glutamine (Sigma), and maintained at 37 °C, 5% CO2 and in a humidified atmosphere. Macrophages were dislodged from the flask substrate with a cell scraper, and resuspended in conditioned
ACCEPTED MANUSCRIPT media. The conditioned media was the supernatant of C3H/10T1/2 cells cultivated without magnesium or with 5 mM of magnesium, as previously described. The reason to choose just the 5 mM concentration of magnesium was based on previous effects of this concentration observed for C3H/10T1/2 results. Raw 264.7 cells were seeded in 96-well plates at a density of 5 × 105 cells per
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mL, and cultured for 3 days. Subsequently, macrophages cultured in conditioned media
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(Cond. 0 or 5 mM of Mg2+) were stimulated with 1.25 µg/mL LPS (Sigma) for 24 h. Macrophages were cultured in DMEM without Mg2+ or with 5 mM of Mg2+, used as
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control groups, and assayed in parallel with conditioned media with 0 or 5 mM Mg2+.
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After this period, the production of IL-10, IL-12 and TNF- was determined by ELISA
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kits (R&D Systems), and proliferation rate was evaluated by MTT assay.
2.9 Evaluation of conditioned media of MSCs on lymphocyte proliferation and cytokine
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production
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Spleens from Swiss mice were used as a source of normal lymphocytes, especially T cells. Three-month-old mice were used in the current study, which was
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approved by the Institutional Animal Care and Use Committee of the Faculty of Pharmaceutical Sciences at the University of São Paulo. The spleens were removed and
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gently dissociated using needles and tweezers, in Petri dishes containing 10 mL of Roswell Park Memorial Institute medium (RPMI) (Vitrocell). The spleen capsule was discarded, and intra-capsular content was resuspended in RPMI. Mononuclear cells were separated by the density gradient method with Ficoll Histopaque® (Sigma). Then, cells were resuspended in Cond. 0 or 5 mM Mg2+. Finally, mononuclear cells were seeded into a 96-well plate at a density of 5 × 105 cells per mL, and cultured for 24 h; after that, they were stimulated with 1.25 µg/mL LPS (Sigma) for 24 h. Cells were
ACCEPTED MANUSCRIPT maintained at 37 °C, 5% CO2 and in a humidified atmosphere. Lymphocytes were cultured in parallel without conditioned media, but cultured in DMEM without Mg2+ or with 5 mM Mg2+, and were assayed and used as control cultures. After this period, lymphoid production of IFN-, IL-4 and IL-10 was determined, and the proliferation
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rate was evaluated by MTT assay.
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2.10 Statistical analysis
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After going through a normality test (Kolmogorov-Smirnov), data sets comparing conditioned media and DMEM at the same concentrations were analyzed by
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Student’s t-test, and the level of significance adopted was 5% (p < 0.05). For data analysis of multiple comparisons among groups, analysis of variance and Bonferroni’s
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post hoc test were performed. Values were expressed as mean ± standard error deviation. Statistical analyses were performed using GraphPad Prism® software version
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5.01 (GraphPad Software Inc., California, USA).
3. Results
To
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3.1 Effect of magnesium on TRPM7 expression in MSCs verify
TRPM7
expression
in
MSCs,
flow
cytometry
and
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immunofluorescence techniques were used; no differences in TRPM7 expression among the Mg2+ concentrations (0, 1, 3 and 5 mM; data not shown for all Mg2+ concentrations tested) were observed. Figure 1A and 1B exemplify comparison of the percentage of MSCs expressing TRPM7, and the median fluorescence intensity comparing cells cultivated without Mg2+ and cells cultivated with 5 mM of Mg2+. In addition, as can be seen in Figure 1D and 1E, the immunofluorescence technique also showed that cells cultivated without or with 5 mM of Mg2+ express TRPM7, but no differences between
ACCEPTED MANUSCRIPT groups were observed. Knowing that TRPM7 is an Mg2+-permeable ion channel and is also a constitutively active cation channel that is tightly regulated by intracellular Mg2+, the intracellular Mg2+ concentration was measured [21]. There were no differences in intracellular Mg2+ in all cells cultivated with different Mg2+ concentrations (data not shown for all Mg2+ concentrations tested). Figure 1C exemplifies the comparison
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between intracellular Mg2+ concentration in cells cultivated without Mg and those
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cultivated with 5 mM of Mg2+ and none difference was observed. However, the extracellular Mg2+ concentration reduced after 24h of culture, especially in cells
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cultivated with 3mM and 5mM of Mg2+, in which it reduced from 3.09 ± 0.07 mM to
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2.25 ± 0.32mM (p≤0.01) and from 5.06 ± 0.05 mM to 3.68 ± 0.57mM (p≤0.01),
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respectively.
3.2 Effect of magnesium on cell viability, proliferation rate and cell cycle in MSCs
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To test whether the Mg2+ concentration affects cell viability, flow cytometry
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using annexin and PI was performed; none of the Mg2+ concentrations tested affected cell viability (Figure 2A).
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The results showed differences in proliferation rate depending on the treatment. Cells stimulated with LPS or TNF- showed higher proliferation rates than cells
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without stimulus. In addition, cells cultivated with higher Mg2+ concentrations showed increased proliferation rates when compared to cells cultured without Mg2+ (Figure 2B), independent of the stimuli. The cell cycle status of MSCs was evaluated in cells cultivated at different concentrations of Mg2+, and stimulated with LPS or TNF-. MSCs not stimulated and cultivated with Mg2+ showed more cells in the G2/S/M cell cycle phase (Figure 2C). In addition, cells cultivated with 5 mM of Mg2+ and stimulated with LPS also showed a
ACCEPTED MANUSCRIPT higher percentage of cells in the G2/S/M cell cycle phase (Figure 2D). Cells cultivated with 5 mM of Mg2+ and stimulated with TNF- also showed a higher percentage of cells in the G2/S/M cell cycle phase, although the results regarding TNF- stimulus also showed that when the cells were cultivated with 3 mM Mg2+, an increased percentage of
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cells in the G2/S/M cell cycle phase was observed (Figure 2E).
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3.3 Effect of magnesium on cytokine, PGE2 and NO production by MSCs
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The cytokine profile evaluated IL-1, IL-6, IL-10 and TGF- production; PGE2 and NO were also evaluated. The cytokine profile, as well as PGE2 and NO
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production, showed that cells not stimulated with TNF- or LPS did not present differences among them, independent of the Mg2+ concentration. However, cells
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stimulated with LPS or TNF-, especially when cultivated with 3 or 5 mM of Mg2+, showed reduced production of IL-1β as well as of IL-6; both are inflammatory
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cytokines (Figure 3A and 3B, respectively). In contrast, the anti-inflammatory cytokine
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IL-10 showed increased production when cells were cultivated with 3 or 5 mM of Mg2+ and stimulated with TNF- (Figure 3C). Furthermore, PGE2, which is an anti-
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inflammatory compound, showed increased production when cells were cultivated with
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Mg2+, especially at 5 mM, and stimulated with LPS or TNF- (Figure 2D). In relation to TGF-β (Figure 2E), the results showed reduced production when cells were cultivated with 3 or 5 mM of Mg2+ and stimulated with TNF-α, although when cells were stimulated with LPS, TGF-β production was higher when cells were cultivated with 3 or 5 mM of Mg2+ compared to production by cells cultivated in the absence of Mg2+. NO production was not different for any treatment (Figure 2F).
3.4 Effect of magnesium on NFB and STAT-3 expression by MSCs
ACCEPTED MANUSCRIPT Given that NFB and STAT-3 have a central role in coordinating the inflammatory response, the expression of proteins was measured in C3H/10T1/2 cells treated with 0, 1, 3 or 5 mM of Mg2+ and stimulated with LPS or TNF-. No statistical differences in NFB and STAT-3 expression were observed among groups without the stimulus, independent of the Mg2+ concentration. For cells stimulated with LPS, a
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reduction in the ratio of phosphorylated and total NFB was observed when cells were
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treated with 1, 3 or 5 mM of Mg2+, compared to cells cultivated without Mg2+ (Figure
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4A). Moreover, cells treated with 1, 3 or 5 mM of Mg2+ and stimulated with TNF- also
cultivated without Mg2+ (Figure 4A).
