Preconditioning of murine mesenchymal stem cells synergistically enhanced immunomodulation and osteogenesis

Abstract

Background: Mesenchymal stem cells (MSCs) are capable of immunomodulation and tissue regeneration, highlighting their potential translational application for treating inflammatory bone disorders. MSC-mediated immunomodulation is regulated by proinflammatory cytokines and pathogen-associated molecular patterns such as lipopolysaccharide (LPS). Previous studies showed that MSCs exposed to interferon gamma (IFN-γ) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) synergistically suppressed T-cell activation.

Methods: In the current study, we developed a novel preconditioning strategy for MSCs using LPS plus TNF-α to optimize the immunomodulating ability of MSCs on macrophage polarization.

Results: Preconditioned MSCs enhanced anti-inflammatory M2 macrophage marker expression (Arginase 1 and CD206) and decreased inflammatory M1 macrophage marker (TNF-α/IL-1Ra) expression using an in-vitro coculture model. Immunomodulation of MSCs on macrophages was significantly increased compared to the combination of IFN-γ plus TNF-α or single treatment controls. Increased osteogenic differentiation including alkaline phosphate activity and matrix mineralization was only observed in the LPS plus TNF-α preconditioned MSCs. Mechanistic studies showed that increased prostaglandin E2 (PGE2) production was associated with enhanced Arginase 1 expression. Selective cyclooxygenase-2 inhibition by Celecoxib decreased PGE2 production and Arginase 1 expression in cocultured macrophages.

Conclusions: The novel preconditioned MSCs have increased immunomodulation and bone regeneration potential and could be applied to the treatment of inflammatory bone disorders including periprosthetic osteolysis, fracture healing/nonunions, and osteonecrosis.

Keywords: Immunomodulation; Macrophage polarization; Mesenchymal stem cells; Osteogenesis; Prostaglandin E2.

Fig. 1

Immunomodulation of preconditioned MSCs on murine macrophages. a Preconditioning and coculture model. Murine MSCs were preconditioned with IFN-γ plus TNF-α (I + T, 20 ng/ml each), LPS (20 μg/ml) plus TNF-α (L + T), LPS alone (L), or TNF-α alone (T) for 3 days, and cocultured with M1 macrophages for 24 hours. b M2 (Arg1 and CD206) and M1 (TNF-α/IL-1Ra) macrophage marker expression in macrophages measured by quantitative PCR. Data presented as fold-change compared to macrophages cocultured with unstimulated control MSCs. c, d Expression of M2 macrophage markers (Arg1 and CD206) at protein level examined by flow cytometry. ***p < 0.005. Arg1 Arginase 1, IFN interferon, IL1Ra interleukin-1 receptor antagonist, MSC mesenchymal stem cell, TNF tumor necrosis factor

Fig. 2

Increased osteogenic differentiation of MSCs preconditioned with LPS plus TNF-α. MSCs were preconditioned with IFN-γ plus TNF-α (I + T, 20 ng/ml each), LPS (20 μg/ml) plus TNF-α (L + T), LPS alone (L), or TNF-α alone (T) for 3 days in growth media, and replaced by osteogenic media for 3 weeks. The osteogenic differentiation ability was examined by ALP activity at week 2 (a) and bone mineralization (Alizarin Red staining) at week 3 (b). *p < 0.05, **p < 0.01, ***p < 0.005. ALP alkaline phosphatase, MSC mesenchymal stem cell

Fig. 3

Induction of PGE2 secretion in preconditioned MSCs through the COX-2-dependent pathway. MSCs were preconditioned with IFN-γ plus TNF-α (I + T, 20 ng/ml each), LPS (20 μg/ml) plus TNF-α (L + T), LPS alone (L), or TNF-α alone (T) for 3 days, and replaced with fresh media with or without Celecoxib (25 μM) to modulate COX2 signaling. After 24 hours of incubation, the supernatants were collected, and PGE2 production was quantified by ELISA. *p < 0.05, **p < 0.01. COX2 cyclooxygenase-2, MSC mesenchymal stem cell, PGE2 prostaglandin E2

Fig. 4

Immunomodulation ability of preconditioned MSCs mediated by PGE2 secretion. MSC/macrophage cocultured experiments were performed as described in Fig. 1a. Media were supplied with or without 25 μM Celecoxib to modulate COX2 signaling. M2 (Arg1 and CD206) and M1 (TNF-α/IL-1Ra) macrophage marker expression in macrophages was measured by quantitative PCR. Data presented as fold-change compared to macrophages cocultured with unstimulated control MSCs. *p < 0.05, **p < 0.01, ***p < 0.005. Arg1 Arginase 1, IL1Ra interleukin-1 receptor antagonist, MSC mesenchymal stem cell, TNF tumor necrosis factor

Fig. 5

Inhibition of COX-2/PGE2 pathway increased osteogenesis in untreated or preconditioned MSCs. MSCs were preconditioned with LPS plus TNF-α in growth media, and replaced with osteogenic media for 3 weeks. Media were supplied with or without 25 μM Celecoxib during the first week of osteogenesis. Osteogenic differentiation ability examined by ALP activity at week 2 (a) and bone mineralization (Alizarin Red staining) at week 3 (b). *p < 0.05, **p < 0.01. ALP alkaline phosphatase, COX2 cyclooxygenase-2, MSC mesenchymal stem cell