Inflammation and Hypoxia Negatively Impact the Survival and Immunosuppressive Properties of Mesenchymal Stromal Cells In Vitro

MSC were isolated from mouse bone marrow as previously described¹⁴. Cells were grown in low-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% MSC-qualified fetal bovine serum (FBS; Gibco™, Thermo Fisher Scientific) and 1% antibiotic-antimycotic at 37°C in a cell incubator, at 95% humidity, in 5% CO₂ concentration. For experiments, cells were seeded 24 hours before at a density of 25.000 cells/cm² to reach 70–80% confluency. MSCs were cultivated at atmospheric oxygen concentration (21%). To expose the cells to hypoxia, MSC were grown in 2% O₂, 5% CO₂ and 78% humidity in Whitley H35 Hypoxystation (Don Whitley Scientific).

In order to mimic the proinflammatory environment in vitro, MSC were stimulated with IFNγ (20 ng/ml) and TNFα (1, 10 or 20 ng/ml) for 24 to 72 hours, followed by testing the viability and the anti-inflammatory properties of MSC. Recombinant mouse IFNγ and TNFα were purchased from R&D Systems.

Total RNA was extracted using Trizol reagent (ThermoFisher Scientific, 15596026) and the reverse transcription reaction was performed using the High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific, 4374966). Real-time RT-PCR was performed using gene-specific primers and SYBR™ Select Master Mix (Thermo-Fisher Scientific, 4472919).

The nucleotide sequence of each primer can be provided on request. The comparative CT method was used to quantify the data, and 18S rRNA or ACTB mRNA level was used for normalization.

The effect of the proinflammatory cytokines on the MSC cellular index was monitored with the xCELLigence system (Roche Applied Science), as previously described²⁸. Briefly, MSC were detached from cell-culture plates and were seeded at 5.000 cells/well on 16-well E-plates in complete growth medium and cell index was recorded for 24 hours. The next day, the medium was changed with growth medium containing the proinflammatory cytokines (TNFα, IFNγ) at the specific concentration. The cell index was measured for another 2 or 3 days. The instrument read the electric impedance every 10 minutes for the first 3 hours and every hour thereafter.

NO production was detected in the supernatant of MSC cultures by measuring its stable end product nitrite using a Griess reagent according to manufacturer's indications (ThermoFisher Scientific, G-7921). This kit includes two components, A and B. Component B and nitrite form diazonium salt which, in presence of component A, produce an azo dye that can be spectrophotometrically quantified based on its absorbance at 548 nm. Briefly, culture medium was collected at a specific time after cytokine stimulation and was centrifuged at 400 × g for 5 minutes. The culture supernatant was collected and centrifuged again at 2.000 × g for 30 minutes to remove all cellular debris which can interfere with the reaction. A final volume of 130 μl supernatant was transferred to a 96-well plate in triplicate and 20 μl of Griess reagent was added. After 10 minutes of incubation, the absorbance was read at 548 nm with an Infinite M200 microplate reader from Tecan. The nitric oxide synthase inhibitor L-NAME (N omega-nitro-L-arginine methyl ester) was used to downmodulate the NO production in MSC stimulated with proinflammatory cytokines. Nitrite concentration was calculated with reference to a standard curve of six 2-fold serial dilutions of freshly prepared sodium nitrite (0 to 100 μM), and the culture medium alone was used as a blank control. The results are showed as concentration of NO₂ and are expressed in μM.

The results were expressed as the mean ± S.E.M. of at least three independent experiments. Statistical analysis was performed using GraphPad Prism 7 and one-way ANOVA followed by Tukey's post-test was used to assess statistical significance (*p < 0.05, **p < 0.01, ***p < 0.005).

Activated MSC were reported to fulfil their immunosuppressive functions mainly through secretion of immunomodulatory factors⁶. Therefore, the benefits brought by the priming with proinflammatory cytokines over the naïve cells were assayed by determination of IDO1, NOS2, and COX2 expression levels, as major components of the immunomodulatory pathways activated in primed MSC.