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showed a reduction in the ratio of phosphorylated and total NFB in comparison to cells
In addition, the ratio between phosphorylated and total STAT-3 in MSCs was
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significantly higher when cells were cultured with 1, 3 or 5 mM of Mg2+ and stimulated with LPS, in comparison to cells cultivated without Mg2+ (Figure 4B). However, for
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cells not stimulated or cells stimulated with TNF-, no differences among groups were observed in the ratio between phosphorylated and total STAT-3 (Figure 4B).
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3.5 Lymphocyte and macrophage proliferation rates when cultured in conditioned
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media from MSCs cultivated with or without magnesium In order to determine if Mg2+ in MSCs affects the proliferation of immune
cells, macrophages and lymphocytes were cultured in media obtained from MSCs cultivated without Mg2+ or with 5 mM of Mg2+, named conditioned medium (Cond.). Macrophages and lymphocytes were cultivated in conditioned media, and stimulated with LPS or TNF-; according to Figure 5A and 5B, macrophage and lymphocyte proliferation rates were significantly lower when these cells were cultured with conditioned media, compared to DMEM, but an exception is that no differences were
ACCEPTED MANUSCRIPT observed when comparing the lymphocyte proliferation rate between lymphocytes cultivated with DMEM plus TNF- stimulus and lymphocytes cultivated with conditioned media from MSCs cultivated without Mg2+ and stimulated with TNF-. Comparing the proliferation rates between cells cultivated in conditioned media with 0
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or 5 mM Mg2+, no difference was observed.
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3.6 Secretion of immunoregulatory cytokines by lymphocytes and macrophages cultured
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in conditioned media from MSCs cultivated with or without magnesium To test if Mg2+ in MSCs affects the production of cytokines by immune cells,
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lymphocytes and macrophages were cultured for 24 h in MSC conditioned media (Cond. 0 and 5 mM of Mg2+), and stimulated with LPS or TNF-.
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In analysis of the production of TNF- by macrophages, it was observed that cells cultured with conditioned media and challenged with LPS or TNF- showed a
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significant decrease in the production of this cytokine; however, no differences were
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observed between conditioned media with 0 or 5 mM of Mg2+ (Figure 6A). IL-1 production by macrophages showed similar results to those observed for TNF-
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production; however, macrophages stimulated with LPS showed reduced production of
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IL-1 when cultivated with conditioned media 5 mM, compared to conditioned media 0 mM. Moreover, when comparing macrophages cultivated with DMEM without Mg2+ and macrophages cultivated with DMEM with 5 mM of Mg2+, reduced production of IL-1 was also observed (Figure 6B). Additionally, IL-6 production was lower in conditioned media-cultivated cells in comparison to cells cultivated with DMEM. Furthermore, IL-6 production by macrophages was reduced in cells cultivated with conditioned media 5 mM compared to those cultivated with conditioned media 0 mM. Macrophages cultivated with DMEM
ACCEPTED MANUSCRIPT without Mg2+ and macrophages cultivated with DMEM with 5 mM of Mg2+ also showed reduced IL-6 production (Figure 6C). In contrast, IL-10 production was higher in macrophages cultivated in the conditioned media and stimulated with LPS, but no difference in IL-10 production was observed when comparing conditioned media 0 mM and conditioned media 5 mM. However, macrophages cultivated with DMEM and 5
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mM of Mg2+ had higher IL-10 production than macrophages cultivated with DMEM
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without Mg. Cells stimulated with TNF- did not show differences in IL-10 production
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for any treatment (Figure 6D).
When analyzing the capacity of conditioned media to affect cytokine
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production by lymphocytes, we observed that MSC conditioned media did not alter IFN-γ production (Figure 6E). However, IL-4 production by lymphocytes remained
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mostly similar among groups (Figure 5F), and IL-10 production was significantly increased when lymphocytes were cultured in conditioned medium 0 and 5 mM of Mg2+
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compared to cells cultivated in DMEM (Figure 5G), but no differences were observed
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between conditioned media 0 and 5 mM in relation to IL-10 production.
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4. Discussion
Among several minerals, Mg2+ is an essential mineral that plays a critical role
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in the body [1], being able to act in inflammatory/immune responses [7,9]. The literature reports the influence of this mineral in modulating immune cells, especially lymphocytes, as well as neutrophils and eosinophils [22,23]. However, it is not clear how this mineral can modulate MSCs, and the specific means whereby this mineral regulates immunomodulatory aspects of MSCs requires further elucidation. Thus, the goal of this study was to provide additional insight into how variations in Mg2+ concentration in vitro affect some of the immunomodulatory properties of MSCs.
ACCEPTED MANUSCRIPT MSCs are cells with pleiotropic immunomodulatory effects, including direct suppression of allogeneic and mitogenic T cell proliferation [3–6], induction of T cell anergy or apoptosis [7,8] and inhibition of dendritic cell functions, as well as modulation of cytokine production by immune cells [6–8]. The key point in this immunoregulatory ability is imputed by paracrine effects through the secretion of
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soluble factors such as TGF-1, PGE2, NO and cytokines, especially IL-10 [9].
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In recent decades, the interaction of MSCs and Mg2+, and its implications for
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skeletal tissue, have been investigated through several studies that link the use of magnesium-based alloys (due to their high biodegradability and biocompatibility) with
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the biology of MSCs, highlighting mechanisms related to cell differentiation and bone repair [10–12]. Nevertheless, there are still few investigations regarding the ability of
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Mg2+ to modulate MSCs. The biological effects resulting from variation of magnesium concentration on the biology of MSCs, and the impact of this interaction with other
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immune cells, are not completely clear. On the one hand, evidence indicates that cell
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cultures enriched with Mg2+ can assist in the proliferation and differentiation of cells, as well as being able to help in tissue repair and inflammation control [24].
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In this study, MSCs were cultivated with different Mg2+ concentrations; in addition, cells were stimulated with LPS which is a major component of environmental
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microbial products, or TNF- which is a pleiotropic proinflammatory cytokine; both stimuli are classically recognized to activate immediate inflammatory pathways, and stimulate the acute-phase reaction. Mg2+ participates as a cofactor for hundreds of enzymes, and is related to several metabolic and intracellular biochemical processes, as well as being crucial in the synthesis of DNA and in the control of cellular proliferation [1,2]. In this way, our results showed that when MSCs were cultivated with Mg2+,
ACCEPTED MANUSCRIPT especially at a higher dose (5 mM), cellular proliferation rates as well as the percentage of cells in the S/G2/M cell cycle phase were higher, without changes in cell viability. In addition, we tested the expression of TRPM7 to see if different Mg2+ concentrations could affect its expression, as well as to see if intracellular Mg2+ content changes this condition. TRPM7 is a nonselective cation channel and one of the defined
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components of Mg2+ transport which also plays a prominent role in intracellular Mg2+
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homeostasis [25], and it has been associated with cell survival and proliferation [26,27]. The current results showed that MSCs are cells that present receptors of the
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type TRPM7, and also that Mg2+ at the concentrations tested in this study does not
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affect their expression. Free intracellular Mg2+ regulates TRPM7 expression; therefore, we quantified the intracellular Mg2+ content, but no differences in this parameter were
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observed. Although the results did not show changes in the total amount of intracellular magnesium, the results showed reduced values of extracellular magnesium after 24hour
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of culture, especially when MSCs were cultivated in higher magnesium concentrations,
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for example of 5 mM. These results can be explained, since the number of cells increased when they were cultivated with 5 mM of magnesium: The intracellular
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magnesium concentration did not change, but the extracellular magnesium was reduced, which implies that the MSCs consumed magnesium in accordance with their
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proliferation rate.
