Reciprocal interactions of mouse bone marrow-derived mesenchymal stem cells and BV2 microglia after lipopolysaccharide stimulation

Abstract

IntroductionMesenchymal stem cells (MSCs) are immunosuppressive, but we lack an understanding of how these adult stem cells are in turn affected by immune cells and the surrounding tissue environment. As MSCs have stromal functions and exhibit great plasticity, the influence of an inflamed microenvironment on their responses is important to determine. MSCs downregulate microglial inflammatory responses, and here we describe the mutual effects of coculturing mouse bone marrow MSCs with BV2 microglia in a lipopolysaccharide (LPS) inflammatory paradigm.MethodsMouse MSCs were cultured from femoral and tibial bone marrow aspirates and characterized. MSCs were cocultured with BV2 microglia at four seeding-density ratios (1:0.2, 1:0.1, 1:0.02, and 1:0.01 (BV2/MSC)), and stimulated with 1 μg/ml LPS. In certain assays, MSCs were separated from BV2 cells with a cell-culture insert to determine the influence of soluble factors on downstream responses. Inflammatory mediators including nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and chemokine (C-C motif) ligand 2 (CCL2) were measured in cocultures, and MSC and BV2 chemotactic ability determined by migration assays.ResultsWe demonstrated MSCs to increase expression of NO and IL-6 and decrease TNF-α in LPS-treated cocultures. These effects are differentially mediated by soluble factors and cell-to-cell contact. In response to an LPS stimulus, MSCs display distinct behaviors, including expressing IL-6 and very high levels of the chemokine CCL2. Microglia increase their migration almost fourfold in the presence of LPS, and interestingly, MSCs provide an equal impetus for microglia locomotion. MSCs do not migrate toward LPS but migrate toward microglia, with their chemotaxis increasing when microglia are activated. Similarly, MSCs do not produce NO when exposed to LPS, but secrete large amounts when exposed to soluble factors from activated microglia. This demonstrates that certain phenotypic changes of MSCs are governed by inflammatory microglia, and not by the inflammatory stimulus. Nonetheless, LPS appears to "prime" the NO-secretory effects of MSCs, as prior treatment with LPS triggers a bigger NO response from MSCs after exposure to microglial soluble factors.ConclusionsThese effects demonstrate the multifaceted and reciprocal interactions of MSCs and microglia within an inflammatory milieu.

Regulation of Mouse Microglia Activation and Effector Functions by Bone Marrow-Derived Mesenchymal Stem Cells

“…inflammatory (M1-like) into an anti-inflammatory (M2-like) phenotype [26,31,36] mainly through COX-2 mediated production of PGE2 and possibly some other soluble factors [35,37]. Similar PGE2-mediated interaction was also described between other tissue macrophages and MSCs in vitro [12], as well as in vivo [43].…”

“…However, the precise polarization states of microglia in the presence of MSCs under physiological or inflammatory conditions remain largely unknown. In most studies, xenogenic experimental systems were used; that is, cocultures of human MSCs with mouse or rat MGs [26,[31][32][33] and primary microglia were frequently replaced with neoplastic cell lines (BV2 or N9) [34][35][36] or with hippocampal slices [37]. Only two laboratories used autologous mouse [35] or rat [38] primary MGs and MSCs.…”

Paracrine Effects of Mesenchymal Stem Cells-Conditioned Medium on Microglial Cytokines Expression and Nitric Oxide Production

“…These findings are still to be elucidated, as a variety of regulatory mechanisms are involved in controlling iNOS expression [41]. The increase in NO production has also been shown in coculture systems of MSC with T cells [42,43] and microglia [17,18]. In general, the excessive amount of NO can cause neurotoxicity [44,45].…”

“…There were also numerous studies using MSC as a therapeutic tool in various models of neuroinflammation [27,28,29]; MSC demonstrate immunomodulatory effects on microglia [15,16,17,18]. In addition, several studies have shown that MSC-CM accelerates wound healing [30,31], promotes liver regeneration [32] and improves cardiac function following myocardial infarction [33].…”

“…Moreover, MSC show potential for immunotherapy for neurodegenerative diseases as they have been shown to modulate microglial activation [15,16]. Several studies have demonstrated the immunomodulatory effects of MSC on microglial in coculture systems involving cell-cell interactions [15,17,18]. However, there are more obstacles and complex effects produced with cell transplantation in the CNS than in other parts of the body [19,20].…”