EFFECTS OF MESENCHYMAL STEM CELL-CONDITIONED MEDIUM ON THE ACTIVATION OF MICROGLIA

Abstract

Microglia are the resident macrophages in the central nervous system that release pro-inflammatory cytokines and undergo proliferation when activated. Therapy by modulating microglia responses may help in controlling the progression of some neurological diseases. Mesenchymal stem cells (MSC) have been reported to have immunomodulatory properties through their secretions. Hence, the immunomodulatory effects of MSC-conditioned medium (MSC-CM), which contains the secretion of MSC, on microglia were investigated in the present study.

High purity MSC cultures from mouse bone marrow have been established with their typical phenotypes characterized and their multi-lineage potential demonstrated. Subsequently, the medium of MSC cultures was designated as MSC-conditioned medium (MSC-CM). Primary microglial cell and BV2 microglial cell line were used in the present study.

MSC-CM altered microglial morphology wherein the microglia exhibited more and longer processes regardless of LPS induction. This implies the ramified state of the microglia. In the LPS-induced microglia, IL-6 and TNF-alpha expressions were significantly attenuated while IL-10 expression was significantly enhanced by MSC-CM. This shows that MSC-CM has the potential to suppress microglial activation or the activity of activated microglia. MSC-CM significantly reduced NFkB and phosphorylated-JNK/c-Jun expressions, suggesting that MSC-CM may modulate microglia activation via these pathways. Since MSC-CM increased MKP-1 expression, suggesting that the MKP-1 may have inhibited JNK/c-Jun in microglia. MSC-CM has no effect on NO production by microglia. MSC-CM significantly increased BV2 microglial proliferation at 6 and 24-hrs incubation. It was found that MSC secreted higher levels of CXCL12, CXCL1, IL-4, IL-10 and IL-11 compared to LPS-induced microglia. Recombinant proteins of these cytokines were individually incubated with LPS-induced BV2 microglia which aimed to reproduce the effects of MSC-CM on microglia. However, only IL-4 and IL-10 recombinant proteins reduced IL-6, TNF-alpha and iNOS expressions in BV2 microglia.

In conjunction with the study on microglial proliferation, the role of Ect2 in microglia has been elucidated. A microarray analysis shows that Ect2 is highly expressed in amoeboid microglia compared to ramified microglia and MSC-CM increased Ect2 expression in BV2 microglia. Therefore, Ect2 was hypothesized to have a role in microglial activation.

Temporal expressions of Ect2 in the corpus callosum of the developing rat brain showed that Ect2 was expressed in amoeboid microglia and was hardly detectable in ramified microglia. More microglia were found to express Ect2 in rat pups injected with LPS. Ect2 was also expressed in primary microglia and BV2 microglia in vitro, and appeared to be increased in the activated microglia. Ect2 gene-knockdown (siEct2) BV2 microglia appeared as giant multi-nucleated cells, suggesting that Ect2 plays a role in the cytokinesis. Cell cycle analysis showed that siEct2 BV2 microglia were arrested at the G0/G1 phase of the cell cycle. This was confirmed through a decrease in cyclin D, CDK4, CDK6 and E2F-1 and increase in p21. Besides, siEct2 causes a decrease in RhoA and Rac1 expressions in BV2, supporting Ect2 as a guanine nucleotide exchange factor. The use of inhibitors on microglia suggests that Ect2 activity is mediated by the PKC pathway.

The present findings demonstrate that MSC-CM shows immunomodulatory effect on microglial activation. Ect2, whose expression in microglia was increased by MSC-CM, plays a role in microglial cytokinesis and siEct2 inhibits microglial proliferation. This study provides insights into the potential use of MSC-CM to modulate microglial activation.