Aiming to evaluate the influence of Mg on the production of cytokines and
chemokines by MSCs after LPS or TNF- stimulation, levels of IL-1β, IL-6 and IL-10 as well as PGE2, TGF-β and NO were determined; all cytokines and chemokines were significantly affected by Mg2+, especially at 5 mM, confirming that this mineral immunomodulates MSCs. The amount of IL-1β and IL-6, which are classically identified as NFκB-related proinflammatory cytokines, decreased in the presence of 3 or
ACCEPTED MANUSCRIPT 5 mM of Mg2+ after LPS or TNF- stimulus. NFκB is a transcription factor that is involved in the control of a large number of normal cellular and organismal processes, such as immune and inflammatory responses; the current results also showed a reduction in the ratio of phosphorylated NFκB/total NFκB in the presence of Mg2+ in the conditions tested.
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In addition, cells cultivated with Mg2+ at 3 or 5 mM and stimulated with TNF-
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showed increased production of IL-10, which is classically known as an anti-
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inflammatory cytokine as it inhibits the NFκB pathway [28]. Moreover, the results showed an increase in the ratio of phosphorylated STAT-3/total STAT-3, a transcription
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factor able to modulate the expression of anti-inflammatory genes [29,30]. In addition, cells stimulated with LPS showed increased production of TGF-; although a reduction
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of this cytokine was observed when cells were stimulated with TNF-, we can conclude that Mg2+, especially at 3 or 5 mM, is able to modulate the production of this cytokine.
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TGF- is a pleiotropic cytokine with potent regulatory and inflammatory activity [31,32]; being an important immune modulator of T cell activity, it has been shown to play an essential role in establishing immunological tolerance, yet recent studies have
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revealed the proinflammatory roles of TGF- in inflammatory responses which, in part,
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could explain our results [33-35]. Moreover, several soluble factors besides IL-10 and TGF- have been
proposed to mediate the immunosuppressive effect of MSCs, including and highlighting the action of NO and PGE2. Although we did not observe differences among groups for NO production, PGE2, which plays a key role in association of anti-inflammation and immune suppression [36], showed increased production when MSCs were cultivated with 5 mM Mg2+ and stimulated with LPS or TNF-. Based on those findings, the
ACCEPTED MANUSCRIPT cultivation of MSCs with Mg2+, especially at 5 mM, modulates immune responses due to alteration of cytokine and chemokine production. Although the underlying mechanisms of MSC immunomodulation have yet to be elucidated, they are mediated especially by these described soluble factors. To test the action of Mg2+ on the immunoregulatory properties of MSCs, considering that
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MSCs modulate the immune system mainly through the secretion of soluble factors
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[13,21,22], this study focused on understanding the role of MSC soluble factors
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contained in MSC conditioned media on the regulation of macrophage and lymphocyte proliferation and cytokine production.
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IL‐ 10 has been shown to exert a protective effect against inflammation [37,38] due to generalized downregulation of proinflammatory cytokines such as IL‐ 1,
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TNF‐ α and IL‐ 6. The current results showed that conditioned media from MSCs is able to reduce the proliferation rates of macrophages and lymphocytes, but Mg2+ did not
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affect this parameter. In addition, macrophages cultivated in conditioned media from
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C3H/10T1/2 cells showed reduced production of TNF-, IL-1 and IL-6 in contrast to increased IL-10 production. Moreover, it was observed that reduced production of IL-
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1 and IL-6 was more accentuated when macrophages were cultivated in conditioned media from C3H/10T1/2 cultured with 5 mM of Mg2+, which means that MSCs have an
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efficient way to suppress the capacity of macrophages to produce proinflammatory cytokines, and Mg2+ is able to potentiate these effects. Regarding the investigation of cytokine production by spleen lymphoid cells, we observed that the conditioned medium had no effect on IFN-γ production, but increased production of IL-10, although no differences in IFN-γ and IL-10 were observed in cells cultivated in conditioned media from C3H/10T1/2 cultures with 5 mM of Mg2+. In addition, the production of IL-4, a cytokine produced by Th2 cells which
ACCEPTED MANUSCRIPT acts as an anti-inflammatory agent, blocking the synthesis of proinflammatory cytokines, also did not show differences when lymphocytes were cultivated in conditioned media, but interesting results showed that Mg2+, per se, is able to increase IL-4 production. In conclusion, we verified that Mg2+ has anti-inflammatory properties,
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decreasing the levels of IL-1β and IL-6, and increasing levels of IL-10 and PGE2 as
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well as reducing the expression of pNFB/NFB and increasing the expression of pSTAT-3/STAT-3. In addition, MSCs cultured at 5 mM of Mg2+ modulated the
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production profile of cytokines, especially in macrophages showing that Mg2+ is able to
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modulate MSCs, changing their immunoregulatory properties. However, how these immunoregulatory properties are affected by Mg2+ needs further investigation to
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uncover the detailed molecular mechanisms underlying control of this regulation, and thus to understand how the use of this mineral can boost the immunosuppressive effects
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of MSCs.
Funding: This investigation was supported by grants from the Fundação de Amparo a
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Legends
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Pesquisa do Estado de São Paulo – FAPESP (16/16463-8)
Figure 1. Expression of TRPM7 and Intracellular Mg2+ content. (A) Percentage of TRPM7 expression in C3H/10T1/2 cells, (B) Median intensity fluorescence of TRPM7 in C3H/10T1/2 cells, (C) Intracellular Mg2+ content in C3H/10T1/2 cells, (D) Fluorescence microscopy of TRMP7 expression of C3H/10T1/2 cells cultivated without Mg2+ (400x), (E) Fluorescence microscopy of TRMP7 expression of C3H/10T1/2 cells
ACCEPTED MANUSCRIPT cultivated with 5mM of Mg2+ (400x). The results are expressed as the mean SEM of 0mM of Mg2+ (n = 6) and 5mM of Mg2+ (n = 6). Figure 2. (A) Cell viability when C3H/10T1/2 cells are cultured in the presence of different Mg2+ concentrations and stimulated with LPS or TNF-, (B) MTT assay; MSC
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proliferation when C3H/10T1/2 cells are cultured in the presence of different Mg2+ concentrations and stimulated with LPS or TNF-, (C) Cell cycle phases of
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C3H/10T1/2 cells cultured in the presence of different Mg2+ without stimulus, (D) Cell
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cycle phases of C3H/10T1/2 cells cultured in the presence of different Mg2+ stimulated with LPS, (E) Cell cycle phases of C3H/10T1/2 cells cultured in the presence of
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different Mg2+ stimulated with TNF-. The results are expressed as the mean SEM
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(n=6). Significant differences between the treatment groups are illustrated by *(p 0.05), **(p 0.01) and ***(p 0.001).
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Figure 3. Mg2+ effect on (A) IL-1, (B) IL-6, (C) IL-10, (D) PGE2, (E) TGF-and (F) NO secretion by C3H/10T1/2 cells stimulated or not with LPS or TNF-. The results are expressed as the mean SEM (n=6).. Significant differences between the treatment
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groups are illustrated by *(p 0.05), **(p 0.01) and ***(p 0.001).
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Figure 4. Mg2+ effect on the expression of transcription factors by C3H/10T1/2 cells stimulated or not with LPS or TNF-. (A) Results of 6 independent experiments of western blots analysis of p-NFB and NFB, figures are one representative experiment. (B) Results of 6 independent experiments of western blots analysis of p-STAT3 and STAT3, figures are one representative experiment. Results were calculated in relation to the intensity of -actin and expressed in arbitrary units. The results are expressed as the
ACCEPTED MANUSCRIPT mean SEM (n=6). Significant differences between the treatment groups are illustrated by *(p 0.05) and **(p 0.01). Figure 5. Effect of Mg2+ on C3H/10T1/2 cells: Effect of C3H/10T1/2 conditioned media on (A) macrophage lineage and (B) lymphocyte proliferation. Results of 3
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independent experiments, each having an internal duplicate, are expressed as mean ± SEM. Significant differences between the treatment groups are illustrated by *(p 0.05)
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and **(p 0.01).
