The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination

Abstract

Myelin loss is the hallmark of the demyelinating disease multiple sclerosis (MS) and plays a significant role in multiple neurodegenerative diseases. A common factor in all neuropathologies is the central role of microglia, the intrinsic immune cells of the central nervous system (CNS). Microglia are activated in pathology and can have both pro- and anti-inflammatory functions. Here, we examined the effects of the flavonoid agathisflavone on microglia and remyelination in the cerebellar slice model following lysolecithin induced demyelination. Notably, agathisflavone enhances remyelination and alters microglial activation state, as determined by their morphology and cytokine profile. Furthermore, these effects of agathisflavone on remyelination and microglial activation were inhibited by blockade of estrogen receptor α. Thus, our results identify agathisflavone as a novel compound that may act via ER to regulate microglial activation and enhance remyelination and repair.

Keywords: Agathisflavone; Estrogen receptors; Flavonoids; Microglia; Neuroinflammation; Remyelination.

Graphical abstract

Fig. 1

Agathisflavone enhances remyelination and induces oligodendrocyte proliferation in organotypic cerebellar slices culture. Organotypic cerebellar slices from P10-12 Sox10-EGFP mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Photomicrographs showing the cerebellar white matter stained with MBP (red) and NF (blue); scale bar 20 μm. (B, C) Bar graphs showing the NF + axon index (C) and the percentage of MBP+/NF + myelinated axons (D) per constant field of view (FOV). (D) Oligodendrocyte lineage Sox10-EGFP + cells (green), immunostained for the proliferating marker Ki67 (red) and counterstained with Hoechst nuclear dye (blue); scale bar 20 μm. (E, F) Bar graphs showing the number of Sox10+ cells per FOV (E) and the percentage of SOX10+/Ki67+ cells (F) in a constant FOV. (G) Photomicrographs of OPCs immunolabelled for NG2; scale bar 50 μm. (H) Bar graph showing the number of NG2 + OPCs per FOV. Data are expressed as the mean ± SEM (n = 6); *p < 0.05, *** p<0.001, ****p < 0.0001 (comparing controls to treatment groups); ‡p < 0.05, ‡‡‡p < 0.001, ‡‡‡‡p < 0.0001 (comparing LPC+DMSO to LPC+FAB5 and LPC+FAB10), One-way ANOVA followed by Tukey’s post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Agathisflavone increases mature oligodendrocyte cells number and prevents oligodendrocyte apoptosis. Organotypic cerebellar slices from P10-12 Sox10-EGFP mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Oligodendrocyte lineage cells identified by expression of the Sox10-EGFP reporter (green), immunolabelling for CC1 for mature oligodendrocytes (yellow) and active Caspase 3 for apoptotic cells (red), and counterstained with Hoechst nuclear dye (blue); scale bar 50 μm. (B, C) Individual values column graphs showing the percentage of CC1+ /Sox10+ cells (B), Caspase+/SOX10+ cells (C) and Caspase+/CC1+ cells (D) in a constant FOV; data are expressed as mean ± SEM (n = 4–6); *p < 0.05, ****p < 0.0001 (comparing controls to treatment groups); ‡‡‡‡p < 0.0001 (comparing LPC+DMSO to LPC+FAB5 and LPC+FAB10), One-way ANOVA followed by Tukey’s post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Agathisflavone modifies microglial activation state. Organotypic cerebellar slices from P10-12 Sox10-EGFP mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Microglial proliferation was analyzed by immunolabelling for IBA1 (yellow) and Ki67 (red), counterstained with the nuclear dye Hoechst (blue). (B, C) Bar graph showing the number of IBA1+ microglia (B) and the percentage of IBA1+/Ki67+ proliferating microglia (C); data are expressed as the mean ± SEM (n = 5–11) and tested for significance using One-way ANOVA followed by Tukey’s post-hoc test. (D) Photomicrographs and binary and skeletonized IBA + microglia illustrating morphological differences in the different treatment groups; scale bar 50 μm. (E, F, G) Individual values violin plots of microglial soma size per microglial cell (20 microglial cells/image were analyzed) (E) and violin graphs of process endpoints (F) and length (G) per microglial cell; data are expressed as the median ± IQR; *p < 0.05, ****p < 0.0001 (comparing controls to treatment groups); ‡p < 0.05, ‡‡p < 0.01, ‡‡‡p < 0.001, ‡‡‡‡p < 0.0001 (comparing LPC+DMSO to LPC+FAB5 and LPC+FAB10); ††p < 0.01 (comparing LPC+FAB5 to LPC+ FAB10); Kruskal-Wallis test followed by Dunns. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Agathisflavone modulates microglia-oligodendrocyte interactions. Organotypic cerebellar slices from P10-12 Sox10-EGFP mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Photomicrographs of IBA1 immunostaining (red) and SOX10-EGFP+ oligodendrocytes (green) showing oligodendrocytes-microglia contacts in the different treatment groups; scale bar 20 μm. (B) Diagram illustrating microglial processes contacting oligodendrocytes body (Pr-B), or apposition of microglial and oligodendrocyte cell bodies (B—B). (C) Grouped bar graph showing the number of microglial contacts per SOX10+ cells; data are expressed as the mean ± SEM (n = 6), *p < 0.05, **p < 0.01, ***p < 0.001 (comparing control to treatment groups); ‡‡p < 0.01, ‡‡‡p < 0.001 (comparing LPC-DMSO to LPC+FAB5 and LPC+FAB10; two-way ANOVA followed by Tukey’s post-hoc test. (D) Photomicrographs of slices illustrating Sox10-RGFP + oligodendrocytes (green) and immunolabelling for MBP (red) and Iba1 (yellow), showing interrelationships between microglia, oligodendrocytes, and myelinated fibres in the different treatment groups; clusters of IBA1+ microglia around myelin debris and oligodendrocytes are evident following LPCv treatment and are rarely observed in controls or following agathisflavone treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Agathisflavone promotes a microglial polarization from a M1 to a M2 profile. Organotypic cerebellar slices from P10-12 mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Microglial profile analyzed by double immunofluoresecence labelling for the M1 pro-inflammatory marker CD16/32 (red) and M2 anti-inflammatory marker CD206 (green), where co-expression appears yellow; scale bar 50 μm. (B, C) Bar graphs showing the number of CD16/32+, CD206+ and CD206+/CD16/32+ cells (B) and the M1/M2 ratio (C); data are expressed as the mean ± SEM (n = 6), *p < 0.05, **p < 0.01, ****p < 0.0001 (comparing control to treatment groups); ‡‡‡‡p < 0.0001 (comparing LPC-DMSO to LPC+FAB5 and LPC+FAB10); One-way ANOVA followed by Tukey’s post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Agathisflavone modulates transcript levels of neuroinflammatory genes. Organotypic cerebellar slices from P10-12 mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A, B) Heat map showing the expression of neuroinflammatory genes (A) and respective graphs (B) of RT-qPCR analysis showing the expression of neuroinflammatory genes. (C, D) Heat map showing the expression of regulatory factors (C) and respective graphs (D). Data are expressed as the mean ± SEM or median ± IQR (n = 4); *p < 0.05, **p < 0.01, ***p < 0.001 (comparing control to treatment groups); ‡p < 0.05, ‡‡p < 0.01, ‡‡‡p < 0.001, ‡‡‡‡p < 0.001(comparing LPC-DMSO to LPC+FAB5 and LPC+FAB10); ††p < 0.01 (comparing LPC+FAB5 to LPC+FAB10); samples with Gaussian distribution (bar graphs) were analyzed by one-way ANOVA followed by Tukey’s post-hoc test, non-parametric samples (individual values column graphs) by Kruskal-Wallis followed by Dunns.

