The secretome of microglia induced by IL-4 of IFN-γ differently regulate proliferation, differentiation and survival of adult neural stem/progenitor cell by targeting the PI3K-Akt pathway

Abstract

Microglia has been reported to be able to regulate the proliferation, differentiation and survival of adult neural stem/progenitor cells (NSPCs) by modulating the microenvironment, which results in different consequences of adult neurogenesis. However, whether the microglial activation is beneficial or harmful to NSPCs is still controversial because of the complexity and variability of microglial activation phenotypes. In this study, we systematically explored the activation phenotypes of IFN-γ- or IL-4-induced microglia at different time after stimulation, and investigated the effects of the secretome of different phenotype of microglia on the process of proliferation, differentiation and survival of NSPCs. Moreover, the possible molecular pathways of secretory influence on NSPCS were further explored using western blotting. The result showed that IFN-γ and IL-4 differently regulate microglial phenotypes, IL-4 induced a M2-like phenotype, while IFN-γ induced a M1-like phenotype. These phenotypes of microglia can only be maintained for 24 h after removal of IFN-γ or IL-4 intervention. The secretome from IFN-γ- or IL-4-induced microglia also had opposite effects on NSPCs proliferation, differentiation and survival. The secretome from the IL-4-treated microglia promoted NSPCs proliferation, survival and differentiation into neurons and oligodendrocytes, while factors secreted by the INF-γ-treated microglia stimulated the NSPCs differentiation into astrocyte, inhibited the neurogenesis and oligodendrogliogenesis, and induced NSPCs apoptosis. Furthermore, the PI3K-Akt pathway mediates the effects of the secretome from IFN-γ- or IL-4-induced microglia on NSPC proliferation, differentiation, and survival. In conclusion, our results suggested that the secretome of microglia induced by IL-4 of IFN-γ differently regulate proliferation, differentiation and survival of adult neural stem/progenitor cell by targeting the PI3K-Akt pathway. These findings will help further study the biological mechanism of microglia regulating neurogenesis, and provide a therapeutic strategy for neurological diseases by regulating microglial phenotypes to affect neurogenesis.

Keywords: Interferon-gamma, Interleukin-4, Neural stem/progenitor cell; Microglia; Neurogenesis; PI3K/Akt pathway.

Fig. 1

Characterization of microglia under IFN-γ or IL-4 intervention conditions. A The fluorescence micrographs of microglia exposed in PBS, IFN-γ or IL-4 for 24 h. Microglia were stained with IBA1 (red) by immunocytochemistry and nuclei were stained with DAPI (blue). Scale bar is 10 μm. And the histogram shows the area of each microglia. B Levels of mRNA encoding anti-inflammatory markers (Arg-1, YM-1, CD206 and IL-10) and pro-inflammatory markers (TNF-α, iNOS, IL-1β and CCR2) in microglia after treatment with PBS, IFN-γ or IL-4 for 24 h and 48 h. Data are showed Mean ± SEM, n = 4–6, *P < 0.05, **P < 0.01, ***P < 0.001 vs Ctrl group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs IL-4 group

Fig. 2

The expression of pro-inflammatory markers and anti-inflammatory markers in microglia after treatment with IFN-γ or IL-4 for 24 h and 48 h. A and B Fluorescence micrographs of the expression of iNOS and Arg-1 when microglia were exposed in PBS, IFN-γ or IL-4 for 24 h. iNOS and Arg-1 was stained with antibody (green). The microglia were stained IBA1 (red) and the nucleus is labeled by DAPI (blue). Scale bar is 10 μm. The histograms represent quantification of the mean fluorescence intensity (MFI) of iNOS or Arg-1 when microglia were exposed in PBS, IFN-γ or IL-4 for 24 h and 48 h. C The histograms show the intracellular and extracellular protein concentrations of TNF-α and TGF-β in microglia were exposed in PBS, IFN-γ or IL-4 for 24 h and 48 h. Data are showed Mean ± SEM, n = 4–6, *P < 0.05, **P < 0.01, ***P < 0.001 vs Ctrl group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs IL-4 group

Fig. 3

Phenotype maintenance of M1 and M2 microglia after removing IFN-γ or IL-4 intervention. A Experimental scheme to monitor phenotype maintenance of microglia after removal of IFN-γ or IL-4 intervention. B Levels of mRNA encoding anti-inflammatory markers (Arg-1, YM-1, IL-10 and TGF-β) and pro-inflammatory markers (BDNF, TNF-α, iNOS and IL-1β) in microglia at 24 h and 48 h after the removal of the intervening factors. C Fluorescence micrographs of the expression of iNOS and Arg-1 at 24 h and 48 h after the removal of the intervening factors in microglia. The iNOS and Arg-1 was stained with antibody (green) and the nucleus is labeled by DAPI (blue). Scale bar is 10 μm. The histograms represent quantification of the mean fluorescence intensity (MFI) of iNOS or Arg-1. Data are showed Mean ± SEM, n = 4–6, *P < 0.05, **P < 0.01, ***P < 0.001 vs Ctrl group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs IL-4 group