Figure 6. Effect of Mg2+ on C3H/10T1/2 cells: Effect of MSC conditioned media on the
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production of (A) TNF-, (B) IL-1, (C) IL-6 and (D) IL-10 by macrophages and (E) IFN-γ, (F) IL-4 and (G) IL-10 by lymphocytes. The results are expressed as mean ±
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SEM of 3 independent experiments. Comparisons among conditioned media from MSCs cultured with different Mg2+ concentrations: means with different symbols are
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ACCEPTED MANUSCRIPT Highlights
Mg2+ has anti-inflammatory properties.
Mg2+ is able to modulate Mesenchymal Stem Cells, changing their immunoregulatory properties
Mg2+ modulates the production of Mesenchymal Stem cells
Mg2+ modulates the transcription factor NFB of Mesenchymal Stem cells
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IL-1, IL-6, IL-10, PGE2 and TGF- by
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Fabiana da Silva Lima, Amanda Batista da Rocha Romero, Araceli Hastreiter, Amanda Nogueira-Pedro, Edson Makiyama, Célia Colli, Ricardo Ambrósio Fock PII: DOI: Reference:
S0955-2863(17)30978-6 doi:10.1016/j.jnutbio.2018.02.006 JNB 7926
To appear in: Received date: Revised date: Accepted date:
7 November 2017 4 January 2018 5 February 2018
Please cite this article as: Fabiana da Silva Lima, Amanda Batista da Rocha Romero, Araceli Hastreiter, Amanda Nogueira-Pedro, Edson Makiyama, Célia Colli, Ricardo Ambrósio Fock , An insight into the role of magnesium in the immunomodulatory properties of mesenchymal stem cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jnb(2018), doi:10.1016/ j.jnutbio.2018.02.006
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ACCEPTED MANUSCRIPT An insight into the role of magnesium in the immunomodulatory properties of mesenchymal stem cells
Fabiana da Silva Lima1, Amanda Batista da Rocha Romero1, Araceli Hastreiter2,
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Amanda Nogueira-Pedro 2, Edson Makiyama 2, Célia Colli 1 , Ricardo Ambrósio
1
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Fock 1
Department of Food and Experimental Nutrition, Faculty of Pharmaceutical
Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical
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2
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Sciences, University of São Paulo, São Paulo, Brazil.
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Sciences, University of São Paulo, São Paulo, Brazil.
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* To whom correspondence should be addressed. Fock, Ricardo Ambrósio. Laboratory of Experimental Hematology, Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, University of São Paulo. Avenida Lineu Prestes, 580 - Bloco 17. São Paulo, SP, Brazil. 05508-900. Phone: +551130913639. email: [email protected]
Running Title: The role of magnesium in the immunomodulatory properties
ACCEPTED MANUSCRIPT Abstract Magnesium (Mg2+) is a mineral with the ability to influence cell proliferation and to modulate inflammatory/immune responses, due to its anti-inflammatory properties. In addition, mesenchymal stem cells (MSCs) modulate the function of all major immune cell populations. Knowing that, the current work aimed to investigate
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the effects of Mg2+ enrichment, and its influence on the immunomodulatory capacity of
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MSCs. Murine C3H/10T1/2 MSCs were cultivated in media with different
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concentrations of Mg2+ (0, 1, 3 and 5 mM), in order to evaluate the effects of Mg2+ on MSC immunomodulatory properties, cell proliferation rates, expression of NFB and
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STAT-3, production of IL-1, IL-6, TGF-, IL-10, PGE2 and NO, and TRPM7 expression. The results showed that TRPM7 is expressed in MSCs, but Mg2+, in the way
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that cells were cultivated, did not affect TRPM7 expression. Additionally, there was no difference in the intracellular concentration of Mg2+. Mg2+, especially at 5 mM, raised
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proliferation rates of MSCs, and modulated immune responses by decreasing levels of
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IL-1β and IL-6, and by increasing levels of IL-10 and PGE2 in cells stimulated with LPS or TNF-. In addition, MSCs cultured in 5 mM Mg2+ expressed lower levels of
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pNFB/NFB and higher levels of pSTAT-3/STAT-3. Furthermore, conditioned media from MSCs reduced lymphocyte and macrophage proliferation, but Mg2+ did not affect
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this parameter. In addition, conditioned media from MSCs cultured at 5 mM of Mg2+ modulated the production profile of cytokines, especially of IL-1 and IL-6 in macrophages. In conclusion, Mg2+ is able to modulate some immunoregulatory properties of MSCs.
Keywords: Magnesium, mesenchymal stem cells, macrophages and lymphocytes, cytokines, immunomodulation.
ACCEPTED MANUSCRIPT 1. Introduction Magnesium (Mg2+) is one of the most abundant minerals in the human body. Mg2+ participates as a cofactor for hundreds of enzymes, and is related to several metabolic and intracellular biochemical processes; beyond that, it is crucial for the synthesis of DNA and for the control of cellular proliferation [1,2].
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Experimental assays performed in vitro and in vivo using humans and animals
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have shown that Mg2+ is important in the modulation of inflammatory/immune
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responses, by affecting the function of several cells of the immune system [3–5], but little is known about the influence of this mineral on the mesenchymal stem cell (MSC).
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MSCs are known to have an immunomodulatory role, with participation in the regulation of cells from adaptive and innate immune responses [6,7]. In addition, MSCs
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have diverse immune regulatory features, especially linked to their ability to act in immunosuppressing the function of immune cell populations, thus affecting immune
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responses [6–8]. The immunomodulatory properties of MSCs depend on several factors,
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such as the production and secretion of soluble factors, especially cytokines and chemokines [9]. In recent decades, the relationship between MSCs and Mg2+, and its
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implications for skeletal tissue, has been investigated [10–12]. However, the ability of Mg2+ to modulate the immunomodulatory properties of MSCs is still unknown, as is the
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impact of Mg2+ and MSC interactions with other immune cells. Based on the assumption that Mg2+ and MSCs have important roles in the
modulation of inflammatory/immune responses, the current work aimed to evaluate if the effects of Mg2+ supplementation in vitro could influence some immunomodulatory properties of MSCs.
ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1 Mesenchymal cell line and treatments C3H/10T1/2 MSCs were obtained from the American Type Culture Collection (Rockville, MD), and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing a low glucose concentration (Vitrocell, São Paulo, Brazil),
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supplemented with 10% fetal calf serum (Vitrocell), 1% penicillin/streptomycin (Sigma
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Aldrich, Missouri, USA) and 2 mM of L-glutamine (Sigma). C3H/10T1/2 cells were incubated and grown at 37 °C in a 5% CO2 humidified incubator. Cells were harvested
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and plated in six-well culture plates at 5 × 105 cells per mL, and cultivated for 24 hours
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in DMEM without Mg2+ or in the presence of 1, 3 or 5 mM of Mg2+ [13] and as source of magnesium was used magnesium sulfate (MgSO4) [14]. After this period, cells were
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stimulated with 1.25 g/mL of lipopolysaccharide (LPS – Gram-negative bacterial coat component, Sigma, # L2880), which elicits a variety of immune responses and is a
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highly potent trigger of cytokine secretion. In addition, cells were also stimulated for 2
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h with 10 ng/mL of TNF- (R&D Systems, Abingdon, UK, # 410MT), a proinflammatory cytokine that acts directly in the inflammatory response. The dosage
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of LPS and TNF- were previously standardized in our laboratory and this concentration had the potential to amplify the inflammatory response and activate the
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innate immune response without resulting in high percentage of cell death [15-17]. MSC supernatants were collected to measure production of cytokines, prostaglandin E2 (PGE2) and nitric oxide (NO), or for conditioned culture with macrophages or lymphocytes as described below.
