Donor variability among anti-inflammatory pre-activated mesenchymal stromal cells

Abstract

Therapeutic mesenchymal stromal cells (MSCs) are attractive in part due to their immunomodulatory properties, achieved by their paracrine secretion of factors including prostaglandin E2 (PGE2). Despite promising pre-clinical data, demonstrating clinical efficacy has proven difficult. The current studies were designed to develop approaches to pre-induce desired functions from naïve MSCs and examine MSC donor variability, two factors contributing to this disconnect. MSCs from six human donors were pre-activated with interleukin 1 beta (IL-1β) at a concentration and duration identified as optimal or interferon gamma (IFN-γ) as a comparator. Their secretion of PGE2 after pre-activation and secondary exposure to pro-inflammatory molecules was measured. Modulation of tumor necrosis factor alpha (TNF-α) secretion from M1 pro-inflammatory macrophages by co-cultured pre-activated MSCs was also measured. Our results indicated that pre-activation of MSCs with IL-1β resulted in upregulated PGE2 secretion post exposure. Pre-activation with IL-1β or IFN-γ resulted in higher sensitivity to induction by secondary stimuli compared to no pre-activation. While IL-1β pre-activation led to enhanced MSC-mediated attenuation of macrophage TNF-α secretion, IFN-γ pre-activation resulted in enhanced TNF-α secretion. Donor variability was noted in PGE2 secretion and upregulation and the level of improved or impaired macrophage modulation.

Keywords: Donor Variability; Immunomodulation; Interleukin 1 Beta; Macrophages; Mesenchymal Stromal Cells; Prostaglandin E2.

Figure 1

Experimental schematics. (a) MSC culture medium was replaced by medium without or with IL-1β, LPS or both for 1, 6, or 24 hours. (b) Pre-activated cell culture supernatants were replaced by fresh medium for 6 or 24 hours. (c) MSC culture medium was replaced by medium without or with IL-1β for 1 hour. Pre-activated cell culture supernatants were replaced by fresh medium without or with IL-1β, TNF-α, IFN-γ, or LPS for 24 hours. (d) MSC culture medium was replaced by medium without or with IL-1β, TNF-α, IFN-γ, or LPS for 1 hour. Pre-activated cell culture supernatants were replaced by fresh medium without or with the same factor. (e) MSC transwell culture medium was replaced by medium without or with IL-1β or IFN-γ for 1 hour, and then transwells were washed and transferred to macrophage cultures with LPS for 48 hours. (f) MSC culture medium was replaced by medium without or with IL-1β or IFN-γ for 1 hour. Pre-activated cell culture supernatants were replaced by fresh medium without or with IL-1β, TNF-α, IFN-γ, or LPS for 24 hours.

Figure 2

Dose response of MSC PGE2 secretion to IL-1β and LPS. MSCs were cultured without or with increasing doses of (a) IL-1β, (b) LPS, or (c) IL-1β + LPS for 6 (dark grey bars), 24 (light grey bars), or 48 hours (white bars) and then analyzed for PGE2. Data are the mean ± SEM for PGE2 normalized to cell number and medium control (0 ng/mL) at 6 hours for n = 6 replicates. *p < 0.05 compared to 0 ng/mL; ^†p < 0.05 compared to previous dose by ANOVA and Fisher’s LSD; ^‡p < 0.05 compared to No LPS by ANOVA and Fisher’s LSD. Other statistical comparisons were performed using Student’s t test.

Figure 3

MSC PGE2 secretion at the end of the activation period and over time post-activation. MSCs were cultured without or with IL-1β, LPS or both for (a) 1, (b) 6, or (c) 24 hours. These supernatants were collected and analyzed for PGE2. Supernatants of MSCs that were activated by IL-1β, LPS or both for 1 (dark grey bars), 6 (light grey bars), or 24 (white bars) hours were replaced by fresh medium for (b) 6 or (c) 24 hours, which was collected and analyzed for PGE2. Data are the mean ± SEM for PGE2 normalized to cell number for n = 6 replicates. *p < 0.05 compared to medium by Student’s t test; **p < 0.05 by Student’s t test; ***p < 0.05 by ANOVA and Fisher’s LSD; ^†p < 0.05 compared to 1 or 6 hours of pre-activation by ANOVA and Fisher’s LSD.