First, MSC were seeded on tissue culture plates and 24 hours later, they were stimulated with TNFα and IFNγ alone or in combination (20 ng/ml each). After another 24 hours, the cells were imagined using phase contrast microscopy and no major morphological changes were observed under these experimental conditions (Figure 1A). Subsequently, cells were used for RNA extraction and qRT-PCR analysis. The results showed that TNFα and IFNγ had different effects on the expression levels of three important mediators of pathways associated to immunomodulation. Thus, a statistically significant overexpression of IDO1 was obtained in MSC after 24-hour stimulation with IFNγ, while TNFα did not produce any effect, either alone or in the presence of IFNγ (Figure 1B). However, stimulation with TNFα alone significantly increased the expression of NOS2 and COX2 and in the presence of IFNγ the effect was synergistic (Figure 1C–D).

Figure 1

The morphological changes were also followed with xCELLigence system. MSC stimulation with proinflammatory cytokines was performed under the same experimental conditions as described above, and the cell index was determined at 10-min interval for 48 hours. The xCELLigence analysis showed that IFNγ stimulation caused a slight decrease of the cellular index, most likely as a result of reduced cell proliferation. However, TNFα, either alone or in combination with IFNγ, caused a much more abrupt collapse of the cell index (Figure 2A). These results were confirmed by phase contrast microscopy images, which proved morphological alterations of TNFα-stimulated cells and increased mortality in the cytokine mix culture condition (Figure 2B).

Figure 2

As TNFα has important impact on cell index without affecting cell viability within the first 48 hours of the treatment, the next step was to evaluate the effect of an extensive treatment of MSC with different doses of TNFα. To this aim, MSC were stimulated with different concentrations of TNFα (1, 10 and 20 ng/ml) and cell index was continuously recorded for 72 hours, at 1-hour intervals. Of note, the xCELLigence recording showed a significant decrease in cell index even for the lowest dose of TNFα (Figure 2C), which apparently did not impact the cell viability, as concluded by phase contrast microscopy (Figure 2D). However, stimulation of MSC with higher TNFα concentration (10 and 20 ng/ml) for 3 days resulted in significant cell mortality (Figure 2D).

The above results showed that MSC stimulation with TNFα and IFNγ induced overexpression of NOS2, in parallel with an increase in cell mortality which suggested that the NO produced by NOS2 could induce cytotoxicity in cultured cells. In order to test this hypothesis, NO quantification was performed in control MSC cells stimulated for 48 hours with TNFα and IFNγ alone or in combination. The nitric oxide synthase inhibitor L-NAME was used to validate the specificity of the cellular response. NO is a relatively unstable molecule which suffers a spontaneous oxidation under physiological conditions, thus producing nitrite/nitrate, which can be determined in the culture medium by Griess reaction²⁹. Phase contrast microscopy images illustrated that NOS2 inhibition partially reduced the mortality of cells stimulated with TNFα+IFNγ (Figure 3A). The quantification of nitrite in the culture medium was correlated with NOS2 gene expression in the cells and showed that TNFα stimulation was able to produce nitrite accumulation while IFNγ alone had no effect. Moreover, the combination of TNFα and IFNγ produced 4 times more nitrite than TNFα alone. Interestingly, NOS2 inhibition by L-NAME completely reduced nitrite accumulation in TNFα-stimulated cells, while only partially, yet significantly, reversed the increase in nitrite under TNFα+IFNγ stimulation (Figure 3B).

Figure 3

Since hypoxia and inflammation were recently reported to play important roles in modulating the fate of the transplanted cells³⁰. Cell survival and nitrite production were determined in MSC cultured for 24 hours under normoxic (21% O2) or hypoxic (2% O₂) conditions, in the presence or absence of TNFα+IFNγ stimulation (with or without L-NAME). As expected, phase-contrast microscopy images showed that hypoxia alone did not affect cell viability, however the combination of hypoxia with the proinflammatory cytokines resulted in the death of almost all cells (Figure 4A). Regarding NO production, hypoxia alone did not change nitrite level in the culture medium, but when combined with TNFα + IFNγ, it significantly reduced the level of NO production, probably due to increased cell mortality. NOS2 inhibition by L-NAME partially reversed cell survival in the presence of the both cytokines and hypoxic conditions, in addition to the partial reduction of nitrite level in the medium (Figure 4A–B). Taken together, our data suggest that both hypoxia and inflammation impact the cell survival after transplantation, in part by inducing NOS2 over-expression followed by NO-induced cytotoxicity.

Figure 4