2.2 Expression of TRPM7
ACCEPTED MANUSCRIPT The expression of TRPM7 was determined by flow cytometry with antiTRPM7
(Abcam,
Cambridge,
UK,
#
ab109438),
and
checked
by
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immunofluorescence technique (Abcam, # ab7007). Samples were incubated with 1 µg of antibody/106 cells/mL, incubated for 20 min at 25C, and sheltered from light. After this period, the samples were centrifuged at 400 × g for 3 min, the supernatant was
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discarded and the cell pellet was washed twice with phosphate-buffered saline (PBS;
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Sigma). Cells were resuspended in 500 L of PBS, and acquisition was performed in a
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FACSCanto II flow cytometer (Becton & Dickinson, California, USA). Fluorescence measurements were obtained with at least 1 × 104 cells. The data was analyzed using the
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software package FlowJo 7.6® (TreeStar, Oregon, USA). To determine the threshold of fluorochromes, we performed FMO controls for each fluorochrome. In addition,
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samples were verified by fluorescence microscopy, and representative images were
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obtained.
2.3 Intracellular and extracellular magnesium The concentrations of intracellular and extracellular Mg2+ in C3H/10T1/2
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MSCs were determined by atomic absorption spectroscopy (AAnalyst 100, Perkin
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Elmer, Connecticut, USA) [18, 19]. For the intracellular magnesium dosage the cells were lysed by addition of 250 L of demineralized water, while extracellular magnesium of samples of culture medium were first digested in nitric acid (1 : 1; v/v). In each case, an aliquot of 5% lanthanum (III) oxide solution was added to the sample to yield a final concentration of lanthanum equivalent to 0.1% (w/v). Appropriate calibration curves were constructed using magnesium chloride (Titrisol, Merck, Darmstadt, Germany). Accuracy of the employed methods was verified using Seronorm (SeronormTM Trace Elements Serum L-1 and Urine Blank, Sero, Billingstad, Norway)
ACCEPTED MANUSCRIPT certified standard trace elements, and secondary standards for the samples that were digested.
2.4 MSC viability and apoptosis
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C3H/10T1/2 cells (5 × 105) were cultured with or without Mg2+, as described previously, and stimulated for 24 h with 1.25 µg/mL of LPS or with 10 ng/mL of TNF-
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. Then, the cells were harvested in PBS (Sigma) and centrifuged; the pellet was
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resuspended with 50 L of annexin buffer, and incubated with 3 L of annexin-V
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(Becton & Dickinson, # BD 556419) and 5 L of propidium iodide (PI; Becton & Dickinson, # BD 556463) for 20 min, protected from light. After incubation, cells were
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centrifuged and resuspended in 200 L of annexin buffer for data acquisition using a FACSCanto II flow cytometer. At least 1 × 104 cells were acquired. Data analysis was
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performed using FlowJo 7.6® software (Tree Star).
2.5 MSC cell cycle and proliferation
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To assess the effect of Mg2+ on MSC proliferation, cells were cultivated in fetal bovine serum (FBS)-starved media (DMEM with 1% penicillin/streptomycin only).
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After 16 h, 5 × 105 MSCs were cultured with or without Mg2+ as described previously, and stimulated for 24 h with 1.25 µg/mL of LPS or with 10 ng/mL of TNF-. Cells were harvested and fixed with 70% ethanol for 20 min on ice. After centrifugation, the pellet of the cells was resuspended with 4 mg/mL ribonuclease A (USBiological, Massachusetts, USA, # R2011) and 4 L of PI for 45 min at 37 °C, protected from light. Data acquisition and analysis by flow cytometry were performed as described above. Cell cycle status was assessed by quantifying the percentage of histogram regions
ACCEPTED MANUSCRIPT corresponding to G1/G0 and S/G2/M phases. The MSC proliferation rate was determined by MTT colorimetric assay using MTT-tetrazolium (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (Sigma, # M2128)) [20].
2.6 Cytokine, PGE2 and NO production by MSCs
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C3H/10T1/2 cells were cultured in different Mg2+ concentrations as previously
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described. After 24 h, cells were stimulated for 2 h with 1.25 µg/mL of LPS or with 10
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ng/mL of TNF-. C3H/10T1/2 cell supernatants were collected, and the production of IL-1, IL-6, transforming growth factor beta 1 (TGF-), IL-10, PGE2 and NO was
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determined. Cytokine production was assessed by enzyme-linked immunosorbent assay (ELISA) kits (Quantikine ELISA®, R&D Systems) and PGE2 by Prostaglandin E2
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ELISA (Cayman Chemical, Michigan, USA); NO concentrations were measured using a chemiluminescence analyzer (NOATM 280, Sievers, Colorado, USA), sampled in
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triplicate.
2.7 NFB and STAT-3 expression by MSCs
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Expression of NFB and STAT-3 was evaluated in C3H/10T1/2 cells by Western blot technique. C3H/10T1/2 cells were cultured as previously described; when
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confluent, cells were trypsinized and seeded into six-well plates at a density of 5 × 105 cells per mL of MSC medium, without magnesium (0 mM) or with 1, 3 or 5 mM of Mg2+, for a period of 24 h, and were then stimulated for 2 h with 1.25 µg/mL of LPS or with 10 ng/mL of TNF-. Subsequently, MSCs were lysed with RIPA® buffer (Pierce, Illinois, USA, #89900) containing protease and phosphatase inhibitors (0.5 mM PMSF, 50 mM NaF, 10 µg/mL leupeptin and 10 µg/mL aprotinin; Sigma). Protein quantification was performed based on the Bradford method, and a commercial kit
ACCEPTED MANUSCRIPT (BCATM protein assay kit®, Pierce) was used for this aim. Subsequently, sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%) was performed using 20 µg of protein sample, followed by a polyvinylidene fluoride membrane (PVDF®, Amersham Biosciences, Pennsylvania, USA) transfer. A molecular weight standard (Bio-Rad, Philadelphia, USA) was used to compare separated molecular weight fractions. Primary
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antibodies anti-NFB (C-20, # sc-372), -pNFB (Ser 311, # sc-33039), -STAT-3 (C-
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20, # sc-482) and -pSTAT-3 (Ser 727, # sc-8001-R) from Santa Cruz Biotechnology were diluted in TBS-Tween buffer, respectively to 1 : 1000, 10 : 1000, 4 : 1000 and
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4 : 1000, and incubated overnight. Finally, membranes were incubated for 1 h with anti-
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IgG rabbit biotin-conjugated secondary antibody (R&D Systems) diluted to 1 : 10000 in TBS-Tween buffer. Immunoreactive bands were visualized using the ECL detection
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system® (Amersham Biosciences), and images were captured using ImageQuantTM 400® version 1.0.0 (Amersham Biosciences, USA). For standardization and quantification,
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images were analyzed using ImageQuant TL® (Amersham Biosciences). Results were
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normalized to the intensity of β-actin (Sigma, St. Louis, USA, # A3854), which was
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diluted to 3 : 10000 in TBS-Tween buffer.
2.8 MSCs and their effects on macrophage lineage proliferation and cytokine
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production
Macrophages (mouse leukemic monocyte macrophage cell line Raw 264.7,
American Type Culture Collection (Rockville, MD)) were cultured in high glucose DMEM (Vitrocell) supplemented with 10% FBS (Vitrocell), 1% penicillin/streptomycin (Sigma, St. Louis, MO, USA, # P3032 and # S9137) and 2 mM L-glutamine (Sigma), and maintained at 37 °C, 5% CO2 and in a humidified atmosphere. Macrophages were dislodged from the flask substrate with a cell scraper, and resuspended in conditioned
ACCEPTED MANUSCRIPT media. The conditioned media was the supernatant of C3H/10T1/2 cells cultivated without magnesium or with 5 mM of magnesium, as previously described. The reason to choose just the 5 mM concentration of magnesium was based on previous effects of this concentration observed for C3H/10T1/2 results. Raw 264.7 cells were seeded in 96-well plates at a density of 5 × 105 cells per
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mL, and cultured for 3 days. Subsequently, macrophages cultured in conditioned media
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(Cond. 0 or 5 mM of Mg2+) were stimulated with 1.25 µg/mL LPS (Sigma) for 24 h. Macrophages were cultured in DMEM without Mg2+ or with 5 mM of Mg2+, used as
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control groups, and assayed in parallel with conditioned media with 0 or 5 mM Mg2+.
