Effect of Comedications and Endotoxins on Mesenchymal Stem Cell Secretomes, Migratory and Immunomodulatory Capacity

Abstract

Mesenchymal stem cells (MSCs) are becoming an increasingly popular therapeutic option among patients with a broad range of ailments to modulate immunity and induce regeneration. The majority of patients receiving these MSC therapies are on concurrent medication or have ongoing infection. In the present study, we examined the effect of immunosuppressive drugs and lipopolysaccharides (LPS)/endotoxins on the secretory profile, migration towards site of injury, and suppression of lymphocyte proliferation of bone marrow-derived MSCs (BMSCs). Generally, LPS coculture augmented the secretory capacity of BMSCs while exposure to immunosuppressive drugs resulted primarily in no change or attenuated secretion, with some cases of increased secretion, dependent on the cytokine assayed. Among the immunosuppressants evaluated, Hydrocortisone had the most widespread inhibitory effect, while LPS from E. coli O111:B4 had the most potent stimulatory effect. In addition, we also showed that Hydrocortisone or LPS from E. coli O111:B4 affected the migratory and immunosuppressive capacity of BMSCs. Following simulation with Hydrocortisone, BMSC migration was attenuated, and immunosuppressive capacity against T cell proliferation was enhanced, however, the opposite effects were seen with LPS from E. coli O111:B4. Our data suggests that the clinical outcomes of MSC-based therapy are affected by the use of immunosuppressive medication or the presence of endotoxemia in patients.

Keywords: LPS; cell migration; cytokines; immunomodulation; immunosuppressive drugs; mesenchymal stem cells; secretome.

Figure 1

Cytokine secretion profiles for bone marrow-derived MSCs (BMSCs) treated with immunosuppressive medications or lipopolysaccharides (LPS). Heat Map of combined fold change in cytokine secretion for three cell lines (BMSC22, BMSC32 and BMSC34) after 3 days of coculture with immunosuppressants or LPS. EGF—epidermal growth factor, FGF-2—fibroblast growth factor-2, Flt3L—Fms-related tyrosine kinase 3 ligand, G-CSF—granulocyte–colony-stimulating factor, GM-CSF—granulocyte macrophage colony stimulating factor, IFN—interferon, IL—interleukin, MCP—monocyte chemoattractant protein, MDC—macrophage-derived chemokine, MIP—macrophage inflammatory protein, PDGF—platelet-derived growth factor, TGF—transforming growth factor, TNF—tumor necrosis factor, VEGF—vascular endothelial growth factor.

Figure 2

Stimulation with Hydrocortisone or E. coli O111:B4 regulates BMSC migration. The migration capacity of BMSC22, BMSC32 and BMSC34 in presence of Hydrocortisone (A–C) or LPS (D–F) was evaluated by Transwell Assays. BMSCs were seeded on top of Transwells, and after 24 h the cells that had migrated to the lower chamber were fixed, stained and quantified. Representative images of cells stained with DAPI are depicted below. (Scale bar is 50 µM, Magnification is 40×). * p < 0.05 versus 0 µM Hydrocortisone or 0 µg/mL LPS as determined by one-way ANOVA.

Figure 3

Stimulation with Hydrocortisone or E. coli O111:B4 affects BMSC immunosuppressive function. (A) Percentage of T-cells was determined by flow cytometry after coculture with BMSCs in the presence of Hydrocortisone or LPS from E. coli O111-B4. The results of statistical analysis for the percentage of CD3+CD45+ T cells are depicted as the mean ± S.D for the three cell lines (BMSC22, BMSC32 and BMSC34) evaluated. (B) Representative example of gating strategy for determination of T cell population after coculture with BMSC22 in presence of Hydrocortisone or LPS from E. coli O111-B4. (C) Representative images of BMSC22 and PBMC coculture in presence of Hydrocortisone or LPS from E. coli O111-B4. Scale bar: 50 µm. * p < 0.05 versus MSC-PBMC only (3rd column), as determined by one-way ANOVA.

Figure 3

Stimulation with Hydrocortisone or E. coli O111:B4 affects BMSC immunosuppressive function. (A) Percentage of T-cells was determined by flow cytometry after coculture with BMSCs in the presence of Hydrocortisone or LPS from E. coli O111-B4. The results of statistical analysis for the percentage of CD3+CD45+ T cells are depicted as the mean ± S.D for the three cell lines (BMSC22, BMSC32 and BMSC34) evaluated. (B) Representative example of gating strategy for determination of T cell population after coculture with BMSC22 in presence of Hydrocortisone or LPS from E. coli O111-B4. (C) Representative images of BMSC22 and PBMC coculture in presence of Hydrocortisone or LPS from E. coli O111-B4. Scale bar: 50 µm. * p < 0.05 versus MSC-PBMC only (3rd column), as determined by one-way ANOVA.