Fig. 7

Agathisflavone regulates reactive astrogliosis and is neuroprotective. Organotypic cerebellar slices from P10-12 mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. . (A) Photomicrographs illustrating GFAP-EGFP + astrocytes (green) and Hoescht stained nuclei (blue); scale bar 20 μm. (B) Violin graphs showing the mean fluorescence intensity of GFAP in the different treatment groups. (C) Photomicrographs of Purkinje neurons immunolabelled for Calbindin (yellow) and the apoptosis marker cleaved Caspase-3 (red) and counterstained with Hoechst (blue). The panels on the left side show entire cerebellar lobules and the organization of its layers (ML: Molecular layer; PCL: Purkinje cells layer; GL: Granular layer; WM: White matter); scale bar 50 μm. The remaining panels focus on the PCL; scale bar 50 μm. Insets illustrate individual Purkinje cells ; scale bar 20 μm. (D, E) Violin graphs showing the number of Calbindin + cells per FOV (D) and the percentage of Caspase+ /Calbindin + cells (E). (F, G, H) RT-qPCR analysis Cntf (F), Egfr (G) and Gabbr1 mRNA expression in cerebellar slices in the different treatment groups; data are expressed as the mean ± SEM or median ± IQR (n = 5); *p < 0.05, **p < 0.01, ****p < 0.0001 (comparing control to treatment groups); ‡p < 0.05, ‡‡p < 0.01, ‡‡‡‡p < 0.001 (comparing LPC-DMSO to LPC+FAB5 and LPC+FAB10); †p < 0.05, ††p < 0.01 and ††††p < 0.0001 (comparing LPC+FAB5 to LPC+FAB10); samples with Gaussian distribution (bar graphs) were analyzed by One-way ANOVA followed by Tukey’s post-hoc test, non-parametric samples (individual values column graphs) by Kruskal-Wallis followed by Dunns. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Estrogen receptor (ER) activation is required for agathisflavone to inhibit microgliosis and promote remyelination. (A) Root mean square deviation (RMSD) values and rod representation of crystallographic ligand pose (lilac) and the best pose of this ligand generated by DOCK 6.8 (yellow) for each complex. Distances less than 2 Å between the calculated pose and the crystallographic pose indicates that the program was successful in reproducing the experimental data (B) Representation of interactions between agathisflavone (FAB) and retinoic and estrogen receptors; the captions are described in the figure. (C–G) Organotypic cerebellar slices from SOX10-EGFP animals were maintained for 7DIV, then exposed to LPC for 15–17 h, followed by 2 h pretreatment with the selective ER-α antagonist MPP dihydrochloride at 10 nM (1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole-dihydrochloride), or the selective ER-β antagonist PHTPP at 1 μM (4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol), which were kept together with 10 μM FAB for a further 2 DIV. (C) Oligodendrocytes were identified by the Sox10-EGFP reporter (green) and slices were immunolabeled for MBP (red), neurofilament (blue) and Iba-1 (yellow); scale bar: 20 μm. Bar graphs compare LPC and LPC + FAB 10 μM with the effects of the ER antagonists MPP and PHTPP on the NF + axon index (D), the percentage of MBP+/NF + myelinated axons (E), the number of Sox10-EGFP + oligodendrocytes (F) and the number of Iba1+ microglia (G); data are expressed as the mean ± SEM (n = 5); ‡p < 0.05, ‡‡p < 0.01, ‡‡‡‡p < 0.0001 (comparing LPC-DMSO to other treatment groups); ††p < 0.01 and †††p<0.001 (comparing LPC + FAB10 to LPC + FAB10+MPP); &p < 0.05 (comparing LPC+FAB10+MPP to LPC+FAB10+PHTPP); One-way ANOVA followed by Tukey’s post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)