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After this period, the production of IL-10, IL-12 and TNF- was determined by ELISA
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kits (R&D Systems), and proliferation rate was evaluated by MTT assay.
2.9 Evaluation of conditioned media of MSCs on lymphocyte proliferation and cytokine
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production
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Spleens from Swiss mice were used as a source of normal lymphocytes, especially T cells. Three-month-old mice were used in the current study, which was
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approved by the Institutional Animal Care and Use Committee of the Faculty of Pharmaceutical Sciences at the University of São Paulo. The spleens were removed and
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gently dissociated using needles and tweezers, in Petri dishes containing 10 mL of Roswell Park Memorial Institute medium (RPMI) (Vitrocell). The spleen capsule was discarded, and intra-capsular content was resuspended in RPMI. Mononuclear cells were separated by the density gradient method with Ficoll Histopaque® (Sigma). Then, cells were resuspended in Cond. 0 or 5 mM Mg2+. Finally, mononuclear cells were seeded into a 96-well plate at a density of 5 × 105 cells per mL, and cultured for 24 h; after that, they were stimulated with 1.25 µg/mL LPS (Sigma) for 24 h. Cells were
ACCEPTED MANUSCRIPT maintained at 37 °C, 5% CO2 and in a humidified atmosphere. Lymphocytes were cultured in parallel without conditioned media, but cultured in DMEM without Mg2+ or with 5 mM Mg2+, and were assayed and used as control cultures. After this period, lymphoid production of IFN-, IL-4 and IL-10 was determined, and the proliferation
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rate was evaluated by MTT assay.
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2.10 Statistical analysis
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After going through a normality test (Kolmogorov-Smirnov), data sets comparing conditioned media and DMEM at the same concentrations were analyzed by
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Student’s t-test, and the level of significance adopted was 5% (p < 0.05). For data analysis of multiple comparisons among groups, analysis of variance and Bonferroni’s
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post hoc test were performed. Values were expressed as mean ± standard error deviation. Statistical analyses were performed using GraphPad Prism® software version
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5.01 (GraphPad Software Inc., California, USA).
3. Results
To
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3.1 Effect of magnesium on TRPM7 expression in MSCs verify
TRPM7
expression
in
MSCs,
flow
cytometry
and
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immunofluorescence techniques were used; no differences in TRPM7 expression among the Mg2+ concentrations (0, 1, 3 and 5 mM; data not shown for all Mg2+ concentrations tested) were observed. Figure 1A and 1B exemplify comparison of the percentage of MSCs expressing TRPM7, and the median fluorescence intensity comparing cells cultivated without Mg2+ and cells cultivated with 5 mM of Mg2+. In addition, as can be seen in Figure 1D and 1E, the immunofluorescence technique also showed that cells cultivated without or with 5 mM of Mg2+ express TRPM7, but no differences between
ACCEPTED MANUSCRIPT groups were observed. Knowing that TRPM7 is an Mg2+-permeable ion channel and is also a constitutively active cation channel that is tightly regulated by intracellular Mg2+, the intracellular Mg2+ concentration was measured [21]. There were no differences in intracellular Mg2+ in all cells cultivated with different Mg2+ concentrations (data not shown for all Mg2+ concentrations tested). Figure 1C exemplifies the comparison
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between intracellular Mg2+ concentration in cells cultivated without Mg and those
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cultivated with 5 mM of Mg2+ and none difference was observed. However, the extracellular Mg2+ concentration reduced after 24h of culture, especially in cells
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cultivated with 3mM and 5mM of Mg2+, in which it reduced from 3.09 ± 0.07 mM to
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2.25 ± 0.32mM (p≤0.01) and from 5.06 ± 0.05 mM to 3.68 ± 0.57mM (p≤0.01),
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respectively.
3.2 Effect of magnesium on cell viability, proliferation rate and cell cycle in MSCs
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To test whether the Mg2+ concentration affects cell viability, flow cytometry
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using annexin and PI was performed; none of the Mg2+ concentrations tested affected cell viability (Figure 2A).
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The results showed differences in proliferation rate depending on the treatment. Cells stimulated with LPS or TNF- showed higher proliferation rates than cells
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without stimulus. In addition, cells cultivated with higher Mg2+ concentrations showed increased proliferation rates when compared to cells cultured without Mg2+ (Figure 2B), independent of the stimuli. The cell cycle status of MSCs was evaluated in cells cultivated at different concentrations of Mg2+, and stimulated with LPS or TNF-. MSCs not stimulated and cultivated with Mg2+ showed more cells in the G2/S/M cell cycle phase (Figure 2C). In addition, cells cultivated with 5 mM of Mg2+ and stimulated with LPS also showed a
ACCEPTED MANUSCRIPT higher percentage of cells in the G2/S/M cell cycle phase (Figure 2D). Cells cultivated with 5 mM of Mg2+ and stimulated with TNF- also showed a higher percentage of cells in the G2/S/M cell cycle phase, although the results regarding TNF- stimulus also showed that when the cells were cultivated with 3 mM Mg2+, an increased percentage of
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cells in the G2/S/M cell cycle phase was observed (Figure 2E).
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3.3 Effect of magnesium on cytokine, PGE2 and NO production by MSCs
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The cytokine profile evaluated IL-1, IL-6, IL-10 and TGF- production; PGE2 and NO were also evaluated. The cytokine profile, as well as PGE2 and NO
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production, showed that cells not stimulated with TNF- or LPS did not present differences among them, independent of the Mg2+ concentration. However, cells
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stimulated with LPS or TNF-, especially when cultivated with 3 or 5 mM of Mg2+, showed reduced production of IL-1β as well as of IL-6; both are inflammatory
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cytokines (Figure 3A and 3B, respectively). In contrast, the anti-inflammatory cytokine
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IL-10 showed increased production when cells were cultivated with 3 or 5 mM of Mg2+ and stimulated with TNF- (Figure 3C). Furthermore, PGE2, which is an anti-
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inflammatory compound, showed increased production when cells were cultivated with
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Mg2+, especially at 5 mM, and stimulated with LPS or TNF- (Figure 2D). In relation to TGF-β (Figure 2E), the results showed reduced production when cells were cultivated with 3 or 5 mM of Mg2+ and stimulated with TNF-α, although when cells were stimulated with LPS, TGF-β production was higher when cells were cultivated with 3 or 5 mM of Mg2+ compared to production by cells cultivated in the absence of Mg2+. NO production was not different for any treatment (Figure 2F).
3.4 Effect of magnesium on NFB and STAT-3 expression by MSCs
ACCEPTED MANUSCRIPT Given that NFB and STAT-3 have a central role in coordinating the inflammatory response, the expression of proteins was measured in C3H/10T1/2 cells treated with 0, 1, 3 or 5 mM of Mg2+ and stimulated with LPS or TNF-. No statistical differences in NFB and STAT-3 expression were observed among groups without the stimulus, independent of the Mg2+ concentration. For cells stimulated with LPS, a
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reduction in the ratio of phosphorylated and total NFB was observed when cells were
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treated with 1, 3 or 5 mM of Mg2+, compared to cells cultivated without Mg2+ (Figure
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4A). Moreover, cells treated with 1, 3 or 5 mM of Mg2+ and stimulated with TNF- also
cultivated without Mg2+ (Figure 4A).
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showed a reduction in the ratio of phosphorylated and total NFB in comparison to cells
In addition, the ratio between phosphorylated and total STAT-3 in MSCs was
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significantly higher when cells were cultured with 1, 3 or 5 mM of Mg2+ and stimulated with LPS, in comparison to cells cultivated without Mg2+ (Figure 4B). However, for
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cells not stimulated or cells stimulated with TNF-, no differences among groups were observed in the ratio between phosphorylated and total STAT-3 (Figure 4B).