Fig. 4

The intracellular and extracellular protein expression of microglia after removal of IL-4 or IFN-γ intervention. A The intracellular protein expression (TGF-β, Arg-1 and iNOS) in IL-4- and IFN-γ-treated microglia at 0 h, 24 h and 48 h after removal of intervention. B The extracellular protein (IL-10, TGF-β, TNF-α and IL-1β) in IL-4- and IFN-γ-treated microglia at 0 h, 24 h and 48 h after removal of intervention. Data are showed Mean ± SEM, n = 4–6, *P < 0.05, **P < 0.01, ***P < 0.001 vs Ctrl group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs IL-4 group

Fig. 5

Effects of the secretome from IL-4- or IFN-γ-treated microglia on proliferation of NSPCs. A Experimental scheme to monitor microglia-conditioned medium on neurosphere size of adult NSPCs. B Micrographs of adult NSPCs treated with PBS, IL-4 or IFN-γ or co-cultured with PBS-, IFN-γ- or IL-4-treated microglia for 24 h. The histograms represent the number of neurospheres in each well and the neurosphere size. C Experimental scheme to monitor microglia-conditioned medium on proliferation of adult NSPCs. D Fluorescence micrographs of BrdU⁺ cells from NSPCs exposed to PBS, IL-4 or IFN-γ or microglia-conditioned medium for 24 h. The BrdU⁺ cells were stained with antibody (green) and nucleus is labeled by DAPI (blue). Scale bar is 10 μm. The histogram represents quantification of percentage of BrdU⁺ cells from adult NSPCs. Data are showed Mean ± SEM, n = 4–6, *P < 0.05, **P < 0.01 vs PBS group, ^##P < 0.01, ^###P < 0.001 vs IL-4 group

Fig. 6

Effects of the secretome from IL-4- or IFN-γ-treated microglia on differentiation of NSPCs. A Experimental scheme to monitor microglia-conditioned medium on differentiation of adult NSPCs. B Fluorescence micrographs of MAP2⁺ cells from NSPCs differentiate in PBS, IL-4 or IFN-γ or microglia-conditioned medium for 7 days. The MAP2⁺ cells were stained with antibody (red) and nucleus is labeled by DAPI (blue). Scale bar is 10 μm. The histogram represents quantification of percentage of MAP2⁺ cells from adult NSPCs. C Fluorescence micrographs of NG2⁺ cells from NSPCs differentiate in PBS, IL-4 or IFN-γ or microglia-conditioned medium for 7 days. The NG2⁺ cells were stained with antibody (green) and nucleus is labeled by DAPI (blue). Scale bar is 10 μm. The histogram represents quantification of percentage of NG2⁺ cells from adult NSPCs. D Fluorescence micrographs of GFAP⁺ cells from NSPCs differentiate in PBS, IL-4 or IFN-γ or microglia-conditioned medium for 7 days. The GFAP⁺ cells were stained with antibody (green) and nucleus is labeled by DAPI (blue). Scale bar is 10 μm. The histogram represents quantification of percentage of GFAP⁺ cells from adult NSPCs. Data are showed Mean ± SEM, n = 4–6, *P < 0.05, **P < 0.01 vs PBS group, ^###P < 0.001 vs IL-4 group

Fig. 7

Effect of secretome from from IL-4- or IFN-γ-treated microglia on survival and proliferation via PI3K/Akt pathway in adult NSPCs. A Fluorescence micrographs of apoptotic cells from NSPCs differentiate in PBS, IL-4 or IFN-γ or microglia-conditioned medium for 7 days. The apoptotic cells were stained with Cleaved-caspase 3 (green) by immunocytochemistry and nucleus is labeled by DAPI (blue). Scale bar is 10 μm. B The histogram represents quantification of percentage of CC-3⁺ cells. Data are showed Mean ± SEM, n = 4–6, *P < 0.05 vs PBS group, ^###P < 0.001 vs IL-4 group

Fig. 8

Effects of the secretome from IFN-γ- or IL-4-induced microglia on PI3K/Akt signaling pathway during NSPCs proliferation or differentiation. A–D Western blotting showing the levels of PI3K, Akt, and phospho-Akt (p-Akt) in NSPCs under different treatment conditions. Levels of PI3K and Akt were normalized to those of β-actin, and levels of p-Akt was normalized to those of Akt. Data are showed Mean ± SEM, n = 4–6, *P < 0.05, **P < 0.01, ***P < 0.001 vs PBS group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs IL-4 group