Figure 4

Sensitivity of pre-activated MSCs to pro-inflammatory secondary stimuli. MSCs were cultured (a) without (black bars) or with (grey bars) IL-1β for 1 hour, (b) without (black bars) or with (grey bars) IL-1β, TNF-α, IFN-γ, or LPS, or (c) without (black bars) or with IL-1β (grey bars) or IFN-γ (white bars) for 1 hour. Pre-activated cell culture supernatants were replaced by fresh medium without or with IL-1β, TNF-α, IFN-γ, or LPS for 24 hours. PGE2 was quantified in the resulting supernatants. Data are the mean ± SEM for secreted PGE2 level normalized to cell number for n = 9 replicates. *p < 0.05 compared to MSCs without pre-activation by Student’s t test; **p < 0.05 by Student’s t test.

Figure 5

Pre-activated MSC modulation of macrophages. (a) MSCs in transwells were cultured without or with IL-1β or IFN-γ for 1 hour, and then transwells were washed and transferred to macrophage cultures with LPS for 48 hours. Co-culture supernatants were analyzed for (a) TNF-α and (b) PGE2. Data are the mean ± SEM for secreted level normalized to the LPS control for n = 9–19 replicates. *p < 0.05 compared to LPS by Student’s t test. **p < 0.05 determined by Student’s t test.

Figure 6

Response of pre-activated MSCs from multiple donors to secondary pro-inflammatory stimuli. MSCs were cultured without (black bars) or with IL-1β (grey bars) or IFN-γ (white bars) for 1 hour. PGE2 was quantified 24 hours after pre-activated cell culture supernatants were replaced by fresh medium without or with IL-1β, TNF-α, IFN-γ, or LPS. Data are the mean ± SEM for PGE2 normalized to cell number for n = 3 (Donors 2–6) or n = 9 (Donor 1) replicates.

Figure 7

Two donor variability in pre-activated MSC response to secondary pro-inflammatory stimuli. (a) Box and whisker plots display the minimum, first quartile, median (second quartile), third quartile, maximum, and outliers in the normalized PGE2 level secreted by the donors for each activation condition. (b) The interquartile range (IQR) is the difference between the first and third quartile and is plotted for the fold change in PGE2 compared to no pre-activation for each activation condition in the order of highest to lowest (grey bars are IL-β pre-activated; white bars are IFN-γ pre-activated).

Figure 8

Pre-activated MSC modulation of macrophages. (a) MSCs in transwells were cultured without or with IL-1β or IFN-γ for 1 hour, and then transwells were washed and transferred to macrophage cultures with LPS for 48 hours. (b) Co-culture supernatants were analyzed for TNF-α (grey bars) and PGE2 (black circles/line). Data are the mean ± SEM for secreted level the LPS control for n = 3 (Donors 2–5) or n = 9–19 (Donor 1) replicates.

Figure 9

Donor variability in pre-activated MSC modulation of macrophages. Box and whisker plots display the minimum, first quartile, median (second quartile), third quartile, maximum, and outliers in the (a) TNF-α level (normalized to LPS) and (b) PGE2 (normalized to LPS) present in the co-cultures for each activation condition. (c) Comparison of percent improvement in TNF-α reduction imparted by pre-activation. Improvement in attenuation of TNF-α was determined using the percent change in relative TNF-α level for IL-1β pre-activated (grey bars) and IFN-γ pre-activated (white bars) MSC conditions compared to no pre-activation for each donor. Negative values indicate impaired attenuation of TNF-α. *p < 0.05 compared to donor 1, **p < 0.05 compared to donor 5, ***p < 0.05 compared to donor 6 by ANOVA with Fisher’s LSD.