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3.5 Lymphocyte and macrophage proliferation rates when cultured in conditioned
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media from MSCs cultivated with or without magnesium In order to determine if Mg2+ in MSCs affects the proliferation of immune
cells, macrophages and lymphocytes were cultured in media obtained from MSCs cultivated without Mg2+ or with 5 mM of Mg2+, named conditioned medium (Cond.). Macrophages and lymphocytes were cultivated in conditioned media, and stimulated with LPS or TNF-; according to Figure 5A and 5B, macrophage and lymphocyte proliferation rates were significantly lower when these cells were cultured with conditioned media, compared to DMEM, but an exception is that no differences were
ACCEPTED MANUSCRIPT observed when comparing the lymphocyte proliferation rate between lymphocytes cultivated with DMEM plus TNF- stimulus and lymphocytes cultivated with conditioned media from MSCs cultivated without Mg2+ and stimulated with TNF-. Comparing the proliferation rates between cells cultivated in conditioned media with 0
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or 5 mM Mg2+, no difference was observed.
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3.6 Secretion of immunoregulatory cytokines by lymphocytes and macrophages cultured
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in conditioned media from MSCs cultivated with or without magnesium To test if Mg2+ in MSCs affects the production of cytokines by immune cells,
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lymphocytes and macrophages were cultured for 24 h in MSC conditioned media (Cond. 0 and 5 mM of Mg2+), and stimulated with LPS or TNF-.
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In analysis of the production of TNF- by macrophages, it was observed that cells cultured with conditioned media and challenged with LPS or TNF- showed a
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significant decrease in the production of this cytokine; however, no differences were
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observed between conditioned media with 0 or 5 mM of Mg2+ (Figure 6A). IL-1 production by macrophages showed similar results to those observed for TNF-
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production; however, macrophages stimulated with LPS showed reduced production of
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IL-1 when cultivated with conditioned media 5 mM, compared to conditioned media 0 mM. Moreover, when comparing macrophages cultivated with DMEM without Mg2+ and macrophages cultivated with DMEM with 5 mM of Mg2+, reduced production of IL-1 was also observed (Figure 6B). Additionally, IL-6 production was lower in conditioned media-cultivated cells in comparison to cells cultivated with DMEM. Furthermore, IL-6 production by macrophages was reduced in cells cultivated with conditioned media 5 mM compared to those cultivated with conditioned media 0 mM. Macrophages cultivated with DMEM
ACCEPTED MANUSCRIPT without Mg2+ and macrophages cultivated with DMEM with 5 mM of Mg2+ also showed reduced IL-6 production (Figure 6C). In contrast, IL-10 production was higher in macrophages cultivated in the conditioned media and stimulated with LPS, but no difference in IL-10 production was observed when comparing conditioned media 0 mM and conditioned media 5 mM. However, macrophages cultivated with DMEM and 5
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mM of Mg2+ had higher IL-10 production than macrophages cultivated with DMEM
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without Mg. Cells stimulated with TNF- did not show differences in IL-10 production
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for any treatment (Figure 6D).
When analyzing the capacity of conditioned media to affect cytokine
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production by lymphocytes, we observed that MSC conditioned media did not alter IFN-γ production (Figure 6E). However, IL-4 production by lymphocytes remained
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mostly similar among groups (Figure 5F), and IL-10 production was significantly increased when lymphocytes were cultured in conditioned medium 0 and 5 mM of Mg2+
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compared to cells cultivated in DMEM (Figure 5G), but no differences were observed
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between conditioned media 0 and 5 mM in relation to IL-10 production.
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4. Discussion
Among several minerals, Mg2+ is an essential mineral that plays a critical role
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in the body [1], being able to act in inflammatory/immune responses [7,9]. The literature reports the influence of this mineral in modulating immune cells, especially lymphocytes, as well as neutrophils and eosinophils [22,23]. However, it is not clear how this mineral can modulate MSCs, and the specific means whereby this mineral regulates immunomodulatory aspects of MSCs requires further elucidation. Thus, the goal of this study was to provide additional insight into how variations in Mg2+ concentration in vitro affect some of the immunomodulatory properties of MSCs.
ACCEPTED MANUSCRIPT MSCs are cells with pleiotropic immunomodulatory effects, including direct suppression of allogeneic and mitogenic T cell proliferation [3–6], induction of T cell anergy or apoptosis [7,8] and inhibition of dendritic cell functions, as well as modulation of cytokine production by immune cells [6–8]. The key point in this immunoregulatory ability is imputed by paracrine effects through the secretion of
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soluble factors such as TGF-1, PGE2, NO and cytokines, especially IL-10 [9].
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In recent decades, the interaction of MSCs and Mg2+, and its implications for
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skeletal tissue, have been investigated through several studies that link the use of magnesium-based alloys (due to their high biodegradability and biocompatibility) with
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the biology of MSCs, highlighting mechanisms related to cell differentiation and bone repair [10–12]. Nevertheless, there are still few investigations regarding the ability of
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Mg2+ to modulate MSCs. The biological effects resulting from variation of magnesium concentration on the biology of MSCs, and the impact of this interaction with other
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immune cells, are not completely clear. On the one hand, evidence indicates that cell
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cultures enriched with Mg2+ can assist in the proliferation and differentiation of cells, as well as being able to help in tissue repair and inflammation control [24].
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In this study, MSCs were cultivated with different Mg2+ concentrations; in addition, cells were stimulated with LPS which is a major component of environmental
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microbial products, or TNF- which is a pleiotropic proinflammatory cytokine; both stimuli are classically recognized to activate immediate inflammatory pathways, and stimulate the acute-phase reaction. Mg2+ participates as a cofactor for hundreds of enzymes, and is related to several metabolic and intracellular biochemical processes, as well as being crucial in the synthesis of DNA and in the control of cellular proliferation [1,2]. In this way, our results showed that when MSCs were cultivated with Mg2+,
ACCEPTED MANUSCRIPT especially at a higher dose (5 mM), cellular proliferation rates as well as the percentage of cells in the S/G2/M cell cycle phase were higher, without changes in cell viability. In addition, we tested the expression of TRPM7 to see if different Mg2+ concentrations could affect its expression, as well as to see if intracellular Mg2+ content changes this condition. TRPM7 is a nonselective cation channel and one of the defined
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components of Mg2+ transport which also plays a prominent role in intracellular Mg2+
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homeostasis [25], and it has been associated with cell survival and proliferation [26,27]. The current results showed that MSCs are cells that present receptors of the
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type TRPM7, and also that Mg2+ at the concentrations tested in this study does not
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affect their expression. Free intracellular Mg2+ regulates TRPM7 expression; therefore, we quantified the intracellular Mg2+ content, but no differences in this parameter were
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observed. Although the results did not show changes in the total amount of intracellular magnesium, the results showed reduced values of extracellular magnesium after 24hour
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of culture, especially when MSCs were cultivated in higher magnesium concentrations,
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for example of 5 mM. These results can be explained, since the number of cells increased when they were cultivated with 5 mM of magnesium: The intracellular
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magnesium concentration did not change, but the extracellular magnesium was reduced, which implies that the MSCs consumed magnesium in accordance with their
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proliferation rate.
Aiming to evaluate the influence of Mg on the production of cytokines and
chemokines by MSCs after LPS or TNF- stimulation, levels of IL-1β, IL-6 and IL-10 as well as PGE2, TGF-β and NO were determined; all cytokines and chemokines were significantly affected by Mg2+, especially at 5 mM, confirming that this mineral immunomodulates MSCs. The amount of IL-1β and IL-6, which are classically identified as NFκB-related proinflammatory cytokines, decreased in the presence of 3 or
ACCEPTED MANUSCRIPT 5 mM of Mg2+ after LPS or TNF- stimulus. NFκB is a transcription factor that is involved in the control of a large number of normal cellular and organismal processes, such as immune and inflammatory responses; the current results also showed a reduction in the ratio of phosphorylated NFκB/total NFκB in the presence of Mg2+ in the conditions tested.
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In addition, cells cultivated with Mg2+ at 3 or 5 mM and stimulated with TNF-
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showed increased production of IL-10, which is classically known as an anti-
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inflammatory cytokine as it inhibits the NFκB pathway [28]. Moreover, the results showed an increase in the ratio of phosphorylated STAT-3/total STAT-3, a transcription
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factor able to modulate the expression of anti-inflammatory genes [29,30]. In addition, cells stimulated with LPS showed increased production of TGF-; although a reduction
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of this cytokine was observed when cells were stimulated with TNF-, we can conclude that Mg2+, especially at 3 or 5 mM, is able to modulate the production of this cytokine.
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TGF- is a pleiotropic cytokine with potent regulatory and inflammatory activity [31,32]; being an important immune modulator of T cell activity, it has been shown to play an essential role in establishing immunological tolerance, yet recent studies have
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revealed the proinflammatory roles of TGF- in inflammatory responses which, in part,
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could explain our results [33-35]. Moreover, several soluble factors besides IL-10 and TGF- have been
proposed to mediate the immunosuppressive effect of MSCs, including and highlighting the action of NO and PGE2. Although we did not observe differences among groups for NO production, PGE2, which plays a key role in association of anti-inflammation and immune suppression [36], showed increased production when MSCs were cultivated with 5 mM Mg2+ and stimulated with LPS or TNF-. Based on those findings, the
ACCEPTED MANUSCRIPT cultivation of MSCs with Mg2+, especially at 5 mM, modulates immune responses due to alteration of cytokine and chemokine production. Although the underlying mechanisms of MSC immunomodulation have yet to be elucidated, they are mediated especially by these described soluble factors. To test the action of Mg2+ on the immunoregulatory properties of MSCs, considering that
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MSCs modulate the immune system mainly through the secretion of soluble factors
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[13,21,22], this study focused on understanding the role of MSC soluble factors
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contained in MSC conditioned media on the regulation of macrophage and lymphocyte proliferation and cytokine production.
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IL‐ 10 has been shown to exert a protective effect against inflammation [37,38] due to generalized downregulation of proinflammatory cytokines such as IL‐ 1,
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TNF‐ α and IL‐ 6. The current results showed that conditioned media from MSCs is able to reduce the proliferation rates of macrophages and lymphocytes, but Mg2+ did not
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affect this parameter. In addition, macrophages cultivated in conditioned media from
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C3H/10T1/2 cells showed reduced production of TNF-, IL-1 and IL-6 in contrast to increased IL-10 production. Moreover, it was observed that reduced production of IL-
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1 and IL-6 was more accentuated when macrophages were cultivated in conditioned media from C3H/10T1/2 cultured with 5 mM of Mg2+, which means that MSCs have an
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efficient way to suppress the capacity of macrophages to produce proinflammatory cytokines, and Mg2+ is able to potentiate these effects. Regarding the investigation of cytokine production by spleen lymphoid cells, we observed that the conditioned medium had no effect on IFN-γ production, but increased production of IL-10, although no differences in IFN-γ and IL-10 were observed in cells cultivated in conditioned media from C3H/10T1/2 cultures with 5 mM of Mg2+. In addition, the production of IL-4, a cytokine produced by Th2 cells which
ACCEPTED MANUSCRIPT acts as an anti-inflammatory agent, blocking the synthesis of proinflammatory cytokines, also did not show differences when lymphocytes were cultivated in conditioned media, but interesting results showed that Mg2+, per se, is able to increase IL-4 production. In conclusion, we verified that Mg2+ has anti-inflammatory properties,
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decreasing the levels of IL-1β and IL-6, and increasing levels of IL-10 and PGE2 as
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well as reducing the expression of pNFB/NFB and increasing the expression of pSTAT-3/STAT-3. In addition, MSCs cultured at 5 mM of Mg2+ modulated the
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production profile of cytokines, especially in macrophages showing that Mg2+ is able to
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modulate MSCs, changing their immunoregulatory properties. However, how these immunoregulatory properties are affected by Mg2+ needs further investigation to
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uncover the detailed molecular mechanisms underlying control of this regulation, and thus to understand how the use of this mineral can boost the immunosuppressive effects
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of MSCs.
Funding: This investigation was supported by grants from the Fundação de Amparo a
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Legends
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Pesquisa do Estado de São Paulo – FAPESP (16/16463-8)
Figure 1. Expression of TRPM7 and Intracellular Mg2+ content. (A) Percentage of TRPM7 expression in C3H/10T1/2 cells, (B) Median intensity fluorescence of TRPM7 in C3H/10T1/2 cells, (C) Intracellular Mg2+ content in C3H/10T1/2 cells, (D) Fluorescence microscopy of TRMP7 expression of C3H/10T1/2 cells cultivated without Mg2+ (400x), (E) Fluorescence microscopy of TRMP7 expression of C3H/10T1/2 cells
ACCEPTED MANUSCRIPT cultivated with 5mM of Mg2+ (400x). The results are expressed as the mean SEM of 0mM of Mg2+ (n = 6) and 5mM of Mg2+ (n = 6). Figure 2. (A) Cell viability when C3H/10T1/2 cells are cultured in the presence of different Mg2+ concentrations and stimulated with LPS or TNF-, (B) MTT assay; MSC
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proliferation when C3H/10T1/2 cells are cultured in the presence of different Mg2+ concentrations and stimulated with LPS or TNF-, (C) Cell cycle phases of
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C3H/10T1/2 cells cultured in the presence of different Mg2+ without stimulus, (D) Cell
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cycle phases of C3H/10T1/2 cells cultured in the presence of different Mg2+ stimulated with LPS, (E) Cell cycle phases of C3H/10T1/2 cells cultured in the presence of
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different Mg2+ stimulated with TNF-. The results are expressed as the mean SEM
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(n=6). Significant differences between the treatment groups are illustrated by *(p 0.05), **(p 0.01) and ***(p 0.001).
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Figure 3. Mg2+ effect on (A) IL-1, (B) IL-6, (C) IL-10, (D) PGE2, (E) TGF-and (F) NO secretion by C3H/10T1/2 cells stimulated or not with LPS or TNF-. The results are expressed as the mean SEM (n=6).. Significant differences between the treatment
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groups are illustrated by *(p 0.05), **(p 0.01) and ***(p 0.001).
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Figure 4. Mg2+ effect on the expression of transcription factors by C3H/10T1/2 cells stimulated or not with LPS or TNF-. (A) Results of 6 independent experiments of western blots analysis of p-NFB and NFB, figures are one representative experiment. (B) Results of 6 independent experiments of western blots analysis of p-STAT3 and STAT3, figures are one representative experiment. Results were calculated in relation to the intensity of -actin and expressed in arbitrary units. The results are expressed as the
ACCEPTED MANUSCRIPT mean SEM (n=6). Significant differences between the treatment groups are illustrated by *(p 0.05) and **(p 0.01). Figure 5. Effect of Mg2+ on C3H/10T1/2 cells: Effect of C3H/10T1/2 conditioned media on (A) macrophage lineage and (B) lymphocyte proliferation. Results of 3
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independent experiments, each having an internal duplicate, are expressed as mean ± SEM. Significant differences between the treatment groups are illustrated by *(p 0.05)
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and **(p 0.01).
Figure 6. Effect of Mg2+ on C3H/10T1/2 cells: Effect of MSC conditioned media on the
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production of (A) TNF-, (B) IL-1, (C) IL-6 and (D) IL-10 by macrophages and (E) IFN-γ, (F) IL-4 and (G) IL-10 by lymphocytes. The results are expressed as mean ±
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SEM of 3 independent experiments. Comparisons among conditioned media from MSCs cultured with different Mg2+ concentrations: means with different symbols are
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ACCEPTED MANUSCRIPT Highlights
Mg2+ has anti-inflammatory properties.
Mg2+ is able to modulate Mesenchymal Stem Cells, changing their immunoregulatory properties
Mg2+ modulates the production of Mesenchymal Stem cells
Mg2+ modulates the transcription factor NFB of Mesenchymal Stem cells
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IL-1, IL-6, IL-10, PGE2 and TGF- by
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